Friedrich Miescher Institute Report 2005 / 2006

Report 2005 / 2006

Friedrich Miescher Institute
 Contents
2 Introduction

Neurobiology 6 Silvia Arber Neuronal circuit formation

in the developing spinal cord
9 Pico Caroni Plasticity of neuronal circuits

13 Andreas Lthi Cellular mechanisms of learning and memory

16 Andrew Matus Molecular mechanisms of synaptic plasticity

19 Denis Monard Control of endogenous extracellular proteases

in development and brain function

23 Thomas Oertner Physiology and plasticity of individual synapses

26 Botond Roska Structure and function of local neural circuits

Epigenetics 30 Rafal Ciosk Mechanisms of totipotency in the germline

33 Witold Filipowicz Mechanisms of RNA interference

and microRNA function in mammalian cells

37 Susan Gasser Functional organization of the nucleus

41 Helge Grosshans Regulation of animal development

by microRNAs

44 Patrick Matthias Regulation of gene expression by POU proteins,

their coactivators and chromatin regulators

47 Frederick Meins Plant epigenetics and RNA silencing

50 Antoine Peters Epigenetic programming in the mammalian

germ line and pre-implantation embryos

53 Dirk Schbeler Dynamics and propagation of epigenetic states

Growth Control 58 Joy Alcedo Neural mechanisms that influence

the aging process

61 Ruth Chiquet Cell communication in growth control

and differentiation

65 Brian A. Hemmings Protein kinase function in development

and cancer

69 Jan Hofsteenge Molecular and functional aspects of

covalent protein modifications

72 Nancy Hynes The molecular basis of breast cancer

76 Yoshikuni Nagamine Regulation of the plasminogen activator

system with emphasis on the DExH
helicase RHAU

79 Administration and services

80 Acknowledgements
81 Publications and dissertations
88 Lectures and seminars
96 Teaching
97 Technology platforms
100 Students and postdocs
104 Visiting the FMI

FMI Report 2005/2006 1

 The Friedrich Miescher Institute The FMI is
devoted to fundamental biomedical research.
We employ new technologies to explore
basic molecular mechanisms of cells and
organisms in health and disease.
NOVELTY AT THE FMI
One of our goals at the FMI is to pursue innovative
research on topics of biomedical interest. A con-
tinuous stream of discoveries of pathways and
components controlling cell growth and differen-
tiation will be necessary to turn the watermill of
drug development. With this in mind, the FMI has
sought to attract bright young minds and to give
them strong core funding and outstanding tech-
nological support. The last few years have seen the
arrival of seven new group leaders and a major ex-
pansion in live imaging facilities, bioinformatics,
histology and the establishment of a protein struc-
ture platform. Indeed, although the FMI has cele-
brated its 35th year, signs of aging are few. Even our
neighbourhood has novelty to offer in the form of
two new university institutes that have joined us
on the evolving Rosental campus.
FMI research in the field of epigenetics has been
enhanced by the appointment of two new group
leaders, Rafal Ciosk and Helge Grosshans. Rafal
arrived from the Fred Hutchinson Cancer Research
Center, Seattle and will work on the molecular basis
of totipotency. Helge was previously at Yale and is
interested in the molecular mode of action and de-
velopmental roles of microRNAs. Both will exploit
the powerful genetics and developmental system
of the nematode C. elegans. This expansion fosters
a creative environment at the FMI in which analysis
of a model organism like the worm is skilfully ex-
ploited to address previously intractable questions
of biomedical relevance.
Botond Roska arrived from Harvard in 2005 and
his laboratory is the latest in a series of FMI groups
using high-resolution optical imaging, electrophys-
iology and in vivo tagging of neuronal cells. His
research using the mammalian retina as a model
system sharpens the focus of our neurobiology
unit on neuronal circuitry. Network function will
also be the topic of Rainer Friedrich, who will join
us from the Max Planck Institute in Heidelberg in
2007 to continue his groundbreaking studies on
the physiology of Zebrafish olfaction.
New group leaders arriving in 2006 include Mo-
2 FMI Report 2005/2006
hamed Bentires-Alj from Harvard Medical School,
whose work on protein tyrosine phosphatases in
cancer and metastasis will synergize with studies of
metastasis in the Hynes laboratory. Nicolas Thom,
a structural biologist from the Memorial Sloan
Kettering Cancer Center, will pursue work on pre-
mature aging syndromes linked to cancer and ge-
nomic instability. A protein structure facility will
be established at the FMI under his guidance.
Fang-Lin Sun, a group leader working on the dy-
namics of chromatin structure in transcriptional
gene regulation during development, has left the
FMI after almost 6 years to return to China. Lin
will take up a position at the School of Medicine,
Tsinghua University, Beijing. We wish him good
luck and much success in his further career.
The broad approach at the FMI to the regulation
of the human genome and proteome is increasingly
dependent upon high-throughput techniques to
quantify changes in gene expression and protein
modification and to relate gene expression to the
genome-wide distribution of specific chromatin
marks. The novel MeDIP assay of Dirk Schbeler
at the FMI is one example of how these approaches
can provide an overview of genome-wide events.
Such techniques require the means to integrate and
analyse large amounts of data and the know-how
to model the changes and their functional impact
on cells. This requires specialized instruments, but
equally important is the availability of highly trained
technical staff. The last two years have seen con-
siderable expansion at the FMI in the hardware,
software and the personnel of our central technical
services, especially analytical and preparative fluo-
rescence activated cell sorting, mass spectrographic
protein analysis, tissue preparation and histology,
and the fluorescence imaging of living cells and con-
focal microscopy. For the first time, we have dedica-
ted a chapter of this Report to our technical facilities.
Since November 2004, the FMI has enjoyed the
presence of the Centre for Biomedical Research of
the University of Basel here in what is becoming
known as the Biopark Rosental. As part of the uni-
versity Department of Clinical and Biological Sci-
ences, the Centre is led by Gerhard Christofori and
hosts 120 research scientists in the fields of oncology
and immunology, cell plasticity and tissue repair.
Further good news for the FMI (and for research
in Basel in general) came with the decision of the
ETH in Zurich to locate a new Centre of Biosystems
Science and Engineering next door to the FMI.
This will be one of the key links in the Swiss Ini-
tiative in Systems Biology Systems X and will be
directed by Renato Paro. Renatos interest in epige-
netics strongly reinforces one of our major research
themes here at the FMI.
As a central component of our institute we now
count about 180 students and postdocs pursuing
PhDs or postgraduate training. Apart from their
studies and research contributions, the students
and postdocs at the FMI are engaged pro-actively
in science education and career development. They
are to be congratulated for their organisation of
frequent lectures and workshops, including the
impressive series of Students Science Colloquia
that have so far featured 20 internationally renowned
speakers, including several Nobel laureates.A further
student initiative in 2005 was the Career Guid-
ance Conference in Life Sciences under the motto
of aspire,advance,achieve.Over 450 students from
as far away as Strasbourg and Geneva participated
in this highly successful event.
In the context of expanding links between current
and past members of the FMI, we held the first ever
FMI Alumni Day on March 15, 2006. Former FMI
collaborators working in Switzerland met during
the 1-day event, which included talks by alumni
and current junior group leaders. The afternoon
saw a lively round-table discussion chaired by Susan
Gasser on the subject of Fundamental Research and
Pharmaceutical Developments. The enthusiasm
shown by the alumni and current FMI members
alike guarantees a repeat performance.
Many honours have been bestowed on FMI
members over the last two years. We congratulate
both Ruth Chiquet and Patrick Matthias on being
named Titular professor of the University of Basel.
Denis Monard has been elected the incumbent
Susan Gasser
President of the Swiss Academy of Sciences and
Brian Hemmings received both the prestigious
Novartis Corporate Research Award and the lu-
crative Swiss Bridge Award for scientific excellence.
Silvia Arber was elected to the Swiss Academy
of Medical Sciences and as an EMBO member,
and also received the Schellenberg Prize for her
research on neuro-muscular junctions. Witold
Filipowicz was elected to the Polish Academy of
Sciences and to Academia Europea. Finally, FMI
postdocs also feature in the awards list with Oliver
Tschopp winning the Swiss Diabetes Association
Prize 2005, Yann Humeau together with Andreas
Lthi receiving the Pfizer Research Prize in Neu-
roscience 2006 and Ramesh Pillai taking both
the Scaringe Prize 2005 of the International RNA
Society and the Prize for Young Biochemists from
the Federation of European Biochemical Societies.
As the FMI looks forward to an exciting future
of research, it is also grateful for the continuing
support and advice from Paul Herrling of the No-
vartis Research Foundation and former FMI Di-
rector Max Burger. While we embrace the power-
ful possibilities before us, it is useful to remember
that nothing in science would ever be achieved if
it were not for the fact that we stand on the shoul-
ders of giants.
Susan Gasser Patrick King
Director Editor
FMI Report 2005/2006 3
 Neurobiology The formation of neuronal
connections during development and their
maintenance in adulthood are crucial
determinants of nervous system function
in health and disease. We use molecular
and genetic techniques to explore the
cellular mechanisms that determine how
circuits are first made and later modified
to support animal behaviour.

Silvia Arber
Neuronal circuit formation
in the developing spinal cord

Pico Caroni
Plasticity of neuronal circuits

Andreas Lthi
Cellular mechanisms of learning
and memory

Andrew Matus
Molecular mechanisms of
synaptic plasticity

Denis Monard
Control of endogenous extracellular
proteases in development and
brain function

Thomas Oertner
Physiology and plasticity of
individual synapses

Botond Roska
Structure and function of local
neural circuits

4 FMI Report 2005/2006

FMI Report 2005/2006 5
Neurobiology

Silvia Arber
Neuronal circuit
formation in
the developing
spinal cord

INTRODUCTION
The innervation of neuronal targets is a precisely
timed process that requires the pathfinding of ax-
ons to specific targets, the growth and branching
of axons in the vicinity of their targets and the for-
mation of stable synaptic connections. The aim
of our studies is to understand the molecular and
mechanistic basis of the establishment of specific
neuronal connections within a circuit of intercon- GROUP LEADER
nected neurons. A deep knowledge of the logic of Silvia Arber
how neuronal circuits are assembled during de- silvia.arber@fmi.ch
velopment and which molecules are involved in the
establishment of neuronal circuits may contribute TECHNICAL/RESEARCH
to our understanding of the functioning of the ASSOCIATES
mature nervous system. Monika Mielich
We aim to elucidate the principles of neuronal Markus Sigrist
circuit formation in the developing vertebrate
spinal cord. In the spinal monosynaptic reflex cir- POSTDOCTORAL
cuit (Fig. 1), many details of early neuronal speci- FELLOWS
fication as well as mature connectivity are already Simon Hippenmeyer*
well understood. We focus mainly on the develop- Ina Kramer*
ment of motor neurons in the ventral horn of the David Ladle
spinal cord and proprioceptive afferents (Ia affer- Stan Takumi Nakanishi
ents) in dorsal root ganglia (DRG) that establish
monosynaptic connections with motor neurons. PhD STUDENTS
It is thought that many of the decisions taken by Simon Dalla Torre
these neurons during early development, when Andreas Friese
they start extending axons and dendrites, are con- Jun Lee
trolled by cell-intrinsic factors and are determined Vera Niederkofler*
even at stages before they exit the cell cycle. Dif- Rishard Salie*
ferent classes of transcription factors have been Anna Stepien
shown to control sequential steps in the differen- Eline Vrieseling
tiation hierarchy of motor neurons in the devel-
oping spinal cord. In addition to specification of UNDERGRADUATES
neurons by cell-intrinsic cues, signaling interac- Jennifer Henaghan*
tions with cues encountered by axons en route to Roland Huber
the target or from the target region itself also have Celia Lngle*
important roles in the specification of later steps Thomas Portmann*
of neuronal connectivity (Hippenmeyer et al.2004). Claudia Suenderhauf*
To unravel the cascades of molecules controlling
neuronal circuit formation, we combine gain- and
loss-of-function mouse genetics, light microscope *left the group
imaging of fluorescently labeled neuronal sub-
populations, electrophysiological analysis and gene
expression profiling.

6 FMI Report 2005/2006

FUNCTION OF ETS TRANSCRIPTION FACTORS la Afferent
IN THE ASSEMBLY OF THE VERTEBRATE MONO- Neuron in DRG
SYNAPTIC REFLEX CIRCUIT
S. Hippenmeyer, I. Kramer, D. Ladle, C. Lngle, Muscle
Spindle
T. Portmann, M. Sigrist, E. Vrieseling

Previously, we identified two molecular pathways

linking specific peripherally derived signals to in-
duction of the ETS transcription factors Pea3 and
Er81 in defined subsets of motor neurons (Pea3)
and dorsal root ganglia (DRG) sensory neurons
(Er81). Furthermore, these retrograde signals from
an intermediate target region act selectively to con-
trol progressive specification and differentiation of
distinct neuronal subpopulations through the ac-
tivation of the ETS transcription factors Pea3 and
Er81 (Hippenmeyer et al. 2004). The studies clearly
demonstrated that neurons are not completely
predetermined intrinsically with respect to the
establishment of their target innervation, but re-
quire retrograde signals from their intermediate
target region to further control their specification
and differentiation. The results provide molecular
evidence for the existence of such signaling path-
ways and show that specific peripherally derived
signals are required for the induction of ETS tran-
scription factors in defined subsets of motor neu-
rons and DRG sensory neurons.
Recently we asked how the temporal precision of
the expression and activity of an ETS transcription
factor controls the assembly of a neuronal circuit.
We showed that upregulation of ETS transcription
factor signaling specifically at a late stage of propri-
oceptive DRG sensory neuron differentiation is im-
Motor
Neuron
Extrafusal
Muscle Fiber
Fig.1. Schematic represen-
RUNX TRANSCRIPTION FACTORS AND DRG tation of the mature spinal
NEURON SPECIFICATION monosynaptic reflex circuit.
Motor neurons project to
I. Kramer, M. Sigrist extrafusal muscle fibers
and receive direct synaptic
Neuronal differentiation involves the acquisition input from Ia afferent sen-
of many specialized molecular properties essential sory neurons
for later neuronal function. The emergence of dis-
tinct subpopulations of DRG neurons is controlled
by the selective activation of distinct transcriptional
programs. However, the transcriptional mecha-
nisms controlling the fragmentation of neuronal
classes into subtypes of primary sensory neurons
remain obscure.
In a screen to identify genes selectively expressed
by functionally distinct subpopulations of DRG
neurons, we identified two members of the Runx
family of transcription factors as markers for TrkC+
proprioceptive afferents (Runx3) and TrkA+ cuta-
neous afferents (Runx1) (Kramer et al. 2006; Fig.3).

Wild-type Mutant

A B
portant for the appropriate developmental func-
tion (Hippenmeyer et al. 2005). Premature ETS
signaling interferes with establishment of neuronal
projections, acquisition of terminal neuronal traits
and dependence on neurotrophic support (Fig. 2).
In contrast, late expression of the identical ETS
transcriptional regulator in the same neuronal lin-
eage can substitute for ETS gene function and pro-
mote neuronal differentiation. Together, the findings
suggest that DRG sensory neurons undergo a tem-
poral developmental switch, revealed by distinct
responses to ETS transcription factor signaling at
sequential steps of neuronal maturation.
Fig.2. Culture of DRG
Using genetic approaches in the mouse, we exam- isolated from wild-type em-
ined whether Runx transcription factor activity bryos (A) or mutant em-
contributes to the acquisition of selective profiles bryos with premature ETS
signaling (B) cultured
of neurotrophic factor receptor and neuropeptide
without neurotrophic sup-
expression in different subpopulations of DRG port (left) or in presence
sensory neurons, traits associated with function- of NGF (right). Mutant
ally distinct neuronal subpopulations that ensure DRG neurons survive and
their maturation.We found that runt domain tran- grow axons in the absence
scription factor signaling is essential for the emer- of neurotrophic support
gence of key aspects of subpopulation character
in sensory neurons, apparently by repressing alter-
nate traits. Specifically, we found that Runx3 con-
trols the establishment of a proprioceptor pheno-

FMI Report 2005/2006 7

Neurobiology

pressed in complementary patterns in the nervous

A B
Runx3
Parv
Fig.3. Runx transcription
factor expression in defined
subpopulations of DRG
neurons. In embryonic neu-
rons, Runx3 expression
coincides with Parvalbumin
and marks proprioceptive
afferents (A). In contrast,
Runx1 expression does not
overlap with Parvalbumin
and marks cutaneous affer-
ents (B)
Runx1
Parv
type by promoting a selective TrkC phenotype
through erosion of TrkB expression, whereas Runx1
expression within TrkA+ cutaneous afferents re-
presses the expression of the neuropeptide CGRP.
Our findings thus provide evidence that Runx
transcription factor signaling controls three criti-
cal elements of DRG neuronal phenotype: neuro-
trophin sensitivity, peptidergic neurotransmitter
profile and axonal targeting. Runx proteins serve
as key transcriptional intermediaries in the as-
signment of the functional features of DRG sen-
sory neuron subclasses that underlie the early steps
of somatosensory processing.
FUNCTIONAL ANALYSIS OF REPULSIVE
GUIDANCE MOLECULE (RGM) FAMILY MEMBERS
V. Niederkofler, R. Salie, M. Sigrist
system, including the spinal cord and superior col-
liculus. Functional studies in the mouse revealed
a role for mRGMa in controlling cephalic neural
tube closure but not anterior-posterior targeting of
RGC axons to their stereotypic termination zones
in the superior colliculus (Niederkofler et al. 2004).
The third member of the family (mRGMc) is
expressed in skeletal muscles. Moreover, mouse
mRGMc is selectively expressed within the liver by
periportal hepatocytes (Niederkofler et al. 2005).
Our recent analysis of mRGMc mutant mice re-
vealed that mRGMc is involved in the control of
iron metabolism and its mutation leads to juvenile
hemochromatosis, a frequently fatal disease with
early onset of iron accumulation and absence of
hepcidin expression. Moreover, mice mutant in
mRGMc fail to express hepcidin in response to di-
etary or injected iron, thus providing a molecular
explanation for the severe iron accumulation ob-
served in mRGMc mutant mice. In contrast, these
mice retain the ability to upregulate hepcidin ex-
pression in response to acute inflammation induced
by LPS or its downstream products IL-6 or TNF a.
We also showed that mRGMc expression in wild-
type mice upon induction of inflammation is se-
lectively downregulated in the liver but not in
skeletal muscle. These results together with previ-
ous observations suggest that inflammation in-
duces temporary elimination of iron sensing by
downregulation of hepatic mRGMc. Our data also
suggest that mRGMc plays an essential role in the
regulation of hepcidin expression selectively through
the iron-sensing pathway and define a key role for
mRGMc in dietary iron sensing. Moreover, mRGMc
mutant mice provide a useful animal model for the
iron overload disease juvenile hemochromatosis.

Repulsive Guidance Molecule (RGM) has been im-
plicated in the control of the topography of reti-
nal ganglion cell (RGC) axon termination zones
along the anterior-posterior axis of the chick tec-
tum. The molecular identification of RGM re-
cently opened the way to functional studies in the
mouse. We found that there are three mouse pro-
teins homologous to chick RGM. Two members
of this gene family (mRGMa and mRGMb) are ex-
This work was carried out in collaboration with
P. Caroni (Friedrich Miescher Institute, Basel), J. A.
Hassell (McMaster University, Ontario), C.E. Hen-
derson (Columbia University, New York), T.M. Jessell
(Columbia University, New York), K. M. Murphy
(Washington University, St. Louis), A.Pierani (Ecole
Normale Suprieure, Paris) and W. D. Snider (Uni-
versity of North Carolina, Chapel Hill).

Selected publications
Hippenmeyer S, Kramer I, Arber S (2004)
Control of neuronal phenotype: what tar-
gets tell the cell bodies. Trends Neurosci
27:482-488
Hippenmeyer S, Vrieseling E, Sigrist M,
Portmann T, Laengle C, Ladle DR,
Arber S (2005)
A developmental switch in the response
of DRG neurons to ETS transcription factor
signaling. PLoS Biol 3:e159
Kramer I, Sigrist M, de Nooij JC, Salie R, Niederkofler V, Arber S (2005)
Taniuchi I, Jessell TM, Arber S (2006) Patterning molecules: multitasking in the
A role for Runx transcription factor signal- nervous system. Neuron 45:189-192
ing in dorsal root ganglion sensory neuron
diversification. Neuron 49:379-393
Niederkofler V, Salie R, Arber S (2005)
Hemojuvelin is essential for dietary iron
sensing, and its mutation leads to severe
iron overload. J Clin Invest 115:2180-
2186

8 FMI Report 2005/2006

Pico Caroni
Plasticity of
neuronal circuits

INTRODUCTION
Transmission of information in the nervous sys-
tem takes place at unique intercellular contact sites,
the synapses. We investigate regulatory mecha-
nisms that control the formation, maintenance
and turnover of synaptic connections (anatomical
plasticity). We are particularly interested in dis-
covering and understanding mechanisms that de-
termine the plasticity of defined neuronal circuits, GROUP LEADER
as they may inform us about principles of learning, Pico Caroni
adaptation, and resilience to disease in the nervous pico.caroni@fmi.ch
system.
To capture specific plasticity that might go un- TECHNICAL/RESEARCH
detected using more reductionistic strategies, we ASSOCIATES
rely on detailed anatomical maps of how individ- Corinna Schneider
ual defined neurons are inserted into the circuit, Lan Xu
and on experimental approaches that allow us to
monitor and manipulate precisely defined ele- POSTDOCTORAL
ments of the circuit. FELLOWS
Applying this approach to the neuromuscular Ewa Bednarek
system, we investigate mechanisms underlying Tamara Brown*
intrinsic and age-dependent differences in the Alexandre Ferrao Santos
anatomical plasticity of defined synapses, as well Smita Saxena
as mechanisms of selective vulnerability to disease
in neurodegeneration. We find that the arrange- PhD STUDENTS
ment of neuromuscular subsystems differing in Michael Abanto
plasticity and vulnerability properties is such that Yuichi Deguchi
selective manipulation and molecular analysis are Ivan Galimberti
feasible up to the level of identified cell genomics Nadine Gogolla
and proteomics. This type of approach also guides Sharon Lefler*
our research on anatomical plasticity in the hippo- Sarah Rdiger
campus, a brain structure that plays a critical role Anirban Sadhu*
in learning and memory. We are discovering spe- Ivana Samarzija*
cific life-long synaptic rearrangements influenced Claudia Vittori
by experience in an axonal projection with major Stefan Wacha
functional significance to hippocampal function.
We apply combinations of mouse genetics, be-
havioral analysis, neuroanatomy, live imaging, tis- *left the group
sue and cell culture, cell biology, proteomics, and
single cell genomics. To visualize neurons and their
subcellular components in situ, we use transgenic
mice expressing targeted fluorescent chimeric pro-
teins in single neurons.

FMI Report 2005/2006 9

Neurobiology
INTRINSIC MECHANISMS CONTROLLING
NERVE GROWTH
T. Golub, S. Wacha
Process outgrowth and synaptic rearrangements
coincide with the expression of specific sets of
genes in neurons, including the GAP43-like pro-
teins GAP43, CAP23, MARCKS, and MacMAR-
CKS. Functional studies in vivo have provided
strong evidence that GAP43-like proteins are in-
trinsic determinants of anatomical plasticity, con-
ferring competence for morphogenetic processes
to cells that express them. GAP43 is particularly
effective in augmenting stimulus-induced nerve
sprouting and synaptic growth, whereas CAP23
stabilizes dynamic structures. We found that the
combined expression of GAP43 and CAP23 in
adult neurons is sufficient to confer competence
for lesion-induced regeneration of axons in the
adult. Within cells, GAP43-like proteins accumu-
late at lipid rafts, where they codistribute with the
dentrites
Fig.1. Hippocampal mossy
fiber connectivity onto lipid second messenger PI(4,5)P2, and modulate
pyramidal neurons in CA3. surface accumulation and trafficking of rafts in
Left: Mossy fiber axons response to calcium/calmodulin and PKC.
(thin white lines) and two
We recently investigated how PI(4,5)P2-rich rafts
pyramidal neuron dendrites
(with spines) expressing
accumulate and organize to influence protrusive
surface GFP. Arrows: motility at the cell surface. We showed that signals
cream, LMTs; red, LMT triggering lamellipodial motility at the cell edge in-
contacting long segment duce rapid local accumulation of dynamic choles-
of a dendrite. Right: terol- and PI(4,5)P2-rich raft-based plasmalemmal
Schematic drawing of domains with unique turnover properties for acy-
same CA3 field. LMTs of
lated raft components and PI(4,5)P2. We further
the same complex have
the same color; bar 4 m
showed that the patches capture and stabilize MT
plus ends through patch-associated IQGAP1. MTs
in turn promoted the clustering of raft patches into
spatially focused and temporally stable domains,
restraining and polarizing motility. Taken together,
our results suggest a two-step model for local con-
trol of motility and polarization in which: 1) local
signaling at the cell surface induces raft patching
through PI(4,5)P2 and Cdc42 to promote motility;
2) clustering of the patches into more stable plat-
forms through MTs and PKA polarizes and orga-
10 FMI Report 2005/2006
nizes motility. As there are extensive similarities
among the molecular requirements for processes
involving sustained polarized signaling at the cell
surface, these principles for local control of signal-
ing and polarization through raft assembly and or-
ganization likely apply to further cellular processes.
MECHANISMS CONTROLLING ANATOMICAL
PLASTICITY AT NEUROMUSCULAR JUNCTIONS
A.F. Santos, M. Abanto
The formation of synapses during development
and their modification in the mature organism are
thought to involve reciprocal interactions between
pre- and postsynaptic cell types. Although much is
known about molecules associated with pre- and
postsynaptic structures in the mature state, a clear
understanding of the initial steps in synapse for-
mation requires a more precise analysis of the in-
dividual contributions of pre- and postsynaptic
partners to this process. Nerve-muscle synapses
(NMJs) are well suited to studies of the relative
roles of pre- and postsynaptic elements in the for-
mation of synaptic connections, because of the rel-
ative accessibility of presynaptic motor neurons
(MN) and postsynaptic muscles.
We found that skeletal muscles can be subdi-
vided into two previously unrecognized subtypes,
designated FaSyn and DeSyn muscles, which ex-
hibit marked differences in the rate of assembly
of stable synapses. The differences reflect muscle-
intrinsic, nerve-independent, programs of (post-
synaptic) focal AChR clustering. While NMJs on
FaSyn and DeSyn muscles exhibit a comparable
anatomical organization in postnatal mice, treat-
ments that challenge synaptic stability in young
adult mice result in the selective remodeling and
disassembly of NMJs on DeSyn muscles, whereas
those on FaSyn muscles remain stable.
We recently exploited the differences between
DeSyn and FaSyn muscles to investigate the mole-
cular mechanisms controlling remodeling at the
NMJ. We showed that nerve sprouting and synapse
remodeling are controlled locally through an in-
hibitory mechanism involving the assembly state
of the postsynaptic apparatus. Thus, disruption of
the postsynaptic apparatus was sufficient to induce
sprouting in any muscle. We further showed that
induction of plasticity depended on altering the
permissiveness state at individual NMJs and mus-
cle fibers. In postnatal mice, permissiveness was
conferred gradually by a process triggered by the
absence of miniature events, limited to young adult
DeSyn muscles, and involving mTOR1 and the
proteasome. These results from experimentally ac-
cessible NMJs may provide a conceptual frame-
work to account for how synaptic activity and con-
tact-mediated mechanisms interface to regulate
anatomical plasticity at individual synapses.
ANATOMICAL PLASTICITY IN ADULT HIPPO-
CAMPAL CIRCUITS AND ITS REGULATION BY
EXPERIENCE
I. Galimberti, N. Gogolla, Y. Deguchi, S. Ruediger
Sustained rearrangements of synaptic connections
can provide mechanisms to alter connectivity in
neuronal circuits and encode experience in the
brain. Recent time-lapse imaging studies in neo-
cortex indicated the appearance and disappear-
ance of fractions of synaptic structures in the adult
and showed that the frequency of these events
can be influenced by sensory experience. However,
these studies imaged groups of either pre- or post-
synaptic elements within small regions of neuropil
and, thus, could not assign complete sets of
synapses by individual identified presynaptic neu-
rons to their postsynaptic targets. Consequently, it
was unclear to what extent synapse rearrangement
processes in the adult produce net alterations
in the numbers of synaptic connections between
identified synaptic partners. For the same reasons,
it was also not known whether and under what
circumstances repeated rearrangement processes
can lead to incremental shifts of connectivity in the
adult. To address these questions, we focus on well-
characterized circuitry that has been implicated
in experience-related anatomical plasticity and
which is accessible to large-scale repeated imaging
over long periods.
We recently exploited transgenic mice express-
ing membrane-targeted GFP in only few neurons
(Thy1-mGFP s ) and high-resolution imaging to in-
vestigate the connectivity of hippocampal Large
Mossy fiber Terminals (LMTs) in fixed mouse tis-
sue and organotypic slice cultures. We found that
LMTs are highly heterogeneous in vivo and in slice
cultures and that many are connected through 10-
to 200-m processes to satellite LMTs that can CELL-SPECIFIC PATHWAYS OF SELECTIVE AX-
contact distinct pyramidal neurons in CA3 (Fig. 1).
LMTs are thus components of local presynaptic
terminal arborization complexes (LMT-Cs) by
mossy fibers, exhibiting varying degrees of diver-
gence with respect to their local targets in CA3.
We then showed that LMT-Cs exhibit pronounced
long-term rearrangements in the adult and pro-
vided evidence for two distinct types of rearrange-
ments: 1) life-long gradual growth of the largest
LMT-Cs along pyramidal cell dendrites; 2) dra-
matic increase in the complexity of many LMT-Cs
in mice housed in an enriched environment (EE).
We finally showed that subsets of LMT-Cs exhibit
comparable rearrangements and growth over
weeks and months in slice cultures, that these
anatomical rearrangements reflect functional re-
arrangements in the local connectivity of LMT-Cs
with pyramidal neurons, that heterogeneities in
plasticity and growth reflect local properties of in-
dividual LMT-Cs, and that LMT-C maintenance
LMT-Cs
groth
(age)
Pyramidal dendrite
complexity
(EE)
Fig.2. Local rearrangements
and growth are regulated by synaptic activity, bring changes in functional
mGluR2-sensitive transmitter release from LMTs, connectivity between mossy
and PKC. Taken together, these results demonstrate fiber LMT-Cs and CA3 pyra-
midal neurons. Grey box:
the existence of sustained local rearrangements
dendrite; blue and green
of connectivity by defined terminal arborization ovals: LMT-Cs. Left: Blue
structures regulated by activity in the adult. and green activation pat-
The expansion and activity-regulated diver- terns elicit weak postsynap-
gence of LMT-C subsets along pyramidal cell tic responses. Upper right:
dendrites in CA3 throughout life might result in Potentiation of blue re-
increased focusing of information flow from indi- sponse through expansion.
Lower right: Potentiation
vidual mossy fibers to a local segment of the asso-
of blue response through
ciative network in CA3, mediating the emergence satellites
of microcircuits of preferentially interconnected
neurons (Fig. 2). In addition to its effects on LMT
size distribution, EE specifically increased the fre-
quency of satellites by LMT-Cs and the lengths and
complexities of postsynaptic thorny excrescences.
The increased complexity of LMT-Cs and thorns
under EE conditions might support hippocampal
learning by providing more opportunities for lo-
cal convergence of co-active terminals onto pyra-
midal neurons in CA3 (Fig. 2).
ONAL VULNERABILITY IN MOTONEURON DISEASE
S. Saxena, S. Pun
Just as experience-dependent rearrangements in
neuronal circuitry may mediate learning and the
acquisition of new skills, losses in circuitry may
play a central role in neurodegenerative and psy-
chiatric diseases. Our studies are based on the hy-
pothesis that selective losses of axons and synapses
represent key early processes in diseases affecting
the nervous system, and that disease progression
involves increasing failure by neural systems to
cope with disease through plasticity and repair.
In neurodegenerative diseases, years of slowly
progressing and clinically undetectable alterations
and losses set the stage for devastating clinical
phases, when treatments have produced disap-
pointing results. Synapses are lost early in disease,
but the mechanisms underlying this vulnerability
are poorly understood. It is not clear whether par-
FMI Report 2005/2006 11
Neurobiology

A B
Fig.3. Quantitative topo-
graphic map of motor units
innervating LGC muscle
in mouse. A Nerves (green),
NMJs (red), compartments
(lat., int., med.) and sub-
compartments (l1-2, i1-3,
m1-3) in mouse LGC.
B Arborization of axons
mGFP (green) innervating
medial compartment of
LGC. RITC-a-Bungarotoxin
(red) visualizes all NMJs;
bar 1 mm
ticular synapses are lost selectively, gradually or
abruptly, and whether it is synapses, axons or den-
drites that are targeted by disease. The complexi-
ties of adult brain circuitry pose formidable chal-
lenges to elucidating mechanisms of early disease
progression. However, in animal models of moto-
neuron (MN) disease, more accessible synapses be-
tween MNs and muscles are also lost early on and
in reproducible patterns. MN disease models thus
provide uniquely advantageous systems to investi-
gate pathways of vulnerability and disease pro-
gression in neurodegeneration. Such information
is essential for early detection and the development
of more effective treatments against these diseases.
Transgenic mice expressing human SOD1 point
mutant proteins associated with familial amy-
otrophic lateral sclerosis (FALS) develop paralytic
MN disease closely resembling human ALS. Mice
expressing high levels of human SOD1(G93A)
provide a particularly valuable model of FALS due
to their remarkably predictable pattern of disease
progression. The mice develop first clinical signs
of MN disease at postnatal day (P) 80-90 and die
at P1365. Recent studies have established that
many peripheral synapses between MNs and mus-
cles (NMJs) are lost in SOD1(G93A) mice from
P50 on, before detectable losses of motor axons
in ventral roots exiting the spinal cord and long
before any clinical sign of disease. In addition, a
detailed study revealed reproducible differences in
the timing of denervation of individual muscles,
and distinct topographic patterns of denervation
in individual hindlimb muscles, suggesting that
NMJs were being lost according to defined albeit
still unknown principles. Considering possible un-
derlying principles, one possibility seems to be that
the predictable patterns of losses might reflect dif-
ferences between MNs and/or muscle fibers. Thus,
MNs innervating skeletal muscle fibers are subdi-
vided into the functional subtypes fast fatigable
(FF), fast fatigue-resistant (FR) and slow (S), which
exhibit distinct excitability and recruitment prop-
erties and establish motor units (consisting of one
MN and all the muscle fibers it innervates) with
markedly distinct fatigue and force properties.
We recently exploited a combination of Thy1-
transgenic mice expressing GFP fusion proteins in
only a few neurons, together with established his-
tological procedures, to quantitatively map the in-
nervation of hindlimb muscle compartments by
MNs and their functional subtypes in the mouse
(Fig. 3). We then applied these maps and proce-
dures to elucidate principles and mechanisms of
early disease progression in FALS mice. Our results
identified axons of first FF and then FR MNs as
being selectively vulnerable at well-defined times
early in disease and showed that, where present, S
MN axons resist and compensate through sprout-
ing and reinnervation. Axonal vulnerability in-
volved absence of neurofilament adjustments,
synaptic vesicle stalling, loss of synaptic vesicles
from all peripheral synapses, and upregulation of
Bcl2A1-a in MNs; this was followed by Lactacystin-
sensitive pruning of all peripheral synapses and
terminal axon branches. The axonal vulnerability
process could be alleviated by peripheral applica-
tions of CNTF. Since early defects in axonal trans-
port have also been associated with Alzheimer's
and Huntington's disease, these findings may re-
flect general principles of disease progression and
treatment in neurodegeneration, independent of
the molecular pathways triggering particular forms
of disease.

Selected publications
De Paola V, Holtmaat A, Knott G, Song S,
Wilbrecht L, Caroni P, Svoboda K (2006)
Cell type-specific structural plasticity of
axonal branches and boutons in the adult
neocortex. Neuron 49:861-875
Golub T, Caroni P (2005)
PI(4,5)P2-dependent microdomain assem-
blies capture microtubules to promote
and control leading edge motility. J Cell
Biol 169:151-165
Santos AF, Caroni P (2004)
Assembly, plasticity and selective vulnera-
bility to disease of mouse neuromuscular
junctions. J Neurocytol 32:849-862

Galimberti I, Gogolla N, Alberi S, Santos Pun S, Santos AF, Saxena S, Lefler S,

AF, Muller D, Caroni P (2006)
Long-term rearrangements of hippocampal
mossy fiber connectivity regulated in the
adult by experience. Neuron 50:749-763
Caroni P (2006)
CNTF-sensitive selective vulnerability
and pruning of phasic motoneuron axons
in motoneuron disease. Nature Neurosci
3:408-419

12 FMI Report 2005/2006

Andreas Lthi
Cellular mechanisms of
learning and memory

INTRODUCTION
Experience-dependent changes in behavior are
mediated by long-term functional modifications
in brain circuits. To study the underlying synaptic
and cellular mechanisms, we are using classical
(Pavlovian) fear conditioning, a simple form of as-
sociative learning that is particularly suitable for
studying in rodents. Behavioral and in vivo elec-
trophysiological experiments suggest that associa- GROUP LEADER
tive synaptic plasticity in the lateral amygdala, a Andreas Lthi
key structure for emotional learning and memory, andreas.luethi@fmi.ch
underlies classical fear conditioning. These exper-
iments established the strongest link between sy- TECHNICAL/RESEARCH
naptic plasticity and behavioral learning described ASSOCIATES
so far in mammals. Ronald Knig*
A thorough understanding of the cellular mech- Christian Mller
anisms underlying changes in behavior requires a
detailed knowledge of the anatomical and func- POSTDOCTORAL
tional properties of the relevant neuronal network FELLOWS
elements. Principal neurons in the lateral amyg- Guillaume Casassus
dala receive converging thalamic and cortical sen- Ingrid Ehrlich
sory afferents that are simultaneously active dur- Christine Gebhardt*
ing fear conditioning. We found recently that both Francois Grenier
afferents exhibit input-specific associative synaptic Cyril Herry
plasticity mediated by distinct molecular mecha- Yann Humeau*
nisms. We are also interested in the functional role
of distinct inhibitory circuits. PhD STUDENTS
Using a combination of in vitro and in vivo elec- Stphane Ciocchi
trophysiology, imaging, molecular biology and be- Elodie Fourcaudot
havioral approaches, we investigate activity- and Karin Loretan*
experience-dependent changes in the neural cir- Verena Senn
cuitry of the mouse amygdala with the ultimate Hamdy Shaban*
goal of understanding network function and plas-
ticity subserving this simple form of associative UNDERGRADUATES
learning. Dorothea Hmmerer*
Magalie Klaey*

GUEST SCIENTIST
Heidrun Bchli*

*left the group

FMI Report 2005/2006 13

Neurobiology
Thalamic spine Cort. stim.
Cortical spine Thal. stim.
Fig.1. Two-photon functional
imaging of Ca2+ transients
in dendrites and indivi-
dual spines of LA projection
neurons. Neurons were
filled with a red, Ca2+ insen-
sitive dye revealing spine
morphology. The green,
Ca2+-sensitive dye shows a
rise in spine Ca2+ elicited
by stimulation of thalamic
or cortical afferents to the
lateral amygdala
14 FMI Report 2005/2006
INPUT-SPECIFIC MECHANISMS OF
EXCITATORY SYNAPTIC PLASTICITY IN THE
LATERAL AMYGDALA
E. Fourcaudot, H. Shaban, G. Casassus, C. Gebhardt,
Y. Humeau
Projection neurons in the lateral amygdala (LA) re-
ceive converging thalamic and cortical sensory af-
ferents that are simultaneously active during fear
conditioning. We found that simultaneous activa-
tion of cortical and thalamic afferents specifically
induces associative, NMDA-receptor-dependent
long-term potentiation (LTP) at cortical but not
at thalamic inputs. Both induction and expression
of heterosynaptic, associative LTP are mediated
by presynaptic mechanisms involving activation
of presynaptic NMDA receptors and an increase
in the probability of release (Humeau et al. Nature
426:841-845, 2003). We are now examining the
molecular mechanisms by which NMDA receptor
activation increases the probability of release at
cortical afferent synapses. Our results show that
activation of the cAMP/PKA signal transduction
pathway is required for induction of heterosynap-
tic, associative LTP. Moreover, we are studying pos-
sible downstream targets of PKA mediating long-
term alterations in presynaptic function.
Thal. stim.
Cort. stim.
Our results show a dynamic interaction between
simultaneously active thalamic and cortical affer-
ents to the LA, raising the question of where the
dendritic spines contacted by thalamic and cor-
tical afferents are located on the dendritic arbor of
LA projection neurons. Using electrophysiological
recordings and 2-photon confocal imaging of
LA projection neurons, we showed that the mor-
phologies of spines located on the same dendrite
specifically match presynaptic inputs from thala-
mic or cortical afferents (Fig. 1). Large spines con-
tacted by thalamic afferents exhibited larger Ca2+
transients during action potential backpropaga-
tion than small spines contacted by cortical affer-
ents. Accordingly, induction of Hebbian plasticity,
dependent on postsynaptic spikes, was restricted
to thalamic afferents. This synapse-specific effect
involved activation of R-type voltage-dependent
Ca2+ channels preferentially located at thalamic in-
puts. Thus, afferent-specific mechanisms of post-
synaptic,associative Hebbian plasticity in LA projec-
tion neurons depend on local, spine-specific mor-
phological and molecular properties, rather than on
global differences between dendritic compartments.
ROLE OF PRE- AND POSTSYNAPTIC INHIBITION
DURING INDUCTION OF SYNAPTIC PLASTICITY
H. Shaban, K. Loretan, C. Herry, S. Ciocchi,
G. Casassus and Y. Humeau, in collaboration with
B. Bettler (University of Basel), R. Shigemoto
(NIPS, Okazaki, Japan), K. Kaupmann and H. van
der Putten (Novartis, Basel)
There is converging evidence that postsynaptic in-
duction of LTP at thalamic afferents to the LA is
necessary for the acquisition of fear conditioning.
Consistent with the notion that the LA is a brain
structure tightly controlled by local GABAergic in-
hibition, we found that LTP of excitatory synaptic
transmission at thalamic afferents to the LA can-
not be induced in the presence of postsynaptic
GABAA receptor-mediated inhibition. To over-
come this inhibitory constraint, neuromodulators
such as dopamine (DA) transiently suppress post-
synaptic inhibition, thereby enabling LTP induction
(Bissire et al. Nature Neurosci 6:587-592, 2003).
Conversely, DA also increases the excitability of in-
hibitory interneurons in the LA. Unlike D1 recep-
tor-mediated transmission in other brain areas,
this involves activation of the protein tyrosine
kinase Src. Thus, DA appears to orchestrate the ac-
tivity of populations of interneurons in the LA by
a D1-dependent, non-canonical signal transduc-
tion pathway.
To examine the role of presynaptic inhibition
during induction of presynaptic LTP at cortical af-
ferents, we used a combined genetic and electro-
physiological approach in mice. Lack of a specific
GABAB receptor subtype, GABAB(1a,2), unmasks
a non-associative, NMDA receptor-independent
form of presynaptic LTP at cortico-amygdala af-
ferents. Suppression of GABA release by a -opioid
receptor agonist decreases GABAB receptor activa-
tion and shifts the balance between associative and
non-associative plasticity. At the behavioral level,
unmasking of non-associative LTP by genetic loss
of GABAB(1a) is accompanied by a generalization of
conditioned fear to non-conditioned stimuli.
Thus, depending on the level of GABAergic activ-
ity, presynaptic inhibition through GABAB(1a,2) re-
ceptors serves as a constraint on the induction of
homosynaptic plasticity, which appears important
for preventing generalization of conditioned fear.
IN VIVO ANALYSIS OF LEARNING-INDUCED
NEURONAL PLASTICITY
C. Herry, F. Grenier, V. Senn and S. Ciocchi,
in collaboration with K. Lingenhhl and H.R. Olpe
(Novartis, Basel)

To study the plasticity of excitatory and inhibitory

circuits during fear conditioning and extinction,
we use in vivo electrophysiological techniques that
allow recording from single neurons in freely mov-
ing mice. Conditional changes in the tone-evoked
firing rate of projection neurons in the lateral
nucleus of the LA are thought to underlie the aqui-
sition of auditory fear conditioning. Moreover, ex-
periments in rats have demonstrated that this in-
crease in tone-evoked activity in some neurons is
not reversed during extinction of conditioned fear,
suggesting that long-term fear memories could be
stored within the LA. Since genetic mouse models
are increasingly important to study the molecular
and cellular mechanisms underlying fear learning,
we investigate conditional changes in tone-evoked
neuronal activity during fear conditioning and
extinction in the mouse LA using combined elec-
trophysiological and immunohistochemical tech-
niques. Our results show that LA projection neurons
specifically increase their activity to the condi-
tioned but not to the non-conditioned tone. This
provides the first evidence for conditional changes
of tone-evoked activity of single LA neurons fol-
lowing fear conditioning in mice. We are currently
using immunohistochemical analysis of activity-
dependent immediate-early gene (IEG) induction
to further identify amygdala neurons and circuits
activated during distinct behaviors.
In the past, only extracellular recording tech-
niques were applied to assess auditory processing
in the amygdala in vivo. Extracellular recordings,
however, only allow recording of the output (spikes)
of single neurons. To analyze auditory processing
at the synaptic level, we also use in vivo intracellular
recording techniques to investigate how auditory
stimuli are processed in the amygdala (Fig. 2).
Fig. 2. In vivo intracellular
TRANSLATIONAL MODELS OF AMYGDALA FUNCTION recordings from mouse
LA neurons. Left: Vertical
C. Herry, in collaboration with E. Seifritz (University approach of microelectrode
of Bern) and K. Scheffler (University of Basel) to basolateral amygdala
(BLA). Center: Spontaneous
Unpredictable aversive events have devastating activity of a BLA neuron at
physiological, behavioral and emotional conse- rest. Upper right: Morpho-
quences. It has been suggested that individuals aim logical reconstruction of
at reducing unpredictability in the sensory envi- recorded neuron filled with
ronment to minimize stress and anxiety but it is Neurobiotin; bars from left
unclear whether unpredictability per se promotes to right 1 s, 10 mV, 20 m
anxiogenic states. Results from a translational ap-
proach to this question show that perception
of unpredictable time-series of neutral auditory
stimuli in mice and humans activates the amyg-
dala, a central component of the brain's fear sys-
tem, and affects emotional behavior. Exposing
mice to auditory sound pulse sequences with un-
predictable pulse timing increases expression of
the immediate early gene c-fos and enhances sin-
gle-unit activity in the basolateral amygdala. At the
behavioral level, unpredictable but not predictable
auditory stimulation induces anxiety-like behavior
on the elevated plus-maze and avoidance in a place
preference task. In healthy humans, functional mag-
netic resonance imaging revealed that unpredict-
ably compared with predictably timed sound
pulses cause sustained neural activity in amygdala.
Moreover, unpredictable sound pulse stimulation
was associated with anxiety-like behavior quanti-
fied by enhanced attention to emotional faces. The
results identify temporal unpredictability as an im-
portant feature of the sensory environment influ-
encing emotional behavior. They expand current
concepts of animal and human amygdala function
to include a more general role of anticipating po-
tential threat and controlling emotional behavior
in an unpredictable sensory environment.

Selected publications
Herry C, Triffilieff P, Micheau J, Lthi A,
Mons N (2006)
Extinction of auditory fear conditioning re-
quires MAPK/ERK activation in the basolat-
eral amygdala. Eur J Neurosci 24:261-269
Humeau Y, Lthi A (2006)
Dendritic calcium spikes induce bi-direc-
tional synaptic plasticity in the lateral
amygdala. Neuropharmacology (in press)
Humeau Y, Herry C, Kemp N, Shaban H,
Fourcaudot E, Bissire S, Lthi A (2005)
Dendritic spine heterogeneity determines
afferent-specific Hebbian plasticity
in the amygdala. Neuron 45:119-131
Lortan K, Bissire S, Lthi A (2004)
Dopaminergic modulation of spontaneous
inhibitory network activity in the lateral
amygdala. Neuropharmacology 47:631-639
Shaban H, Humeau Y, Herry C, Casassus G,
Shigemoto R, Ciocchi, S, Barbieri S,
van der Putten H, Kaupmann K, Bettler B,
Lthi A (2006)
Generalization of amygdala LTP and condi-
tioned fear in the absence of presynaptic
inhibition. Nature Neuroscience
9:1028-1035

FMI Report 2005/2006 15

Neurobiology

Andrew Matus
Molecular mechanisms
of synaptic plasticity

INTRODUCTION
With my retirement due in April 2007, this last re-
port from my group is an opportunity to reflect on
our work over the past 29 years at the FMI and to
consider what implications our results may have
for the future. Our mission has been to explore the
molecular mechanisms regulating morphological
changes in the synaptic connections of brain neu-
ronal circuits, with the ultimate aim of gaining GROUP LEADER
new insights into the cellular basis of long-term Andrew Matus
memory. We began at an obvious place by seeking aim@fmi.ch
to identify structural proteins involved in building
synapses during brain development. First, we TECHNICAL/RESEARCH
worked out methods for isolating postsynaptic ASSOCIATES
densities and, using the then new method of SDS Heike Brinkhaus
gel electrophoresis, for identifying candidate mol- Urs Mller
ecules (Walters and Matus 1975). Subsequently, we
raised specific antibodies for immunohistochem- POSTDOCTORAL
ical staining to locate sites of action of these mol- FELLOWS
ecules in the brain. Indeed the rationale for setting Martin Verkuyl
up my group at the FMI was the potential of new Pingwei Zhao*
immunohistochemical methods for solving the
mystery of how specific assemblies of proteins form PhD STUDENT
and maintain the distinctive functional structures Andreas Birbach*
of neurons their axons, dendrites and synapses.
The exploitation of emerging technologies has UNDERGRADUATES
been a constant theme in our work and a major Christian Mller*
source of the discoveries we have been fortunate Reto Zwahlen*
to make. In a wider context, it is clear that the de-
velopment of new technology platforms, ranging
from DNA sequencing to live-cell microscopy, has *left the group
been by far the most important source of novel
data in biomedical research over the past quarter
century. Indeed, the inherent flexibility of the
Friedrich Miescher Institute, as part of an inde-
pendent research foundation, remains a key ad-
vantage for the rapid adoption of cutting edge ap-
proaches.

16 FMI Report 2005/2006

It was the opportunity to apply monoclonal anti-
bodies to locate synaptic molecules at the begin-
ning of my work at the FMI, and more recently to
use green fluorescent protein (GFP) as an autoflu-
orescent genetic tag to study the dynamics of in-
dividual proteins in living neurons that led to our
most useful, and exciting, discoveries. In the first
case, the precision of clonally derived antibodies
showed us how axons, dendrites and synapses form
via microdifferentiation of distinct cytoplasmic
compartments (Matus et al. 1983). In the second,
time-lapse imaging of a single structural protein,
actin, revealed unexpectedly that the dendritic
spines, which are small protrusions forming part
of most excitatory synapses in the brain, are not
just morphologically plastic but are capable of
rapid and continuous changes in shape (Fischer et
al. 1998).
The work on actin was part of a project, pursued
throughout the existence of the group, based on
the idea that the regulated assembly of cytoskele-
tal filaments is a key mechanism in neuronal plas-
ticity. Our approach was to examine the influence
of accessory proteins that regulate filament as-
sembly. An early discovery was that microtubule-
associated protein 2 (MAP2 ), which promotes mi-
crotubule assembly, is specifically associated with
dendrites,implying that the differential distribution
of cytoskeletal molecules is a determinant of the
morphological distinction between axon and den-
drite (Matus 1988). More recently, we have found
that the small actin-binding protein profilin stabi-
lizes the morphology of dendritic spines (Acker-
mann and Matus 2003). The targeting of profilin
to spines, which initiates this activity, is triggered
by NMDA-type receptors for glutamate, the neu-
rotransmitter at excitatory synapses. This class of
receptor has long been implicated in long-term
memory, and a recent study has demonstrated a
correlation between fear-conditioned learning and
the number of excitatory synapses showing high
levels of profilin in their dendritic spines (Lam-
precht et al. Nature Neurosci 9:481-483, 2006). Al-
though preliminary, these data suggest that the role
of profilin in regulating dendritic spine plasticity
is part of the molecular mechanism underlying
learning and memory.
Our most recent results described below suggest
that profilin also has a role in regulating activity-
dependent changes in gene transcription, a further
phenomenon known to be essential for long-term
memory.
ACTIVITY-DEPENDENT ACCUMULATION OF
PROFILIN IN NEURONAL NUCLEI
A. Birbach, M. Verkuyl
In transfected hippocampal neurons under resting
conditions, profilin 2-GFP fluorescence is strong
in the cytoplasm and relatively weak in the nucleus
(Fig. 1A). When NMDA-type glutamate receptors
are activated, the distribution changes and profilin
2-GFP rapidly accumulates in the nucleus (Fig.1B).
At the same time, there is a marked increase in the
number of brightly labelled GFP-fluorescent spots
along neuronal processes (Fig.1B, bottom row), in-
dicating accumulation of profilin 2 in dendritic
spines. When the stimulus is removed, profilin lev-
els decrease in the nucleus and increase in the cy-
toplasm (Fig. 1C). Thus, the nuclear targeting is re-
versible but profilin 2-GFP fluorescence in spines
remains high (Fig. 1C). These events appear to
A B C
Fig.1. Activating postsynap-
depend on the export of a profilin-actin complex tic glutamate receptors
from the nucleus via a known pathway (Stven et targets profilin to dendritic
al. EMBO J 22:5928-5940, 2003), since profilin with spines and neuronal nuclei.
Profilin 2-GFP distribution
point mutations that lower its affinity for actin
in hippocampal neurons
leads to its accumulation in nuclei independent of in dissociated cell culture.
activity. Experiments with slices of hippocampus Confocal plane: top, level
from mature mice demonstrated that reversible of cell nucleus; bottom,
targeting of profilin 2 to the nucleus also occurs in bottom of culture to show
pyramidal cells in organized brain tissue (Birbach neuronal processes.
et al. 2006). A, B, C: profilin 2-GFP be-
fore, during and after
Together these results suggest that profilin func-
NMDA receptor stimulation
tions in two distinct modes in pyramidal neurons,
both dependent on the activation of NMDA re-
ceptors. In one there is a long-lasting accumulation
of profilin 2 in dendritic spines, while the other
involves rapid and reversible accumulation in nu-
clei. Data from other laboratories implicate these
events in long-term memory and gene transcrip-
tion. In the amygdala, the location of synapses
involved in fear-related learning, there is a large
increase in profilin-labelled dendritic spines after
fear conditioning (Lamprecht R et al. Nature Neu-
rosci 9:481-483, 2006). Profilin also binds to a novel
transcriptional repressor, p42POP, concentrated in
the brain, reducing its repressive effect in an in vitro
transcription reporter assay (Lederer M et al. J Cell
Sci 118:331-41, 2005). In the immediate future, we
will attempt to identify neuronal genes whose ex-
pression is modulated by profilin action on p42POP.
FMI Report 2005/2006 17
Neurobiology

CONCLUSIONS AND OUTLOOK

Our data indicate that activation of NMDA recep-

Fig. 2. A section from area
CA1 of mouse hippocampus,
stained with specific anti-
bodies, showing that profilin
2 levels in the nucleus
vary between neighbouring
neurons. Inset: higher
magnification; bar 10 m
PROFILIN LEVELS IN BRAIN NUCLEI
U. Mller, M. Verkuyl
To approach the possible physiological signifi-
cance of profilin in the nucleus, we have begun ex-
amining its levels in neuronal nuclei of the brain.
Initial results from immunocytochemical staining
show that high-level profilin 2 in the hippocam-
pus is restricted to the nuclei of pyramidal neurons
(Fig. 2). It is these cells in the hippocampus that
are rich in dendritic spines and which show NMDA
receptor-dependent targeting of profilin 2 to spines
and nuclei in our in vitro experiments. In addition,
we have observed considerable heterogeneity in
nuclear levels of profilin 2 between hippocampal
neurons in sections of mouse brain. This suggests
that activity-dependent changes in nuclear profilin
2 levels occur in brain nuclei.
tors leads to simultaneous changes in profilin dis-
tribution at synapses, where its level increases in
postsynaptic dendritic spines, and in the cell body,
where it accumulates in the nucleus. In dendritic
spines, it acts to suppress actin dynamics and stabi-
lize dendritic spine morphology (Ackermann and
Matus 2003). At present, we can only guess at its
function in the nucleus, but its binding to p42POP,
a brain-enriched nuclear protein acting as a tran-
scriptional repressor in vitro, suggests a role in reg-
ulating gene expression.
Many questions remain unanswered. What are
the stimuli that trigger the changes in profilin dis-
tribution at the two locations? Are they the same
or distinct? How are they related to the stimuli
required for memory-related changes in signal
strength, long-term potentiation and long-term
depression? Do the genes whose expression is reg-
ulated via profilin subsequently act at the synapses
whose dendritic spines have been simultaneously
tagged by that same protein.And what functions,
if any, do those gene products have in long-term
memory?
As always in scientific research, new data raise
fresh questions, leading researchers ever onwards
with the promise of limitless expanding vistas. But
there is a limit, whether imposed by society or biol-
ogy, that sooner or later replaces one group leader
with another. As another Basel biologist recently
pointed out, the chance to pass on to a younger
generation the opportunity to pioneer new paths
of enquiry is one that senior scientists should wel-
come (G. Schatz. Jeff 's View. Elsevier, Amsterdam,
2006).
After enjoying the outstanding research support
provided by the Friedrich Miescher Institute over so
many years, my long-term memories will be only
of gratitude and exceptional good fortune. Besides,
the path leading away from the laboratory door no
doubt also holds challenges and surprises.

Selected publications
Ackermann M, Matus A (2003)
Activity-induced targeting of profilin and
stabilization of dendritic spine morphology.
Nature Neurosci 6:1194-1200
Birbach A, Verkuyl JM, Matus A (2006)
Reversible, activity-dependent targeting
of profilin to neuronal nuclei. Exp Cell Res
312:2279-2287
Fischer M, Kaech S, Knutti D, Matus A
(1998)
Rapid actin-based plasticity in dendritic
spines. Neuron 20:847-854
Matus A (1988)
Microtubule-associated proteins: their
potential role in determining neuronal mor-
phology. Annu Rev Neurosci 11:29-44
Matus A, Huber G, Bernhardt R (1983)
Neuronal microdifferentiation. Cold Spring
Harbor Symp Quant Biol 48:775-782
Walters BB, Matus A (1975)
Tubulin in postsynaptic junctional lattice.
Nature 257:496-498

18 FMI Report 2005/2006

Denis Monard
Control of endogenous
extracellular proteases
in development
and brain function

INTRODUCTION
The question of the importance of extracellular
proteolytic activity for cellular behavior both in
vitro and in vivo has been neglected for a long time,
partly because of the difficulties in precisely dis-
secting complex proteolytic cascades. For example,
serine proteases activate latent forms of some met-
alloproteases that subsequently boost the activity
of other members of this family. These potent am- GROUP LEADER
plification systems degrade components of the ex- Denis Monard
tracellular matrix, trigger activation or inactiva- denis.monard@fmi.ch
tion of specific classes of receptors and generate
biologically active factors by specific cleavage of TECHNICAL/RESEARCH
their latent forms. Locally secreted inhibitors con- ASSOCIATES
trol the onset, the efficiency, and the duration of Sabrina Djaffer
these proteolytic explosions. Our group has con- Elisabeth Fries Schmid*
tributed to this field of research by characterizing Maria Rita Meins
a neurite-promoting factor as protease nexin-1 Eliza Pandini Figueiredo Moreno
(PN-1), one of, if not the most potent endogenous
serine protease inhibitor. POSTDOCTORAL
Our aim remains to dissect the in vivo relevance FELLOWS
of the adequate control of endogenous serine pro- Gianluca Civenni*
tease activity by studying mice lacking PN-1 or by Brengre Fayard
overexpressing it in neurons. To efficiently localize Anne-Catherine Feutz*
and quantitatively monitor in vivo PN-1 expres- Maria Maddalena Lino
sion, we also generated reporter mice by inserting Slobodanka Orolicki
an internal ribosomal entry site-b-galactosidase Tanuja Rohatgi
construct at the end of the PN-1 coding sequence, Catherine Vaillant Alibert
thus maintaining intact the entire 5 regulatory
region of the gene (PN-1 KI mice). PhD STUDENTS
Mirna Kvajo*
Xiaobiao Li*

UNDERGRADUATES
Cdric Fischer*
Colette Maurer*
Antonia Rosenstiel*
Hlne T*

*left the group

FMI Report 2005/2006 19

Neurobiology
Fig.1. PN-1 deficiency
impairs maturation
of Bergmann glia cells.
P10 cerebellar sections of
PN-1+/+ and PN-1-/- mice
immunostained for GFAP.
PN-1-/- Bergmann glia
show higher GFAP levels,
increased thickness and
larger end-feet of proces-
ses than wild type. Glial
processes of mutant
Bergmann glia cells are
thicker and irregular
with enlarged end-feet at
the pial surface (insets).
IGL inner external granular
layer; EGL outer external
granular layer; bar 40 m MODULATION OF SONIC HEDGEHOG SIGNALING
and 15 m IN CEREBELLAR DEVELOPMENT BY PN-1 AND
20 FMI Report 2005/2006
ENDOGENOUS CONTROL OF PROTEOLYTIC ACTIVITY
We focus on two main questions:
A. Does endogenous control of proteolytic ac-
tivity influence the fate of precursor cells? Very
strong PN-1 expression is detected in structures
rich in precursor/progenitor cells, both during
development and in the adult. Consequently, we
study its role in the proliferation, migration and
differentiation of cells, focusing mainly on cere-
bellar development and the skin. As some of the
effects are mediated through the interaction of PN-
1/protease complexes with LRP-receptors, we also
investigate pathways of PN-1 internalization.
B. Is endogenous control of brain proteolytic ac-
tivity required for proper formation, maintenance
and functioning of neuronal circuits? We investi-
gate the effects of an altered balance between ser-
ine proteases and their inhibitors on the develop-
ment, maintenance and activity of given neuronal
circuits. We have shown that recovery is delayed
following sciatic nerve injury in mice lacking PN-1
expression. Focusing on defined neuronal circuits,
we perform biochemical, histological and immu-
nocytochemical analyses in the whiskers-to-cor-
tical barrel field pathway, the amygdala and the
neuromuscular junction. We are also examining
appropriate correlations at the behavioral level.
internalization of PN-1 stimulated the Ras-ERK
signaling pathway.
ITS RECEPTOR LRP
C. Vaillant, H. T and S. Taieb, in collaboration
with O. Michos and R. Zeller (DKBW Center of Bio-
medicine, University of Basel)
Development of the postnatal cerebellum relies on
tight regulation of cell number by morphogens
that control the balance between cell proliferation,
survival and differentiation. Given the PN-1 ex-
pression profile during the development of the
cerebellum, we analyzed its role in the prolifera-
tion and differentiation of cerebellar granular
neuron precursors (CGNPs), focusing on possible
modulation of their main mitogenic factor, sonic
hedgehog (SHH). Our studies showed that PN-1
interacts with the low-density lipoprotein recep-
tor-related proteins (LRPs) to antagonize SHH-in-
duced CGNP proliferation and inhibits activity of
the SHH target Gli. PN-1 binding to LRPs inter-
feres with SHH-induced cyclin D1 expression and
activates PKA, which is known to antagonize SHH.
CGNPs isolated from PN-1-deficient mice exhibit
enhanced basal proliferation rates due to over-ac-
tivation of the SHH pathway and show higher sen-
sitivity to exogenous SHH. In vivo, PN-1 deficiency
alters expression of SHH target genes. CGNPs en-
try into differentiation is delayed, which results in
enlargement of the outer external granular layer
and alteration of Bergmann glia maturation (Fig. 1).
PN-1 deficiency also leads to overproduction of
CGNPs and enlargement of the internal granular
layer in certain cerebellar lobes during late devel-
opment and in adulthood (Fig. 2). We propose that
PN-1 contributes to the shaping of the cerebellum
by promoting cell cycle exit.
SWITCH IN SIGNAL TRANSDUCTION
PATHWAY UPON PN-1 INTERNALIZATION IN
LRP1-DEFICIENT CELLS
X. Li
The internalization of the PN-1 protease com-
plexes is thought to be mediated by LRP1. How-
ever, both wild-type (WT) and LRP1-/- mouse
embryonic fibroblasts (MEF) internalize PN-1.
Receptor associated protein (RAP) interfered with
PN-1 uptake only in WT MEF cells, indicating that
another receptor mediates PN-1 uptake in the ab-
sence of LRP1. In LRP1-/- MEF cells, inhibitor sen-
sitivity and kinetic values (t1/2 at 45 min) of PN-1
uptake indicated a similarity to syndecan-1-medi-
ated endocytosis. In these cells, PN-1 uptake was
increased by over-expression of full-length synde-
can-1 and decreased by RNA interference target-
ing this proteoglycan. Most important, in contrast
to PKA activation known to be triggered by LRP1-
mediated internalization, syndecan-1-mediated
PN-1 MEDIATED REGULATION OF CELL FATE IN
SKIN AND APPENDAGES
A.C. Feutz and S. Taieb, in collaboration with
Y. Barrandon (University of Lausanne)
Hair follicle development and cyclic growth is
tightly regulated by interactions between a subset
of mesenchymal cells forming the dermal papilla
and follicular epithelial cells. PN-1 shows cyclic
expression in both mesenchymal structures of hair
follicle, namely the dermal papilla (DP) and the
dermal sheath (DS). Here, we show during anagen
that PN-1 colocalizes with thrombin in dermal
compartments and controls its activity. Thrombin
activity increased in follicles of PN-1 knockout
(KO) mice and its protein accumulated in DP. Cul-
tured DP cells lacking PN-1 showed a thrombin-
dependent overgrowth. In addition, increased ex-
pression of smooth muscle cell actin protein
(SMA) correlated with a thrombin-dependent
gain in contractile properties. These two in vitro
features indicate that PN-1 protects DP cells from
acquiring the characteristics of myofibroblastic
DS cells. Both growth and contraction are under
the control of the PI3K/Akt pathway, suggesting
that they are linked or coordinated. The relevance
of our in vitro data was confirmed by detecting
global overactivation of the PI3-kinase pathway
and increased expression of SMA protein in PN-1
KO hair follicles. Moreover, SMA expression was
reinforced in trailing dermal sheath cells and ap-
peared in DP during catagen, indicating that DP
to DS-like transition correlates with natural dis-
appearance of PN-1 expression during the hair fol-
licle cycle. In PN-1-/- mice, change in cell differ-
entiation was associated with decreased ability of
DP cells to sustain stimulation of the survival path-
way in cultured keratinocytes. Such a deficiency
could be reproduced by pretreatment of WT DP
cells with thrombin. Our study demonstrates that
PN-1 control of thrombin activity is involved in
maintaining both the identity and functional in-
tegrity of DP. It strongly suggests that thrombin
links control of DP cell differentiation and the
switch in exocrine activity during the follicular
cycle.
DELAYED STRUCTURAL AND FUNCTIONAL
RECOVERY FOLLOWING SCIATIC NERVE CRUSH
IN MICE LACKING PN-1
M. Lino, in collaboration with S. Atanasoski and
U. Suter (ETH Zurich)
To assess the role of PN-1 during peripheral nerve
regeneration, we performed nerve crush experi-
ments in adult PN-1 KO mice and their WT litter-
mates and studied synapse re-innervation at dif-
ferent times post-lesion. Twelve days after crush,
PN-1 KO mice showed a significant delay in
synapse re-innervation compared with WT ani-
mals. The re-innervation delay was accompanied
by both a reduction in proliferation and increased
Schwann cells apoptosis. Increased fibrin deposits
were present in the injured sciatic nerve of PN-1
KO mice. Furthermore, tPA activity and mature
BDNF amounts increased in PN-1 KO mice after
nerve crush, which may explain both the delayed
re-innervation and the increased apoptosis. To test
whether the re-innervation delay was due to PN-1
absence from Schwann cells or the nerve, we over-
A B
C D
Fig.2. PN-1 deficiency leads
expressed PN-1 exclusively in the nerve in the PN- to cerebellar expansion.
1 KO background. PN-1 presence in the nerve was Wild-type (A, B) and mutant
not sufficient to rescue the delay in re-innervation. (C, D) adult cerebellar mid-
sagittal sections were stained
These results indicate that Schwann cell-derived
for Ptc-1 (Sonic Hedgehog
PN-1 is crucial for re-innervation, by influencing receptor) mRNA. Mutant
Schwann cell proliferation and rate of apoptosis. cerebella show global enlarge-
ment of IGL in zones facing
ANXIETY-LIKE BEHAVIOR AND IMPAIRED FEAR the cerebellum external side.
EXTINCTION IN MICE WITH ALTERED CONTROL IGL facing deep fissures is
OF EXTRACELLULAR BRAIN PROTEOLYTIC not modified. B, D Higher
magnification of lobe VI; bars
ACTIVITY
A, C 300 m; B, D 100 m
M. Meins, E. Moreno, C. Fischer and S. Orolicki,
in collaboration with C. Herry, S. Ciocchi and
A. Lthi (FMI)
Regulation of serine proteases has been implicated
in the modulation of synaptic plasticity. PN-1 is
expressed in specific neuronal subpopulations in
the adult central nervous system. The in vivo ex-
pression of PN-1 is regulated by neuronal activity
(Kvajo et al. 2004). This event is mimicked in vitro
upon NMDA receptor stimulation in cultured hip-
pocampal slices. In mice lacking PN-1 (PN-1-/-),
the brain homogenate proteolytic profile is altered
and NMDA receptor mediated transmission is re-
duced (Luthi et al. 1997; Kvajo et al. 2004). To ex-
amine the in vivo consequences of these changes,
we compared cognitive and emotional responses
of PN-1-/- mice to their WT littermates. No mem-
ory impairments were detected. In contrast PN-1-
/- mice displayed enhanced anxiety and avoidance
behavior. Cued fear conditioning was not changed
but fear extinction was drastically impaired. At the
FMI Report 2005/2006 21
Neurobiology

Fig. 3. PN-1 is prominently

expressed (reporter mouse,
blue X-gal staining) in some
areas of the amygdala
cellular level, c-FOS immunoreactivity, indicative
of neuronal activity, did not increase in the PN-1-/-
basolateral amygdala. Of potential interest is the
prominent expression of PN-1 in GABAergic neu-
rons of the central amygdala and intercalated cell
masses (Fig. 3). These latter cells may modulate
extinction behavior through their projections to
the basolateral and central amygdala. As these
neurons receive glutamatergic innervation from
higher brain centers, the lack of fear extinction in
the PN-1-/- mice could be explained by reduced
NMDA sensitivity. To investigate this hypothesis,
we are analyzing downstream effectors (pCAMKII,
c-FOS, pMAPK) of NMDA receptor activation in
the intercalated cells. These results not only cor-
roborate the importance of the control of prote-
olytic activity in synaptic plasticity but also sug-
gest that PN-1 -/- mice are an interesting model
for studying brain structures and molecular mech-
anisms involved in fear extinction.

Selected publications
De Castro Ribeiro M, Badaut J, Price M,
Meins M, Bogousslavsky J, Monard D, Hirt
L (2006)
Thrombin in ischemic neuronal death. Exp
Neurol 198:199-203
Kvajo M, Albrecht H, Meins M, Hengst U,
Troncoso E, Lefort S, Kiss JZ, Petersen CCH,
Monard D (2004)
Regulation of brain proteolytic activity con-
trols the function of NMDA receptors. J Neu-
rosci 24:9734-9743
Li X, Herz J, Monard D (2006)
Activation of ERK signaling upon alternative
protease nexin-1 internalization mediated by
syndecan-1. J Cell Biochem (in press)
Luthi A, Putten H, Botteri FM, Mansuy IM,
Meins M, Frey U, Sansig G, Portet C, Schmutz
M, Schroder M, Nitsch C, Laurent JP, Monard
D (1997)
Endogenous serine protease inhibitor modu-
lates epileptic activity and hippocampal long-
term potentiation. J Neurosci 17:4688-4699

22 FMI Report 2005/2006

Thomas Oertner
Physiology and plasticity
of individual synapses

INTRODUCTION
Neurons in the brain communicate with each other
through synapses. These change their strength de-
pending on the temporal patterns of activity in
pre- and postsynaptic cells, a process thought to be
crucial for processing and storage of information
in the brain. It is not known, however, whether
all synapses change their transmission character-
istics according to the same rules. Should we view GROUP LEADER
synapses as independent units of information pro- Thomas Oertner
cessing or do neighboring synapses cooperate, e. g., thomas.oertner@fmi.ch
during induction of plasticity? Do synapses switch
between potentiated and depressed states or do TECHNICAL/RESEARCH
they display a continuum of stable states? To address ASSOCIATES
such questions, we focus on the connection be- Daniela Gerosa Erni
tween hippocampal CA3 pyramidal cells and CA1 Ursin Stauss*
pyramidal cells, a well-defined system for synaptic
plasticity. Most of these synapses are formed on POSTDOCTORAL
dendritic spines, which show a rich, dynamic di- FELLOW
versity of shape and size. The functional implica- Tobias Rose
tion of this diversity is of great interest to us.
To examine the properties of individual synapses PhD STUDENTS
in intact brain tissue, we use two-photon laser Niklaus Holbro
scanning microscopy to optically record the am- Asa Mller-Grunditz
plitude of postsynaptic calcium transients in den- Yan-Ping Zhang
dritic spines. This allows us to monitor the activ-
ity of individual, identified synapses over several UNDERGRADUATES
hundred stimulations and to measure synaptic pa- Matthew Mikulski*
rameters, e.g., the time constants of facilitation and Lei Tian*
depression. We can distinguish changes in presy-
naptic release probability from changes in the am- GUEST SCIENTIST
plitude of the postsynaptic response. At the same Jim Yu-Hsiang Tiao*
time, motility and growth of new spines can be
plotted. Acute hippocampal slices and organotypic
slice cultures of the rodent brain are used to pre- *left the group
serve the local environment of the synapses scru-
tinized. In vitro transfection techniques are also
applied to study morphology and function of gene-
tically modified neurons within a network of wild-
type cells. Optical monitoring and manipulation
of the number of fluorescently tagged proteins in
individual synapses should give insight into regu-
latory mechanisms governing synaptic transmis-
sion.

FMI Report 2005/2006 23

Neurobiology
A B
Fig.1. Particle-mediated
gene transfer visualizing
and stimulating individual
synapses. A Pyramidal
cells in a hippocampal slice
culture expressing a light
gated channel (ChR2).
B, C High magnification of
potential synaptic contacts
between the axon of a cell
expressing ChR2 (red)
and the dendrite of a cell
expressing CaMKII-GFP
(green). Switching excita-
tion wavelength (B 810 nm,
C 980 nm) selectively
highlights pre- or postsy-
naptic cells
24 FMI Report 2005/2006
LONG-TERM POTENTIATION OF INDIVIDUAL
SYNAPSES
YP. Zhang
In connections between pyramidal cells in the hip-
pocampus, particular patterns of synchronized ac-
tivity in pre- and postsynaptic cells lead to long-
lasting changes of the transmission characteristics
of the synaptic connection. The postsynaptic cur-
rent, measured by electrophysiology, typically re-
sults from the simultaneous activity of many
synapses. However, the specificity of these activ-
ity-induced changes is unknown: Do only directly
stimulated synapses change their potency or are
neighboring synapses also affected? Is the growth
of new spines both in vivo and in vitro triggered
by certain patterns of local synaptic activity? To ad-
C tivated enzymes to the active synapse, they permit
dress such questions, we collect information about
the temporal and spatial distribution of synaptic
activity. Thus, we may find out whether the location
of a synapse on the dendritic tree relative to other
inputs is of special importance for the induction
of functional and morphological plasticity.
Until now, long-term plasticity has been studied
mainly by looking at the average response of many
synapses. At the level of somatic currents (EPSCs),
the magnitude of the induced long-term plasticity
varies gradually with the stimulation intensity. It is
possible, however, that individual synapses switch
in an all-or-nothing fashion between a potentiated
and a non-potentiated state, assuming different
synapses have different induction thresholds for
plasticity.A binary behavior would make them more
robust information storage devices in spite of small
random fluctuations in transmission characteris-
tics caused e.g., by glutamate receptor turnover.We
wish to know whether all synapses responds in the
same fashion to a particular stimulation pattern, and
whether the changes are graded or all-or-nothing.
One strategy to examine potentiation at indi-
vidual synapses is to monitor the fluorescence of
GFP-labeled aCaMKII in individual dendritic
spines. Glutamate receptor activation leads to phos-
phorylation of aCaMKII molecules, which sub-
sequently translocate from the dendrite to the
postsynaptic density of active synapses where they
phosphorylate AMPA and NMDA receptors. We
are investigating whether this translocation can be
used as an indicator of potentiation and depoten-
tiation. To stimulate individual identified axons
in a slice culture, we transfect neurons with a light-
activated cation channel, channelrhodopsin-2
(Fig.1). Under these conditions, brief pulses of blue
light are used to induce single action potentials
with millisecond precision. Measuring electrical
responses in a postsynaptic cell, we can relate
changes in aCaMKII concentration to changes in
synaptic strength induced by various stimulation
patterns.
CALCIUM-SILENT SYNAPSES ON CA1
PYRAMIDAL CELLS
A. Grunditz, L. Tian
Dendritic spines can be viewed as tiny bioreactors.
By restricting the action of second-messenger-ac-
synapse-specific functional modifications. It is less
clear, however, whether the spine neck resistance
also provides significant electrical isolation of the
synapse from its parent dendrite. If so then changes
in spine neck morphology could dramatically af-
fect attenuation of the postsynaptic potential, giv-
ing a fast and efficient mechanism for synaptic
plasticity. To address this question, the ideal would
be to measure depolarization of the spine head di-
rectly, e.g., using voltage-sensitive dyes. While this
is not yet possible, spine calcium measurements
give us an indirect clue. Under current clamp con-
ditions, we can infer spine head depolarization
from the calcium signal amplitude due to the volt-
age-dependence of the NMDA receptor, the main
gateway for spine calcium. A single action poten-
tial in the presynaptic axon is sufficient to trigger
calcium influx in most spines, indicating a sur-
prisingly strong depolarization of the spine head.
In a subset of spines, however, NMDA receptors
are active only when the entire dendrite is depo-
larized. We are especially interested in the proper-
ties of these calcium-silent spines because of their
potential role in synaptic plasticity. Our optical
recording technique allows us to investigate corre-
lations between release probability, calcium signal
amplitude and morphology of individual spines.
We found that calcium-silent spines have altered
short-term plasticity, showing that pre- and post-
synaptic properties of single synapses are matched.
As well as the calcium imaging experiments, we
use biophysical models of spines to understand the
complex interplay of glutamate receptors and volt-
age-gated channels in the spine head.
SUBCELLULAR DISTRIBUTION OF GABAB
RECEPTOR VARIANTS
YP. Zhang
Although our research is focused mainly on exci-
tatory synapses, we have started to investigate G-
protein coupled GABAB receptors, which induce
slow inhibitory potentials in the dendrite by acti-
vating K+-channels. GABAB activation also leads to
inhibition of voltage-gated calcium channels in
presynaptic terminals and in spines. Multiple sub-
types of GABAB receptors exist but little is known
about their subcellular distribution or specific
function. In collaboration with B. Bettler, we in-
vestigate specific targeting of GFP-labeled GABAB
receptor variants in organotypic slice cultures.
Receptors containing the GABAB1a subunit were
found in presynaptic terminals and on the dendrite
but were largely excluded from dendritic spines. In
contrast, GABAB1b-GFP was detected at the base of
many spines but not in the axon. This differential
distribution may underlie the functional diversity
demonstrated in electrophysiological studies of
knockout mice (Vigot et al. Neuron 50:589-601,
2006).
CONTRIBUTION OF INTRACELLULAR CALCIUM
STORES TO POSTSYNAPTIC CALCIUM TRANSIENTS
N. Holbro
Calcium uptake into intracellular stores influences
the duration of synaptically induced calcium tran-
sients and could potentially influence synaptic
plasticity.A minority of dendritic spines is equipped
with a specialized form of endoplasmic reticulum
termed spine apparatus. To identify those spines
unambiguously, we look for synaptopodin, a pro-
tein associated with the spine apparatus (Fig. 2).
The spine apparatus may serve as a calcium sink
or as a calcium source and influence synaptic plas-
ticity accordingly. There is conflicting evidence
about activity-dependent calcium release from in-
tracellular stores in dendritic spines, possibly due
to subtle differences in preparation or recording.
These intracellular processes are not trivial to
study, because whole-cell patch-clamp recordings
can compromise the function of second messen-
ger systems, which might also affect activation of
intracellular stores. To study the interplay between
intracellular calcium stores and synaptic calcium
influx, alternative and less invasive recording tech-
niques are needed.
In collaboration with O. Griesbeck, we use ge-
netically expressed calcium indicators based on
troponin C to measure intracellular calcium dy-
namics. These indicators are based on fluorescence
resonance transfer (FRET) between CFP and YFP.
Calcium ions binding to the troponin-linker cause
a conformational change in the molecule, bring-
ing the energy-donor (CFP) closer to the energy-
acceptor (YFP). As a result, CFP fluorescence de-
creases and YFP fluorescence increases simul-
taneously. The YFP/CFP ratio can be used as an
indicator of intracellular calcium changes. Tech-
niques for non-invasive optical calcium measure-
ments are at an experimental stage but have great
potential for monitoring the activity of neural cir-
cuits in culture and in intact animals. They can be
targeted to certain cell types and even to specific
subcellular compartments, e.g., the postsynaptic
density. In the future, we wish to use genetic cal-
cium indicators to visualize postsynaptic calcium
currents and thus differentiate between active and
inactive synapses.
This work was carried out in collaboration with
B. Bettler (Pharmazentrum, University of Basel),
O. Griesbeck (MPI of Neurobiology, Martinsried), Fig. 2. Detecting synap-
A. Matus (FMI, Basel) and T. Meyer (Stanford Uni- topodin inside a dendritic
versity). spine (yellow spot) of a
pyramidal cell filled with
neurobiotin (red) by observ-
ing 3 orthogonal planes
through the spine center
using 3-D datasets acquired
by 2-photon microscopy.
Bottom: synaptically evoked
spine calcium signals from
20 trials. Note frequent
failure of synaptic transmis-
sion (flat lines typical
of small CNS synapses)

Selected publications
Nimchinsky EA, Yasuda R, Oertner TG,
Svoboda K (2004)
The number of glutamate receptors
opened by synaptic stimulation in single
hippocampal spines. J Neurosci
24:2054-2064
Oertner TG, Matus A (2004)
Calcium regulation of actin dynamics in
dendritic spines. Cell Calcium 37:477-482
Oertner TG, Sabatini BL, Nimchinsky EA,
Svoboda K (2002)
Facilitation at single synapses probed with
optical quantal analysis. Nature Neurosci
5:657-664
Yasuda R, Nimchinsky EA, Scheuss V,
Pologruto TA, Oertner TG, Sabatini BL,
Svoboda K (2004)
Imaging calcium dynamics in small
neuronal compartments. Science STKE
2004 (219), pl5

FMI Report 2005/2006 25

Neurobiology

Botond Roska
Structure and function
of local neural circuits

INTRODUCTION
The function of the brain can be studied at many
different hierarchical levels. We are interested in
how neurons interact in local neuronal networks
to compute behaviorally relevant functions. We
use the mammalian retina as a model system be-
cause the input, the dynamically changing light
pattern, is well defined and can be easily manipu-
lated experimentally. Moreover, the activity of each GROUP LEADER
neuron can be recorded during retinal light stim- Botond Roska
ulation. Our experimental approach is inter-dis- botond.roska@fmi.ch
ciplinary: we combine physiological, molecular,
viral and computational approaches to reveal the TECHNICAL/RESEARCH
structure and function of retinal circuits. We use ASSOCIATES
molecular techniques to genetically identify cell Brigitte Gross Scherf
types in the network and label them using trans- Yukiko Shimada
genic technologies. The connections of labeled cells
are revealed using trans-synaptic viruses. Next we POSTDOCTORAL
study the function of a genetically isolated circuit FELLOWS
with physiological and two-photon laser imaging David Balya
tools. Finally, we use computational methods to Pamela Lagali
predict the behavior of an isolated circuit in nat- Thomas Mnch
ural conditions.
PhD STUDENTS
This brief report introduces the work of Botond Roska, Sandra Siegert
a new group leader at the FMI who arrived recently Tim Viney
from Harvard University.
UNDERGRADUATE
Volker Busskamp

GUEST SCIENTIST
Daniel Hillier

26 FMI Report 2005/2006

INTERACTION OF INHIBITION AND EXCITATION
DURING IMAGE MOVEMENT IN GENETICALLY
IDENTIFIED SUBTYPES OF RETINAL GANGLION
CELLS

The inner plexiform layer of the mammalian retina

is comprised of about 10 different strata, formed
by the dendritic arborizations of a dozen different
classes of ganglion cell. Each stratum incorporates
a different neural representation of the visual world
that is computed through interactions between
excitatory inputs from subclasses of bipolar cells
and inhibitory inputs from subclasses of amacrine
cells. We are interested in understanding how im-
age motion is represented in the different strata
and how local circuits compute the measured rep-
resentation. We record from subtypes of mice gan-
glion cells labeled with green fluorescent protein
(GFP). GFP-expressing ganglion cells are visualized
with two-photon laser microscopy. Patch clamp
techniques are used to record the spiking output
as well as the inhibitory and excitatory inputs dur-
ing retinal stimulation with moving images. Com-
paring the inhibitory and excitatory inputs with
the spiking output during image movement allows
us to ask about the role of inhibitory amacrine cells
in shaping the neural representation of a moving
object.
To understand the functional role of individual
cell types in the local circuit of the GFP-expressing
ganglion cells, we express a light-activated channel
fused to a fluorescent marker randomly and spar-
sely in bipolar and amacrine cells. While recording
from genetically identified ganglion cells, we stim-
ulate the presynaptic amacrine and bipolar cells
locally. This approach allows us to identify the cells
types that are functionally connected to the gan-
glion cell of interest.
STRUCTURE AND FUNCTION OF PROJECTIONALLY
IDENTIFIED RETINAL GANGLION CELLS
Fig.1. Amacrine cells in a
cells. We combine transsynaptic pseudorabies virus mouse retina after in vivo
(PRV) labeling, two-photon laser microscopy and electroporation with a
electrophysiological techniques to light up the plasmid that expresses
membrane-bound GFP
local circuit of ganglion cell subtypes in the retina
and record light-evoked activity from the labeled
cells. We inject GFP-expressing PRV into brain re-
gions where only subtypes of ganglion cells send
their axons. The transsynaptic, retrogradely sprea-
ding PRV labels ganglion cells as well as their synap-
tic partners in the retina, making it possible to cor-
relate structure and function in the local circuit.
DEVELOPMENT OF CELL IDENTITY
The mammalian retina contains a large number of
different cells types. In order to understand how
different these cell types are and how they might
change their role in the local circuit during devel-
opment, we correlate physiology, morphology and
gene expression in genetically identified subsets of
amacrine and ganglion cells during the develop-
ment of retinal circuits.
Our work is carried out in collaboration with Zsolt
Boldogkoi (University of Szeged), Lynn Enquist
(Princeton University), Connie Cepko (Harvard Uni-
versity) and Karl Deisseroth (Stanford University)

Many parts of the nervous system contain myriad

cell types organized into a large number of paral-
lel circuits. In order to understand the structure
and function of different cell types in a particular
local circuit, the neurons in that circuit must be
labeled and activity recorded from the identified

Selected publications
Balya D, Roska B (2005)
Retina model with real time implementa-
tion. International Symposium on Circuits
and Systems ISCAS 2005, Kobe, Japan,
pp 5222-5225
Roska B, Werblin FS (2001) Roska B, Molnar A, Werblin FS (2006)
Vertical interactions across ten parallel Parallel processing in retinal ganglion cells:
stacked representations in the mammalian how integration of space-time patterns
retina. Nature 410:583-587 of excitation and inhibition form the spiking
output. J Neurophysiol 95:3810-3822
Roska B, Werblin FS (2003)
Global shifts in natural scenes block spiking
in specific ganglion cell types. Nature
Neurosci 6:600-608

FMI Report 2005/2006 27

 Epigenetics Epigenetic modifications are
potentially heritable but reversible alterations
in gene expression. They determine cell
fate, help maintain genome integrity, and
play a role in diseases, including cancer and
neuronal disorders. Our interdisciplinary
approach exploits various model organisms
to examine the molecular mechanisms
underlying epigenetic regulation.

Rafal Ciosk
Mechanisms of totipotency in the germline

Witold Filipowicz
Mechanisms of RNA interference and
microRNA function in mammalian cells

Susan Gasser
Functional organization of the nucleus

Helge Grosshans
Regulation of animal development by
microRNAs

Patrick Matthias
Regulation of gene expression by
POU proteins, their coactivators and
chromatin regulators

Frederick Meins
Plant epigenetics and RNA silencing

Antoine Peters
Epigenetic programming in the mammalian
germ line and pre-implantation embryos

Dirk Schbeler
Dynamics and propagation of epigenetic
states

28 FMI Report 2005/2006

FMI Report 2005/2006 29
Epigenetics

Rafal Ciosk
Mechanisms of
totipotency
in the germline

INTRODUCTION
Germline cells eventually develop into sperm and
eggs. Like embryonic stem cells, germ cells give rise
to most cell types through a property called totipo-
tency that may one day be utilized to treat degen-
erative diseases such as Parkinson or juvenile dia-
betes. Loss of totipotency in human germ cells can
result in unusual tumors called teratomas, which
are a mix of various cell types such as teeth, hair or GROUP LEADER
bone. These tumors are the most frequent cancer Rafal Ciosk
type in the ovary and also occur in testes, but little rafal.ciosk@fmi.ch
is known about how they form or how totipotency
is normally maintained in the germline. TECHNICAL/RESEARCH
We use Caenorhabditis elegans as a model system ASSOCIATE
to study these problems. In principle, maintenance Mathias Senften
of germ cell totipotency could be achieved by in-
sulating germ cells from external cues that induce PhD STUDENT
non-germ cell fates. However, when C. elegans germ Bjrn Biedermann
cells are abnormally released from the gonad,
they invade somatic tissues but maintain germ cell UNDERGRADUATE
identity. There is also evidence that germ cells are Elzbieta Kowalska
insulated from internal cues that might specify
somatic fates. A master regulator of somatic differ-
entiation in the embryo, the transcription factor
PAL-1/Caudal, is occasionally expressed in the
wild-type germline, but its expression is not suffi-
cient to induce somatic differentiation. These ob-
servations suggest that germ cells have a mecha-
nism(s) preventing inappropriate developmental
programs. Our previous recent results suggested
that two conserved KH-domain translational re-
pressors, MEX-3 and GLD-1, have partially re-
dundant functions in the germline. To test this,
we constructed a mex-3 gld-1 double mutant and
found, in addition to defects in gametogenesis, that
many gonad cells did not resemble germ cells. By
several criteria, including expression of molecular
markers and cellular morphology, these cells trans-
differentiated into various somatic cell types, in-
cluding muscles, neurons, and intestinal cells. This
worm teratoma requires entry of germ cells into
meiosis and coincides with the disappearance of
germline-specific protein/RNA structures called P
granules. Our findings implicated RNA regulation
in the maintenance of germline totipotency and
established a genetically tractable model for study-
ing teratomas.

30 FMI Report 2005/2006

CAENORHABDITIS ELEGANS GERMLINE AS A
MODEL FOR STUDYING TOTIPOTENCY
The C.elegans hermaphrodite gonad contains germ
cells in a linear sequence of developmental stages
(Fig. 1A). Many events in germline development,
such as the switches between mitosis/meiosis
and spermatogenesis/oogenesis, involve transla-
tional regulation by the GLD-1 protein. GLD-1 is
expressed primarily in the central gonad and is
a member of the STAR family of KH-domain,
RNA-binding proteins that includes mammalian
Quaking and Sam68. MEX-3, a distinct KH-do-
main protein, is expressed in a complementary
pattern. The functions of human MEX-3 orthologs
remain unknown. Previous work showed that
MEX-3 and GLD-1 share target mRNAs and that
mutants lacking GLD-1 express MEX-3 abnormally
throughout the germline (Ciosk et al. 2004).
These finding raised the possibility that MEX-3
compensates in part for the lack of GLD-1.We con-
structed and examined mex-3 gld-1 double mu-
tants and found that germ cells in the absence
of MEX-3 and GLD-1 transdifferentiate into var-
ious somatic cell types, including muscle, neurons,
and intestinal cells (Fig. 1B ; Ciosk et al. 2006). The
ectopic muscles contained ultrastructures resem-
bling those found in normal muscles, expressed
muscle-specific markers, and contracted. The neu-
rons expressed a neuronal-specific GFP reporter
and had extensive processes similar to normal neu-
rons (Fig. 1C).
GERM CELL TRANSDIFFERENTIATION
The molecular pathways that induce teratomas are
not well understood. For our analysis of the worm
teratoma we focused on muscle differentiation.
Specification of most muscle precursors in normal
embryogenesis involves the conserved transcrip-
tional regulator PAL-1/Caudal and downstream
factors such as HLH-1/MyoD (Fig. 2A). HLH-1 is
not expressed in wild-type germ cells, but it is ex-
pressed in many germ cells in the mex-3 gld-1 go-
nad. Because depletion of PAL-1 from mex-3 gld-1
gonads caused a dramatic reduction in number of
both HLH-1-expressing cells and ectopic body
muscles (Fig. 2B), body muscles in the mex-3 gld-1
gonad appear to differentiate through a pathway
that produces most muscles in normal embryo-
genesis.
Both MEX-3 and GLD-1 participate in transla-
tional repression of pal-1 mRNA in wild-type go-
nads. However, because PAL-1 is occasionally ex-
pressed in wild-type meiotic germ cells without
inducing somatic differentiation, inappropriate
expression of PAL-1 on its own is unlikely to in-
duce transdifferentiation. Moreover, mex-3 gld-1
gonads depleted of PAL-1 contain only a few body
muscles but numerous neurons and pharyngeal
cells (Fig. 2B and not shown), suggesting that ad-
ditional, PAL-1 independent pathways of somatic
differentiation contribute to transdifferentiation.
We propose that transdifferentiation in the mex-3
gld-1 germline involves both (1) the abnormal ex-
pression of factors such as PAL-1 that normally
regulate somatic differentiation in embryos, and
(2) a defect that allows germ cells to respond to
these factors. Similar to ovarian teratomas, worm
teratoma requires the entry of germ cells into
meiosis. GLD-1 is required for wild-type meiotic
germ cells to progress beyond the pachytene stage
of meiosis to diakinesis. One possibility is that a
prolonged, aberrant pachytene stage makes germ
cells sensitive to factors such as PAL-1.
A
B C
Fig.1. Loss of MEX-3 and
THE MOLECULAR FUNCTION OF MEX-3/GLD-1 GLD-1 leads to transdiffer-
IN TOTIPOTENCY entiation of germ cells into
somatic cell types. A Wild-
type gonad expressing
Because MEX-3 and GLD-1 regulate translation of
MEX-3 (yellow) and GLD-1
diverse mRNAs, abnormal expression of specific (blue). Germ nuclei at
target mRNA(s) may promote transdifferentiation. pachytene predominate in
One known GLD-1 target encodes a component the central region; asterisk
of the histone H3 methyltransferase. Because LET- distal zone of proliferating
418/Mi-2, a component of the nucleosome remo- stem cells. B Micrograph
deling and histone deacetylase complex, has been of 1-day-old mex-3 gld-1
adult gonad with clusters of
shown to prevent expression of germline proteins
apparent neurons (green)
in somatic cells (Unhavaithaya et al. 2002), MEX- or muscles (red) (image of
3/GLD-1-dependent regulation of chromatin mo- boxed region shown at left);
difiers may prevent germ cells from adopting so- asterisk distal end of gonad.
matic fates. The P granule defects in mex-3 gld-1 C Neuronal cluster in a
mutants might also contribute to transdifferenti- mex-3 gld-1 gonad with long
ation. P granules contain multiple maternally pro- processes (arrowheads)
duced mRNAs and regulators of RNA metabolism.
In yeast and mammalian somatic cells, cytoplasmic
structures with some similarity to P granules have
a role in mRNA silencing and decay (Sheth and
Parker 2003). Thus, P granule defects might lead
to the release and inappropriate expression of com-
ponent mRNAs, resulting in transdifferentiation.
FMI Report 2005/2006 31
Epigenetics

2A OUTLOOK

Our findings so far raise several important ques-

2B tions: How do MEX-3 and GLD-1 normally main-
tain germline totipotency? How is totipotency con-
trolled in germ cells at other development stages?
Is the commitment to a new developmental fate
irreversible? Are the mechanisms that maintain
germline identity conserved? We will be addressing
these questions in our future research at the FMI.

This brief report introduces the work of Rafal Ciosk,

a new group leader at the FMI who arrived recently
from the Fred Hutchinson Cancer Research Center,
Seattle. The work was performed in collaboration
with James Priess.

Fig. 2. Ectopic muscles

in mex-3 gld-1 gonads
differentiate to muscles in
normal embryogenesis.
A Muscle differentiation
in normal embryogenesis
involves PAL-1/Caudal
and downstream targets
like HLH-1/MyoD.
B Mock-depleted (top)
and PAL-1-depleted
(bottom) mex-3 gld-1 go-
nads; bar 50 m

Selected publications
Ciosk R, DePalma M, Priess JR (2004)
ATX-2, the C. elegans ortholog of ataxin 2,
functions in translational regulation in the
germline. Development 131:4831-4841
Ciosk R, DePalma M, Priess JR (2006)
Translational regulators maintain totipo-
tency in the Caenorhabditis elegans
germline. Science 311:851-853
Good K, Ciosk R, Nance J, Neves A,
Hill RJ, Priess JR (2004)
The T-box transcription factors TBX-37 and
TBX-38 link GLP-1/Notch signaling to
mesoderm induction in C. elegans embryos.
Development 131:1967-1978
Sheth U, Parker R (2003)
Decapping and decay of messenger RNA
occur in cytoplasmic processing bodies.
Science 300:805-808
Unhavaithaya Y, Shin TH, Miliaras N, Lee J,
Oyama T, Mello C (2002)
MEP-1 and a homolog of the NURD
complex component Mi-2 act together to
maintain germline-soma distinctions in
C. elegans. Cell 111:991-1002

32 FMI Report 2005/2006

Witold Filipowicz
Mechanisms of
RNA interference and
microRNA function
in mammalian cells

INTRODUCTION
The epigenetic control of gene expression and post-
transcriptional silencing of genes by RNA inter-
ference (RNAi) and microRNAs (miRNAs) has
emerged recently as an extraordinarily important
and interesting area of molecular biology. These
reactions contribute significantly to the develop-
mental- and tissue specificity of gene expression.
They also exemplify the key role of hundreds of GROUP LEADER
novel non-coding RNAs in the regulation of gene Witold Filipowicz
expression. witek.filipowicz@fmi.ch
During RNAi, double-stranded RNA (dsRNA)
acts as a sequence-specific inducer of mRNA de- TECHNICAL/RESEARCH
gradation. The degradation is mediated by ~20-bp ASSOCIATES
small interfering RNAs (siRNAs) that arise by Caroline Artus
the endonucleolytic processing of dsRNA by the Tabea Zoller
RNase III enzyme Dicer (Fig.1). In their structural
properties siRNAs are related to miRNAs,a new type POSTDOCTORAL
of small ~21-nt regulator. Hundreds of miRNAs FELLOWS
are encoded in the genomes of eukaryotes as Sankar Bhattacharyya
diverse as plants and mammals. Unlike siRNAs, Suvendra Nath Bhattacharyya
which guide endonucleolytic degradation of tar- Michael Doyle
get mRNAs, miRNAs in metazoa generally imper- Maciej Drozdz*
fectly base-pair to the mRNA 3-untranslated Fabrice Kolb*
regions and inhibit protein synthesis (Fig. 1). Ramesh Pillai*
Translationally repressed mRNAs accumulate in Lanja Saleh*
cytoplasmic processing bodies, P-bodies, where Petr Svoboda
they are either stored for future reutilization or
degraded. RNAi and miRNA-mediated regulation PhD STUDENTS
are very complex reactions involving dozens of dif- Jody Filkowski
ferent proteins and intersecting with many other Astrid Haase
cellular pathways. The most intriguing connec- Lukasz Jaskiewicz
tion is that with transcriptional gene silencing, Daniela Schmitter*
TGS. Components of the RNAi machinery are Lasse Sinkkonen
involved in the control of chromatin structure Kaifu Tang*
and/or DNA methylation but details of these reac-
tions remain poorly understood, particularly in UNDERGRADUATES
mammals. Marcin Dembinski
Our research focuses on the mechanism and a Christine Ender*
biological function of RNAi and miRNAs and on Regula Habermacher
the role of the RNAi apparatus in TGS in mam- Adam Kole*
malian cells. Luca Schenk*

*left the group

FMI Report 2005/2006 33

Epigenetics
ROLE OF DICER IN RNAi AND miRNA REACTIONS
L. Jaskiewicz, M. Doyle, T. Zoller, F. Kolb
Dicer is a key enzyme in the RNAi and miRNA
pathways required for the biogenesis of both com-
ponents. Human Dicer is a large ~220 kDa multi-
domain protein. It contains RNA helicase/ATPase,
DUF283 and PAZ domains, two neighboring
RNase III-like domains (RIIIa and RIIIb), and a
C-terminal dsRNA-binding domain (dsRBD). The
RNase III domains resemble the catalytic domain
of the bacterial RNase III, which also has specificity
for dsRNA. Our previous work with different mu-
tants of Dicer indicated that the enzyme functions
as an intramolecular dimer of RIIIa and RIIIb
domains, assisted by two RNA-binding domains,
dsRBD and PAZ, the latter being involved in the
recognition of the 3-overhang end. Contrary to
previous speculation, we found that Dicer has only
Fig.1. Schematic represen-
one processing center containing two independent
tation of RNAi and miRNA
pathways
RNA cleavage sites and generating products with
2-nt 3-overhangs.Various mutants of Dicer are now
being used to characterize its in vivo and in vitro
interactions with other proteins participating in
the assembly of the RNA-induced silencing com-
plex (RISC; Fig. 1), such as Argonaute (Ago) and
TRBP. Using purified recombinant Dicer, we found
that the enzyme also binds to siRNAs in vitro (col-
laboration with Erik Sontheimer, Northwestern
University, Evanston, Illinois), consistent with the
idea that Dicer plays a role in downstream steps of
RNAi in addition to its processing function. Pre-
vious cell fractionation and immunolocalization
experiments indicated that Dicer resides mainly
in the cytoplasm. However, the reported involve-
ment of RNAi machinery in chromatin transac-
tions prompted us to re-investigate the localization
of Dicer and other RNAi components in mam-
malian cells.
34 FMI Report 2005/2006
PROTEINS INTERACTING WITH DICER AND THE
RISC ASSEMBLY
A. Haase, L. Jaskiewicz and F. Kolb, in collaboration
with T. Hobman (University of Alberta Edmonton) and
A. Gatigniol (McGill University, Montreal)
Several proteins interacting with Dicer have been
identified. These include members of the Argonaute
family, small proteins containing two dsRNA Bind-
ing Domains (dsRBDs) and the ortholog of human
Fragile X Mental Retardation protein, dFMR1, in
Drosophila. More recently, Dicer was also found to
be the core component of RISC essential for mRNA
target cleavage. The Argonaute family is highly
conserved in most eukaryotes and is divided into
two subgroups: Ago and Piwi/Hiwi. Ago proteins
are components of RISC, mediating gene silencing
through mRNA cleavage and translational repres-
sion. The PAZ domains of the Ago and Dicer pro-
teins were originally thought to mediate their in-
teraction. However, we have found that it is the
PIWI domain of human proteins hAgo2 and Hiwi
that binds directly to the Dicer RNase III domain.
To identify new proteins associating with Dicer,
we raised monoclonal and polyclonal antibodies
against human Dicer. The antibodies, which effi-
ciently immunoprecipitate Dicer from extracts of
different cultured cells, were used to identify inter-
acting proteins by combining two-dimensional gel
electrophoresis and mass spectrometry. We identi-
fied TRBP [HIV-1 transactivating response (TAR)
RNA-binding protein] as a protein partner of hu-
man Dicer and demonstrated it to be essential for
optimal RNA silencing mediated by siRNAs and
endogenous miRNAs. Previously TRBP, which
contains three dsRBD domains, has been assigned
several different functions, including inhibition
of the interferon (IFN)-induced dsRNA-regulated
protein kinase PKR, modulation of HIV-1 gene
expression, and control of cell growth. The TRBP-
Dicer interaction now established raises the possi-
bility of cross-talk between RNAi and the IFN/PKR
pathways. We are using immunoprecipitation and
other approaches to identify additional proteins
interacting with Dicer. We are also mapping the
interaction between Dicer, Ago and TRPB, and use
recombinant proteins in experiments aimed at the
reconstitution of the catalytically active RISC com-
plex.
ROLE OF RNAi MACHINERY IN CHROMATIN
SILENCING IN MAMMALS
P. Svoboda, L. Sinkkonen and T. Zoller, in collabo-
ration with R. Schultz (University of Pennsylvania,
Philadelphia)
In various organisms, factors involved in RNAi also
participate in the formation and maintenance of
heterochromatin and in TGS. This connection,
which has been established best for Schizosaccha-
A
romyces pombe and plants, also appears to be con-
served to some degree in mammals, but details of
RNAi roles in DNA methylation and other chro-
matin modifications in mammals remain largely
unknown. To determine whether dsRNA-directed
DNA methylation operates in mammalian cells, we
used bisulfite sequencing to analyze DNA in mouse
oocytes constitutively expressing long dsRNA
against the Mos gene. Long dsRNA induces efficient
Mos mRNA knockdown but not CpG and non-
CpG DNA methylation of the endogenous Mos
sequence in oocytes and early embryos. These data
demonstrate that dsRNA does not directly induce
DNA methylation in trans of this sequence in the
cells investigated. We have also generated stable
human cell lines in which Dicer can be knocked-
B
down by expression of siRNA hairpins. The effect
of Dicer expression on the chromatin status of these
cels lines is being investigated.

EFFECTS OF LONG dsRNA IN MAMMALIAN CELLS

J. Filkowski, P. Svoboda

Mammalian cells contain several pathways respon-

ding to long dsRNA. In addition to RNAi, they in-
clude the dsRNA-dependent adenosine deamina-
tion editing pathway, the PKR-induced interferon
response leading to general inhibition of trans-
lation and degradation of mRNA through RNAse
L, and two more recently described defense path-
ways mediated by Toll-like Receptor 3 (TLR3) and
by RNA helicase RIG-1. We are investigating how
dsRNA expression in the cell nucleus triggers these
pathways and which conditions lead to induction of
the sequence-specific pathways. As model systems
we use HeLa cells and mouse transgenic lines ex-
pressing long dsRNA hairpins and appropriate re-
porters.
MECHANISM OF TRANSLATIONAL REPRESSION
BY MAMMALIAN miRNAs
R. Pillai, S. N. Bhattacharyya, C. Artus, D. Schmitter,
F. Kolb and C. Ender, in collaboration with E. Bertrand
(Institute of Molecular Genetics, Montpellier)
miRNAs are ~21-nt RNAs involved in the regula-
tion of development, differentiation and other
processes in eukaryotes. In metazoa, they base-pair
to the mRNA 3-UTR, forming imperfect helices
with mismatches and bulges. To find out whether
miRNA-mRNA duplexes have specific features re-
cognized by factors mediating the repression, we
directly tethered Ago proteins, as the best charac-
terized components of miRNPs, to the mRNA
3UTR. Tethering of Ago proteins inhibited mRNA
translation by a mechanism similar to that induced
by miRNAs, indicating that miRNAs function as
guides positioning repressive factors on the 3-UTR.
Fig. 2. Starvation stress-
We also used mRNA reporters whose translation induced relocation from P-
is regulated by either endogenous let-7 miRNP or bodies is specific for CAT-1
by Ago tethering to investigate at which step miR- mRNA. A In Huh7 cells on
a rich medium, CAT-1
NAs inhibit protein synthesis. Let-7 miRNPs or Ago
mRNA (red) is enriched in
tethering inhibited translation initiation.m7G-cap- P-bodies (P-body protein
independent translation initiated at internal ribo- Dcp1a is expressed as a
some entry site (IRES) was not subject to repres- GFP fusion; red). Upon star-
sion, suggesting that miRNPs interfere with re- vation, CAT-1 mRNA exits
cognition of the cap. The repressed mRNA, Ago P-bodies (lower panels).
proteins and miRNAs were all found to accumulate B But miR-122 co-localizes
with P-bodies in fed and
in P-bodies.We believe that relocalization of repres-
starved Huh7 cells
sed mRNAs to P-bodies is a consequence rather than
a cause of the repression. However, the P-body lo-
calization could contribute to repression by main-
taining the mRNA in an environment unfavorable
for translation. Many mRNAs targeted by miRNAs
are also subject to considerable degradation. It is
likely that the degradation is a consequence of the
mRNA relocalization to P-bodies, which contain
mRNA catabolic enzymes. We are investigating de-
tails of the degradation process using cell lines in
which expression of Ago proteins is down-regu-
lated.

FMI Report 2005/2006 35

Epigenetics

REVERSIBILITY OF TRANSLATIONAL REPRESSION

BY miRNAs
S.N. Bhattacharyya and R. Habermacher,
in collaboration with E. Closs (Johannes Gutenberg
University, Mainz)

There are numerous examples of specific mRNAs

undergoing microRNA-mediated repression but it
is not known whether the repression is reversible.
We have obtained evidence that cationic amino
acid transporter 1 (CAT-1) mRNA is the subject of
repression by the liver-specific miRNA miR-122
in human hepatocarcinoma Huh7 cells. Consistent
with our previous results on the let-7 miRNA-
mediated repression, both the endogenous CAT-1
miRNA and miR-122 are localized in P-bodies. In-
terestingly, the repressed CAT-1 mRNA or reporters
bearing its 3UTR can exit P-bodies under con-
ditions of cellular stress and re-associate with poly-
somes and re-enter translation. The derepression
requires binding of HuR, an AU-rich-element-
binding protein, to the 3UTR of CAT-1 mRNA.
These data provide evidence that P-bodies func-
tion in the storage of miRNA-repressed mRNAs
and demonstrate that mRNAs can be relieved of
the miRNA-induced inhibition under specific cel-
lular conditions. We propose that proteins inter-
acting with the 3UTR generally act as modifiers
altering the potential of miRNAs to repress gene
expression. These data also support a two-step
mechanism of miRNA action, with the localization
to P-bodies being a secondary event following the
inhibition of translation.

Selected publications
Bhattacharyya S, Habermacher R,
Martine U, Closs EI, Filipowicz W (2006)
Relief of microRNA-mediated translational
repression in human cells subjected to
stress. Cell 125:1111-1124
Filipowicz W (2005)
RNAi: the nuts and bolts of the RISC ma-
chine. Cell 122:17-20
Haase A, Jaskiewicz L, Zhang H, Lain S, Zhang H, Kolb FA, Jaskiewicz L, Westhof E,
Sack R, Gatignol A, Filipowicz W (2005) Filipowicz W (2004)
TRBP, a regulator of cellular PKR and Single processing center models for
HIV-1 virus expression, interacts with Dicer human Dicer and bacterial RNase III. Cell
and functions in RNA silencing. EMBO Rep 118:57-68
6:961-967
Pillai R, Bhattacharyya S, Artus C,
Zoller T, Cougot N, Basyuk E, Bertrand E,
Filipowicz W (2005)
Inhibition of translational initiation by
let-7 microRNA in human cells. Science
309:1573-1576

36 FMI Report 2005/2006

Susan Gasser
Functional organization
of the nucleus

INTRODUCTION
My laboratory pursues two general research themes.
The first concerns epigenetic regulation of gene
expression and genome duplication. Our area of
expertise is the long-range three-dimensional or-
ganization of chromatin in the interphase nucleus
and the impact of such structural aspects on in-
herited patterns of gene expression. The second
theme is that of genome instability during DNA GROUP LEADER
damage. Here we focus on double-strand breaks Susan Gasser
and replication fork instability, which impose con- susan.gasser@fmi.ch
trols over cell cycle progression through checkpoint
signaling. The link between the two themes is our TECHNICAL/RESEARCH
commitment to combine genetic systems with ASSOCIATES
quantitative live imaging techniques. Our geneti- Razel Arpagaus
cally tractable model organism was initially bud- Vronique Kalck
ding yeast but now includes the worm Caenorhab- Thierry Laroche*
ditis elegans. Important insights arising during the Monique Thomas
last two years are summarized below. Monika Tsai-Pflugfelder

POSTDOCTORAL
FELLOWS
Jennifer Cobb*
Olivier Fritsch*
Pierre Meister
Brietta Pike
Kenji Shimada
Angela Taddei*
Haico Van Attikum
Elisa Varela Sanz

PhD STUDENTS
Ruchi Chauhan
Anna Maria Friedel
Lutz Gehlen
Fabrizio Martino
Shigeki Nagai
Thomas Schleker
Heiko Schober

UNDERGRADUATES
Sabine Rohner*

GUEST SCIENTISTS
Yukako Oma*
Takahito Yoshida*

*left the group

FMI Report 2005/2006 37

Epigenetics
Fig.1. Nuclear pore associa-
tion confers optimal expres-
sion levels for an inducible
yeast gene. 4D time-lapse
tracking of lacop-tagged
HXK1 in yeast nuclei under
different induction condi-
tions. HXK1 activation co-
incides with re-localization
to nuclear pores
38 FMI Report 2005/2006
NUCLEAR ORGANIZATION AND SILENCING
A. Taddei, H. Schober, S. Nagai, F. Martino,
F. Cubizolles
Eukaryotic genomes are distributed on linear chro-
mosomes in the interphase nucleus, an organelle
surrounded by a characteristic double membrane
studded with proteinaceous pores. The DNA fiber
is wrapped around histones that bear distinguish-
ing marks and is complexed with repressor pro-
teins that help regulate accessibility. However, in
addition to a local three-dimensional structure,
chromatin subdomains assume a non-random dis-
tribution within interphase nuclei. This organiza-
tion differs from tissue to tissue in multicellular
organisms.
Our goal is to examine the functional implica-
tions of the long-range organization of chromatin.
We are beginning to understand the general rules
that determine interphase chromosome position
and dynamics and we have shown that the nuclear
envelope and its associated proteins help determine
both local and higher-order chromatin structure.
In budding yeast, factors that bind silent chro-
matin (SIR proteins) have a second role of an-
choring repressed chromatin at the nuclear enve-
lope. This SIR-dependent pathway is redundant,
with a second anchoring pathway working through
the DNA end-binding complex yKu. Neither path-
way involves nuclear pore proteins directly, and si-
lencing and anchoring functions can be separated
in both yKu and Sir4 proteins. Importantly, we find
that anchoring of genes at the nuclear envelope is
critical for repression only when SIR proteins are
sequestered at telomeres and are, therefore, limit-
ing in the nucleus. Furthermore, microarray assays
in yeast cells that have lost both anchoring path-
ways showed that many internal genes are misreg-
ulated due to mislocalization of silencing factors
(Sir2, Sir3 and Sir4).
Using a recombination technique that allows ex-
cision of a ring of chromatin from a chromosome
in living cells, we have shown that inactive or silent
chromatin can position itself autonomously at
the nuclear envelope. This positioning requires the
interaction of Sir4, an integral component of the
repressed chromatin, with a perinuclear protein
called Esc1 (Enhances silent chromatin 1). Esc1 is
a large, acidic protein bound to the inner face of
the nuclear envelope. The second Ku-dependent
pathway anchors both silent chromatin and tel-
omeric ends in a complex that requires the RNA
component of telomerase. We have shown that
telomere length maintenance is perturbed when
telomeres are not appropriately anchored.
Apart from yKu,the chromatin-anchoring factors
at the yeast nuclear envelope are not conserved in
higher eukaryotes.Nonetheless,our studies shed light
on a rationale for the presence in all eukaryotic nu-
clei of heterochromatic foci in which repressed chro-
matin is sequestered away from active chromatin.
The reconstitution of chromatin structures that
require a higher-order folding of the nucleosomal
fiber is being pursued with the use of a baculovi-
ral expressed SIR 2-3-4 complex. This repressive
complex includes Sir2, an NAD-dependent histone
deacetylase that forms a homotrimer when ex-
pressed alone but a 1:1:1 heterotrimer when ex-
pressed with Sir3 and Sir4. The assembly of Sir pro-
teins into silent chromatin correlates with a signi-
ficant reduction in both protein and telomere dif-
fusion rates in vivo (in collaboration with T. Mis-
teli, NIH). This suggests that the nucleosomal fiber
forms a distinct complex with these proteins, which
we hope to reconstitute in vitro (in collaboration
with D. Rhodes, MRC, Cambridge).
CHROMATIN MOBILITY AND GENE EXPRESSION
AT NUCLEAR PORES
A. Taddei, G. Van Houwe, V. Kalck, F. Hediger
Most yeast chromatin moves continuously in a
random walk (radius of constraint = 0.60.1 m),
except when it is anchored as a silent domain
(Fig. 1). The excision of a sequence from its chro-
mosomal context greatly enhances its mobility.
We have now found that the binding of an ATP-
dependent nucleosome remodeler or of a strong
transcriptional activator can enhance chromatin
mobility. This argues that events leading up to
transcription involve enhanced dynamics that co-
incide with the unfolding of the nucleosomal fiber.
Recently, a novel type of pore-mediated anchor-
ing was detected for the inducible yeast gene
HXK1, which is found in a subtelomeric region of
chromosome 6. The transcriptional activation of
HXK1 by growth on a non-glucose carbon source
led to its relocalization to nuclear pores. This re-
location required the 3UTR of the gene, which is
essential for efficient message processing and ex-
port. On the other hand, induction of the same gene
by another transcription factor moved the locus
away from the nuclear periphery. Thus the mode
of induction can influence both the mobility and
the site of transcription of a gene. When we inter-
fered with HXK1 localization by antagonizing as-
sociation with pore proteins or by tethering to the
nuclear periphery, accumulated transcript levels
were reduced or increased, respectively. From this,
we conclude that nuclear position has an active role
in determining optimal gene expression levels.
MAINTENANCE OF GENOME INTEGRITY
Maintenance of the genetic information and its ac-
curate transmission to offspring is primordial to
all stable forms of life. Correct replication requires
accurate duplication of chromosomes in S phase,
reformation and proper length maintenance of the
protective chromosomal cap at telomeres, as well
as efficient systems for the repair of DNA damage.
Furthermore, accurate mitotic transmission of
chromosomes requires their ordered condensation
and segregation into sister chromatids. Finally,
the genome must be protected from random in-
sertions by viral or other invasive agents. Naturally,
the mechanisms that prevent or facilitate these
events are of central interest to biomedical research.
Checkpoints are mechanisms that impose delays
in the cell cycle in response to DNA damage or de-
fects in DNA replication, to allow time for the re-
pair of such lesions and to ensure that mitotic
transmission is error-free. Failure to delay the cell
cycle in the presence of damage converts an easily
reparable DNA lesion into one far more dangerous
that can lead to genomic instability or cell death.
Not surprisingly, mutations in checkpoint proteins
are correlated with increased risk of cancer and with
enhanced cellular transformation rates.
Hereditary tumor-associated mutations affect
genes such as ATM and BRCA1 but also BLM,
which encodes a member of the RecQ family of he-
licases. BLM, like its yeast homologue Sgs1, plays
an important role during DNA replication, the
stage during which most spontaneous and induced
damage occurs in dividing cells. We have addressed
the question of how cells respond to the stalling of
replication forks, and specifically how the Sgs1 he-
licase preserves genomic integrity.
THE CELLULAR RESPONSE TO STALLED
REPLICATION FORKS
J. Cobb, A. Friedel, T. Schleker, K. Shimada
By artificially stalling synchronously moving repli-
cation forks with hydroxyurea (HU), we are able
to study the maintenance of DNA polymerases at
stalled replication forks in budding yeast. The sta-
bilization of such enzymes in a mode that allows
reinitiation requires both the ATR homologue
Mec1 and the RecQ helicase Sgs1. We found that
these two proteins function in a synergistic manner
to suppress gross chromosomal rearrangements,
independently of the downstream checkpoint ki-
nase Rad53. The Sgs1 pathway of polymerase sta-
bilization requires Top3 and Sgs1 helicase activity
and its function is at least partially dependent on
the recombination factor Rad51. These effects are
additive with the polymerase stabilization medi-
ated by Mec1 and a polymerase-associated factor,
Mrc1. The downstream checkpoint kinase Rad53
has quite a different role: this kinase is needed for
keeping the MCM helicase associated with the
stalled fork. The dissociation of MCM helicase
from poly-merases under conditions of damage or
polymerase stalling also leads to replication fork
collapse and strand breaks, and ultimately to ge-
nomic instability. A model of how these mecha-
nisms may synergise at stalled replication forks is
shown in Figure 2.
Fig.2. Synergism between
Using a quantitative chromatin-immunoprecip- yeast ATR kinase Mec1 and
itation approach, we have detected abundant lev- RecQ helicase Sgs1 pre-
els of histone H2A phosphorylation on a C-termi- vents stalled replication fork
collapse. Sgs1 interacts
nal serine (called gH2aX in mammalian cells) at
with RPA to promote stable
HU-arrested replication forks. This histone mod- binding of DNA polymerase
ification is catalyzed by the checkpoint kinase ATR alpha. Mec1/Ddc2 kinase
and it also occurs at double-strand breaks. Sur- acts directly on RPA, Mrc1
prisingly, high levels of H2A phosphorylation were and possibly Sgs1 itself.
detected at yeast telomeres during normal growth, Rad53 kinase ensures that
as well as on HU. The effect of these and other his- the MCM complex remains
fork associated
tone modifications on polymerase stability at both
normal and telomere-associated replication forks
is being examined.
THE DYNAMICS OF DNA STRAND BREAKS
H. van Attikum, O. Fritsch, L. Gehlen, T. Schleker,
K. Shimada, M. Tsai
The budding yeast INO80 complex is a conserved
ATP-dependent nucleosome remodeler contain-
ing actin-related proteins Arp5 and Arp8. Yeast
cells lacking Ino80, Arp5 or Arp8 are hypersen-
FMI Report 2005/2006 39
Epigenetics
Fig. 3. Integrated gene ar-
rays in C. elegans. Univer-
sally expressed lacI-GFP
displays inserted chromoso-
mal arrays of genes in living
worms and allows tracking
of gene mobility and posi-
tion as cells differentiate.
Inset at higher magnifica-
tion
Selected publications
Cobb JA, Schleker T, Rojas V, Bjergbaek L,
Tercero JA, Gasser SM (2005)
Replisome instability, fork collapse and
gross chromosomal rearrangements arise
synergistically from Mec1 kinase and
RecQ helicase mutations. Genes Dev
19:3055-3069
40 FMI Report 2005/2006
sitive to general DNA-
damaging agents and to
specific double-strand
breaks (DSB) induced
by the HO endonucle-
ase. Thus, it was pro-
posed that the remod-
eling complexes play
a direct role in DSB
repair.Using chromatin
immunoprecipitation,
we have shown that
Ino80, Arp5, and Arp8
are recruited to an HO-
induced DSB. Phos-
phorylated H2A also
accumulates at these
sites and the recruitment of Ino80 is compromised
in cells lacking the H2A S129 phospho-acceptor
site. Finally, we have demonstrated that conversion
of the DSB into single-stranded DNA is compro-
mised in arp8 and H2A mutants, which impair
INO80 activity or recruitment at the site of dam-
age. These results implicate INO80-mediated chro-
matin remodeling in the processing of DSBs for
checkpoint activation and repair.
Actin related proteins (ARPs), together with
conventional actin, are integral components of all
known chromatin-remodeling complexes and of
some histone acetyltransferase (HAT) complexes
as well. It has been shown that Arp6 co-fraction-
ates with SWR1-chromatin remodeling complex
(SWR1-C), which facilitates the exchange reaction
of histone H2A to the H2A variant Htz1. Moreover,
Arp5 and Arp8 are part of the INO80 complex,
which is recruited to DSBs. The chromosome-wide
distribution of Arp6 and Swr1 subunits has been
determined, as has the chromosome-wide distrib-
ution of Ino80 and Arp5 and Arp8 (in collabora-
tion with M. Harata, Tohoku University, Sendai,
Japan). Preliminary evidence suggests that Arp6
and other actin-related proteins play a role in mod-
ulating the subnuclear position of the chromoso-
mal sites at which they bind. Given that both the
INO80 complex and the SWR1 complex are re-
covered at DSB, we are examining the importance
Cubizolles F, Martino F, Perrod S,
Gasser SM (2006)
A homotrimer-heterotrimer switch in Sir2
structure differentiates rDNA and telomeric
silencing. Mol Cell 21:825-836
Gartenberg MR, Neumann FR, Laroche T,
Blaszczyk M, Gasser SM (2004)
Sir-mediated repression can occur indepen-
dently of chromosomal and subnuclear
contexts. Cell 119:955-967
of actin-related proteins and their ability to alter
chromatin position during DNA repair.
PHARYNGEAL FATE DETERMINATION
AND NUCLEAR ORGANIZATION IN C. ELEGANS
P. Meister, B. Pike, M. Thomas
Much of the initial work on the functional impact
of nuclear organization has been performed in
budding yeast or in mammalian cells, yet it would
be optimal to pursue these questions in a multicel-
lular organism that is readily modified genetically.
C. elegans provides such a convenient system. We
are studying a conserved developmental program
of transcriptional changes, namely the determina-
tion of mesoderm and endoderm. In worms, this
occurs during the early steps of pharyngeal differ-
entiation and is dependent on the conserved FoxA
transcription factor PHA-4.
Because changes in replication patterns often
precede changes in transcription patterns, one part
of the project requires the observation, description,
and functional characterization of DNA repli-
cation in C. elegans. This is done by following
GFP-PCNA by live fluorescence imaging. Other
approaches for visualizing gene positioning will be
an adaptation of the tagging techniques achieved
by the insertion of lac operators, co-expression of
GFP-lac repressor, and the coupling of this label-
ing technique to markers of the nuclear envelope
(Fig. 3).
During the endoderm-mesoderm commitment
process, a large set of genes becomes determined
to remain silent or to become active. Studying gene
replication patterns and subnuclear positioning
may allow us to relate changes in expression pat-
terns with both nuclear localization and replica-
tion timing. Unlike the yeast nucleus, the worm nu-
cleus has lamins and conserved lamin-associated
proteins, such as emerin, Man1 and BAF, which are
likely candidates for fastening DNA to the nuclear
envelope. In this way, the general principles derived
from the study of yeast are being tested in a more
complex, differentiating organism.
Taddei A, Van Houwe G, Hediger F, Kalck V,
Cubizolles F, Schober H, Gasser SM (2006)
Nuclear pore association confers optimal
expression levels for an inducible yeast
gene. Nature 441:774-778
van Attikum H, Fritsch O, Hohn B,
Gasser SM (2004)
INO80 recruitment by H2A phosphorylation
links ATP-dependent chromatin remodeling
with DNA double-strand break repair.
Cell 119:777-788
Helge Grosshans
Regulation of
animal development
by microRNAs

INTRODUCTION
MicroRNAs (miRNAs) constitute a large family of
noncoding RNAs that regulate the expression of
numerous target genes in plants and animals. In-
dividual miRNAs have been implicated in diverse
developmental processes such as cell differentia-
tion, apoptosis, and stem cell division.
Using the nematode Caenorhabditis elegans as a
model system, we explore the basic biology of an- GROUP LEADER
imal miRNAs. We are particularly interested in the Helge Grosshans
let-7 miRNA, which controls terminal differentia- helge.grosshans@fmi.ch
tion of certain cells in C.elegans. In humans, where
the developmental role of let-7 is unknown, let-7 TECHNICAL/RESEARCH
has been implicated in cancer. Strikingly, lessons ASSOCIATE
learnt from C. elegans can be directly relevant to Monika Fasler
our understanding of human biology and disease.
For instance, we identified several novel, physio- POSTDOCTORAL
logically important targets of C. elegans let-7 em- FELLOW
ploying a combination of computational sequence Ingo Bssing
analysis and experimentation. Based on this work,
we subsequently identified the RAS proto-oncogene PhD STUDENT
as a target of human let-7 miRNA and gathered Xavier Ding
evidence implicating let 7 as a tumor suppressor
gene in the lung. UNDERGRADUATE
We are investigating let-7 as both an important Keovilay Chanthavinout
developmental regulator and a model miRNA. To
understand let-7 function and, thus, to learn about
miRNA biology in general, we are identifying co-
factors, targets, regulators, and biogenesis factors
of this miRNA. At the same time, we have begun
to investigate additional miRNAs implicated in de-
velopmental regulation.

This report introduces the work of a new research

group at the FMI. Some of the results described
were obtained at the Department of Molecular, Cel-
lular and Developmental Biology, Yale University,
New Haven.

FMI Report 2005/2006 41

Epigenetics
Fig.1. An miRNA (let-7;
blue) recognizes partially
complementary sites in
the 3' untranslated region
(UTR) of target mRNA
(lin-41; black). Binding to
target mRNA prevents ac-
cumulation of the encoded
protein by a poorly charac-
terized mechanism (?), let-7. Using RNAi, we found that ten further genes
perhaps involving inhibition
of translation
42 FMI Report 2005/2006
THE C. ELEGANS let-7 microRNA REGULATES
SEVERAL TARGET GENES AT THE LARVAL-
TO-ADULT TRANSITION
H. Grosshans and F.J. Slack, in collaboration with
T. Johnson and M. Gerstein (Yale University, New Haven)
After hatching, the nematode C. elegans develops
through four larval stages before entering the sex-
ually mature, adult stage. Major changes occur in
many tissues at the larval-to-adult transition; this is
known as the larval-to-adult (L/A) switch.Through
forward genetics, the let-7 microRNA (miRNA) has
been identified as a key factor in the regulation of
this event. For instance, a subset of epidermal cells
called the seam cells divide in a stereotypic, stem-
cell-like pattern during larval development, but
exit the cell cycle and fuse at the L/A transition.
Lack of let-7 prevents terminal seam cell differen-
tiation so that these cells re-iterate larval develop-
mental programs and continue to divide.
let-7 is first expressed during late larval devel-
opment, resulting in repression of the lin-41 and
hbl-1 target genes through an antisense mechanism
(Fig. 1). In the absence of functional let-7, its tar-
get genes are overexpressed, preventing correct ex-
ecution of the L/A switch. The observation that
inactivation of lin-41 or hbl-1 through mutation
or RNAi can partially suppress let-7 mutant phe-
notypes demonstrates the physiological impor-
tance of their regulation by let-7.
We exploited the fact that let-7 binds to partially
complementary sequences in its target mRNAs
(Fig. 1) by using computational sequence analysis
to predict additional let-7 targets. The resulting
candidate target list was queried for suppression of
the let-7 phenotypes to identify physiologically im-
portant targets through genetic interaction with
mors. Expression of let-7 was lower in lung tumors
in addition to lin-41 and hbl-1 could suppress let-7
mutant phenotypes.
The putative let-7 binding sites of the novel let-7
targets are located in the 3 untranslated regions
(UTRs) of the target transcripts. To demonstrate
that these 3UTRs are sufficient to induce let-7-
dependent target gene regulation, we individually
fused five of these sequences to reporter genes ex-
pressed in the seam cells or the intestine. We chose
these tissues as they are sites of endogenous let-7
expression, allowing us to avoid ectopic (over-)ex-
pression of the miRNA. In all five cases, the reporter
gene was efficiently down-regulated in the presence
of wild-type but not mutant let-7 (Fig. 2).
The new target genes are mostly transcription fac-
tors, including the nuclear hormone receptor daf-12
and the forkhead transcription factor pha-4/hnf-3.
Strikingly, let-7 has the potential to coordinate de-
velopmental decisions across multiple tissues, as it
acts in at least three tissues (seam cells, intestine,
and ventral nerve cord) to regulate different tran-
scription factors and cell signaling molecules. We
are now working towards identifying the full com-
plement of let-7 target genes using computational,
genetic, and candidate gene approaches. This work
is the basis for understanding the function of let-7
in terminal cell differentiation and the L/A switch.
Let-7 REGULATES THE RAS PROTO-ONCOGENE
IN C. ELEGANS AND HUMANS
H. Grosshans, S.M. Johnson and F.J. Slack, in collab-
oration with J. Shingara, M. Byrom, R. Jarvis, A. Cheng,
E. Labourier and D. Brown (Ambion, Austin, USA)
Some miRNAs are highly conserved in sequence in
different animals. One example is let-7, which is
identical in C. elegans and humans. While let-7 is
known to control terminal differentiation of a sub-
set of epidermal cells in C.elegans, its role in human
development is not understood. However, recent
work has implicated human let-7 in lung cancer.
Our computational sequence analysis had pre-
dicted let-60/ras as a target of C. elegans let-7. We
validated this target by demonstrating that knock-
down of let-60/ras by RNAi suppresses let-7 mutant
phenotypes and that its 3UTR is sufficient to reg-
ulate the expression of a reporter gene in a let-7-
dependent manner.
let-60/ras is homologous to the human RAS
oncogene and we observed that the RAS 3UTR
also contains putative let-7 binding sites. Experi-
mental validation for RAS as a let-7 target was pro-
vided by our observation that modulation of let-7
levels affected RAS expression in vitro. Moreover,
a reporter gene carrying only the 3UTR of RAS
was similarly responsive to altered let-7 levels.
To examine the relevance of this observation in
vivo, we assessed let-7 expression in human tu-
than in normal lung tissue from the same patients,
indicating that let-7 targets may be overexpressed
in these tumors. Consistent with this notion, we
found that the tumors underexpressing let-7 also
overproduced RAS protein. As RAS is an impor-
tant oncogene in the lung, our work suggests that
let-7 functions as a tumor suppressor gene in the
lung by regulating RAS expression.
MEDIATORS OF let-7
X. Ding, M. Fasler, H. Grosshans
MicroRNAs interact with their target mRNAs
through base-pairing to complementary se-
quences, but the mechanistic details of how base-
pairing achieves target repression are not under-
stood (Fig. 1). Using an RNAi-based suppressor
screen, we identified genetic interaction partners
of C. elegans let-7. These let-7 suppressors include
known targets of let-7 and putative novel targets,
as indicated by the presence of let-7-binding sites
in their 3UTRs. A second class of suppressors does
not contain such binding sites and their molecu-
lar nature suggests that these genes are part of the
miRNA machinery. We are focusing our efforts on
elucidating the precise function of genes in this
second class. Already, we have shown some of these
genes to have developmental phenotypes consistent
with a role in let-7 function, i.e., they affect the tim-
ing of seam cell differentiation. Moreover, our pre-
liminary results indicate that a let-7 target reporter
gene is susceptible to reduced levels of these puta-
tive let-7 mediators, while a related reporter gene
with a 3UTR lacking let-7 binding sites is not.
These factors thus appear to suppress the let-7 mu-
tation by specifically repressing let-7 target genes.
This work provides first insights into the mecha-
nism of action employed by let-7 in vivo.
Let-7 REGULATORY NETWORKS
K. Chanthavinout, M. Fasler and H. Grosshans,
in collaboration with M. Zavolan (Biozentrum, Basel)
Previous efforts to elucidate the developmental
roles of miRNAs have typically looked at miRNAs
and their targets in isolation. However, reports
from C. elegans and plants have suggested a com-
plex interplay between miRNAs, their targets, and
the factors regulating miRNA expression and ac-
tivity. Moreover, distinct miRNAs can function re-
dundantly. Therefore, to understand the develop-
mental role of an miRNA requires a comprehensive
view of its interaction network. We will analyze
the interaction network of C. elegans let-7. By fo-
cusing on this well-studied miRNA, we can exploit
a wealth of already accumulated data. Its conser-
vation in sequence and, at least partially, function
A B
C
D
Fig. 2. Reporter gene assay
from C.elegans to humans further suggests that we for posttranscriptional gene
will be able to learn general lessons on miRNA regulation. A The reporter
function in animals. To unravel the let-7-depen- gene is transcribed in seam
cells during all development
dent network in C. elegans development, we are
stages; activity is visualized
using genetic screens as well as computational as blue staining.
tools that we are currently developing in collabora- B, C Reporter gene activity
tion with M.Zavolan (Biozentrum, Basel) to iden- is seen in a first larval stage
tify let-7 targets, co-factors, and regulators. Our re- (B, arrows) but is repressed
sults will improve our understanding of a develop- in adults (C). D When let-7
mentally important and model miRNA and provide is mutated, reporter gene
expression persists in adults
a basis for studying other miRNAs.
(arrows); thus, regulation
depends on let-7
DEVELOPMENTAL ROLES OF miRNAS
I. Bssing, H. Grosshans
Although individual miRNAs have been identified
as important regulators of animal development,
the functions of most miRNAs remain unknown.
Similarly, while miRNAs are thought to regulate
large parts of animal transcriptomes, few miRNA
targets have been established.
To obtain new insights into the genetic regula-
tion of developmental programs, we have begun to
investigate a subset of C.elegans miRNAs that have
developmentally regulated expression patterns but
no known function. We will examine the loss-of-
function and overexpression phenotypes of these
miRNAs and correlate them with their temporal and
spatial expression patterns. To identify the molec-
ular basis of the mutant phenotypes, we will also
predict targets for these miRNAs and validate them
in vivo. We expect that some of the miRNAs under
investigation participate in the control of cell fates
and, thus, will offer new insights into mechanisms
of development.

Selected publications
Grosshans H (2006)
An in vivo perspective on microRNAs:
lessons from the worm. In: Clarke NJ,
Sanseau PX (eds) MicroRNAs: biology,
function and expression. DNA Press,
Eagleville, USA (in press)
Grosshans H, Slack FJ (2002) Johnson SM, Grosshans H, Shingara J,
Micro-RNAs: small is plentiful. J Cell Biol Byrom M, Jarvis R, Cheng A, Labourier E,
156:17-22 Reinert KL, Brown D, Slack FJ (2005)
RAS is regulated by the let-7 microRNA
Grosshans H, Johnson T, Reinert KL, family. Cell 120:635-647
Gerstein M, Slack FJ (2005)
The temporal patterning microRNA let-7
regulates several transcription factors at
the larval to adult transition in C. elegans.
Dev Cell 8:321-330

FMI Report 2005/2006 43

Epigenetics

Patrick Matthias
Regulation of gene
expression by POU proteins,
their coactivators
and chromatin regulators

INTRODUCTION
The complex interplay between signal transduc-
tion cascades and genetic hierarchies of transcrip-
tion factors forms a molecular code that is unique
to each cell-type and defines its properties and fate.
In this context, our laboratory studies the mecha-
nisms of cell-specific gene expression in a geneti-
cally tractable system and concentrates on the trans-
criptional and epigenetic control of lymphoid cell GROUP LEADER
development and function (Fig. 1). We study sev- Patrick Matthias
eral transcription regulators that play a role in this patrick.matthias@fmi.ch
process, such as the POU family transcription fac-
tors Oct-1 and Oct-2 and their coactivator OBF-1. TECHNICAL/RESEARCH
It is a goal of the laboratory to understand the mode ASSOCIATES
of regulation and the respective biological roles of Chun Cao
these factors, as well as their mechanisms of action. Gabriele Matthias
Previous studies from our group have identified Adam Mizeracki*
several critical functions of OBF-1 for the normal
development and function of the lymphoid system. POSTDOCTORAL
In a second complementary direction we analyse FELLOWS
proteins involved in chromatin regulation and in Boris Bartholdy*
particular in the control of acetylation. Evidence Fabien Cubizolles
has accumulated that acetylation of histones rep- Camille Du Roure
resents a crucial epigenetic annotation of chro- Markus Kaller
matin that contributes significantly to the regula- Alexander Karnowski*
tion of gene expression. In addition, non-histone Yu Zhang*
protein acetylation plays a role in an increasing
range of cellular processes. Histone deacetylases PhD STUDENTS
(HDACs) remove acetyl groups from histone N Alain Bordon
terminal tails -as well as from other proteins- and Mathieu Dalvai
thereby contribute to the formation of a more con- So Hee Kwon
densed chromatin and to the modulation of gene Teppei Yamaguchi
expression. However, the biological role of HDACs
in mammals is still poorly understood, but it is clear UNDERGRADUATES
that these proteins are deregulated in many forms Frederick Vath*
of cancer. Recent findings suggest that HDACs also Matthias Wrobel*
play a role in other diseases. Thus, they represent
valuable potential therapeutic targets. GUEST SCIENTIST
To investigate the function of these different reg- Fatima Cavaleri*
ulatory proteins, we use a broad range of methods,
including generation and analysis of transgenic or
conditionally targeted mice together with molecu- *left the group
lar biology, biochemistry, functional genomics and
proteomics.

44 FMI Report 2005/2006

DISSECTION OF THE ROLES OF Oct-1, Oct-2
AND OBF-1
M. Dalvai, G. Matthias, A. Mizeracki and P. Matthias,
in collaboration with S. Massa and A. Rolink
(University of Basel) and F. Cavaleri and H.R. Schler
(Max Planck Institute, Mnster)
The transcription factors Oct-1 and Oct-2 belong
to the POU protein family, a subclass of the home-
odomain proteins containing a bipartite DNA-
binding domain, the POU domain. Whilst Oct-1
is a ubiquitous protein, Oct-2 is mostly restricted to
B lymphocytes and also to some T cells and to cells
in the nervous system. Early work has suggested
that Oct-1 and Oct-2 are critical for the control of
immunoglobulin (Ig) genes transcription by bind-
ing to the highly conserved octamer motif of Ig
promoters, thereby recruiting one or more tran-
scriptional coactivators. One of these coactivators
is the B cell-specific protein OBF-1 (aka OCA-B or
Bob1), which interacts with Oct-1 or Oct-2 and
plays an essential role in controlling the immune
response. However, individual knockouts for Oct-2
and OBF-1 gave superficially similar phenotypes
with respect to B cell development and immuno-
globulin gene transcription, suggesting that the two
factors are redundant in vivo. Surprisingly, mice
lacking both B cell specific factors still had IgM-
positive B cells although in reduced numbers,
and largely unaffected transcription of Ig genes.
Mice lacking the ubiquitous factor Oct-1, consti-
tutively or conditionally, have now been generated.
We found that Oct-1 is an essential protein, the ab-
sence of which leads to an early embryonic lethal
phenotype. Oct-1-deficient embryos die around
gastrulation and also show a defect in the forma-
tion of extra-embryonic tissues. Mouse embryo
fibroblasts and ES cells devoid of Oct-1 have been
established and are used to examine the cellular
function of this transcription factor. In these two
cellular contexts, no impairment in cell prolifera-
tion is observed in the absence of Oct-1 but the
cells show a strikingly altered response to stress.
The role of Oct-1 was also evaluated in B cells, by
crossing conditionally targeted Oct-1 mice with a
mouse line expressing Cre in a pan-hematopoietic
manner. No strong effect of Oct-1 ablation in B cells
was seen in these mice; however, other hematopoi-
etic lineages were affected by the lack of the Oct-1
protein.
THE PHYSIOLOGICAL ROLES OF OBF-1: NOVEL
TARGET GENES AND THE FUNCTION OF ISOFORMS
B. Bartholdy, A. Bordon, C. Du Roure, P. Matthias
We showed previously that absence of the coacti-
vator OBF-1 leads to multi-focal defects in the lym-
phoid system. A major defect in OBF-1-/- mice is a
severely impaired immune response correlated
with an incapacity to develop germinal centres
(GC) in secondary lymphoid organs. So far, how-
ever, only a few OBF-1 molecular targets have been
identified that can help explain the known pheno-
types. To address this question, we have generated
transgenic mice that constitutively express OBF-1
in the lymphoid compartment with the goal of
identifying genes that are deregulated in this set-
ting. This forced expression of OBF-1 was found
to have significant consequences for the develop-
ment of the B and T cell compartments and the
proportions of several lymphoid cell subsets are
strongly altered in these mice. RNA profiling ex-
periments with cells from wild-type or transgenic
Fig.1. Genetic switch regu-
mice allowed the identification of several genes that lating early B cell specifica-
are deregulated and are potential novel OBF-1 tar- tion in bone marrow. Devel-
get genes. In particular, the Ets transcription fac- opmental steps affected
by mutation of specific tran-
tor Spi-B, which is known to be important for the
scription factors are indica-
formation of GCs, was shown to be directly regu- ted. In some cases, mutation
lated by OBF-1 in the spleen. Furthermore, using of two factors is required for
an inducible system, it was demonstrated that a developmental phenotype,
OBF-1 is required for physiological induction of e.g. the Aiolos/OBF-1 dou-
the Spi-B gene. ble mutants (Matthias and
In addition, OBF-1 comprises two isoforms, one Rolink, Nature Rev Immunol
2005)
of which is nuclear and the other is generated from
a precursor and is localized in the cytoplasmic and
at the membrane owing to myristoylation of its N-
terminus. To define the biological role of these dif-
ferent isoforms, we used BAC transgenic technol-
ogy to generate mice expressing selectively one or
the other isoform at normal physiological levels.
This approach will allow us to define the specific
functions of the OBF-1 isoforms.
AIOLOS AND OBF-1 CONTROL THE pre-B
TO IMMATURE B CELL TRANSITION IN EARLY
B CELL DEVELOPMENT
A. Karnowski, C. Cao, G. Matthias and P. Matthias,
in collaboration with J. Skok (University College
London) and L. Martensson (Babraham Institute)
The Aiolos gene encodes a zinc finger protein that
interacts with Ikaros proteins to form a higher or-
der complex with chromatin remodelling and hi-
stone deacetylase activities. Aiolos mutant mice
show spontaneous GC formation in the absence of
antigen challenge and their B cells readily enter an
FMI Report 2005/2006 45
Epigenetics

ANALYSIS OF HISTONE DEACETYLASE FUNCTION

Y. Zhang, T. Yamaguchi, SH. Kwon, G. Matthias,
A. Mizeracki, F. Cubizolles and P. Matthias,
in collaboration with C. Caron and S. Khochbin
(Institut Albert Bonniot, Grenoble), L. Schaeffer
(Ecole Normale Suprieure, Lyon) and C. Seiser
(University of Vienna Biocenter)

To better understand the role of protein acetyla-

Fig.2. HDAC-6 is mostly
cytoplasmic and partially
co-localizes with the micro-
tubule network. Exponen-
tial NIH3T3 cells double
stained for HDAC-6 (green,
anti-HDAC-6 antibody),
beta-tubulin (red, anti-beta
tubulin antibody) and DNA
(blue, DAPI)
tion in mammals, we have begun a genetic analysis
of several histone deacetylases (HDACs). HDAC-1
activated state. Because Aiolos-/- and OBF-1-/- mice
is known to be essential during mouse embryoge-
have an opposite phenotype with respect to GC
nesis. To circumvent this, conditionally targeted
formation, we intercrossed them to define whether
mice were generated to test the in vivo role of this
these two proteins function in the same pathway.
protein and analysis of the function of HDAC-1 in
During these studies, we discovered that mice lack-
the immune system is currently underway. In ad-
ing Aiolos have symptoms that recapitulate human
dition, knockout MEFs were generated. Unlike
systemic lupus erythematosus (SLE), a complex
HDAC-1 -/- ES cells, they do not show a prolifera-
systemic autoimmune disease of multiple origins.
tion defect, which may indicate that HDAC-1 plays
Strikingly, in this model, development of all the
different roles in stem cells and differentiated cells.
SLE symptoms requires OBF-1 function and GCs
HDAC-6 is a class II histone deacetylase with a
fail to develop in the double mutant mice.
unique primary structure comprising two con-
In addition, in the absence of both factors a block
served hdac domains. Unlike most other HDACs,
in early B cell differentiation was seen at the tran-
it is not found in the cell nucleus but is localized
sition between pre-B and immature B cells (Fig.1).
mostly in the cytoplasm, where it partly co-localizes
Therefore, normal passage from the pre-B to the
with the microtubule network (Fig. 2). We showed
immature B cell stage requires function of both
that HDAC-6 can deacetylate microtubules both
Aiolos and OBF-1. We searched for deregulated
in vitro and in vivo and it is, therefore, a tubulin
genes in compound mutant mice and identified
deacetylase (TDAC) regulating microtubule acety-
several novel target genes. Interestingly, we found
lation. While it has been known for a long time that
that components of the pre-B cell receptor, such as
microtubules can be acetylated, the significance of
the lambda5 and VpreB genes, were not properly
this modification for microtubule function is not
downregulated in double deficient pre-B cells and
understood.
this is likely to be the cause of the developmental
We performed a structure /function analysis of
block observed. Furthermore, we found that the
HDAC-6 and tested the activity of mutant proteins
sub-nuclear relocalization and the bi- or mono-
in vitro and in vivo. The organization of HDAC-6
allelic expression of the lambda 5 gene were specif-
is critical for the activity of this enzyme and two
ically altered in the different mutant B cells.
intact hdac domains are essential. Based on these
observations, we speculate that the deacetylation
reaction in general may require two catalytic do-
mains. In addition, cells and mice lacking HDAC-6
function were generated. Surprisingly, HDAC-6 -/-
mice are viable and appear normal. Microtubules
in these mice are heavily hyperacetylated, con-
firming that tubulin is a physiological substrate of
HDAC-6 and also demonstrating that the absolute
level of tubulin acetylation is not critical for the
homeostasis.

Selected publications
Matthias P, Rolink AG (2005)
Transcriptional networks in mature and
developing B cells. Nature Rev Immunol
5:497-508
Bartholdy B, Du Roure C, Bordon A,
Emsli D, Corcoran LM, Matthias P (2006)
The Ets factor Spi-B is a direct critical
target of the coactivator OBF-1. Proc Nat
Acad Sci USA (in press)
Sun J, Matthias G, Mihatsch MJ,
Georgopoulos K, Matthias P (2003)
Lack of the transcriptional coactivator
OBF-1 prevents the development of
systemic lupus erythematosus-like pheno-
types in Aiolos mutant mice. J Immunol
170:1699-1706
Zhang Y, Gilquin B, Khochbin S, Matthias P
(2006)
Two catalytic domains are required for
protein deacetylation. J Biol Chem
281:2401-2404
Zhang Y, Li N, Caron C et al. (2003)
HDAC-6 interacts with and deacetylates
tubulin and microtubules in vivo.
EMBO J 22:1168-1179

46 FMI Report 2005/2006

Frederick Meins
Plant epigenetics and
RNA silencing

INTRODUCTION
During development, cells and tissues become
progressively committed to specific fates. This re-
markable process, called determination, results
from epigenetic modifications, i.e., stable but po-
tentially reversible alterations in gene expression.
Epigenetic states can be inherited by somatic cells
and in cases such as genomic imprinting and
epimutation are even transmitted to the next sex- GROUP LEADER
ual generation. Epigenetic mechanisms involving Frederick Meins Jr.
DNA methylation, chromatin modification, and frederick.meins@fmi.ch
RNA silencing are important in human diseases,
including cancer, and play a key role in the mainte- TECHNICAL/RESEARCH
nance of genomic integrity, defense against viruses, ASSOCIATES
and transgenerational inheritance. We exploit the Jrme Ailhas
powerful advantages of Arabidopsis genetics to elu- Estelle Arn
cidate epigenetic mechanisms conserved in both Martin Regenass*
plants and animals. Heike Schley*
Our major interest is RNA silencing, which refers
collectively to RNA cleavage, translational repres- POSTDOCTORAL
sion, and DNA methylation guided by ca. 21-26 nu- FELLOWS
cleotide-long small RNAs (smRNAs). These smR- Konstantina Boutsika Muckenschnabel
NAs are generated by Dicer or Dicer-like (DCL) Quanan Hu*
activities from large, double-stranded RNAs (dsR- Corinne Keichinger*
NAs) or stem-loop precursors. Plants have evolved Azeddine Si-Ammour
a network of redundant RNA-silencing pathways Franck Vazquez
with dedicated functions and coordinate regula-
tion (Fig. 1). The small interfering RNA (siRNA) PhD STUDENTS
or RNA interference (RNAi) pathway functions Todd Blevins
primarily in defense against foreign nucleic acids and Claudia Kutter
viruses. Endogenous microRNA (miRNA), trans- Yang Ping Lee
acting siRNA (tasiRNA), and natural antisense
siRNA (nasiRNA) pathways help regulate develop- UNDERGRADUATES
ment, hormonal signaling, and stress responses. Fi- Simona Bieri
nally, the repeat-associated siRNA (rasiRNA) path- Maja Brenner*
way targets RNA-directed methylation (RdDM) of Sina Henrichs*
DNA repeats implicated in transcriptional gene Elzbieta Kowalska*
silencing (TGS) and epigenetic regulation of chro- Adrian Lutz*
matin. During the past two years, we have identified Magali Perret
mutants and novel smRNAs that provide insight Dominik Ziegler*
into how these pathways function in development,
virus defense, and epigenetic inheritance.
*left the group

FMI Report 2005/2006 47

Epigenetics
Fig.1. A current model
showing major smRNA path-
ways in plants. The sites of
action of several silencing-
related Arabidopsis genes
(green boxes) are indicated
48 FMI Report 2005/2006
NOVEL ARABIDOPSIS MUTANTS AFFECTING
RNAi, VIRUS DEFENSE AND miRNA REGULATION
K. Boutsika, F. Vazquez, E. Arn, J. Ailhas
and E. Kowalska, in collaboration with F. Di Serio
(Istituto di Virologia Vegetale, CNR, Bari, Italy)
We are functionally characterizing EMS loss-of-
function mutations of five Arabidopsis ESI genes
that result in spontaneous, constitutive RNAi of
a green fluorescent protein reporter gene. ESI1 en-
codes a 5 3 exonuclease, XRN4, that helps de-
grade 3-fragments generated by miRNA/siRNA-
guided cleavage of mRNA.Intriguingly, esi1 mutants
show pronounced variation in fertility, organ
placement, and organ number, suggesting that ESI1
has an important role in developmental home-
ostasis. In contrast, esi4 mutants regularly show
a specific leaf abnormality, increased accumula-
tion of miRNAs, and decreased expression of some
miRNA targets. These mutants also show enhanced
expression of key genes in each of the known sm-
RNA pathways. This raises the possibility that ESI4
is a general suppressor of RNA silencing affecting
pathways regulating leaf morphogenesis.
RNA silencing is a major defense mechanism of
plants against virus infection. Similar mechanisms
help protect mammals against infection by viruses
including influenza, HIV and SV40. As a counter-
defense, some viruses encode suppressor proteins
that block RNA silencing and function in both
plants and mammals. At least two ESI genes have
virus-defense related functions. The 2b protein en-
coded by Cucumber Mosaic Virus (CMV) sup-
presses systemic spread of RNAi. Studies of esi1
and esi2 mutants infected with CMV show that an
endogenous, silencing-suppression pathway exists
in Arabidopsis. This pathway is activated by CMV
2b, requires ESI2, and is down-regulated by ESI1.
PRODUCTION OF siRNAs DERIVED FROM
DNA VIRUSES
T. Blevins, A. Si-Ammour and C. Kutter,
in collaboration with T. Hohn and M. Pooggin
(University of Basel)
The Arabidopsis Dicer-like genes DCL1-DCL4
and the putative RNA-dependent RNA polymerase
genes RDR2 and RDR6 are believed to have dedica-
ted functions in specific silencing pathways (Fig.1).
Our studies of deficiency mutants infected with the
DNA viruses, Cauliflower Mosaic Virus (CaMV)
and Cabbage Leaf Curl Virus (CaLCuV), assign
functions to all known Dicer-like activities in the
production of viral siRNAs. DCL4, DCL2, and
DCL3 are required for biogenesis of 21-, 22- and
24-nt viral siRNAs, respectively; whereas, DCL1,
which is a component of the miRNA pathway, is
required for processing of 21-nt siRNAs from a
highly structured viral RNA. Accumulation of
viral siRNAs was not affected in rdr2 or rdr6
mutants, indicating that RDR2-DCL3 and RDR6-
DCL4 are not obligate partners for siRNA biogen-
esis. None of the dcl mutants showed increased
susceptibility to CaMV or CaLCuV. Apparently, re-
dundant RNA-silencing pathways for viral defense
exist or RNA silencing is not sufficient for defense
against these viruses.
MULTIPLE SMALL RNA PATHWAYS ASSOCIATED
WITH METHYLATION OF DNA REPEATS
T. Blevins, A. Si-Ammour, F. Vazquez, J. Ailhas,
S. Henrich
Our studies of rasiRNA production and DNA
methylation in deficiency mutants identified mul-
tiple RdDM pathways targeting different classes of
Arabidopsis DNA repeats. The 180-bp centromeric
repeat Atcon and the 160-bp pericentromeric re-
peat ATAR3 are targeted for DNA methylation by
the DCL3-RPD1/2-RDR2 pathway described for
the SINE element AtSN1 (Fig.1). This pathway also
depends on the maintenance methylase MET1 and
the chromatin-remodeling factor DDM1. Methy-
lation of the major 5S rDNA repeat did not depend
on DCL3 and rasiRNA production, but did require
RPD1 and RDR2, known to be essential for dsRNA
production. Moreover, the double mutants ddm1/
rpd1 and ddm1/rdr2 showed additive effects on
methylation, suggesting that 5S rDNA repeats are
targeted by parallel DDM1-dependent and dsRNA-
dependent pathways. Finally, TGS of transgenes
depends on a third, distinct silencing pathway that
requires DDM1, but not DCL3, RPD1, or RDR2.
TRANSGENERATIONAL EFFECTS ON RNA SILENCING
A B
Q. An, T. Blevins, F. Meins Jr and J. Ailhas,
in collaboration with F. Mette (Leibnitz Institute,
Gatersleben, Germany)

Transgenerational inheritance of epigenetic states

has important developmental consequences rele-
vant to cell specification, disease, and evolution.
We have developed a model for studying trans-
generational effects using Arabidopsis transgenes
as reporters for RNAi. Earlier, we showed that RNAi
in plants is a form of epimutation. Although com-
petence for RNAi persists over many generations,
the resultant silent state is erased during early de-
velopment of the next generation. Unexpectedly,
one transgenic line showed two forms of silencing
(Fig.2). Plants shifted stochastically between these
heritable states in successive generations. Eventu-
ally, some progeny of silent plants reverted to the
high-expressing state, indicating that competence
for RNAi is not always transmitted faithfully to the
next generation. Thus, the same transgene can ex-
hibit multiple epigenetic states differing in devel-
opmental regulation and transgenerational sta-
bility. Maintenance of RNAi depends on a critical,
threshold level of target-gene expression. Target-
gene transcription declined in lineages showing
eventual loss of silencing, suggesting that these
transgenerational effects could result from TGS.
Support for this hypothesis came from studies of
another transgenic line showing that, over a period
of 3-7 sexual generations, a reporter gene silenced
by RNAi can also become the target of TGS. The
shift from RNAi to TGS results from progressive
methylation of the promoter region of the trans-
gene and requires DDM1 for maintenance. Rever-
tants of the transcriptionally silent reporter gene
in a ddm1 background exhibit systemic, stochastic
RNAi. These results establish a clear link between
RNAi and TGS. The same transgene is the target of
a dynamic interplay of RNAi and TGS that slowly
shifts from generation to generation. We are cur-
rently testing the hypothesis that RNAi triggers
progressive TGS, which in turn reduces compe-
tence for RNAi.
Fig.2. Early, stable silencing
BIOGENESIS AND FUNCTION OF PLANT miRNAs (A) and late, unstable silen-
cing (B) of the same yellow-
A. Si-Ammour, C. Kutter, E. Arn, S. Bieri fluorescent protein (YFP)
transgene in progeny of a
miRNAs important for developmental regulation
homozygous Arabidopsis
are often derived from multiple genetic loci and transformant. Red autofluo-
have multiple targets. We currently focus on the rescence of UV-illuminated
biogenesis and function of two simple miRNA plants indicates YFP-gene
pathways targeting Arabidopsis genes. miR393 tar- RNAi
gets the cleavage of RNAs encoding TIR1 and
AFB1-AFB3. These F-box proteins are auxin re-
ceptors and components of SCF ubiquitination
complexes important for auxin-regulated gene ex-
pression. We have identified the major MIR393 lo-
cus and shown that miR393 biosynthesis depends
on the canonical DCL1-HYL1-dependent pathway.
Paradoxically, although miR393-deficient mutants
do not show increased accumulation of its target,
TIR1a mRNA, over-expression of miR393 pheno-
copies the altered auxin responses of a tir1a defi-
cient mutant. This suggests that miR393 could have
additional effects at the level of translational re-
pression or involve other forms of regulation. The
novel miRAt275 is encoded at a single locus and
has a single target, namely, mRNA encoding one
member of a transcription-factor family important
for regulating organ specification and flower de-
velopment. Studies with loss-of-function mutants
of the target and mutants over-expressing miRNA-
resistant target RNA suggest that miRAt275 func-
tions in adaxial cell specification and leaf morpho-
genesis.

Selected publications
Akbergenov R, Si-Ammour A, Blevins T,
Amin I, Kutter C, Vanderschuren H,
Zhang P, Gruissem W, Meins F Jr, Hohn T,
Pooggin MM (2006)
Molecular characterization of gemini-
virus-derived small RNAs in different plant
species. Nucl Acids Res 34:462-471
Di Serio F, Schb H, Iglesias A, Tarina C,
Bouldoires E, Meins F Jr (2001)
Sense- and antisense-mediated gene silenc-
ing in tobacco is inhibited by the same viral
suppressors and is associated with accumu-
lation of small RNAs. Proc Natl Acad Sci
USA 98:6506-6510
Glazov E, Phillips K, Budziszewski GJ,
Schb H, Meins F Jr, Levin JZ (2003)
A gene encoding an RNase D exonu-
clease-like protein is required for posttran-
scriptional silencing in Arabidopsis.
Plant J 35:342-349
Klahre U, Crt P, Leuenberger SA,
Iglesias VA, Meins F Jr (2002)
High molecular weight RNAs and small
interfering RNAs induce systemic posttran-
scriptional gene silencing in plants.
Proc Natl Acad Sci USA 99:11981-11986
Meins F Jr, Si-Ammour A, Blevins T (2005)
RNA silencing systems and their relevance
to plant development. Annu Rev Cell Dev
Biol 21:297-318

FMI Report 2005/2006 49

Epigenetics

Antoine Peters
Epigenetic programming
in the mammalian germ
line and pre-implantation
embryos

INTRODUCTION
In mammals, the generation of totipotent embry-
onic cells from two distinct and highly differenti-
ated gametes is a prerequisite for successful devel-
opment. Totipotency enables embryonic cells to
proliferate and ultimately differentiate into a mul-
titude of distinct cell types, each with specialized
functions. The identity of each of these descendant
cells results from the interplay between transcrip- GROUP LEADER
tion factor-based and epigenetic regulatory mech- Antoine Peters
anisms translating the genetic information and antoine.peters@fmi.ch
directing somatic development.
Nuclear transfer experiments demonstrate that TECHNICAL/RESEARCH
genome-wide epigenetic marks characteristic of a ASSOCIATE
fully differentiated nucleus can be reset in a pre- Frdric Zilbermann
implantation embryo. The low developmental suc-
cess rate of such cloned embryos, however, under- POSTDOCTORAL
scores the efficacy of epigenetic erasure and sub- FELLOWS
sequent programming of parental genomes natu- Philip Hublitz
rally occurring during male and female gametoge- Pawel Pelczar*
nesis. For DNA methylation, genome wide patterns Rmi Terranova
are erased and reset during early germ cell and pre- Shihori Yokobayashi
implantation embryonic development. Similarly,
other epigenetic marks like histone methylation and PhD STUDENTS
acetylation as well as chromatin-associated factors Urszula Brykczynska
undergo extensive changes during early gameto- Eszter Posfai
genesis and embryogenesis. Mareike Puschendorf
Using conditional mouse mutants, we study the Tianke Wang
in vivo role of various histone and DNA methyl-
transferases and histone deacetylases for epigenetic UNDERGRADUATES
programming in primordial germ cells, maturing Anthony Kelly*
oocytes, developing male germ cells and pre-im- Carolin Kolb
plantation embryos. In particular, we aim to de-
termine the level of the maternal and paternal con- *left the group
tributions of certain epigenetic modifications for
regulating genome-wide activation of gene tran-
scription in the two-cell embryo and directing cell
fate decisions during subsequent pre-implantation
embryonic development. These studies will contri-
bute to our understanding of the regulatory role of
chromatin in defining and maintaining pluripo-
tency versus directing differentiation, in condi-
tions of health and disease.

50 FMI Report 2005/2006

EPIGENETIC PROGRAMMING IN THE MATERNAL
GERM LINE AND PRE-IMPLANTATION EMBRYOS
M. Puschendorf, A. Peters
During somatic development of multicellular or-
ganisms, different cells and tissues acquire distinct
programs of gene expression. Epigenetic modifiers
are thought to regulate this process by providing
each cell-type with a unique epigenetic signature.
For most cells, these epigenetic marks become fixed
once the cell differentiates or exits the cell cycle and
are thought to mediate a cellular memory of the
differentiated state. In the pre-implantation em-
bryo, however, the paternal and maternal genomes
undergo major epigenetic reprogramming events.
For example, the paternal genome undergoes
genome-wide active DNA demethylation and ac-
quires various histone methylation modifications
de novo. In contrast, the maternal genome gradu-
ally loses DNA methylation by DNA replication
while retaining histone modifications and chro-
matin-bound proteins inherited from the oocyte.
These epigenetic (re)programming events may be
needed for the establishment of the totipotent state,
for the initiation of embryonic gene expression at
the two-cell stage and for directing lineage-specific
development in the pre-implantation embryo.
The Trithorax (TrxG) and Polycomb (PcG)
groups of proteins have been identified for their
role in faithfully maintaining transcriptionally ac-
tive or repressed states of key developmental reg-
ulatory genes, providing an epigenetic mechanism
of cellular memory. Members of both groups are
highly conserved and contain distinct histone
methyltransferase (HMT) activities, raising the
possibility that TrxG/PcG-mediated cellular mem-
ory functions, in part, through the methylation of
different lysine residues in the N-termini of histone
H3 and H1. PcG proteins function in two multi-
protein complexes, PRC1 (Polycomb Repressive
Complex 1) and PRC2/3. Enhancer of zeste 2 (Ezh2)
is an HMT present in PRC2/3 and confers tri-
methylation at H3 lysine 27 (H3K27me3). Recent
data suggest that the H3K27me3 modification
contributes to the targeting of PRC1 to chromatin
and maintenance of the repressed state. Genetic
loss-of-function studies show that Ezh2 and other
PRC2/3 and PRC1 members are required for gas-
trulation during murine embryogenesis. Impor-
tantly, maternal deletion of Ezh2 leads to growth
retardation of offspring. In this project, we investi-
gate the maternal and zygotic role of Ezh2 for epi-
genetic programming of the pre-implantation em-
bryo.
Immunofluorescence analyses (IF) show that
most active and repressive histone methylation
marks are present in the oocyte and are transmitted
to the embryo (Fig. 1). In contrast, the paternal
genome becomes de novo methylated at different
lysine residues and to different degrees (mono-, di-
or tri-methylation) in a temporally and spatially
coordinated manner after the protamine to histone
exchange has occurred (see below). RT-PCR, West-
ern blot and IF analyses show that different PRC1
and PRC2/3 members are maternally provided to
the embryo. We are currently using pre-implanta-
tion embryos that are maternally and/or zygoti-
cally deficient for Ezh2 to address the develop-
mental function of this HMT, to identify primary
target genes (by genome-wide gene expression
analysis) and to determine the regulatory cross-
talk between Polycomb and DNA methylation-
based repressive pathways.
Fig.1. Epigenetic asymmetry
ROLE OF HISTONE METHYLATION IN PATERNAL between the parental
TRANSMISSION OF EPIGENETIC INFORMATION genomes in mouse embryo.
Maternally, mono- and tri-
U. Brykczynska, A. Peters methylation states at histone
H3 lysine 27 are transmitted
Mammalian gametes differ considerably in their via the anaphase-II chromo-
potential to transmit histone-encoded epigenetic somes (fertilization) to the
information. As in somatic cells, chromatin in pro-nuclear (PN) stage em-
oocytes is organized in a nucleosomal configura- bryo. Paternally, H3K27me1
tion. In developing male germ cells, however, most is newly established prior
histones (85 % in humans and 99 % in mice) are to DNA replication (PN1-3)
and H3K27me3 during
exchanged for smaller, extremely arginine-rich
replication (PN4-5). Left:
proteins (protamines) that mediate chromatin paternal pronucleus; right:
condensation and direct the structural reorgani- maternal pronucleus
zation of the sperm nucleus into its characteristic
elongated form, required for motility. After fertil-
ization, protamines are again replaced by histones
that subsequently acquire post-translational mod-
ifications. The mechanisms by which HMTs are
specifically targeted to distinct regions of the pa-
ternal genome are currently unknown. We propose
that paternally inherited modified histones influence
the acquisition of modifications on newly deposited
FMI Report 2005/2006 51
Epigenetics

differentially methylated, harboring allelic DNA

A B
Fig. 2. A Histone modifica-
tions at imprinted genes in
the Kcnq1 imprinting
cluster in extra-embryonic
tissues. Green/blue arrows:
mono-allelic expression
at maternal and paternal
alleles. Hexagons, triangles,
squares: histone and DNA
modifications; active marks:
H3K9ac; H3K4me2; repres-
sive marks: H3K9me2,
H3K27me3; DNA methyla-
tion. Regular arrows: genetic
interactions; dashed arrows:
enzyme-target relations.
B Trophectodermal stem cells
shows mono-allelic expres-
sion of Osbpl5, Cd81 and
Kcnq1ot1
histones in cis. As such, they could direct zygotic
transcriptional activation of the paternal genome.
To test this hypothesis, we examined whether
mature spermatozoa contain methylated histones.
Western blot analysis performed on motile murine
and human epididymal sperm revealed the presence
of several methylation marks on retained histones.
We are currently performing chromatin immuno-
precipitation (ChIp) experiments on sperm chro-
matin to identify sequences associated with his-
tones and their covalent modifications.
EPIGENETIC REGULATION OF IMPRINTING
R. Terranova, S. Yokobayashi, A. Peters
In placental mammals, some genes are expressed
or silent depending on whether they are inherited
from the mother or the father. To date, approxi-
mately 80 of these so-called imprinted genes have
been identified, many of them involved in growth
and development of the placenta and embryo or
postnatally in neurological processes. Genomic
imprinting implies that each gamete, male or fe-
male, provides information that directs the tran-
scriptional state of an allele after fertilization. Cor-
rect establishment and maintenance of these allele-
specific transcriptional states is vital, as deregula-
tion of imprinted gene expression is associated
with developmental defects, cancer and behavioral
syndromes in humans.
Most imprinted genes are clustered in large chro-
mosomal domains and are controlled by cis-act-
ing Imprinting Control Regions (ICRs). ICRs are
methylation inherited from the maternal or pater-
nal gamete. Following acquisition during either
oogenesis or spermatogenesis, these DNA methy-
lation marks are maintained in the zygote and are
reliably transmitted throughout development. Ul-
timately, the epigenetic features at ICRs are trans-
lated in different ways to ensure proper parental
allele-specific silencing of neighboring genes. At
each generation, imprints are reset in the germ cell
lineage upon primordial germ cell reprogramming.
The factors and molecular mechanisms that read the
parental imprints and establish and maintain im-
printed silencing during embryonic and extra-em-
bryonic development are gradually being unraveled.
One of the most intensively studied imprinted
gene clusters, the so-called Kcnq1 cluster on the
distal end of mouse chromosome 7, is homologous
to the locus for Beckwith-Wiedemann syndrome
on human chromosome 11p15.5. In this cluster, a
non-coding RNA Kcnq1ot1 is transcribed from the
paternal allele, whereas the maternal allele is DNA
methylated and silent. Several studies have demon-
strated the importance of the Kcnq1ot1 non-cod-
ing RNA for silencing paternal alleles within the
Kcnq1 cluster. Moreover, all repressed alleles are
associated with repressive histone modifications
such as H3K9me2 and H3K27me3 (Fig. 2a).
Using the Kcnq1 imprinting cluster as a model
system, we aim to examine the role and interrela-
tionship of non-coding RNAs, DNA methylation,
histone methylation and the recruitment of chro-
matin-associated proteins in both the establishment
(during gametogenesis and pre-implantation de-
velopment) and maintenance (during post-im-
plantation development) of allele-specific silenc-
ing. We use mice conditionally deficient for DNA
and histone methyltransferases to assess their func-
tion in genomic imprinting and we have estab-
lished an RNA-FISH-based method to detect loss
of imprinting in cultured cells and early embryos
(Fig. 2b). We use DNA polymorphisms between
parental alleles and the ChIP methodology to an-
alyze the chromatin status at both alleles of im-
printed genes and to investigate changes in chromatin
organization and gene expression in chromatin
modifier-deficient mice.

Selected publications
Peters AHFM, Schbeler D (2005)
Methylation of histones: playing Memory
with DNA. Curr Opin Cell Biol 17:230-238
Peters AHFM, Kubicek S, Mechtler K,
O'Sullivan R, Derijck AAHA, Perez-Burgos L,
Kohlmaier A, Opravil S, Tachibana M,
Shinkai Y, Martens JHA, Jenuwein T (2003)
Partitioning and plasticity of repressive
histone methylation states in mammalian
chromatin. Mol Cell 12:1577-1589
Peters AHFM, Mermoud JE, O'Carroll D,
Pagani M, Schweizer D, Brockdorff N,
Jenuwein T (2002)
Histone H3 lysine 9 methylation is an
epigenetic imprint for facultative hetero-
chromatin. Nature Genetics 30:77-80
Peters AHFM, O'Carroll D, Scherthan H,
Mechtler K, Sauer S, Schfer C,
Weipoltshammer K, Pagani M, Lachner M,
Kohlmaier A, Opravil S, Doyle M, Sibilia M,
Jenuwein T (2001)
Loss of the Suv39h histone methyltrans-
ferases impairs mammalian heterochromatin
and genome stability. Cell 107:323-337

52 FMI Report 2005/2006

Dirk Schbeler
Dynamics and propagation
of epigenetic states

INTRODUCTION
Chromatin is the complex of DNA and proteins in
which the genetic material is packaged inside the
cells of organisms with nuclei. Regulatory signals
entering the nucleus encounter chromatin and
the rate-limiting biochemical responses that lead
to activation of gene expression involve alterations
in chromatin structure. Nucleosomal histones are
highly modified at multiple residues and their GROUP LEADER
modification status appears to be connected with Dirk Schbeler
a variety of measurable characteristics of a gene lo- dirk@fmi.ch
cus, such as DNA methylation and timing of DNA
replication. Such epigenetic information can re- TECHNICAL/RESEARCH
strict or enhance sequence-specific recognition of ASSOCIATE
DNA and, thus, manifests a plastic epigenome that Christiane Wirbelauer
specifies cellular responses to external and internal
cues. Current models suggest that this epigenetic POSTDOCTORAL
information specifies cellular identity and potency FELLOWS
and that it behaves dynamically during cellular dif- Tim-Christoph Roloff
ferentiation and transformation. Indeed misregu- Michal Weber
lation of proteins involved in epigenetic regulation
of chromatin and DNA have been linked to many PhD STUDENTS
human diseases, including cancer. Stan Oliver Bell
Our research is aimed at understanding the hier- Fabio Mohn
archy and crosstalk between different constituents Michaela Schwaiger
of epigenetic information, their maintenance and
the regulation of reprogramming events. UNDERGRADUATES
The many players involved in epigenetic regula- Elena Aritonovska
tion point to a complex and intertwined regulatory Ozren Bogdanovic*
network, as exemplified by the increasing number David Wittig*
of described covalent histone modifications. Using
Drosophila and mammalian cellular models, we *left the group
combine a molecular approach with a genome-wide
experimental readout using DNA microarrays.
This enables us to describe the epigenome and its
dynamics in an unbiased and comprehensive fash-
ion. Our goal is to understand the control of epi-
genetic gene regulation and its role in cellular re-
programming events that occur during develop-
ment and disease.

FMI Report 2005/2006 53

Epigenetics
A promoter and is linked to the process of chromatin
B does not occur simultaneously during the S-phase
Prom. 1-2.5 kb
Fig.1. Nuclear and chromo-
somal distribution of histone
H3 variants and euchromatic
histone modifications.
A Immunostaining for ecto-
pically expressed histone
H3 (*H3) or histone H3.3
(*H3.3). Counterstaining
with DAPI highlights bright
staining centromeric hete-
rochromatin. B Distribution
of histone variants and
selected active chromatin
marks along the body of
active genes
54 FMI Report 2005/2006
CHROMATIN STATES AND CHROMATIN MODIFIERS
AT ACTIVE GENES
C. Wirbelauer, O. Bell, E. Aritinovska
A large number of histone modifications have been
described recently. These are assumed to be in-
volved in all DNA-templated events, yet we are only
just beginning to understand their individual and
combinatorial functions. Is the encoded informa-
tion used to direct gene activity to particular genes?
Or does it recruit enzymatic activity that is gener-
ally coupled to processes such as transcription,
replication or DNA repair? To gain insights into
3 end
the chromosomal complexity of various histone
tail modifications, we have mapped the genomic
distribution of five different histone modifications
known to be involved in gene activation. By study-
ing the presence of histone H3 and H4 acetylation
and H3 methylation at lysines 4 and 79 in 6000
genes in Drosophila, we assembled the first global
chromatin map in a metazoan genome, and this
yielded a surprising result. All analyzed chromatin
marks were present at all active genes, suggesting
that their function is coupled to transcription rather
than specifying sites of differential regulation
(Schbeler et al. 2004). Based on this observation,
we asked whether coinciding euchromatic histone
modifications could be predetermined by tran-
scription-coupled incorporation of the histone
variant H3.3. We generated cell lines expressing hi-
stone variants and analyzed variant deposition in re-
lation to histone modifications in detail and also
during gene induction. We showed that H3.3
incorporation occurs throughout active genes,
suggesting a model in which transcription causes
nucleosomal displacement along the transcribed
gene followed by the incorporation of nucleosomes
containing H3.3 (Wirbelauer et al. 2005). However,
the tested euchromatic histone modifications
showed a bias towards the 5 end distinct from that
of H3.3 (Fig. 1) and we hypothesize that H3.3 does
not predetermine chromatin states but serves as a
system to regain nucleosomal density following
transcription-coupled displacement.
Our current efforts are aimed at studying the
individual contribution of chromatin marks on ac-
tive genes and we have started to analyze the function
and distribution of the methylation of lysine 36 of
histone H3. This modification is also enriched at all
active genes but it is enriched downstream of the
compaction following nucleosomal disruption by the
polymerase. We have cloned the enzymes respon-
sible for lysine 36 methylation in Drosophila and are
currently defining their function in vitro and in vivo.
PLASTICITY AND REGULATION OF DNA
REPLICATION TIMING
M. Schwaiger
Replication of the genome before mitotic cell divi-
sion is a regulated process that ensures the fidelity
of DNA duplication. Chromosome duplication
of the cell cycle, rather different parts of the genome
replicate at different times. DNA replication initi-
ates at defined locations throughout the chromo-
somes, called origins of replication (ORIs). Exper-
imental evidence suggests that the choice of origins
used during S-phase can vary as can their time of
firing. As a consequence, the pattern of replica-
tion timing along the chromosomes can vary widely,
making the process of DNA duplication highly
dynamic. So far, however, little is known about
the extent of this dynamic change, its regulation or
biological function. The relationship between re-
plication timing and gene expression has been the
subject of some speculation. Early microscopic
studies showed that genetically inactive hetero-
chromatin replicates later during S-phase than
euchromatin, which contains most of the genes. Us-
ing a microarray-based functional genomics ap-
proach, we generated a genome-wide map of repli-
cation timing for Drosophila and showed that active
genes are more likely to be early replicating than in-
active genes (Schbeler et al. 2002).
Since this global interplay between replication
and transcription was only observed in Drosophila
but not in budding yeast, we hypothesize that it re-
flects the more complex organization of the
genomes of higher eukaryotes. Indeed, we observed
a similar relationship in the replication timing of
the human chromosome 21 in different cell types
(White et al. 2004). To better understand the un-
derlying regulation, ongoing efforts are aimed at
identifying genomic regions that switch their repli- A B
cation timing during development. We hypothe-
size that late replication is enforced through
changes in chromatin structure and, therefore, we
are applying a candidate gene approach to identify
proteins modulating replication timing.

DNA METHYLATION IN NORMAL AND

TRANSFORMED CELLS
M. Weber, F. Mohn and T. Roloff, in collaboration with
C
E. Oakeley and M. Rebhan (FMI)

The reversible methylation of cytosines occurs fre-

quently in mammalian genomes and leads to tran-
scriptional repression. DNA methylation is thought
to be critically involved in defining cell identity by
blocking expression of non-lineage specific genes.
This model predicts that cellular reprogramming
during development and transformation coincides
with dynamic changes in this epigenetic mark.
To study the distribution and plasticity of DNA
methylation in a quantitative fashion, we developed
Me-DIP (methylated-DNA-immunoprecipitation),
an approach to identify DNA methylation indepen-
dent of sequence context by immunocapturing of
methylated cytosines, which can be combined with
analysis using existing microarray platforms (Fig.2).
Using this assay, we determined the DNA methy-
lation state of the complete human genome, thus
enabling us to generate the first global epige-
nome map of differential DNA methylation. We
found that DNA methylation in primary non-trans-
formed human cells is more abundant in gene-rich
regions.Furthermore,the inactive X-chromosome of
females has a very different pattern. Against current
dogma, the inactive x shows reduced methylation
outside of promoters, while only promoters become
methylated and inactivated. Together these findings
revise the simple model that DNA methylation
leads to heterochromatinization and instead suggest
a more complex, context-dependent function in
finetuning transcription (Weber et al. 2005).
By also studying epigenetic changes in colon
cancer, we showed that alterations in DNA methy-
Fig. 2. Genome-wide detec-
lation of transformed cells occur in very defined tion of DNA methylation.
regions, suggesting a localized and not global mis- A Methylated DNA immuno-
regulation. Furthermore, we provide evidence that precipitation enriches
genomic DNA that is epige-
cancer-specific hypomethylation occurs preferen-
netically modified by
tially in gene-poor regions. A parallel analysis of methylation of cytosines
6000 promoters provided a quantitative view of and quantifies this mark
promoter methylation and identified a number of by PCR or micro-array.
novel genes that are epigenetically misregulated in B Comparison of the methy-
cancer, all of which are putative disease markers lation of 6000 CpG island
and therapeutic targets. As an extension of this pi- promoter sequences in
primary and transformed
lot study, we are currently examining the methy-
cells identifies sequences
lation status of all human promoters not restricted aberrantly methylated in
to CpG islands. Our goal is to define a complete set cancer. C Bioinformatics
of reprogrammed cis-regulatory regions, which identification of sequences
should give us further insights into the context- characteristic of methy-
dependent functions of this epigenetic mark and lated DNA
the underlying targeting mechanisms.
Using bioinformatics approaches, we are asking
whether epigenetically reprogrammed genes share
sequence motifs, regulation, chromosomal posi-
tion, proximity to repeats and other genomic fea-
tures. Furthermore, we are using sequence com-
parisons to the chimpanzee genome to define how
epigenetic modifications of the DNA have contri-
buted to the genome divergence between humans
and apes.

Selected publications
Schbeler D (2006)
Dosage compensation in high resolution:
global up-regulation through local recruit-
ment. Genes Dev 20:749-753
Schbeler D, MacAlpine DM, Scalzo D,
Wirbelauer C, Kooperberg C, van Leeuwen F,
Gottschling DE, O'Neill LP, Turner BM,
Delrow J, Bell SP, Groudine M (2004)
The histone modification pattern of active
genes revealed through genome-wide
chromatin analysis of a higher eukaryote.
Genes Dev 18:1263-1271
Weber M, Davies JJ, Wittig D, Oakeley EJ, Wirbelauer C, Bell O, Schbeler D (2005)
Haase M, Lam WL, Schbeler D (2005) Variant histone H3.3 is deposited at sites
Chromosome-wide and promoter-specific of nucleosomal displacement throughout
analyses identify sites of differential DNA transcribed genes while active histone mod-
methylation in normal and transformed ifications show a promoter-proximal bias.
human cells. Nature Genet 37:853-862 Genes Dev 19:1761-1766
White EJ, Emanuelsson O, Scalzo D,
Royce T, Kosak S, Oakeley EJ, Weissman S,
Gerstein M, Groudine M, Snyder M,
Schbeler D (2004)
DNA replication-timing analysis of human
chromosome 22 at high resolution and
different developmental states. Proc Natl
Acad Sci USA 101:17771-17776

FMI Report 2005/2006 55

 Growth Control Our growth control program
strives at a comprehensive understanding
of signaling circuits and mechanisms
regulating the growth, division and death
of cells. A deeper knowledge of these
processes should further the development
of innovative, mechanism-based thera-
peutics to counter many human diseases.

Joy Alcedo
Neural mechanisms that influence
the aging process

Ruth Chiquet
Cell communication in growth control
and differentiation

Brian A. Hemmings
Protein kinase function in development
and cancer

Jan Hofsteenge
Molecular and functional aspects of
covalent protein modifications

Nancy Hynes
The molecular basis of breast cancer

Yoshikuni Nagamine
Regulation of the plasminogen
activator system with emphasis on
the DExH helicase RHAU

56 FMI Report 2005/2006

FMI Report 2005/2006 57
Growth Control

Joy Alcedo
Neural mechanisms
that influence
the aging process

INTRODUCTION
Aging is a multi-factorial process that involves
an interaction between the environment and the
gene activities of an animal. This interaction ap-
pears to be mediated by the sensory system, since
we have found that the lifespan of the nematode
Caenorhabditis elegans is influenced by both mu-
tations and treatments that inhibit the function
of its sensory neurons. We have shown that defects GROUP LEADER
in specific sensory neurons extend lifespan and Joy Alcedo
that the ability of some of these neurons to inhibit joy.alcedo@fmi.ch
longevity depends, surprisingly, on the activities
of other sensory neurons that promote longevity. TECHNICAL/RESEARCH
We have also shown that these neurons influence ASSOCIATES
lifespan through signaling pathways that control Martin Regenass
animal physiology. Indeed, one of these pathways, Monique Thomas
the insulin/IGF-1 signaling pathway, has been pro-
posed to act on the aging process by coordinately POSTDOCTORAL
regulating the expression of many genes affecting FELLOW
animal longevity. Thus, through its sensory system Wolfgang Maier
modulating the activities of pathways like insulin/
IGF-1 signaling, it is possible that the animal has PhD STUDENTS
devised a relatively rapid means to adjust its rate of Bakhtiyor Adilov
aging in response to the quality of its environment. Astrid Kleinert
Just how the sensory system elicits physiological Ivan Ostojic
changes that ultimately determine animal lifespan
is at present unclear. By using a combination of UNDERGRADUATE
genetic, cellular and molecular approaches, we Collin Ewald
plan (1) to identify sensory and neuroendocrine
pathways that influence lifespan, (2) to determine
whether the sensory control of lifespan involves
circuits at the neuronal and/or non-neuronal lev-
els, and (3) to examine whether these mechanisms
are conserved in other animals. Our finding that
putative sensory genes influence lifespan provides
an excellent opportunity to elucidate this process.

58 FMI Report 2005/2006

IDENTIFICATION OF SENSORY AND NEUROENDO-
CRINE SIGNALING PATHWAYS THAT INFLUENCE
LIFESPAN
W. Maier, B. Adilov, A. Kleinert, M. Regenass,
M. Thomas, J. Alcedo
This work was initiated in the group of Cynthia
Kenyon at the University of California, San Fran-
cisco.
The lifespan of C. elegans is influenced by specific
gustatory and olfactory neurons (Fig.1), suggesting
that its lifespan can be altered by the perception of
environmental stimuli. One possibility is that these
neurons influence lifespan by sensing food. Inter-
estingly, we have found that not every gustatory
neuron affects lifespan, suggesting that the animal
does not adjust its rate of aging in response to all
food-related signals, but rather to more specific
sensory cues. Consistent with this hypothesis, the
appearance of sensory-impaired animals differs
from that of calorically-restricted animals, which
have a general decrease in food intake.
The nature of the environmental stimuli, the
molecular basis of their detection, and the mech-
anisms integrating sensory information with sig-
naling pathways that regulate longevity are at pre-
sent unknown. To address these questions, we have
been conducting a screen based on RNA-mediated
interference in order to identify chemosensory re-
ceptors and neuropeptides and their receptors that
influence lifespan. To date, we have identified a
putative olfactory G protein-coupled receptor that
inhibits longevity. Interestingly, this receptor is also
expressed in longevity-inhibiting neurons.
Besides the insulin/IGF-1 pathway, the activity of
which is modulated by gustatory neurons (Fig.2a),
signals from the reproductive system also regulate
worm lifespan. The germ line of C. elegans gener-
ates a signal that inhibits longevity and this signal
is antagonized by another signal from the somatic
gonad that promotes longevity (Fig. 2b). We have
found that the somatic gonad signal requires
the activity of certain olfactory but not gustatory
neurons (Fig. 2b). This suggests that C. elegans
longevity is modulated by environmental cues that
are perceived and integrated in a complex manner
by specific sensory neurons. Presently, we are in-
vestigating whether the olfactory receptor we have
identified and shown to inhibit longevity interacts
with the somatic gonad signal and/or the insulin/
IGF-1 pathway to affect lifespan.
As we continue to identify genes involved in the
sensory influence on lifespan, we will also study
their expression patterns using GFP-fusion re-
porter constructs to determine in which neuronal
or non-neuronal cells they function. Furthermore,
we will test whether the functions of these genes
are interdependent or involve the insulin/IGF-1
Fig.1. Gustatory neurons
pathway and the signaling pathways from the re- of C. elegans filled with flu-
productive system. These approaches may enable orescent dye. The dendrites
us to define functional circuits between the longe- of the neurons extend to
the tip of the head. Cilia of
vity-inhibiting or longevity-promoting neurons
these dendrites are directly
and their target tissues. exposed to the environment
Finally, we will determine whether these genes (From the cover of Neuron
influence lifespan by affecting other processes, e.g., 41[1])
behavior. Although sensory mutants do not look
like or behave like animals that are calorically re-
stricted (a treatment that also extends lifespan),
it is still possible that sensory genes affect animal
feeding behavior. Sensory mutants, compared with
wild type, may exhibit a different preferential pro-
file for food products that influence lifespan. A
further possibility is that the genes we identify also
control animal appetite. For example, mammals
are known to eat in excess when presented with
sweet or salty food, whereas they will avoid bitter
food even when starved. In mammals, taste infor-
mation is relayed to the hypothalamus, a region of
the brain regulating endocrine function within the
whole animal. Thus, taste perception may regulate
the release of neuropeptides controlling appetite
and/or metabolism. Furthermore, taste perception
appears to be modulated by physiological cues since
leptin, a hormone known to suppress appetite, in-
hibits the responses of taste receptor cells to sweet
substances but not to salty or bitter substances.
This modulation of taste perception by leptin may
be involved in regulating animal feeding behavior,
possibly as a feedback mechanism. Hence, the sen-
sory system of the worm may control the release
of neuropeptides, including insulin-like peptides,
which regulate appetite and/or metabolism and
consequently lifespan. To test this hypothesis, we
will compare the feeding behavior, such as appetite
and foraging behavior, of wild-type and sensory
mutant animals on different food sources, while
correlating the effects of the different food sources
on the lifespan of these animals.
FMI Report 2005/2006 59
Growth Control
THE TEMPORAL REQUIREMENT FOR THE SENSORY
INFLUENCE ON LIFESPAN
J. Alcedo and W. Maier, in collaboration with
C. Kenyon (University of California, San Francisco)
In addition to determining which cells are involved,
we will also define the critical period during an an-
imal's life when its sensory system can influence
lifespan. To do this, we have been using a temper-
ature-sensitive mutant previously isolated in the
Kenyon lab from an unbiased lifespan mutagene-
sis screen. We have shown that this mutant affects
A B
Fig. 2. A Certain gustatory
neurons inhibit and others the normal development of the sensory cilia, which
promote longevity. Both are important in the chemotactic responses of the
classes appear to modulate worm to environmental stimuli. We have found
insulin/IGF-1 signaling to
that the temperature-sensitive defect in the sen-
affect worm lifespan.
B Worm gonad precursor
sory cilia correlates with the temperature-sensitive
cells. Germline cells (green lifespan phenotype. At the restrictive temperature,
circles) inhibit longevity; the mutant exhibits a defect in its sensory cilia and
the somatic gonad (blue lives longer than wild-type worms, whereas at the
circles) promotes longevity permissive temperature the mutant exhibits wild-
in an olfactory neuron- type sensory cilia and wild-type lifespan. Since the
dependent manner
defect in the sensory cilia of this mutant is rever-
sible, we have performed temperature-shift exper-
iments to determine the temporal requirement for
this gene in the regulation of worm lifespan. We
found that the product of this sensory gene must
be present late in larval development in order to
influence lifespan. Since specific sensory neurons
seem to modulate the activities of different signal-
ing pathways in their effects on lifespan, we are also
determining through laser ablations whether spe-
cific neurons also function at different stages of de-
velopment to affect lifespan.
Selected publications
Alcedo J, Kenyon C (2004) Alcedo J, Ayzenzon M, Von Ohlen T,
Regulation of C. elegans longevity by Noll M, Hooper JE (1996)
specific gustatory and olfactory neurons. The Drosophila smoothened gene encodes
Neuron 41:45-55 (and Cover) a seven-pass membrane protein, a putative
receptor for the Hedgehog signal. Cell
Alcedo J, Noll M (1997) 86:221-232
Hedgehog and its Patched-Smoothened
receptor complex: a novel signalling
mechanism at the cell surface. Biol Chem
378:583-590 (and Cover)
60 FMI Report 2005/2006
THE SENSORY INFLUENCE ON LIFESPAN
APPEARS TO BE CONSERVED IN DROSOPHILA
I. Ostojic and J. Alcedo, in collaboration with W. Boll
and M. Noll (University of Zurich)
Many biological processes have been conserved
between the worm and higher organisms. For ex-
ample, the insulin/IGF-1 pathway also regulates
the lifespan of flies and mice. Furthermore, in
mammals, gustatory and olfactory information are
relayed to neurons in the hypothalamus, which
controls neuroendocrine function, physiology and
behavior. Thus, the sensory systems of other ani-
mals might also influence their lifespan.
We have been testing this hypothesis by deter-
mining whether Drosophila sensory mutants also
have lifespan phenotypes. So far, we have found that
flies missing a subset of taste bristles live longer.
These taste bristles are located in the fly labella and
are innervated by gustatory neurons that extend
processes to the subesophageal ganglion (SOG), a
region in the brain that relays sensory information.
For example, the SOG interneurons project axons
to or near the insulin-producing cells in the fly
brain. Hence, we are currently testing whether flies
missing labellar taste bristles have altered insulin
expression and exhibit phenotypes other than al-
tered lifespan, e.g., feeding behavior and further
phenotypes associated with decreased insulin/
IGF-1 signaling.
We have also found that flies lacking most of
their taste bristles do not live as long as flies lack-
ing only labellar taste bristles. As in C. elegans, it
is possible that Drosophila has both taste neurons
that promote and taste neurons that inhibit
longevity. If this is the case, the activities of these
two neuron classes may either antagonize each
other or the activity of the longevity-inhibiting
neurons may require the activity of the longevity-
promoting neurons to influence lifespan. Together
our findings suggest that the Drosophila sensory
system, like that of C. elegans, also influences lifes-
pan and involves both positive and negative sen-
sory inputs.
Alcedo J, Zou Y, Noll M (2000)
Posttranscriptional regulation of
Smoothened is part of a self-correcting
mechanism in the Hedgehog signaling
system. Mol Cell 6:457-465
Ruth Chiquet-Ehrismann
Cell communication
in growth control and
differentiation

INTRODUCTION
Cells in tissues constantly sense their environment
via cell surface receptors that interact with ligands
on neighboring cells or with molecules of the ex-
tracellular matrix (ECM). These interactions in-
fluence the differentiation status of cells by controll-
ing intracellular signaling pathways that regulate
cellular gene expression programs. Perturbations
in these interactions lead to multiple diseases such GROUP LEADER
as cancer or degenerative diseases. Ruth Chiquet-Ehrismann
For the proper functioning of connective tissues, ruth.chiquet@fmi.ch
the integrity and maintenance of the link between
the ECM of a given tissue with cellular receptors TECHNICAL/RESEARCH
and the cytoskeleton of the cells synthesizing the ASSOCIATES
tissue-specific ECM is essential. This is exempli- Marianne Brown-Ldi
fied by muscle, in which mutations in proteins of Jacqueline Ferralli
the cytoskeleton, transmembrane proteins linking
it to the ECM, or ECM proteins themselves can lead POSTDOCTORAL
to muscular dystrophies, or by skin, in which such FELLOWS
mutations cause blistering diseases. Florence Brellier
In our lab, we focus on several proteins impor- Stefano Canevascini*
tant for connective tissue function. Tenascin-C and Krzysztof Drabikowski*
tenascin-W are present in tendon and bone ECM Laurent Gelman
as well as in the stromal compartment of many Silke Maier
tumors. Therefore, we analyze the anti-adhesive
and tumor growth-promoting functions of the PhD STUDENTS
tenascin family of ECM proteins and attempt Michaela Brosig
to identify their interaction partners and cellular Iwona Bucior*
receptors. Agrin is present in the basal lamina of Martin Degen
muscle as well as at neuromuscular junctions, Ana Hrus
where it regulates the clustering of acetylcholine Daniela Kenzelmann
receptors. We delineate certain aspects of agrin Samantha Nunes*
function in the model organism Caenorhabditis el- Agnieszka Trzebiatowska
egans.
Recently, we discovered a novel cell surface re- UNDERGRADUATES
ceptor family that we termed teneurins. Teneurins Gordon Lau*
are transmembrane proteins with an intracellular Enrico Martina*
domain involved in signal transduction and a large Thomas Walpen*
extracellular part involved in cell-cell interactions.
We analyze teneurin function in vitro by molecu- GUEST SCIENTISTS
lar and cell biological experiments and in vivo us- Matthias Chiquet
ing C. elegans. Bradley Ian Morrison*
Richard Tucker*

*left the group

FMI Report 2005/2006 61

Growth Control
Fibronection
Fig.1. F-actin staining of
fibroblasts plated on fibro-
nectin reveals well-spread
cells with numerous stress
fibers. In contrast, the
morphology of cells on a
tenascin-W substratum
is irregular with actin-rich
processes
62 FMI Report 2005/2006
Tenascin-W
TENASCINS IN CANCER
F. Brellier, M. Degen, T. Walpen
A special connective tissue compartment, the tumor
stroma, surrounds and penetrates solid tumors.
Evidence is accumulating that this tissue influences
cancer development and has an impact on malig-
nancy. Cells of the stromal compartment express
pro-proliferative paracrine signals to epithelial cells
and stimulate angiogenesis. Therefore, novel ap-
proaches for cancer therapy are being developed
based upon the antagonism of tumor stroma func-
tion.
The importance of the tumor stroma in cancer
development prompts the characterization of this
tissue compartment. One prominent ECM protein
specific to the tumor stroma is tenascin-C. Inter-
estingly, tenascin-C is both expressed around an-
giogenic vessels in many tumors and promotes
angiogenesis in cell culture studies. Furthermore,
tenascin-C addition to a fibronectin substratum
stimulates cancer cell growth in vitro. Thus,
tenascin-C is one of the molecules that mediate
pro-tumorigenic effects of the tumor stroma.
Recently, we described the induction of a second
member of the tenascin family, tenascin-W, in the
stroma of mouse mammary tumors. Since the hu-
man orthologue had not been analyzed, we cloned
human tenascin-W cDNA, characterized the re-
combinant protein, and raised antibodies. We
found that tenascin-W is elevated in many tumor
samples but not in corresponding normal tissues.
In breast cancers, tenascin-W was more prevalent
in low-grade tumors. In contrast to tenascin-C,
tenascin-W did not interfere with cancer cell ad-
hesion to fibronectin and fibroblasts could adhere
to a tenascin-W substratum, albeit with a mor-
phology distinct from that of cells plated on fi-
bronectin (Fig. 1). We postulate, therefore, that
tenascin-W is one of the stromal factors influenc-
ing cancer development.
MECHANOTRANSDUCTION IN CONNECTIVE
TISSUES
M. Brosig, M. Chiquet, L. Gelmen, S. Maier
Mechanical forces are essential for connective tis-
sue homeostasis. The ECM plays a key role in the
transmission of forces generated either by the or-
ganism (e.g. muscle contraction) or applied exter-
nally (e.g. gravity). The expression of specific ECM
proteins such as collagens and tenascin-C is influ-
enced by mechanical stimuli. The precise mecha-
nisms by which mechanical strains are translated
into chemical signals and lead to differential gene
expression are, however, not fully understood.
Cell-matrix adhesion sites are good candidates for
hosting a mechanosensory switch as they trans-
mit forces from the ECM to the cytoskeleton and
vice versa by physically linking the cytoskeleton to
the ECM. Integrins, transmembrane proteins lo-
cated to these adhesion sites, are involved in trans-
mission of mechanical signals.
The aim of this project is to unravel the intra-
cellular signaling pathways triggered by mecha-
nical stimulation of fibroblasts that result in
changes in gene expression and protein synthesis
as the basis for tissue remodeling. In embryo fibro-
blasts, the mRNA for the ECM protein tenascin-C
is induced by cyclic strain. This response is at-
tenuated by inhibiting Rho-dependent kinase
(ROCK). The small GTPase RhoA and its down-
stream target ROCK regulate the dynamics of
the actin cytoskeleton. We have demonstrated the
crucial importance of this signaling pathway for
controlling tenascin-C expression. Cyclic strain
activates RhoA and stimulates fibroblast contrac-
tion. Chemical activators of RhoA synergistically
enhance the effects of cyclic strain on cell contrac-
tility. Interestingly, tenascin-C mRNA levels per-
fectly matched the extent of RhoA/ROCK-medi-
ated actin contraction. First, chemical activation
of RhoA alone induced tenascin-C mRNA to an
extent similar to strain alone. Second, RhoA-acti-
vating drugs in combination with cyclic strain
caused a superinduction of tenascin-C mRNA,
which was again suppressed by ROCK inhibition.
Third, disruption of the actin cytoskeleton abol-
ished induction of tenascin-C mRNA by chemical
RhoA activators as well as by cyclic strain or
their combination. Lastly, myosin II-dependent
actin contractility was shown to be required
for tenascin-C induction by cyclic strain. We are
now testing whether RhoA/ ROCK-controlled ac-
to-myosin contractility has a mechanosensory
function in fibroblasts that correlates with the ex-
pression of other genes. Previous RhoA/ROCK ac-
tivation, either by chemical or by mechanical
signals, might render fibroblasts more sensitive to
external mechanical stress, e.g. during wound
healing and regeneration.
AGRIN IN C. ELEGANS
S. Canevascini, A. Hrus, G. Lau
Agrin is a heparan sulfate proteoglycan important
for development and tissue integrity in vertebrates.
A splice form enriched in muscles is secreted into
the basal lamina where it binds laminins and a-dy-
stroglycan and stabilizes the connection between
the ECM and the muscle cytoskeleton. Neuronal-
specific splice forms expressed in cholinergic neu-
rons are secreted into the synaptic clefts at the neu-
romuscular junctions and induce acetylcholine
receptor clustering on the postsynaptic membrane.
Finally, increasing evidence suggests that agrin in-
teracts with growth factors to mediate intercellular
signaling.We investigate agrin function at a genetic
level and isolate novel interacting factors using
C. elegans, which has a single agrin gene encoding
a protein of 1474 amino acids that shares high ho-
mology to its vertebrate homologues. In the adult
worm, agrin-GFP transcriptional fusions resulted
in strong expression of the marker in four neurons
in the head and in the nine pharyngeal epithelial
cells. Monoclonal and polyclonal antibodies raised
against the C-terminus of the protein most strongly
stain the basement membrane of the pharynx; this
staining is absent in worms carrying agrin muta-
tions. We are now analyzing the interaction of agrin
with its cellular receptor dystroglycan and the con-
sequences of agrin mutations on pharynx function.
TENEURINS IN VERTEBRATES
M. Brown-Luedi, J. Ferralli, S. Nunes-Radimerski
and D. Kenzelmann, in collaboration with R.P. Tucker
(University of California, Davis)
Teneurins are a family of four transmembrane pro-
teins with important functions in development.
Teneurin-1 and -2 are prominently expressed in
non-overlapping populations of neurons in the
developing visual system of the chicken. Their ex-
pression in alternating layers within the developing
optic tectum is particularly striking. Teneurin-2
is first found in the optic fiber layer of the retina,
the optic tract and stratum opticum of the optic
tectum on day 7 of embryo development, at a time
near the completion of retinal ganglion cell neurite
outgrowth that coincides with the onset of synap-
togenesis within the optic tectum. This is followed
a few days later by the appearance of teneurin-2
immunostaining in various mesenchephalic and
thalamic nuclei making up the thalamofugal visual
system. Finally, immunostaining is prevalent in
those parts of the forebrain that receive input from
the thalamofugal visual centers in the diecephalon
and midbrain, as well as in the hippocampus. This
pattern is in striking contrast to the distribution of
teneurin-1 mRNAs in the embryonic chicken brain,
which are concentrated in the laminae and nuclei
of the tectofugal visual system as well as in the cere-
bellum and spinal cord. These related, but almost
completely non-overlapping patterns of expression
suggest that teneurins play a role in cell-cell recog-
nition and/or signaling events during the develop-
ment of specific neuronal networks.
In addition to neuronal expression, teneurins are
found at sites of pattern formation. This is seen
most clearly in the developing chicken limb, where
teneurin-2 is expressed in the apical ectodermal
ridge and teneurin-4 in the zone of polarizing
activity. Teneurin-4 expression in the ZPA is tran-
sient and is soon replaced by expression in the
anterodistal mesenchyme of the limb bud. Avian
teneurin-4 is also expressed in the ectoderm of the
pharyngeal clefts and teneurin-2 in pharyngeal arch
mesenchyme. In contrast to the other teneurins,
teneurin-1 transcripts were never seen in non-neu-
ronal tissues of chicken embryos.
Teneurin signaling
Fig. 2. Model of the
These results point to roles for teneurins in ner- structure, cleavage and
vous system development and other morpho- interactions of teneurins
genetic events. To begin to understand these roles,
we transfected full-length teneurin-2 constructs
into neuroblastoma Nb2a cells under culture con-
ditions promoting differentiation into neuron-like
cells. Teneurin-2 expression led to the formation of
numerous actin-rich filopodia and enlarged growth
cones. Cell transfection studies also suggest that
teneurins mediate cell-cell adhesion through homo-
philic interactions.HT1080 cells permanently trans-
fected to express teneurin-2 constructs containing
the YD repeats (seeFig.2) aggregate in vitro,but con-
trol cell lines do not.Moreover,full-length teneurin-
2 in transiently transfected Nb2a cells is concen-
trated at points where adjacent cell bodies touch.
Teneurins harbor several unique domains, such
as the intracellular domain, a segment with con-
served cysteines following EGF-like repeats as well
as the YD repeats (Fig. 2). It will be interesting to
determine their structures as well as their contri-
bution to teneurin function. Some of these do-
mains may participate in homophilic interactions
but the existence of additional ligands needs inves-
tigation. Our current working model for teneurin
action is presented in Figure 2. It includes the
possible release of active neuropeptides from the
FMI Report 2005/2006 63
Growth Control
Ten-1 mutant phenotype
Fig.3. Left: Penetrance of
the phenotypes of Ten-1
mutant worms. Right: The
well-organized gonad of
a wild-type compared
with a Ten-1 mutant worm
Selected publications
Drabikowski K, Trzebiatowska A,
Chiquet-Ehrismann R (2005)
ten-1, an essential gene for germ cell devel-
opment, epidermal morphogenesis, gonad
migration, and neuronal pathfinding in Cae-
norhabditis elegans. Dev Biol 282:27-38
64 FMI Report 2005/2006
C-terminus, termed TCAP (reported by D.A. Love-
joy), homophilic interaction through the extra-
cellular domains distal of the EGF-like repeats and
receptor dimerization through cysteine bridges
between the EGF-like repeats two and five. The in-
tracellular domain is linked to the cytoskeleton
through its interaction with the adaptor protein
CAP/ponsin. After proteolytic release of the intra-
cellular domain, this part of the protein may in-
teract with further nuclear proteins like MBD1 and
zic to influence gene transcription.
This type of direct signaling from a cell surface
receptor to the nucleus by regulated intramem-
branous proteolysis has been found for many re-
ceptor proteins and has probably been most thor-
oughly analyzed in the case of Notch signaling. The
proposed mechanism of action of teneurins by
cleavage of the intracellular domain and its trans-
location to the nucleus needs to be studied more
rigorously. The cleavage site and the protease(s)
must be identified and the function of the intra-
cellular domain within the nucleus elucidated.
TEN-1 IS REQUIRED FOR THE FORMATION OF
THE SOMATIC GONAD
K. Drabikowski, A. Trzebiatowska
C. elegans TEN-1, also a member of the teneurin
family, was proposed to act as a receptor that sig-
nals directly to the nucleus. Its intracellular do-
main is cleaved and translocates to the nucleus
Nunes-Radimerski S, Ferralli J, Choi KD,
Minet A, Chiquet-Ehrismann R (2005)
The intracellular domain of teneurin-1
interacts with MBD1 and CAP/ponsin
resulting in subcellular co-distribution
and translocation to the nuclear matrix.
Exp Cell Res 305:122-132
Ruiz C, Huang W, Hegi ME, Lange K,
Hamou M-F, Fluri E, Oakeley EJ,
Chiquet-Ehrismann R, Orend G (2004)
Differential gene expression analysis reveals
activation of Wnt and MAPK signaling and
downregulation of tropomyosin-1 by
tenascin-C. Cancer Res 64:7377-7385
where it may influence gene expression. Little is
known, however, about the cleavage mechanism
and target genes.
C. elegans has a single teneurin orthologue un-
der the control of alternative promoters. The up-
stream promoter is active in the somatic gonad,
some muscle cells, the gut and a number of neu-
rons. Expression from the downstream promoter
is mainly detected in the nervous system. TEN-1
is a type II transmembrane protein consisting of
a short intracellular domain, a transmembrane do-
main, and a long, highly conserved extracellular
part containing eight EGF-like repeats, a cysteine
rich region and YD repeats.
To determine the function of TEN-1 in C. ele-
gans, we investigated the two loss-of-function mu-
tants ten-1(ok641) and ten-1(tm651). The former
carries an in-frame deletion of four EGF-like re-
peats and part of the cysteine rich region. The lat-
ter has a deletion that removes the transmembrane
domain and introduces a frameshift mutation, re-
sulting in the loss of all but the first few amino acids
of the protein. In both mutants, transcripts for the
predicted truncated proteins were detected at a
level comparable to the wild-type form. Both ten-1
mutants show a pleiotropic phenotype similar to
that observed after reduction of ten-1 expression
by RNAi. Mutant worms are sterile due to various
defects in the somatic gonad and ectopic germline
formation in the proximity of the vulva (Fig. 3).
Time-course analysis of germline development
showed that these abnormalities may result from
an early defect in somatic gonad formation. We
partially rescued the phenotype of ten-1(ok641)
mutant worms by injecting cosmid F36A3 carry-
ing the entire genomic region of the ten-1 gene.
Since such an extrachromosomal array is likely to
be silenced in the germline, gonadal defects appear
to be the result of impaired signaling in the somatic
gonad.
Scherberich A, Tucker RP, Degen M,
Brown-Luedi M, Andrs AC,
Chiquet-Ehrismann R (2005)
Tenascin-W is found in malignant mammary
tumors, promotes alpha8 integrin-depen-
dent motility and requires p38MAPK activ-
ity for BMP-2 and TNF-alpha induced ex-
pression in vitro. Oncogene 24:1525-1532
Tucker RP, Chiquet-Ehrismann R (2006)
Teneurins: a conserved family of transmem-
brane proteins involved in intercellular
signaling during development. Dev Biol
290:237-245
Brian A. Hemmings
Protein kinase
function in development
and cancer

INTRODUCTION
The molecular tapestries of signal transduction
pathways in living organisms from humble yeast
to the rather less humble human brain are being
revealed as complex networks. This revelation is
driven in part by the genomics revolution. Genomes
are being sequenced, open reading frames hunted
down and new signal connections elucidated. It is
now possible to identify in principle all signalling GROUP LEADER
components elaborated by any particular cell. Clas- Brian A. Hemmings
sical signalling pathways are triggered by binding of brian.hemmings@fmi.ch
an extracellular ligand (growth factors, cytokines,
chemokines, hormones, neurotransmitters) to the TECHNICAL/RESEARCH
appropriate plasma membrane receptor. In nor- ASSOCIATES
mal physiology, these signalling pathways interact Peter David Cron
in complex networks to invoke appropriate cellular Deborah Hynx
responses. This involves often subtle and reversible
alterations of multiple protein functions, such as POSTDOCTORAL
intrinsic activity, subcellular localization, half-life, FELLOWS
or interaction of proteins with binding partners. Anne Baudry*
The most common mechanism is phosphorylation Elisabeth Fayard
performed by protein kinases, the largest family of Ekaterina Gresko
enzymes in the human genome. Over 500 different Alexander Hergovich
enzymes can phosphorylate one-third of all intra- Pier Morin
cellular proteins, making kinases the key regula- Arnaud Parcellier
tors in signalling. Accordingly, temporal and spatial Jongsun Park*
regulation of protein kinases is of great importance Lionel Tintignac
in controlling all aspects of cell biology: metabo- Anton Vichalkovski
lism, transcription, cell growth, shape, migration, Zhongzhou Yang*
survival and differentiation.
Our work focuses on the physiological role and PhD STUDENTS
regulation of kinases in eukaryotic signalling path- Samuel Bichsel*
ways, especially their involvement in human dis- Lana Bozulic
ease. Many diseases arise by deregulation of signal Hauke Cornils
transduction pathways leading to inappropriate Bettina Dmmler
signalling. In cancer cells, mutations in key regu- Daniel Heisswolf*
latory enzymes result in permanently up-regulated David Restuccia
proliferation and survival signalling pathways. De- Debora Schmitz
ciphering the processes controlling protein kinase Mario Stegert*
activity will allow development of therapeutics tar- Banu Src
geting specific pathways to alleviate human disease. Oliver Tschopp*
Monika Wnuk*

UNDERGRADUATES
Reto Kohler
Magdalena Paolino*

*left the group

FMI Report 2005/2006 65

Growth Control
Fig.1. hCTMP1 protein local-
ized to the mitochondria.
HeLa cells were transfected
with untagged hCTMP1
in an IRES-GFP construct.
Top: Specific subcellular
localization of CTMP1 stain-
ing (red) with cytoplasm
marker a-tubulin (blue).
Bottom: Localization
of hCTMP1 (green) in mi-
tochondria using anti-Omi/
Htr2a as a marker.
Yellow colour in panel 1f
depicts co-localization
66 FMI Report 2005/2006
PROTEIN KINASE B SIGNALLING PATHWAY
Our laboratory is especially interested in protein
kinase B (PKB, also known as AKT) because it is
the focal point of the PI3Kinase/PTEN signalling
pathway that is activated by many growth factors
and insulin. Significantly, recent DNA sequencing
of tumour samples has shown that several proteins
of the PI3K/PTEN/PKB signalling pathway are
mutated and that this pathway is the most fre-
quently mutated in human cancer. We aim to un-
derstand the isoform-specific functions of PKB in
a physiological setting, its activation mechanisms
and regulation and how deregulation leads to
pathologies, especially cancer.
PKB ABLATION IN MICE
B.A. Dmmler, E. Fayard, D. Hynx, M. Paolino,
Z.Z. Yang, O. Tschopp, A. Baudry
To study the significance and function of the three
PKB isoforms in mammals (PKBa, b and g), we
generated isoform-specific knockout mouse mod-
els. These mice display distinct phenotypes, indi-
cating that the three PKB isoforms have differen-
tial and non-redundant physiological functions.
However, the viability and subtle phenotypes also
suggest that PKB isoforms mutually compensate
in many functions.
To address isoform redundancy, we examined
double-mutant mice, especially PKB a/PKB g and
PKB b/PKB g. Mice deficient in both PKB a and
PKB g showed reduced survival and growth.
PKB a-/- PKBg-/- embryos died between days 11 and
12, with severe defects in the cardiovascular and
nervous systems. Haplo-insufficiency of PKBg in a
PKB a null-background (PKB a-/- PKB g+/-) resulted
in death soon after birth, with multiple defects in
thymus, heart, and skin. However, PKBa+/- PKBg-/-
mice survive normally, showing that PKB a is more
essential than PKB g for development and survival.
To better understand the thymus defects in
PKB a-/- PKBg+/- mice, we analysed the thymus
phenotype of the single PKB knockouts. As only
PKBa-/- young mice showed a significant decrease
in thymus/body weight ratio, we focused our study
on PKBa-/- mice. There was a marked decrease in
the number of thymocytes and peripheral lym-
phocytes in these mice, together with a partial
block in early T cell development and a significant
decrease in the number of mature T cells. This may
reflect the disorganized thymic epithelial struc-
tures observed in PKB a-/- neonates.
In parallel, we generated mice deficient in both
PKB b and PKB g to study the combined contribu-
tions of the PKB b and PKB g isoforms in PKB-re-
lated physiological processes. Surprisingly, PKB b-/-
PKB g-/- mice develop normally and survive with
minimal dysfunctions despite marked reduction of
total PKB in many tissues. This is in sharp contrast
to the lethal phenotypes of PKB a-/- PKB b-/- and
PKB a-/- PKB g-/- mice.In PKB b-/- PKB g-/- mice,PKB a
appears to be sufficient for embryo development
and postnatal survival. However, PKB b-/- PKB g-/-
mice show a ~25 % reduction in body weight and
a reduction in relative size and weight of brain and
testis (20 % and 28 %, respectively). This demon-
strates an in vivo role for PKB b and PKB g in the
determination of whole animal and individual
organ size. In accordance with PKB b being a key
mediator of glucose homeostasis and insulin sig-
nalling, male PKB b-/- PKB g-/- mice exhibited severe
defects in glucose homeostasis and the activation
of total PKB was dramatically reduced in insulin-
responsive tissues. We are currently investigating
the downstream targets of PKB affected by low
PKB levels in various tissues.
THE ROLE OF DNA-PK AND mTOR IN THE
ACTIVATION OF PKB BY S473 HYDROPHOBIC
MOTIF PHOSPHORYLATION
L. Bozulic, B. Surucu, P. Cron, J. Feng, J. Park
Full activation of PKB requires phosphorylation on
T308 by 3-phosphoinositide-dependent kinase-1
(PDK1) and on S473 by an S473 kinase (S473K).
We purified two distinct kinase activities, termed
S473K1 and S473K2. We identified the S473K1 as
PI3 kinase-related kinase (PIKK) family member
DNA-dependent protein kinase (DNA-PK), whilst
the Ser473 kinase activity in the second fraction
was attributed to mammalian target of rapamycin
(mTOR), also a member this family. Accordingly
we set out to elucidate the roles that DNA-PK
and mTOR play in activating PKB to see whether
the phosphorylation of the S473 is stimulus-, sig-
nalling pathway- and/or cell type-specific.We es-
tablished an inducible system for vector-mediated
RNA interference in HEK293 cells to knock down
DNA-PK, mTOR or ATM protein levels. While loss
of ATM did not affect Ser473 phosphorylation
under any conditions tested, mTOR was the main
kinase responsible for Ser473 phosphorylation in
growing cells.In contrast,DNA-PK functioned when
cells were subjected to stress or genotoxic insults.
To further test the role of DNA-PK in activating
PKB under stress and genotoxic insult, we used
mice embryonic fibroblasts (MEFs) from mice
lacking DNA-PK. S473 phosphorylation was
analysed in knockout (KO) and wild-type (WT)
MEFs after serum starvation followed by stimula-
tion with either insulin or IGF-1 and following
g-irradiation. MEFs were also treated with dox-
orubicin, a topoisomerase II inhibitor that intro-
duces double-strand DNA breaks. In all cases, we
observed induction of PKB phosphorylation on
S473 in WT MEFs but not in KO MEFs, support-
ing our in vitro data on a role for DNA-PK in the
activation of PKB under stress and genotoxic insult.
DETERMINING THE ROLE OF PKB IN CANCER
E. Gresko, D.F. Restuccia, P. Morin
After the cloning and identification of human PKB,
a viral oncogenic protein called v-akt was isolated
from a murine thyoma and found to be closely
related to the human kinase. Since then, PKB
deregulation has been increasingly found in a wide
variety of human tumours, often accompanied by
high nuclear translocation of PKB, which is be-
lieved to enable PKB to regulate transcription. Fur-
thermore, a number of observations in recent years
have indicated an important role for PKB in can-
cer. Foremost is the finding that PTEN, the nega-
tive regulator directly upstream of PKB, is the sec-
ond most frequently mutated gene in cancer.
Second is the exponential increase in the identifi-
cation of novel PKB targets, many of which have
been linked to cancer. Pursuing these observations
using mouse models and biochemical techniques,
we are currently generating double-mutant mice
by combining tumour-forming PTEN mouse lines
expressing decreased PTEN with PKB isoform-
specific mutant mice to study their tumour-form-
ing potential.We are also examining the interactions
of PKB in tumours, in particular the identification
of novel PKB targets in the nucleus that may be
involved in aberrant transcriptional regulation in
malignant transformation.
UNDERSTANDING CTMP PROTEIN FUNCTION
L.A. Tintignac, A. Parcellier, P. Cron, M. Hanada
We previously identified human Carboxy Termi-
nal Modulator Protein (hCTMP) protein as a mod-
ulator of PKB activity that can directly bind the
hydrophobic motif of PKB at the membrane and
prevent PKB activation.To elucidate the mechanisms
of this function, we generated a monoclonal anti-
body against hCTMP1 protein.We determined that
endogenous and exogenous hCTMP1 protein in all
cell lines tested to date is predominantly mitochon-
drial (Fig.1).This unexpected finding was confirmed
by cellular fractionation experiments. An in silico
search indicated the presence of an N-terminal mi-
tochondrial targeting signal (MTS) and a protease
cleavage site conserved in mouse, rat, monkey and
human hCTMP1 protein. This combined with site-
directed mutagenesis studies has provided solid ev-
idence that hCTMP1 is targeted to the mitochondria
and then cleaved to a smaller mature form.
NDR PROTEIN KINASE SIGNALLING COMPLEX
The conserved NDR family represents a subclass
of the AGC serine/threonine protein kinases and
consists of mammalian NDR1, NDR2, LATS1 (large
tumour suppressor 1) and LATS2, D. melanogaster
TRC (tricornered) and LATS, C. elegans SAX-1
(sensory axon guidance-1) and LATS, S. cerevisiae
Dbf2p, Dbf20p and Cbk1p, S. pombe Sid2p and
Orb6p, as well as other fungi, protozoan and plant
kinases. The activation segment and the hydro-
phobic motif, common characteristics of AGC
kinases, are present in all NDR kinases. Human, fly
and yeast NDR kinases require phosphorylation
on both the activation segment and the hydro-
phobic motif for full activation. In addition to the
common regulatory elements of AGC kinases,
NDR family members possess two unique features:
a conserved N-terminal regulatory (NTR) domain
and an insert of 30-60 residues separating sub-
domains VII and VIII of the kinase domain.
ACTIVATION OF NDR AND LATS KINASES AT
THE MEMBRANE
A. Hergovich, S. Bichsel, D. Schmitz
Our recent data suggest that the subcellular local-
ization of hMOB1A/B/2 (hMOBs) as well as NDR
plays an important role in the activation process.
Targeting NDR1/2 or its co-activator hMOB to
the membrane results in a substantial increase in
kinase activity. This depends on the direct interac-
tion of hMOB with NDR1/2. Using a chimeric pro-
tein of hMOB1A that allows inducible transloca-
tion of hMOB1A to the membrane, we found that
hMOB1A recruited NDR1/2 to the membrane
within minutes, resulting in rapid activation of
NDR kinases. Our data suggest an in vivo mecha-
nism of NDR1/2 activation through rapid recruit-
ment to the plasma membrane by hMOBs followed
by multi-site phosphorylation.
Strikingly, membrane targeting of hMOB1 also
results in a significant increase in LATS1 activity
with a comparable activation profile, providing
evidence for very similar activation mechanisms
for human NDR1/2 and LATS1/2 (Fig. 2).
FMI Report 2005/2006 67
Growth Control

A B lation activity and hydrophobic motif phosphory-

lation of NDR by Ste20-like kinases to activate
kinase activity.

SUBSTRATES OF THE HUMAN NDR KINASE

R. Kohler, A. Hergovich, R. Tamaskovic

No physiological substrate of human NDR has been

identified to date. We are looking for NDR sub-
strates following two different approaches. Data-
base searches led to the identification of several
candidate proteins that are currently being tested
in vitro. In parallel, we modified the ATP-binding
pocket of NDR1 to alter nucleotide specificity
towards bulky ATP analogues. The mutant NDR
accepts bulky ATP analogues to phosphorylate
crude protein fractions and substrates are identi-
fied by mass spectroscopy.

PHYSIOLOGICAL ROLE OF NDR KINASE IN MICE

H. Cornils, D. Schmitz, D. Hynx, M. Stegert

Fig. 2. NDR kinase family
signalling. A LATS1/2 acti-
vated by MST1/2 in turn
inactivates YAP. RAF1 is a
positive, RASSF1/NORE1
a negative regulator of
MST1/2. MOB1 directly
interacts with LATS1/2 as
a co-activator. B NDR1/2
is activated by MST3 with
MOB1/2 serving as co-
activator. hFurry may serve
as scaffold protein in this
activation. Bold lines vali-
dated interactions, dotted
lines putative, question
marks unknown upstream
regulators, substrates or
direct interaction partners
IDENTIFICATION OF MST3 AS AN HYDROPHOBIC
MOTIF KINASE OF NDR
M. Stegert, A. Hergovich, A. Vichalkovsky
Human NDR1/2 and LATS1/2 are also activated by
phosphorylation through mammalian Ste20-like
(MST) kinases. MST1/2 phosphorylates LATS1/2
in vitro, while MST3 targets NDR1/2. We have
established that MST3 phosphorylates human
NDR1/2 at the membrane. MST3 targets the hy-
drophobic motif phosphorylation site of NDR1/2
but not the activation segment phosphorylation
site. Biochemical studies resulted in the following
model for NDR regulation: hMOBs bind to the
NTR of human NDR1/2 and promote release of
the auto-inhibition of the activation segment lead-
ing to auto-phosphorylation (Ser281 of human
NDR1). hMOBs are recruited to the plasma mem-
brane by an unknown mechanism,bringing NDR1/2
into close proximity to its upstream activators;
MST3 phosphorylates NDR1/2 on the hydropho-
bic motif (Thr444 of human NDR1) resulting in a
fully active NDR kinase.Thus, binding of MOBs to
the N-terminus of NDR elevates auto-phosphory-
Earlier results underline the importance of NDR
kinase family members for higher organisms.
However, the specific physiological role of mam-
malian NDR kinases is still unknown.We are work-
ing on mouse models for NDR1- and NDR2 defi-
ciency to investigate the function of these kinases
in a mammalian system. Mice deficient for NDR1
are fertile and display no developmental defects.
However, aged NDR1 knock-out mice develop tu-
mours at a higher frequency than wild-type litter
mates.
In parallel,the murine NDR2 locus is being condi-
tionally targeted. This will allow us to abolish NDR2
in a time-point- and tissue-specific manner to study
the role of NDR2 at defined developmental stages
and in distinct tissues. As the expression patterns
of NDR1 and 2 overlap, mice lacking NDR1 and 2
will be generated by crossing the single knock-out
animals to exclude mutual compensation by either
isoform. Our genetic studies should provide insights
into the in vivo roles of these protein kinases.

Selected publications
Baudry A, Yang ZZ, Hemmings BA (2006)
PKBa is required for adipose differentiation
of mouse embryonic fibroblasts. J Cell Sci
119:889-897
Hergovich A, Bichsel SJ, Hemmings BA
(2005)
Human NDR kinases are rapidly activated
by MOB proteins through recruitment to
the plasma membrane and phosphorylation.
Mol Cell Biol 25:8259-8272
Hergovich A, Stegert MR, Schmitz D,
Hemmings BA (2006)
NDR kinases regulate essential cell
processes from yeast to humans. Nature
Rev Mol Cell Biol 7:253-264
Stegert MR, Hergovich A, Tamaskovic R,
Bichsel SJ, Hemmings BA (2005)
Regulation of NDR protein kinase by hy-
drophobic motif phosphorylation mediated
by the mammalian Ste20-like kinase
MST3. Mol Cell Biol 25:11019-11029
Tschopp O, Yang ZZ, Brodbeck D,
Dummler BA, Hemmings-Mieszczak M,
Watanabe T, Michaelis T, Frahm J,
Hemmings BA (2005)
Essential role of protein kinase B g
(PKBg/Akt3) in postnatal brain develop-
ment but not in glucose homeostasis.
Development 132:2943-2954

68 FMI Report 2005/2006

Jan Hofsteenge
Molecular and functional
aspects of covalent
protein modifications

INTRODUCTION
Covalent modification of amino acids greatly ex-
tends the repertoire of chemical and, thus, func-
tional properties of proteins. We focus on two
forms of glycosylation and on tyrosine phospho-
rylation of ErbB2, a receptor tyrosine kinase.
Glycosylation is crucial for the development,
growth and survival of an organism. However,
defining the exact function and/or mechanism of GROUP LEADER
action of glycan modifications at the molecular Jan Hofsteenge
level remains a major challenge in glycobiology. The jan.hofsteenge@fmi.ch
importance of glycosylation is shown by the num-
ber of diseases, e.g. neurological disorders, mus- TECHNICAL/RESEARCH
cular dystrophies or lysosomal storage diseases, ASSOCIATES
now known to be due to defects in glycosyltrans- Marianne Grob*
ferases, sugar-transporters, glycosidases or lectins. Dominique Klein
Furthermore, knocking out core glycosyltransfer-
ases nearly always results in a severe, if not lethal, POSTDOCTORAL
phenotype. This is complemented by the discov- FELLOWS
ery of many lectins involved in cellular and phys- Stefano Canevascini
iological processes, e.g. quality control of protein Guillaume De Sampaio*
folding and leukocyte homing. Thus, we begin to Jeremy Keusch
see the enormous coding potential of carbohy-
drates. PhD STUDENTS
We aim to define the biological function of Constanze Heinrich
C-mannosyltryptophan and glucosyl-fucosyl-O- Krisztina Kozma
Ser/Thr (Fig. 1). Both modifications occur close Carsten Krantz
together in thrombospondin type 1 repeats (TSRs). Florence Roux
This module is an essential domain in a range of
proteins with roles in normal physiologyas well as UNDERGRADUATES
in disease, e.g. in the invasion of vector and host Jasmin Althaus*
cells by protozoan parasites. Nominerdene Battsengel*
ErbB2 is one of four tyrosine kinases closely Alexandra Bezler*
related to the EGF receptor and frequently overex- Pascal Hermann*
pressed in cancers. Growth factor binding to het-
erodimers formed with other members of the fam-
ily leads to phosphorylation of tyrosine residues *left the group
in its intracellular domain. These serve as docking
sites for proteins that initiate intracellular signal-
ling pathways. We are studying the temporal dy-
namics of signalling events following growth fac-
tor stimulation and the importance of individual
phosphorylated tyrosine residues for tumor for-
mation.
Part of the group runs the protein- and peptide
analysis facility.

FMI Report 2005/2006 69

Growth Control
C-a
a-mannosyltryptophan
Fig.1. Molecular structure
of the C- and O-linked glyco-
conjugates studied in this
report. The position of
the C -atom of the amino
acid has been indicated
with a red dot
70 FMI Report 2005/2006
MODIFICATION OF THROMBOSPONDIN TYPE 1
REPEATS WITH Glc-Fuc-O-
S. Canevascini, K. Kozma, M. Grob, J. Althaus,
D. Klein and J. Hofsteenge, in collaboration with
R. Chiquet-Ehrismann (FMI)
Previously, we found that TSRs in mammalian pro-
teins can be modified by addition of the di-sac-
charide Glc-Fuc-O-. Our current research in this
area aims at finding the biological function of this
form of glycosylation using C. elegans as a model
system. Before starting genetic analyses, it was nec-
essary to perform biochemical studies to ascertain
that the modification occurs in C. elegans and
to identify the two glycosyltransferases involved.
We have established an in vitro assay for the de-
termination of TSR-O-fucosyltransferase activity
in extracts from cells and entire worms. Using this
assay with cells overexpressing putative O-fucosyl-
transferases from C.elegans, we demonstrated that
the pad-2 gene encodes the fucosyltransferase (PO-
FUT-2) that modifies TSRs. Importantly, product
analysis of isolated peptides by mass spectrometry
showed that the enzyme attaches the fucosyl
residue in the correct position within the TSR.
glucosyl-fucosyl-O-Serine
The fucose residue in TSRs can be extended by
the action of an as yet unidentified glucosyltrans-
ferase (b1,3-GlcT) by the addition of a glucosyl
residue. We have found that C. elegans possesses
this enzyme activity. Using the recombinant PO-
FUT2 preparations described above, we produced
sufficient quantities of pure TSR-fucose to estab-
lish an assay for b1,3-GlcT. Using enzymatic assays
and product analysis by mass spectrometry to ex-
amine candidate genes in a way very similar to that
described for POFUT-2, we have now identified
the human gene encoding b1,3-GlcT.
In collaboration with Dr.S.Mitani (NBRP,Japan),
we obtained a C. elegans mutant, pad-2(tm1756),
that contains a large deletion in the 5 two-thirds
of the gene. The tm1756 strain is homozygous vi-
able and lacks all fucosyltransferase activity to-
wards a TSR. We observed that the ventral part of
the anterior gonad arm in mutant worms leaves the
muscle band prematurely, running with an oblique
angle towards the dorsal muscle band (Fig. 2).
Abnormal gonad shape is generally considered
to result from defects in guidance of the distal tip
cell (DTC). A major cue for the ventral to dorsal
migration of the DTCs results from the repulsive
signal created by ventrally expressed netrin (UNC-
6) that is sensed by two receptors in the DTC,
UNC-5 and UNC-40. The former contains TSRs,
one of which could be O-fucosylated. Interestingly,
the phenotype of pad-2(tm1756) is very similar
to that published for the precocious expression
of UNC-5. Our recent genetic experiments have
shown that the unc-6, unc-5 and unc-40 genes
are epistatic to pad-2. We are currently testing the
hypothesis that pad-2 acts as an inhibitor of the
netrin pathway in the DTC.
Obviously, deletion of the enzymatic activity of
POFUT-2 results in the absence of the entire Glc-
Fuc-O- disaccharide. To examine the role of the
missing glucosyl residue in the pad-2(tm1756)
phenotype, it will be of interest to study the effect
of mutating the activity of b1,3-GlcT.
PROTEIN C-MANNOSYLATION
J. Keusch, C. Krantz and D. Klein, in collaboration
with E. Jacoby and P. Frst (NIBR, Basel)
We are following two approaches to the function
of protein C-mannosylation. First, we are search-
ing for ways to alter the activity of the protein
C-mannosyltransferase (PCMT) in cells and model
organisms, preferably C. elegans. In order to ge-
netically manipulate PCMT activity, we wish to
identify its gene by an expression cloning approach.
Since no suitable cell lines could be found for
this task, we mutagenized an engineered CHO cell
line carrying a C-mannosylation reporter on its
plasma membrane. We obtained several lines lack-
ing C-mannosylation that are otherwise normal
with respect to the level of C-mannosylation re-
porter and the synthesis of the sugar donor
dolichylphosphomannose (MPD). In an alterna-
tive approach, we are looking for chemicals that
inhibit PCMT. Our collaborators have performed
in silico searches on the NIBR small compound li-
braries and identified molecules that might fit the
active site of PCMT. Using the engineered CHO
cell line, we identified 12 compounds that strongly
reduce modification of the C-mannosylation and
are, thus, putative PCMT inhibitors. Interestingly,
the results of further characterization of these
compounds, together with observations made in
mutant CHO lines aberrant in MPD synthesis,
point to the existence of multiple C-mannosylation
pathways, one of which appears to be independent
of MDP.
The second approach to study the function of
protein C-mannosylation is based on our obser-
vation that the patterns of C-mannosylation and
O-fucosylation on the surface of TSRs differ be-
tween proteins. This suggests the existence of a
sugar code that specifies protein-protein inter-
actions. We have started a search for such interac-
tion partners using recombinantly expressed and
differentially glycosylated TSRs as bait.

ErbB2 PHOSPHORYLATION
F. Roux and C. Heinrich, in collaboration with
N. Hynes (FMI)

Our project with the group of Nancy Hynes aims

to study the temporal dynamics of the signalling
events following growth factor stimulation of
ErbB2. Towards that goal, we have implemented
mass spectrometric methods for the analysis of
proteins incorporating arginine that has been dif-
ferentially labelled with stable isotopes (SILAC).
This allows reliable quantitation of complex for-
mation and changes in phosphorylation. In a sec-
ond project, we are studying the importance of in-
dividual phosphorylated tyrosine residues in the
C-terminal tail of the receptor for different aspects
of tumor formation. Using the add-back mutant
T47D cell lines developed in the group of N. Hynes,
we have obtained evidence that the phosphoryla-
tion of one of these tyrosines results in anchorage-
independent growth in a growth factor-dependent
manner. Using the SILAC method, we are eluci-
dating proteins that specifically bind to this phos-
photyrosine residue.
PROTEIN- AND PEPTIDE ANALYSIS FACILITY
R. Portmann, R. Sack and D. Hess, in collaboration
with A. Di Cara and D. Flanders
The main activities of the protein- and peptide-
analysis facility are (1) isolation and comparison of
proteins by 2D-PAGE for proteomic approaches,
(2) identification of isolated proteins of interest
and (3) identification of proteins in mixtures at
ultra-high sensitivity using capillary-LC-MALDI-
MSMS or nano-LC-ESI-MSMS, (4) identification
of post-translational modifications using a com-
bination of the technologies mentioned above.
To provide the FMI with state-of-the-art facili-
ties we continuously modernize equipment and
software tools. Recently, we acquired a MALDI-
TOF/TOF instrument to complement our three
electrospray instruments and also substantially
improve the speed and sensitivity of protein analy-
sis. In addition, all three electrospray instruments
have been interfaced with capillary- or nano-flow
liquid chromatographs, allowing automated data
Fig. 2. C. elegans herm-
acquisition. Finally, in collaboration with the in- aphrodites lacking protein
formatics group, we have automated part of the data O-fucosyltransferase 2
analysis methods and procedures for reporting activity have an abnormal
gonad shape. DIC micro-
analysis results. As in past years, most FMI groups
graphs of the anterior gonad
have made use of the expertise of the facility. arm of pad2(tm1756)
Protein quantification greatly profits from sta- (left) and wild-type (right)
ble isotope labelling using either the SILAC or worms. Dorsal is up and
ITRAC methods. Experiments employing these anterior is to the right.
techniques have been implemented on our linear The shape of the gonad
ion trap mass spectrometer and specialized soft- has been traced by the red
stippled line
ware has been developed in collaboration with the
informatics group.
In the area of posttranslational modification, we
have analysed glycosylation (C- and O-glycosyla-
tion), methylation, acetylation, phosphorylation
and sumoylation. We have studied several protein
complexes including the TRBP-Dicer complex that
functions in RNA silencing (Group Filipowicz)
and a linker histone H1 complex that suppresses
gene transcription (Group Sun). Using more gen-
eral proteomics approaches, we are examining sig-
nalling pathways (F. Roux and C. Heinrich, in col-
laboration with Group Hynes), characterizing the
sperm proteome (Group Peters) and neuro pep-
tides in C.elegans (Group Alcedo) and studying the
effect of microRNA in various systems (Group Fil-
ipowicz).

Selected publications
Furmanek A, Hess D, Rogniaux H,
Hofsteenge J (2003)
The WSAWS motif is C-hexosylated in a
soluble form of the erythropoietin receptor.
Biochemistry 42:8452-8458
Mormann M, Macek B, Gonzalez de Peredo A,
Hofsteenge J, Peter-Katalinic J (2004)
Structural studies on protein O-fucosylation
by electron capture dissociation. Int J Mass
Spectrom 234:11-21
Zanetta J-P, Pon A, Richet C, Timmerman P,
Leroy Y, Bohin J-P, Trinel P-A, Poulain D,
Hofsteenge J (2004)
Quantitative determination of C-mannosyla-
tion of tryptophan residues in glycoproteins.
Anal Biochem 329:199-206

FMI Report 2005/2006 71

Growth Control

Nancy Hynes
The molecular basis
of breast cancer

INTRODUCTION
Cancer results from cumulative alterations in the
genetic make-up of somatic cells that lead to aber-
rant expression, mutation or deletion of proteins
modulating cellular proliferation, differentiation
and survival. Through deregulation of intracellu-
lar pathways, these defects allow cancer cells to
evade signals that normally tightly control cell pro-
liferation and survival. Cancer cells also signal GROUP LEADER
abnormally to their surroundings. One important Nancy Hynes
outcome is the stimulation of neovascular growth, nancy.hynes@fmi.ch
which provides nourishment for the tumor and
promotes metastasis. Receptor tyrosine kinases TECHNICAL/RESEARCH
(RTKs) are often aberrantly activated in human ASSOCIATES
cancers, which impacts on multiple characteristics Susanne Lienhard
of the cancer phenotype. Constitutive activation Francisca Maurer
of the ERBB family is often found in breast cancer,
including epidermal growth factor receptor (EGFR) POSTDOCTORAL
and ERBB2 as well as members of the fibroblast FELLOWS
growth factor receptor (FGFR) family. We are Ali Badache*
studying the intracellular signaling pathways acti- Anne Boulay
vated by these receptors. Targeted approaches are David Cappellen*
used to inactive the receptors and their effects on Subha Susan Jacob*
cancer cell proliferation, survival, migration and Rgis Masson
other characteristics are determined. We also study Thomas Schlange
cross-talk between signaling molecules in cancer Patrizia Sini
cells. ERBB receptors are important mediators of
signaling from other classes of membrane recep- PhD STUDENTS
tors, for example the Wnt/Fz network, which po- Ilja Boschke*
tentially has a role in autocrine ERBB receptor and Julien Dey
downstream signaling pathway activation. Barbara Hnzi
A major goal of our research is to go from a de- Patrick Kaeser
scription of the molecular alterations in breast can- Na Li*
cer cells to an understanding of how these altered Yutaka Matsuda
signal transduction proteins contribute to the meta- Maria Meira
static cancer process. A greater understanding of Ivana Samarzija
the molecular basis of cancer will be essential for Tina Stlzle
the rational development and clinical application
of the new classes of targeted signal transduction UNDERGRADUATES
inhibitors now being used for cancer therapy. Collin Ewald
Sven Falk*
Alexandre Huber*

*left the group

72 FMI Report 2005/2006

RECEPTOR TYROSINE KINASES IN BREAST CANCER
A. Boulay, J. Dey
In breast tumors, amplification of genes encoding
receptor tyrosine kinases (RTK) is an important
mechanism contributing to cancer development.
Genes encoding members of the ERBB/EGFR and
the FGFR families are amplified in specific types of
breast tumors. We are exploring the role of these
receptors in tumor biology using inhibitory mon-
oclonal antibodies and/or small molecule tyrosine
kinase inhibitors (TKIs) selective for ERBB or
FGFR. Our goal is to characterize the RTK-induced
signaling pathways that drive proliferation and
survival of breast tumor cells. Towards this end,
we have found that ERBB2-overexpressing breast
tumor cell lines are very dependent upon the
PI3K/Akt pathway for proliferation, while tumor
cells with FGFR activation are more dependent
upon the MAPK/ERK signaling pathway.
In the clinic, drugs effecting different character-
istics of cancer cells are routinely used in com-
bination. Thus, another important goal of our re-
search is to uncover the combination of signal
transduction inhibitors with the strongest impact
on the malignant phenotype. In addition to RTKs,
breast tumors have alterations in the PI3K/Akt/
mTOR pathway. The mTOR kinase is a central
regulator of cellular responses to multiple stimuli,
including RTK-induced PI3K/Akt activation. In
breast tumors, activating mutations in PIK3A, en-
coding the catalytic subunit of PI3K, or loss of
PTEN, the negative regulator of PI3K activity, are
very frequent and contribute to mTOR activation.
The mTOR complex signals to important protein
synthesis regulators, thereby controlling transla-
tion of mRNAs encoding growth-related proteins.
Thus, combinations of mTOR and RTK inhibitors
are being examined for their impact on breast can-
cer cell proliferation and survival.
TARGETING THE TUMOR-ASSOCIATED
VASCULATURE
P. Sini and I. Samarzija, in collaboration with J. Wood
and colleagues (NIBR Oncology, Basel)
Angiogenesis, the formation of new vessels from
an existing vascular network,sustains tumor growth
and metastasis. Vascular endothelial growth factor
(VEGF), a key factor in inducing tumor-associated
vascularization, transmits the angiogenic signal
by binding VEGF receptors located on the surface
of host endothelial cells (EC). VEGF-mediated ac-
tivation of VEGF receptors exerts potent mitogenic
and survival effects on ECs.Various approaches are
being taken to interfere with the VEGF/VEGF re-
ceptor system, including small molecule TKIs and
VEGF antagonistic antibodies, both of which are in
clinical use. A link between angiogenesis and ERBB
receptor signaling has also been established. ERBB
receptor activation in tumor cells induces the expres-
sion and release of VEGF. Furthermore, ECs express
ERBBreceptorsandwehaveshowninvitrothatEGFR
activation stimulates proliferation and survival.
A B
Fig.1. EGFR is required for
Using the DU145 human prostate cancer xeno- survival of tumor-associated
graft model, effects of selective VEGF receptor and endothelial cells (EC). Sec-
ERBB/EGFR TKIs on tumor growth and angio- tioned tumors from DU145
prostate xenografts immuno-
genesis were evaluated. ERBB inhibition signifi-
stained for EC using CD31
cantly enhanced the antitumor activity of VEGF (red, A and B), for EGFR
receptor inhibition. We also explored the possibil- (green, A) and apoptosis
ity that EGFR inhibition has an effect upon survival using caspase-3 (green, B).
of the tumor-associated vascular endothelium. EC images were overlapped
To this end, EGFR expression and localization with EGFR (A) or caspase-3
were examined in tumor sections. Staining with an images (B); arrows indicate
colocalization (yellow)
EGFR-specific antiserum revealed high receptor
levels on the tumor cells (Fig. 1A, green). Tumor-
associated ECs were stained with an anti-CD31
antibody (Fig. 1A, red). Double staining of tumor
sections with both antisera revealed that ECs lining
the blood vessels also express EGFR (Fig.1A, yellow).
To examine the effect of an EGFR-selective TKI on
survival of the tumor-associated vasculature, tu-
mor sections were prepared from TKI-treated mice
and stained with CD31 (Fig. 1B, red) and activated
caspase-3 (Fig. 1B, green) antisera. Tumors taken
from vehicle-treated mice lacked apoptotic ECs,
whereas tumors from the TKI-treated mice dis-
played co-staining (Fig.1B, yellow). Thus, in the tu-
mor microenvironment it appears that EGFR sig-
naling plays an important role in the survival of
the vasculature. The effects of blocking EGFR may
be indirect via a decrease in the release of VEGF
from the tumor itself and direct via blockade of
EGFR in the tumor-associated endothelium.
MEMO IS A NOVEL ERBB2 EFFECTOR PROTEIN
R. Masson, P. Kaeser, S. Jacob, F. Maurer,
S. Lienhard, M. Meira, B. Hnzi and A. Badache,
in collaboration with D. Hess and M. Rebhan (FMI)
Motility is an important characteristic of metasta-
tic tumor cells and a major goal in our laboratory
has been to identify molecules and pathways con-
FMI Report 2005/2006 73
Growth Control
A The ErbB2 receptor
Fig. 2. A Activation of
ERBB2 leads to phosphory-
lation of tyrosine residues
in its cytoplasmic domain.
Two residues (Y1201,
Y1227) support tumor cell
migration by binding the
indicated signaling proteins.
Memo is a novel P-Y1227-
interacting protein. B In situ
hybridization with a Memo
antisense cRNA probe on an
E13.5 mouse embryo sagit-
tal section
74 FMI Report 2005/2006
tributing to this process. Using ERBB2-dependent
tumor cell migration as a model,we have shown that
two of the autophosphorylated tyrosine residues in
ERBB2s cytoplasmic domain (Tyr 1201 and 1227)
support long-term migration. We used phospho-
peptides corresponding to these receptor regions
as affinity reagents and identified proteins from
tumor cell extracts that bound specifically to the
phospho-peptides. Several proteins were identified
(Fig. 2A) including Shc, PLC g and Crk isoforms
that bind Y1201, and the Y1227 binders Shc and
Memo (mediator of ERB2-driven-cell motility).
Although siRNA-mediated knock-down of each
protein impairs ERBB2-induced cell motility, ana-
lysis of actin and microtubule networks in migrat-
ing cells has revealed interesting differences. Shc
knock-down cells fail to extend actin-based lamel-
lipodia in response to ERBB2 activation, while
Memo knock-down cells form lamellipodia but fail
B E13.5
Memo antisense probe
to extend the microtubule network to the cell cor-
tex. These results suggest that signaling pathways
triggered by specific phospho-tyrosine residues in
ERBB2 cooperate at different levels to induce tu-
mor cell migration.
Memo is a novel protein with an as yet unknown
function. A bioinformatic analysis revealed that
Memo homologs are present in all branches of life.
Memo is expressed in most adult tissues and dur-
ing embryonic development. Figure 2B shows the
results of in situ hybridization with a Memo anti-
sense cRNA probe, carried out on sections of an
E13.5 mouse embryo. Most embryonic tissues stain
positively, with some showing more expression than
others. To explore the physiological role of Memo,
conventional and conditional Memo knock-out
mice strains have been generated.
Considering the importance of Memo in tumor
cell migration, we are exploring its role in tumor
cell metastasis using mammary tumor cells that
rapidly form primary tumors and lung metastases
following injection into the mammary fat pad. Sta-
ble knock-down of Memo expression was achieved
using a Memo-specific shRNA vector. Initial results
suggest that the metastatic spread from primary
tumors to the lungs is impaired in tumor cells with
decreased levels of Memo.
CROSS-TALK BETWEEN THE Wnt PATHWAY AND
ERBB RECEPTORS IN BREAST CANCER
T. Schlange, D. Cappellen, Y. Matsuda,
S. Falk, A. Huber
Wnts are secreted glycoproteins that play impor-
tant roles in normal development. Members of the
Frizzled (Fz) family of transmembrane proteins
are receptors for Wnts. Wnt binding to Fz initiates
a pathway that prevents GSK-3 b from phosphory-
lating b-catenin, leading to its stabilization and
translocation to the nucleus, where it engages tran-
scription factors of the TCF family. This pathway
drives the development of many human cancers.
Unlike colon cancer, in which activating pathway
mutations are often found, breast tumors show lit-
tle evidence of such mutations; however, emerging
evidence suggests that Wnt/Fz signaling is active due,
for example, to loss of expression of the negative
regulator sFRP.We are exploring the role of Wnt/Fz
pathway activation in breast cancer. Our results
suggest that in addition to the canonical pathway
leading to b-catenin stabilization and TCF activ-
ity, Wnts have a role in ERBB receptor activation.
Activation of the ERBB receptors is controlled
by the spatial and temporal expression of their lig-
ands, members of the EGF-family, which are pro-
duced as transmembrane precursors and cleaved by
cell surface proteases, a step that leads to release of
soluble ligands. This cleavage, termed ectodomain
shedding, is an important step in the control of lig-
and availability and receptor activation. ERBB re-
ceptors are constitutively stimulated in many pri-
mary tumors since EGF family ligands are often
co-expressed in the tumor. Thus, it is essential to
understand the mechanisms that control ligand
processing.
The proteases involved in ectodomain shedding
are metalloproteinases. The production of soluble
EGF family ligands occurs in response to diverse
stimuli and was first described following activa-
tion of G protein-coupled receptors (GPCR). In
cells treated with GPCR receptor agonists, metal-
loproteinases that induce cleavage and release of
EGF ligands are stimulated, leading to the rapid
phosphorylation of EGFR. This process, termed
EGFR transactivation, has important biological
implications since it leads to stimulation of intra-
cellular pathways such as MAPK/ERK signaling. We
have shown that ERBB transactivation also results
from the binding of Wnt to Fz (Fig. 3). The mech-
anism appears to be similar to that described for
GPCRs, since it is rapid and blocked by metallo-
proteinase inhibitors. Wnt/Fz-mediated transacti-
vation has been observed in normal mammary cells
and in breast cancer cells and may have important
clinical ramifications.
Some ERBB2-overexpressing breast cancer pa-
tients are resistant to trastuzumab, a recombinant
ERBB2-targeted antibody used clinically in the
metastatic and adjuvant setting for breast cancer
treatment. EGF family ligands may facilitate escape
from trastuzumab through the activation of ERBB
receptor homo- and heterodimers. In fact, we have
shown that trastuzumab cannot block prolifera-
tion of tumor cells with autocrine EGFR activity,
nor can it prevent the ligand-induced formation of
ERBB2-containing heterodimers or the activation
of downstream signaling pathways (Fig.3). In sum-
mary, evidence is accumulating that constitutive
Wnt pathway activity in breast cancer influences not Fig. 3. Wnt activity stimu-
only TCF transcriptional activity, but also pro- tant role in proliferation or maintenance of mam- lates b-catenin stability and
motes ERBB transactivation via an increase in the mary alveolar progenitor cells. Mammary glands TCF transcriptional activity
availability of EGF-related ligands. from females undergoing a second round of preg- and also promotes ERBB
transactivation, via an
nancy showed a strong reduction in the density of
increase in EGF-related lig-
b1 INTEGRIN HAS A ROLE IN MAMMARY GLAND lobulolalveolar units, suggesting a reduction in the ands. The latter may facili-
DEVELOPMENT number of progenitor cells that enable alveolar tate escape from the
expansion. Using the mammary transplantation ERBB2-targeted antibody
N. Li, T. Stlzle
technique, which allows functional identification trastuzumab by ligand-in-
Integrin-extracellular matrix interactions play im- of mammary stem cells by measuring in vivo out- duced activation of ERBB
portant roles in the coordinated integration of ex- growth potential, we showed that epithelium from receptor homo- and het-
erodimers
ternal and internal cues for proper development. b1 integrin mutant glands has a severe impairment
To study the role of b1 integrin in the mammary in ability to repopulate mammary fat pads. This
gland, Itg b1flox/flox mice were crossed with whey suggests that alveolar progenitors have impaired
acidic protein (WAP)Cre transgenic mice, which ability to proliferate in the absence of b1 integrin.
led to specific ablation of b1 integrin in luminal Considering the known role of integrins in the
alveolar epithelial cells. In the b1 integrin mutant maintenance of stem cells in, for example, hair cell
mammary gland, individual alveoli were disorga- follicles, it is tempting to speculate that b1 inte-
nized through alterations in cell-basement mem- grins have a similar role in the mammary gland. Us-
brane associations. Activity of focal adhesion kinase ing this model, we are examining the role of other
(FAK) was also decreased in mutant mammary signaling proteins in mammary stem cell mainte-
glands. Luminal cell proliferation was strongly in- nance. Finally, because integrins have important
hibited in b1 integrin mutant glands, which corre- roles in breast cancer cell biology, we examined the
lated with a specific increase in p21Cip1 expression. effects of knocking-down b1 integrin expression
In a p21Cip1 null background, there was a partial res- in breast cancer cells. In its absence, some cancer
cue of BrdU incorporation, providing in vivo evi- cells arrested in G1 due to up-regulation of p21Cip1.
dence linking p21Cip1 to the proliferative defect ob-
served in b1 integrin mutant glands. Our results
show the essential role of b1 integrin signaling in
mammary epithelial cell proliferation. Further-
more, they suggest that b1 integrin has an impor-

Selected publications
Hynes NE, Lane H (2005)
ERBB receptors and cancer: the complexity
of targeted inhibitors. Nature Rev Cancer
5:341-354
Koziczak M, Hynes NE (2004)
Cooperation between FGFR-4 and ErbB2
in regulation of cyclin D1 translation.
J Biol Chem 279:50004-50011
Li N, Zhang Y, Naylor MJ, Schatzmann F,
Maurer F, Wintermantel T, Schuetz G,
Mueller U, Streuli CH, Hynes NE (2005)
b1 integrins regulate mammary gland pro-
liferation and maintain the integrity of
mammary alveoli. EMBO J 24:1942-1953
Marone R, Hess D, Dankort D, Muller WJ,
Hynes NE, Badache A (2004)
Memo mediates ErbB2-driven cell motility.
Nature Cell Biol 6:515-522
Sini P, Wyder L, Schnell C, O'Reilly T,
Littlewood A, Brandt R, Hynes NE, Wood J
(2005)
The antitumor and antiangiogenic activity
of vascular endothelial growth factor re-
ceptor inhibition is potentiated by ErbB1
blockade. Clin Cancer Res 11:4521-4532

FMI Report 2005/2006 75

Growth Control

Yoshikuni Nagamine
Regulation of the
plasminogen activator
system with emphasis on
the DExH helicase RHAU

INTRODUCTION
The stability of mRNA is one of the important reg-
ulatory steps determining the global level of gene
expression. mRNA stability is regulated via inter-
actions between cis-acting elements in the mRNA
and trans-acting factors recognizing these elements.
Of the cis elements identified so far, the most wide-
spread and studied is the AU-rich element (ARE),
a stretch of 50-100 nt located upstream of the GROUP LEADER
poly(A) signal in the 3untranslated region (3UTR) Yoshikuni Nagamine
that confers instability and is present at any one yoshikuni.nagamine@fmi.ch
time in about 8 % of total mRNAs. The ARE are
classified into at least three groups according to TECHNICAL/RESEARCH
the number and arrangement of a conserved pen- ASSOCIATES
tanucleotide motif AUUA. Various trans factors Sandra Pauli
have been identified that recognize ARE. During Stphane Thiry
the study of the ARE of urokinase-type plasmino-
gen activator mRNA (uPA), the expression level of POSTDOCTORAL
which is high in many metastatic tumors and in- FELLOWS
flammatory cells, we identified a novel DExH RNA Nobuyoshi Akimitsu*
helicase that enhances poly (A) tail shortening and Nives Selak
mRNA decay.
Numerous RNA helicases in a large DExH/D su- PhD STUDENTS
perfamily are engaged in almost all aspects of RNA Katerina Chalupnikova
metabolism from transcription, mRNA splicing, Fumiko Iwamoto
rRNA maturation, nuclear transport, and mRNA Sandra Kleiner*
translation to mRNA stability. Mutation studies in Ching Janice Lai
yeast indicate that each RNA helicase has a specific Simon Lattmann
function and plays an essential role in the cell. Svetlana Shustova
These enzymes contain a helicase core domain Joshi Venugopal*
of 350-400 amino acids consisting of 8-9 highly
conserved sequence motifs flanked by much less UNDERGRADUATES
conserved N- and C-terminal regions of variable Lauren Smith*
lengths. Not all DExH helicases have been shown Morteza Yazdani Shektaei*
to possess double-stranded RNA unwinding activ-
ity. Rather, they appear to exert biological activi- *left the group
ties through modulating protein-RNA interaction.
Mammals express more DExH/D helicases than
yeast and worms, but the functions and regulation
of most of them are as yet unknown. There is
no RHAU homologue in yeasts or Caenorhabditis
elegans.

76 FMI Report 2005/2006

DExH HELICASE RHAU IN uPA mRNA DECAY
H. Tran
The ARE uPA confers instability on otherwise stable
mRNA when inserted into the 3UTR. To elucidate
the mechanism by which ARE uPA enhances mRNA
decay, we searched for ARE uPA-binding proteins in
HeLa cell extracts and found HuR, NFAR1 (NF90)
and a putative DExH box RNA helicase. The first
two proteins were known to bind to the ARE and
stabilize target mRNA.The putative DExH box RNA
helicase has been deposited in a gene database as
DHX36 but nothing is known so far about its
biological features. We termed it RHAU (AU-rich
element associated RNA helicase) and investigated
its role in mRNA metabolism and other aspects of
biology. In vivo and cell-free experiments showed
that RHAU recruits the polyadenyl ribonuclease
PARN and the exosome complex of multiple 3
to 5 exoribonucleases, and enhances poly(A) tail
shortening and mRNA decay. The process depends
upon the ATPase activity of RHAU. So far only uPA
(Hoanh et al. Mol Cell 13:101-111, 2004) and PAI-2
(R.Medcalf,unpublished) mRNAs have been shown
to respond to RHAU.
NF90-DEPENDENT RECOGNITION OF ARE
BY RHAU
N. Akimitsu
Although RHAU was identified in ARE uPA-binding
fractions of HeLa cell extracts, it interacts much
weaker with ARE uPA than do HuR and NF90. RHAU
also does not bind to HuR or NF90. UV cross-link-
ing experiments showed, however, that RHAU
avidly binds to ARE uPA in the presence of NF90 but
not to AREIL2. Interestingly, RHAU binding to
ARE uPA was strongly reduced by addition not only
of ATP but also of the unhydrolysable analogue
ATPgS. This suggests that ATP binding induces
a conformational change in RHAU that depletes
its affinity for ARE and that its binding potential
is restored by ATP hydrolysis, thus ensuring the
efficient cycling (distributive) action of RHAU on
multiple mRNA molecules.
CHANGE IN THE INTRACELLULAR LOCALIZATION
OF RHAU
K. Chalupnikova, F. Iwamoto
Immunocytochemical analysis of ectopic HA-tagged
RHAU revealed a preferential nuclear localization.
Further analysis of transiently expressed EGFP-
RHAU, which allows monitoring in living cells,
also showed RHAU to be predominantly in the nu-
cleus, but it is translocated to the cytoplasm after
serum starvation. Interestingly, oxidative stress by
arsenite treatment that led to translational arrest
resulted in a significant fraction of RHAU in the
cytoplasm, especially in distinct foci stress gran-
ules and P-bodies. Untranslatable mRNAs re-
versibly accumulate in stress granules and many
mRNAs are channeled to degradation in P-bodies.
G4 QUADRUPLEX RESOLVASE ACTIVITY OF RHAU
S. Pauli and Y. Nagamine, in collaboration with
J. Vaughn and S. Akman (Wake Forest University,
Winston-Salem, USA)
While ATPase activity is essential for the mRNA
decay-enhancing activity of RHAU, we did not de-
tect double-stranded RNA-unwinding activity of
RHAU. It fact, many DExH helicases express their
biological activity through modulating protein-
RNA interaction and not by unwinding double-
stranded RNA. Recently, we found that RHAU har-
bors an interesting resolvase activity on guanine
quadruplexes (G4, both DNA and RNA). G4 struc-
Fig.1. Localization of RHAU
tures on which G4 resolvase acts appear in various in stress granules (SG)
places in the genome or in RNA molecules, in- and processing bodies (PB).
cluding telomeres, immunoglobulin heavy chain COS-7 cells transfected
with EGFP-RHAU and EGFP-
switch regions, some target mRNAs of the fragile
vector were cultured with
X mental retardation protein, promoters of proli- or without sodium arsenite
feration-association genes such as c-Myc and (0.5 mM) and stained with
PDGF-A, introns, and the 5/ 3 UTR of many mR- anti-TIA-1 and Dcp1a anti-
NAs. G4 resolvase may be very important for bio- bodies to detect SG and PB,
logical regulation involving these structures.At least respectively
in HeLa cells, more than 70 % of G4 resolvase ac-
tivity is derived from RHAU, suggesting that RHAU
is involved in many biological activities other than
mRNA stability regulation. G4 resolvase activity
is also found in Werner syndrome helicase and
Bloom syndrome helicase, deletion of which leads
to enhanced sensitivity to DNA-damaging agents
and genome instability with consequent acceler-
ated ageing.
FMI Report 2005/2006 77
Growth Control
A PAI-1 GENE REGULATION IN ADIPOCYTES
B and-bound insulin receptor relays two opposing
Fig.2. A Guanine quadru-
plex (G4) resolvase activity
of RHAU. A An example
of G4 structures used for
G4 resolvase assays.
B RNA or DNA G4 incu-
bated at 37 C with differ-
ent amounts of RHAU.
Monomers are obtained
by heating at 95 C
Selected publications
Nagamine Y, Muoz-Cnoves P, Medcalf RL
(2005)
Transcriptional and posttranscriptional
regulation of the plasminogen activator sys-
tem. Thromb Haemost 93:661-675
78 FMI Report 2005/2006
RHAU PARTNERS
S. Pauli
While the helicase core domain of RHAU is highly
conserved among many DExH/D helicases, the
amino and carboxyl terminal regions of RHAU are
much less conserved, raising the possibility that
these regions regulate the activity and intracellu-
lar localization of RHAU through interactions
with various proteins. To examine RHAU regula-
tion, we searched for RHAU-binding partners by
pull-down assays of endogenous RHAU using an
RHAU-specific mouse monoclonal antibody and
mass spectrometry. Many proteins were identified
and interaction with several was confirmed using
various cellular fractions and a specific antibody
for each protein. The most interesting of these pro-
teins are Upf1, Xrn1, eEF1a and NDH II.
Vaughn JP, Creacy SD, Routh ED,
Joyner-Butt C, Jenkins GS, Pauli S,
Nagamine Y, Akman SA (2005)
The DEXH protein product of the DHX36
gene is the major source of tetramolecular
quadruplex G4-DNA resolving activity in
HeLa cell lysates. J Biol Chem 280:38117-
38120
AND OVEREXPRESSION IN INSULIN-RESISTANT
CONDITIONS
J. Venugopal
Blood protein PAI-1 is a specific inhibitor of both
uPA and tissue-type plasminogen activator (tPA).
High expression is conspicuous in obesity and
type-2 diabetes and is one of the major causes
of cardiovascular complications frequently associ-
ated with these diseases. Thus, it is important to
unravel the molecular mechanism by which PAI-1
is upregulated under these conditions. PAI-1 is ex-
pressed in liver, endothelial cells and adipocytes.
In most cases,type-2 diabetes is preceded by adipoc-
ity and, therefore, the adipocytes are considered to
be the main source of PAI-1 expression in this sit-
uation. With respect to PAI-1 gene regulation, lig-
signals, a positive one through the Shc/Ras/Erk
pathway and a negative one through the IRS-1/
PI3K/PKB/E2F pathway. During differentiation of
3T3L1 preadipocytes, PAI-1 gene inducibility is
enhanced in differentiated cells due to reduced po-
tential of E2F activation brought about by reduced
E2F1 protein levels and increased non-phospho-
rylated retinoblastoma protein (pRB) levels. Re-
cent results suggest that plasma membrane cho-
lesterol and caveolin-1 are reduced in obesity.
Subsequently, we found that cholesterol depletion-
induced dysfunction of caveolae, where insulin
receptors reside, impaired only the IRS-1/PI3K
axis. This is responsible for insulin-induced glu-
cose uptake and provides the negative signal to
the PAI-1 promoter, resulting in elevated PAI-1 in-
duction by insulin. This mechanism may account
for PAI-1 upregulation during type-2 diabetes. Us-
ing cell-penetrating peptides that disrupt E2F-pRB
interaction and should release free E2F, thereby
eliciting downstream rejuvenation of the IRS-PI3K-
PKB pathway, we suppressed PAI-1 upregulation
in our insulin-resistance model. Thus the E2F-pRB
interaction may be a novel drug target to amelio-
rate pathological conditions of diabetes.
Venugopal J, Hanashiro K, Yang ZZ,
Nagamine Y (2004)
Identification and modulation of a caveo-
lae-dependent signal pathway that regu-
lates plasminogen activator inhibitor-1
in insulin-resistant adipocytes. Proc Nat
Acad Sci USA 101:17120-17125
Administration
and services Administrative assistance
Isabella Bogdal
Gabriele Gruber
Intellectual property
Mark McGrath
Gaby Schfer
Dawn Hammond
Sara Oakeley Library
Susan Thomas Susanne Krger-Lebus

Animal facilities Media and glassware

Birgit Heller-Stilb Matthias Mller
Marina Claros* Corinne Baumann
Alfred Schweizer Minh-Hoan Do*
Yann Assirelli For Yau Lam
Antonino Condemi* Markus Landolt
Roman Gehring Eva-Marina Libralato
Bjoern Henz* Gieu Sanh Trinh
Patrick Schirmer
Marcel Schuler Microscopy and imaging core facility
Pierre Spenle Patrick Schwarb
Fabien Weider Jens Rietdorf
Laurent Gelman
Bioinformatics support Thierry Laroche*
Michael Rebhan Aaron Ponti
Michael Stadler Sjoerd Van Eeden
Paul Bell*
Matthais Haimel* Monoclonal antibodies
Susanne Schenk Ernst
Building, HSE and security Michel Siegmann
Erich Schlumpf
Office of the Director
Building services Dagmar Baroke
Markus Briker
Pierre Blaes Peptide synthesis
Manfred Wagner Franz Fischer

Cell sorting (FACS) PhD programme

Hubertus Kohler Susan Thomas

DNA sequencing Protein analysis

Maciej Pietrzak Daniel Hess
Reto Portmann
Finance and controlling Ragna Sack
Christian Teuber
Dorothy Searles Radiation safety and biosafety
Patrick King
Functional genomics
Edward Oakeley Single-cell genomics
Herbert Angliker Erik Cabuy
Andrija Tomovic
Transgenic mice production
Grant administration Jean-Franois Spetz
Dorothy Searles Patrick Kopp
Bernard Kuchemann
Histology
Sandrine Bichet
Augustyn Bogucki *left the FMI
Melanie Sticker-Jantscheff*

Human resources
Rudi Unrau
Susan Thomas
Marilyn Vaccaro

Informatics
Dean Flanders
Alessandro Di Cara*
Brett Ellis*
Leandro Hermida
James Melendez*
Alan Naylor
Thomas Nyffenegger
Ingeborg Obergfll
Thibaut Siegmann
Sjoerd Van Eeden

FMI Report 2005/2006 79

 Acknowledgements
Research grants to Syngenta AG
Jan Hofsteenge
The scientific achievements of the FMI de-
scribed in this report were made possible by group leaders
Volkswagen Stiftung
the generous financial support of the Novartis Andreas Lthi
Research Foundation. In addition, the follow- Borderline Personality
Disorder Research Foundation Zurich-Basel Plant Science Centre
ing organisations have awarded research Andreas Lthi Frederick Meins
grants to group leaders and personal fellow-
ships to students and postdoctoral fellows Cancer League Basel-Baselland
during 2004 2006: Brian Hemmings
Yoshikuni Nagamine Personal fellowships
European Commission (FP6) to students and postdocs
Pico Caroni
Witold Filipowicz Australian National Health and
Susan Gasser Research Council
Jan Hofsteenge
Barbara Hohn Boehringer Ingelheim Fund
Nancy Hynes
Antoine Peters Canadian Institutes of Health Research

Fritz Thyssen Stiftung Centre National de la Recherche

Denis Monard Scientifique

Human Frontier Science Foundation European Molecular Biology Organisation

Patrick Matthias
Botond Roska German Research Council

Indo-Swiss Collaboration Human Frontier Science Foundation

in Biotechnology (SNF)
Barbara Hohn Marie Curie Intra-European Fellowship

Leukemia and Lymphoma Society National Center of Competence

Rafal Ciosk in Research

Marie Curie Excellence Grant Netherlands Organization for

Botond Roska Scientific Research

Novartis International AG Roche Research Foundation

George Thomas

Novartis Institutes
for Biomedical Research
Andreas Lthi Collaboration with the
George Thomas
University of Basel
Oncosuisse Collaborative Cancer
Research Projects We are very grateful to the University
Brian Hemmings of Basel, in particular the faculty
Nancy Hynes of the Biozentrum, for their continued
cooperation in research and in our
Roche Research Foundation International PhD Programme.
Brian Hemmings
Andreas Lthi

Stiftung Swiss Bridge

Brian Hemmings
Nancy Hynes

Swiss Cancer League / Oncosuisse /

Swiss Cancer Research
Susan Gasser
Brian Hemmings
Nancy Hynes
George Thomas

Swiss Foundation for Research

into Muscle Diseases
Pico Caroni

Swiss National Science Foundation

Silvia Arber
Matthais Chiquet
Susan Gasser
Andreas Lthi

80 FMI Report 2005/2006

Publications
and dissertations Group Joy Alcedo

Alcedo J, Kenyon C (2004)

Group Pico Caroni

Antoons G, Vangheluwe P, Volders PGA,

Regulation of C. elegans longevity by Holemans P, Ceci M, Condorelli GL,
specific gustatory and olfactory neurons. Wuytak F, Caroni P, Mubagwa K,
Neuron 41:45-55 (and Cover) Sipido KR (2006)
Increased phospholamban phosphoryla-
tion limits the force-frequency response
Group Silvia Arber in the MLP-/- mouse with heart failure.
J Mol Cell Cardiol 40:350-360
Arber S, Wong R (2004)
Neuronal and glial cell biology (Editorial). De Paola V, Holtmaat A, Knott G, Song S,
Curr Opin Neurobiol 14:519-521 Wilbrecht L, Caroni P, Svoboda K (2006)
Cell type-specific structural plasticity of
Bielle F, Griveau A, Narboux-Neme N, axonal branches and boutons in the adult
Vigneau S, Sigrist M, Arber S, Wassef M, neocortex. Neuron 49:861-875
Pierani A (2005)
Multiple origins of Cajal-Retzius cells Galimberti I, Gogolla N, Alberi S,
at the borders of the developing pallium. Santos AF, Muller D, Caroni P (2006)
Nature Neurosci 8:1002-1012 Long-term rearrangements of hippocampal
mossy fiber terminal connectivity
Chen C, Ouyang W, Grigura V, in the adult regulated by experience.
Zhou Q, Carnes K, Lim H, Zhao GQ, Neuron 50:749-763
Arber S, Kurpios N, Murphy TL,
Cheng AM, Hassell JA, Chandrashekar V, Gogolla N, Galimberti I, De Paola V,
Hofmann MC, Hess RA, Murphy KM Caroni P (2006)
(2005) Preparation of organotypic hippocampal
ERM is required for transcriptional slice cultures for long-term live imaging.
control of the spermatogonial stem cell Nature Protocols (in press)
niche. Nature 436:1030-1034
Gogolla N, Galimberti I, De Paola V,
Hippenmeyer S, Vrieseling E, Sigrist M, Caroni P (2006)
Portmann T, Laengle C, Ladle DR, Long-term live imaging of neuronal
Arber S (2005) circuits in organotypic slice cultures.
A developmental switch in the response Nature Protocols (in press)
of DRG neurons to ETS transcription
factor signaling. PLoS Biol 3:e159 Gogolla N, Galimberti I, De Paola V,
Caroni P (2006)
Kramer I, Sigrist M, de Nooij JC, Staining protocol for organotypic
Taniuchi I, Jessell TM, Arber S (2006) hippocampal slice cultures. Nature
A role for Runx transcription factor Protocols (in press)
signaling in dorsal root ganglion sensory
neuron diversification. Neuron Golub T, Caroni P (2004)
49:379-393 Spatial control of actin-based motility
through plasmalemmal PI(4,5)P2-rich
Niederkofler V, Salie R, Arber S (2005) raft assemblies. Biochem Soc Symp
Hemojuvelin is essential for dietary 72:119-127
iron sensing, and its mutation leads to
severe iron overload. J Clin Invest Golub T, Caroni P (2005)
115:2180-2186 PI(4,5)P2-dependent microdomain
assemblies capture microtubules to pro-
Raineteau O, Hugel S, Ozen I, Rietschin L, mote and control leading edge motility.
Sigrist M, Arber S, Gaehwiler BH (2006) J Cell Biol 169:151-165
Conditional labeling of newborn granule
cells to visualize their integration into Golub T, Wacha S, Caroni P (2004)
established circuits in hippocampal slice Spatial and temporal control of signaling
cultures. Mol Cell Neurosci 32:344-355 through lipid rafts. Curr Opin Neurobiol
14:542-550
Salie R, Niederkofler V, Arber S (2005)
Patterning molecules: multitasking Heineke J, Ruetten H, Willenbockel C,
in the nervous system. Neuron Gross SC, Naguib M, Schaefer A,
45:189-192 Kempf T, Hilfiker-Kleiner D, Caroni P,
Kraft T, Drexler H, Wollert KC (2005)
Attenuation of cardiac remodeling after
myocardial infarction by muscle LIM
protein-calcineurin signaling at the
sarcomeric Z-disc. Proc Natl Acad Sci
USA 102:1655-1660

FMI Report 2005/2006 81

Publications and dissertations
Portera-Cailliau C, Weimer RM,
De Paola V, Caroni P, Svoboda K (2005)
Diverse modes of axon elaboration in the
developing neocortex. PLoS Biol 3:e272
Pun S, Santos AF, Saxena S, Lefler S,
Caroni P (2006)
CNTF-sensitive selective vulnerability
and pruning of phasic motoneuron axons
in motoneuron disease. Nature Neurosci
3:408-419
Richards DA, De Paola V, Caroni P,
Gahwiler BH, McKinney RA (2004)
AMPA-receptor activation regulates the
diffusion of a membrane marker in parallel Orend G, Chiquet-Ehrismann R (2006)
with dendritic spine motility in the mouse Tenascin-C induced signaling in cancer.
hippocampus. J Physiol 558:503-512
Richards DA, Mateos JM, Hugel S,
de Paola V, Caroni P, Gahwiler BH,
McKinney RA (2005)
Glutamate induces the rapid formation
of spine head protrusions in hippocampal
slice cultures. Proc Natl Acad Sci USA
102:6166-6171
Willmann R, Pun S, Stallmach L,
Sadavisam G, Santos AF, Caroni P,
Fuhrer C (2006)
Cholesterol and lipid microdomains
stabilize the postsynapse at the neuro-
muscular junction. EMBO J (in press)
Group Ruth Chiquet-Ehrismann
Baumann P, Soric N, Kroese F, Orend G,
Chiquet-Ehrismann R, Uede T, Yagita H,
Sleeman P (2005)
CD24 expression causes acquisition
of multiple cellular properties associated
with tumor growth and metastasis.
Cancer Res 65:10783-10793
Canevascini S (2004)
Genetic analysis of vulva development in
C. elegans. In: Beffa M, Knight J (eds)
Key techniques in practical developmen-
tal biology. Cambridge University Press,
New York, pp 153-166
Drabikowski K, Trzebiatowska A,
Chiquet-Ehrismann R (2005)
ten-1, an essential gene for germ cell
development, epidermal morphogenesis,
gonad migration, and neuronal path-
finding in Caenorhabditis elegans. Dev
Biol 282:27-38
Isler SG, Ludwig CU,
Chiquet-Ehrismann R, Schenk S (2004)
Evidence for transcriptional repression
of SPARC-like 1 (SPARCL1), a gene
downregulated in human lung tumors.
Int J Oncol 25:1073-1079
82 FMI Report 2005/2006
Meloty-Kapella CV, Degen M,
Chiquet-Ehrismann R, Tucker RP (2006)
Avian tenascin-W: expression in smooth
muscle and bone, and effects on calvarial
cell spreading and adhesion in vitro.
Dev Dyn 235:1532-1542
Nunes-Radimerski S, Ferralli J, Choi KD,
Minet A, Chiquet-Ehrismann R (2005)
The intracellular domain of teneurin-1
interacts with MBD1 and CAP/ponsin
resulting in subcellular co-distribution
and translocation to the nuclear
matrix. Exp Cell Res 305:122-132
Cancer Lett (in press)
Ruiz C, Huang W, Hegi ME, Lange K,
Hamou M-F, Fluri E, Oakeley EJ,
Chiquet-Ehrismann R, Orend G (2004)
Differential gene expression analysis
reveals activation of Wnt and MAPK
signaling and downregulation of
tropomyosin-1 by tenascin-C. Cancer
Res 64:7377-7385
Scherberich A, Tucker RP,
Degen M, Brown-Luedi M, Andrs AC,
Chiquet-Ehrismann R (2005)
Tenascin-W is found in malignant
mammary tumors, promotes alpha8 in-
tegrin-dependent motility and requires
p38 MAPK activity for BMP-2 and
TNF-alpha induced expression in vitro.
Oncogene 24:1525-1532
Tucker RP, Chiquet-Ehrismann R (2006)
Teneurins: a conserved family of trans-
membrane proteins involved in inter-
cellular signaling during development.
Dev Biol 290:237-245
Tucker RP, Drabikowski K, Hess J,
Chiquet-Ehrismann R, Adams JC (2006)
Phylogenetic analysis of the tenascin gene Pillai R (2005)
family: evidence of origin early in the
chordate lineage. BMC Evol Biol (in press) for a tiny RNA? RNA 11:1753-1761
Veit G, Hansen U, Keene DR, Bruckner P,
Chiquet-Ehrismann R, Chiquet M,
Koch M (2006)
Collagen XII interacts with avian
tenascin-X through its NC3 domain.
J Biol Chem (in press)
Group Witold Filipowicz
Bhattacharyya SN, Habermacher R,
Martine U, Closs EI, Filipowicz W (2006) 309:1573-1576
Stress induced reversal of miRNA repres-
sion and mRNA P-body localization in
human cells. In: Regulatory RNAs. Cold
Spring Harbor Symposia, vol 71. Cold
Spring Harbor Laboratory Press (in press) a conserved coronavirus enzyme that is
Bhattacharyya S, Habermacher R,
Martine U, Closs EI, Filipowicz W (2006)
Relief of microRNA-mediated trans-
lational repression in human cells sub-
jected to stress. Cell 125:1111-1124
Filipowicz W (2005)
RNAi: the nuts and bolts of the RISC
machine. Cell 122:17-20
Filipowicz W, Jaskiewicz L, Kolb F,
Pillai R (2005)
Post-transcriptional gene silencing by
siRNAs and miRNAs. Curr Opin Cell Biol
15:1-11
Gangloff YG, Mueller M, Dann SG,
Svoboda P, Sticker M, Spetz JF, Um SH,
Brown EJ, Cereghini S, Thomas G,
Kozma SC (2004)
Disruption of the mouse mTOR gene
leads to early post-implantation lethality
and prohibits embryonic stem cell devel-
opment. Mol Cell Biol 24:9508-9516
Haase A, Jaskiewicz L, Zhang H, Lain S,
Sack R, Gatignol A, Filipowicz W (2005)
TRBP, a regulator of cellular PKR
and HIV-1 virus expression, interacts with
Dicer and functions in RNA silencing.
EMBO Rep 6:961-967
Kolb FA, Zhang H, Jaronczyk K, Tahbaz N,
Hobman TC, Filipowicz W (2005)
Human Dicer: purification, properties
and interaction with PAZ PIWI domain
proteins. Meth Enzymol 392:316-336
Kotaja N, Bhattacharyya S, Jaskiewicz L,
Kimmins S, Parvinen M, Filipowicz W,
Sassone-Corsi P (2006)
The chromatoid body of male germ cells:
similarity with P-bodies and presence
of Dicer and microRNA pathway compo-
nents. Proc Natl Acad Sci USA
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LSD1 demethylates repressive
histone marks to promote androgen
receptor dependent transcription. Nature
437:436-439
Peters AHFM, Schbeler D (2005)
Methylation of histones: playing
Memory with DNA. Curr Opin Cell Biol
17:230-238
Puschendorf M, Stein P, Oakeley EJ,
Schultz RM, Peters AH, Svoboda P
(2006)
Abundant transcripts from retrotrans-
posons are unstable in fully grown mouse
oocytes. Biochem Biophys Res Commun
347:36-43
Santos F, Peters AH, Otte AP, Reik W,
Dean W (2005)
Dynamic chromatin modifications
characterise the first cell cycle in mouse
embryos. Dev Biol 280:225-236
Zinner R, Albiez H, Walter J,
Peters AHFM, Cremer T, Cremer M (2005)
Histone lysine methylation patterns in
human cell types are arranged in
distinct three-dimensional nuclear zones.
Histochem Cell Biol 125:3-19
Group Dirk Schbeler
Peters AH, Schbeler D (2005)
Methylation of histones: playing
Memory with DNA. Curr Opin Cell Biol
17:230-238
Schbeler D (2005)
Editorial: From genetic diversity to
chromosomal biology. an array of tasks
beyond expression. Chromosome Res
13:223-224
Schbeler D (2006)
Dosage compensation in high resolution:
global up-regulation through local
recruitment. Genes Dev 20:749-753
Schbeler D, Elgin SC (2005)
Defining epigenetic states through
chromatin and RNA. Nature Genet
37:917-918
Schbeler D, Turner BM (2005)
A new map for navigating the yeast
epigenome. Cell 122:489-492
Schwaiger M, Schbeler D (2006)
A question of timing: emerging links
between transcription and replication.
Curr Opin Genet Dev 16:177-183
Weber M, Davies JJ, Wittig D, Oakeley
EJ, Haase M, Lam WL, Schbeler D
(2005)
Chromosome-wide and promoter-specific
analyses identify sites of differential
DNA methylation in normal and trans-
formed human cells. Nature Genet
37:853-862
White EJ, Emanuelsson O, Scalzo D,
Royce T, Kosak S, Oakeley EJ,
Weissman S, Gerstein M, Groudine M,
Snyder M, Schbeler D (2004)
DNA replication-timing analysis of
human chromosome 22 at high
resolution and different developmental
states. Proc Natl Acad Sci USA
101:17771-17776
Wilson IM, Davies JJ, Weber M,
Brown CJ, Alvarez CE, MacAulay C,
Schbeler D, Lam WL (2006)
Epigenomics: mapping the methylome.
Cell Cycle 5:155-158
Wirbelauer C, Bell O, Schbeler D
(2005)
Variant histone H3.3 is deposited at sites
of nucleosomal displacement throughout
transcribed genes while active histone
modifications show a promoter-proximal
bias. Genes Dev 19:1761-1766
Dissertations Group Brian Hemmings
FMI students successfully defended Bichsel S (2005)
the following dissertations since the last Mechanism of action of NDR
Report: protein kinase by the hMOB1 protein.
PhD thesis, University of Basel
Stegert MR (2005)
Group Silvia Arber
Functional characterization of the
mammalian NDR1 and NDR2 protein
Hippenmeyer S (2004)
kinases and their regulation by the
Molecular mechanisms of neuronal
mammalian STE20-like kinase MST3.
circuit assembly in the vertebrate spinal
PhD thesis, University of Basel
cord. PhD thesis, University of Basel
Yang ZZ (2004)
Kramer I (2005)
Towards an understanding of protein
Molecular pathways of proprioceptive
kinase B (PKB/Akt) function in mouse
dorsal root ganglion (DRG) sensory
development. PhD thesis, University
neuron specification. PhD thesis,
of Basel
University of Basel
Laengle C (2004)
Group Nancy Hynes
Consequences of premature EWS-Pea3
expression in chick and mouse spinal
Li N (2005)
cord. MSc thesis, University of Basel
b1 integrins regulate mammary gland
proliferation and maintain the
Niederkofler V (2005)
integrity of mammary alveoli. PhD thesis,
Identification and functional charac-
University of Basel
terization of the RGM family in mouse.
PhD thesis, University of Basel
Group Andreas Lthi
Salie R (2005)
Mouse RGMs: a three protein family
with diverse function and localization. Shaban H (2005)
GABAB receptor-mediated modulation
PhD thesis, University of Basel
of synaptic plasticity in the lateral
amygdala. PhD thesis, University of Basel
Group Pico Caroni
Ferrao Santos A (2006) Group Andrew Matus
Regulation of anatomical plasticity
at neuromuscular junctions. PhD thesis, Birbach A (2006)
The actin-binding protein profilin II
University of Basel
in neuronal plasticity. PhD thesis,
University of Basel
Golub T (2005)
Regulation of the leading edge motility
by PI (4,5)P2-dependent lipid
microdomains. PhD thesis, University Group Denis Monard
of Basel
Kvajo M (2004)
Sadhu A (2006) Modulation of neuronal plasticity
Role of the neuronal protein Cap23 by extracellular serine proteases and their
inhibitors Proteolytic control of NMDA
in the maturation and maintenance of
receptors. PhD thesis, University of Basel
dendritic arbors in-vivo. PhD thesis,
University of Basel
Li X (2006)
Alternative signaling pathways
triggered by different mechanisms of
Group Ruth Chiquet-Ehrismann serpin endocytosis. PhD thesis,
University of Basel
Nunes-Radimerski S (2005)
Signalling by teneurin-1. PhD thesis,
University of Basel
Group Yoshikuni Nagamine
Walpen T (2005) Kleiner S (2005)
Promoter analysis of the tenascin-W Isoform-specific roles of the adaptor
gene. MSc thesis, University of Basel protein ShcA in cell signaling.
PhD thesis, University of Basel
Venugopal J (2006)
Caveolar dysfunction leads to signal
transduction defects that are critical for
obesity-driven disorders. PhD thesis,
University of Basel
FMI Report 2005/2006 87
 Lectures
and seminars Lectures I.W. Mattaj
EMBL
Heidelberg, Germany
The Ran GTPase as a spatial regulator
Hubert Bloch Lectures in mitosis
The following lectures and seminars were
given by visitors to the FMI: G. Dreyfuss
P. Matzinger
Howard Hughes Medical Institute
National Institutes of Health
Philadelphia, USA
Bethesda, USA
The survival of motor neuron protein:
Immunity is a conversation, not a war
an assembly machine for RNA-protein
complexes
K. Nasmyth
IMP Vienna
M. Kirschner
Vienna, Austria
Harvard Medical School
How do cells hold sister chromatids
Boston, USA
together during mitosis and meiosis?
Timing and order of the cell cycle
E. Neher
Max Planck Institute
Students Colloquia* Gttingen, Germany
The breathing-zipper model of
A. Aguzzi
exocytosis
University Hospital of Zurich
Zurich, Switzerland M. Radman
Molecular biology of prion diseases Necker Medical School
Paris, France
K. Benenson
The fidelity of DNA replication and
Harvard University recombination processes
Cambridge, USA
Biomolecular automata: from concepts M. Raff
to applications University College
London, UK
M. Capecchi
and H.W. Denker
Howard Hughes Medical Institute University of Essen
Salt Lake City, USA Essen, Germany
Gene targeting into the 21st century: Embryonic stem cell research:
modeling human diseases from hope or hype? a debate
cancer to europsychiatric disorders
U. Schibler
W. Gehring
University of Geneva
University of Basel Geneva, Switzerland
Basel, Switzerland Biological clocks: the mammalian
New perspectives on eye development circadian timing system
and evolution of photoreception
R. Schroeder
A. Grace
University of Vienna
University of Pittsburgh Vienna, Austria
Pittsburgh, USA Proteins involved in guiding
Prefrontal cortical-amygdala interactions RNA folding
and the effects of chronic stress
D. Tollervey
M. Grunstein
University of Edinburgh
University of California Edinburgh, UK
Los Angeles, USA Making and breaking RNA
Chromosomal structures and gene
activity J. Wood
IMD Business School
A. Hershko
Lausanne, Switzerland
Technion Israel Institute of Technology The nature of leadership
Haifa, Israel
The ubiquitin system and some of its S. Zeki
roles in cell cycle control University College
London, UK
T. Hunt
The disunity of consciousness
Cancer Research UK
London, UK
The role of cell cycle transitions *organised by the Student
Representatives
B. Lahn
University of Chicago
Chicago, USA
Probing the genetic basis of human
brain evolution

88 FMI Report 2005/2006

 M. Beato D. Brazil
Seminars University of Barcelona University College
Barcelona, Spain Dublin, Ireland
Crosstalk between nuclear receptors Mechanisms and models of diabetic
R. Ahmadian
and kinase cascades in gene regulation nephropathy, a microvascular
Max Planck Institute
complication of diabetes
Dortmund, Germany
M. Bentires-Alj
Rho GTPases in human disease:
Harvard Medical School K. Brueckner
mechanisms, networks and future
Boston, USA Harvard Medical School
perspectives
Role of Gab2 in breast cancer Boston, USA
development and metastasis Drosophila models for cancer
S. Akman
development
Wake Forest University Health Sciences
R. Benton
Winston-Salem, USA
Rockefeller University E. Bucher
G4-DNA resolution by RHAU/DHX36
New York, USA Wageningen University
How flies smell: a molecular dissection The Netherlands
A. Arcaro
of Drosophila odorant receptors On the discovery of suppressors of RNA
University of Zurich
silencing of plant- and mammal-
Zurich, Switzerland
N. Berteaux infecting viruses and the use of RNA
The role of lipid rafts in PI3K signaling
INSERM, University of Lille silencing in multiple virus resistance
Lille, France
P. Arlotta
Regulation and function of the D. Bulavin
Harvard Medical School
H19 mRNA-like non-coding RNA and Institute of Molecular and Cell Biology
Boston, USA
of the 91H antisense RNA Singapore
Molecular development and repair
Control of tumor surveillance
of corticospinal motor neuron circuitry
E. Bertolino mechanisms by Wip1 phosphatase
Howard Hughes Medical Institute
C. Arrieumerlou
Chicago, USA G. Capitani
Biozentrum, University of Basel
Control of immunoglobulin VH University of Zurich
Basel, Switzerland
gene expression and rearrangements Zurich, Switzerland
Using RNAi screens to probe the
by STAT5 through the regulation High-accuracy homology modeling
signaling network induced upon
of chromatin accessibility: molecular of a medically important enzyme:
Shigella infection in epithelial cells
coupling of proliferation and methodology, functional insights and
differentiation interplay with experiment
S. Ashraf
Harvard University
A. Besson G. Capitani
Cambridge, USA
Howard Hughes Medical Institute University of Zurich
A role for microRNA/RISC pathway in
Seattle, USA Zurich, Switzerland
long-term memory in Drosophila
p27Kip1, nuclear tumor suppressor Glutamate decarboxylase and
and cytoplasmic oncogene? sphingosine phosphate lyase:
J. Back
from prokaryotic acid resistance to
IGBMC-ULP
C. Bock eukaryotic growth control
Illkirch, France
MPI for Informatics
The myelo-lymphoid differentiation
Saarbrcken, Germany R. Cartlidge
factor PU.1 determines the self-renewal
CpG island methylation explained by University of Dundee
capacity of erythroid progenitor cells
DNA sequence, structure and repeats Dundee, UK
Linking PKB and RSK to tRNA
H. Baier
Z. Boldogkoi methylation
University of California
Szeged University
San Diego, USA
Szeged, Hungary S. Chattarji
Genetic analysis of vision in zebrafish
Transsynaptic gene targeting by National Centre for Biological Sciences
pseudorabies virus vectors Bangalore, India
A. Bairoch
The synaptic basis of anxiety in the
Swiss Institute of Bioinformatics
T. Bonhoeffer amygdala
Geneva, Switzerland
Max Planck Institute
Do we still need manual annotation
Martinsried, Germany A. Chedotal
of proteins? Yes, indeed!
How activity changes synapses in CNRS
the mammalian brain Paris, France
K. Ballmer
Molecular control of neuronal
Paul Scherrer Institute
E. Boye migration in the cerebellar system
Villingen, Switzerland
University of Oslo
Signaling in angiogenesis: the role of
Oslo, Norway M. Chekulaeva
VEGF receptors
Regulation of the G1/S transition EMBL
in fission yeast Heidelberg, Germany
R. Baumeister
Translational repression via mRNA
Bioinformatics and Molecular Genetics
S. Brachmann oligomerization: regulation of oskar
Freiburg, Germany
Harvard University mRNA during Drosophila oogenesis
Parkin, peptides and (insulin) signaling.
Cambridge, USA
C. elegans models to study age-related
The in vivo role of phosphoinositide
diseases and mechanisms
3-kinase regulatory subunit p85

D. Braguer
Universit de la Mditerrane
Marseille, France
Microtubule dynamics and signal
transduction in tumor progression

FMI Report 2005/2006 89

Lectures and seminars

P. Chene G. Dreyfuss W. Fischle

Novartis Pharma University of Pennsylvania The Rockefeller University
Basel, Switzerland Philadelphia, USA New York, USA
Importance of the tetramerisation The survival of motor neurons protein: Effector mechanisms of histone
domain for the properties of p53 mutants an assembly machine for RNA-protein modifications
complexes
M. Chiquet M. Forstner
University of Berne C. du Roure Novartis Institutes for Biomedical Research
Berne, Switzerland Lymphocyte Development Group, MRC Basel, Switzerland
Gene regulation by mechano- London, UK Addressing the challenges ahead: ways
transduction in connective tissue Using B-lymphocytes to deliver to integrate biochemistry, biophysics,
therapeutic proteins structural biology and cell biology
G. Christofori
DKBW, University of Basel S. Eaton P. Fraser
Basel, Switzerland Max Planck Institute The Babraham Institute
Cell adhesion, angiogenesis and Dresden, Germany Cambridge, UK
metastasis Lipoprotein carriers for lipid-linked Nuclear dynamics and transcriptional
morphogens regulation
G. Christofori
DKBW, University of Basel M. Eilers R. Friedrich
Basel, Switzerland University of Marburg Max Planck Institute
Distinct mechanisms of tumor cell Marburg, Germany Heidelberg, Germany
metastasis Transcriptional regulation and Dynamic processing of odor-evoked
transformation by Myc activity patterns by neuronal circuits
R. Ciosk in the olfactory bulb
Fred Hutchinson Cancer Research Center F. Eisenhaber
Seattle, USA Institute of Molecular Pathology G. Fuellen
Maintaining totipotency in the Vienna, Austria University of Bielefeld
C. elegans germline Prediction of localization signals Bielefeld, Germany
and posttranslational modifications Homology and its uses for protein
D.W. Cleveland from protein sequence function prediction, species tree
Ludwig Institute for Cancer Research inference and interaction data analysis
San Diego, USA A. Engel
Guarding the genome: epigenetic Biozentrum, University of Basel R. Gainetdinov
centromere inheritance and mitotic Basel, Switzerland Duke University Medical Center
checkpoint signaling 3D EM: future prospects for Basel Durham, USA
and Systems X Monoaminergic neurotransmission:
S. Cohen lessons from dopamine transporter
EMBL L. Enquist knockout mice
Heidelberg, Germany Princeton University
MicroRNA functions Princeton, USA C. Garcia-Echeverria
Live imaging of trans-synaptic viruses Novartis Pharma AG
A. Delogu in neurons Basel, Switzerland
Ludwig Boltzmann Institute for Cancer Therapeutic approaches to disrupt
Research E. Fabre IGF-IR in tumour cells
Vienna, Austria Pasteur Institute
Gene repression by Pax5 in B Paris, France J. Gautier
lymphocytes Nuclear pore complexes and Columbia University
subtelomeric double strand break New York, USA
B. Dickson repair and silencing Regulation of DNA replication initiation
Research Institute of Molecular Pathology by checkpoint kinases and oncogenes
Vienna, Austria R. Faessler
Wired for sex: genetic specification Max Planck Institute for Biochemistry D.M. Gilbert
of sexual behavior in Drosophila Martinsried, Germany SUNY Upstate Medical University
Genetic analysis of integrin function Syracuse, USA
A. Donaldson in mice Spatial and temporal control of
University of Aberdeen mammalian chromosome replication
Aberdeen, UK H. Fennema during the cell-cycle and development
Chromosome replication and positioning Invitrogen Corp.
in S. cerevisiae NV Leek, The Netherlands P. Gnczy
New technologies for fluorescent ISREC
A. Dorr live cell imaging and flow cytometry Epalinges, Switzerland
EPFL Mechanisms of centrosome duplication
Lausanne, Switzerland D. Finke in C. elegans embryos
Integration site selection of lentiviral DKBW, University of Basel
vectors in the mouse genome Basel, Switzerland G. Goodall
Lymphoid tissue development: University of Adelaide
role of cellular crosstalk and migration Adelaide, Australia
Cap independent translation: are
W. Fischle cellular IRESs physiologically relevant?
The Rockefeller University
New York, USA
Dynamic readout of stable histone
modifications

90 FMI Report 2005/2006

Y. Goto P. Hawkins P. Hublitz
University of Pittsburgh Science Research Council University of Freiburg
Pittsburgh, USA Swindon, UK Freiburg, Germany
Neural systems of temporal information PI3K signaling in neutrophils The novel INHAT repressor GRIM1
processing: implication for schizophrenia
J.B. Hays J. Huelsken
F. Grosse Oregon State University ISREC
Leibniz Institute for Age Research Corvallis, USA Epalinges, Switzerland
Jena, Germany Mismatch repair in plant and human Wnt signalling in tissue homeostasis
The role of CDC45 during initiation and cells and cancer
elongation of human DNA replication
M. Hegi E. Izzauralde
H. Grosshans University of Lausanne EMBL
Yale University Lausanne, Switzerland Heidelberg, Germany
New Haven, USA Molecular profiles in glioma and Post-transcriptional regulation of gene
The developmental roles and mechanism associations with clinical parameters expression: molecular mechanisms
of action of microRNAs and interaction networks
N. Heintz
M. Gutierrez Rockefeller University S. Jacob
Max Planck Institute New York, USA Novartis Pharma
Dortmund, Germany Understanding the cellular and molecular Basel, Switzerland
Insights into the bioactive conformation complexities of the mammalian brain Structural biology of protein tyrosine
of antitumoral marine compound kinases to support drug discovery
didemnins by using spirolactam b-turn I. Hellmann
mimetics Max Planck Institute M. Jantsch
Leipzig, Germany University of Vienna
M. Husser Mutation and selection in the human Vienna, Austria
University College genome RNA editing by adenosine deamination:
London, UK novel targets, novel functions
A dendritic switch for synaptic T. Hensch
plasticity in neocortical pyramidal cells Harvard University T. Jenuwein
Cambridge, USA Research Institute of Molecular Pathology
E. Hafen Critical period specification by Otx2 Vienna, Austria
University of Zurich homeo-protein in postnatal visual cortex The epigenome in the context of the
Zurich, Switzerland post-genomic era
Insulin signaling and growth control W. Herr
in Drosophila University of Lausanne J. Jiricny
Lausanne, Switzerland University of Zurich
R. Hahnloser HCF-1: coordinating the human Zurich, Switzerland
University of Zurich chromosome cycle Mismatch repair defects in colon cancer
Zurich, Switzerland and in DNA damage signaling
Neurophysiological explorations at J. Herz
the sensory-motor interface of a learned The University of Texas S. Junker
vocal behavior Dallas, USA University of Aarhus
How lipoprotein receptors shape Aarhus, Denmark
A. Hajnal your brain Epigenetic silencing contributes
University of Zurich to loss of B cell identity in Hodgkin/
Zurich, Switzerland P. Heun Reed-Sternberg cells of classical
Signal transduction during C. elegans Lawrence Berkeley National Laboratory Hodgkin's disease
vulval development: how RAS and notch Berkeley, USA
talk to each other The role of epigenetics in centromere I. Kadurin
identity and chromosome organization University of Wrzburg
R.S. Haltiwanger Wrzburg, Germany
State University of New York M. Higdon Regulation of acid-sensing ion channels
New York, USA Applied Precision by associated proteins
Regulation of notch signaling with Issaquah, USA
glycosylation DeltaVision RT Restoration Imaging S. Khochbin
System featuring the world's first real- Institute Albert Bonniot
C. Handschin time restoration imaging Grenoble, France
Dana Farber Cancer Institute Molecular basis of genome
Boston, USA K. Hofmann re-organization during post-meiotic
Molecular mechanisms in the Memorec Biotech GmbH maturation of male germ cells
pathogenesis of type 2 diabetes in Cologne, Germany
skeletal muscle The ubiquitin/proteasome system J. Kirberg
an evolutionary perspective Max Planck Institute of Immunology
H.O. Handwerker Freiburg, Germany
University of Erlangen G. Hollaender BKLF over-expression affects class
Erlangen, Germany DKBW, University of Basel switch recombination and final B cell
Pain mechanisms Basel, Switzerland maturation
Cellular and molecular aspects of
M. Harata thymus organogenesis F. Kirchhoff
Tohoku University Max Planck Institute of Experimental
Sendai, Japan Medicine
Involvement of the actin-related Gttingen, Germany
protein Arp6 in nuclear organization Transgenic expression of fluorescent
proteins to study glial function in situ

FMI Report 2005/2006 91

Lectures and seminars

T. Klausberger L. Landmann T. Marquardt

Medical Research Council DKBW, University of Basel The Salk Institute
Oxford, UK Basel, Switzerland La Jolla, USA
Hippocampal circuits and network Mechanisms of cholestasis Receptor-based interactions underlying
oscillations selective axon targeting
H. Lane
H. Knaut Novartis Pharma U. Martens
Harvard University Basel, Switzerland University Hospital
Cambridge, USA Targeting the mTOR pathway in human Freiburg, Germany
Orchestrating organ formation cancer: from concept to application Telomerase as a target for cancer therapy

A. Konnerth J. Langowski S. Martin

University of Munich German Cancer Research Center Columbia University
Munich, Germany Heidelberg, Germany New York, USA
Single shock LTP and LTD: a model The genome as a flexible polymer chain Regulation of actin assembly by
or online learning microtubule plus ends
D. Leahy
J. Kremers John Hopkins University J. Martin-Perez
Novartis Pharma AG Baltimore, USA Instituto de Investigaciones Biomedicas
Basel, Switzerland Structural basis for EGF/ErbB receptor Madrid, Spain
The correlation between visual signaling and ErbB-targeted cancer Role of Src kinases on prolactin cell
perception and responses of cells in therapies signaling
the lateral geniculate nucleus
J. Lingner B. Martoglio
J. Krijgsfeld ISREC Novartis Pharma AG
University of Utrecht Lausanne, Switzerland Basel, Switzerland
Utrecht, The Netherlands Telomerase and the mechanisms of Expanding the repertoire of signaling
Quantitative proteomics of fruit fly telomere length homeostasis through regulated intramembrane
development by metabolic labeling with proteolysis; unraveling functions of
stable isotopes M. Londei presenilin-type aspartic proteases
Novartis Pharma AG
A. Krishnan Basel, Switzerland J. Mascarenhas
Bioinformatics Institute Auto-reactive T cells and autoimmunity Biozentrum, University of Basel
Singapore Basel, Switzerland
Along the protein sequence, structure, M.P. Longhese Mapping of the laminin binding site
function path. a computational approach Universit degli Studi di Milano-Bicocca on the NtA domain of agrin
Milan, Italy
G. Krupp DNA damage detection and checkpoint A. Matter
Artus GmbH activation Novartis Institute for Tropical Diseases
Hamburg, Germany Singapore
ExpressArt the future of mRNA R. Lovell-Badge The challenges of tropical diseases
amplification National Institute for Medical Research
Mill Hill, UK A. Matter
S. Kubicek Intrinsic and extrinsic control of stem Novartis Institute for Tropical Diseases
IMP cells in the nervous system Singapore
Vienna, Austria Fight against HIV in the Third World
Counteracting histone lysine methylation K. Luger
HMTase inhibitors and demethylases Howard Hughes Medical Institute U. Mayer
Fort Collins, USA Centre d'Immunologie Marseille-Luminy
N. Kunert Histone chaperones and nucleosome Marseille, France
German Cancer Research Center dynamics Analysis of the molecular mechanisms
Heidelberg, Germany underlying the activity of the Ets-1/
DNA methylation in Drosophila Y. Machida USF-1 transcription factor complex
is mediated by Dnmt2 and regulated Nagoya University
by IPOD Nagoya, Japan R. Mazumder
The MAP kinase cascade that controls Georgetown University
C. Kunz plant cytokinesis Washington, USA
DKBW, University of Basel Identification of functionally important
Basel, Switzerland W. Maier experimental targets based on evolutionary
Viewing DNA repair from the perspective MPI for Brain Research analysis of proteins and genes
of an unusual DNA glycosylase Frankfurt, Germany
The molecular mechanism of NMDA R. Medcalf
R. Lahav receptor activation Monash University
University of Lausanne Victoria, Australia
Lausanne, Switzerland P.W. Manley Tissue-type plasminogen activator in
A melanocyte developmental gene as Novartis Institutes for Biomedical Research the brain: good or bad?
target for melanoma therapy Basel, Switzerland
Superglivec AMN107: a novel
Bcr-Abl kinase inhibitor for the treatment
of chronic myelogenous leukemia

92 FMI Report 2005/2006

M.F. Mette U. Mller P.J. Parker
IPK MIT Cancer Research UK
Gatersleben, Germany Cambridge, USA London, UK
RNA-directed transcriptional gene Towards a self-replicating system from Protein kinase C signalling in space
silencing in Arabidopsis thaliana: catalytic RNA and time
a model system for the study of
epigenetic regulation V. Murthy R. Paro
Harvard University ZMBH
J. Michaud Cambridge, USA Heidelberg, Germany
The Walter and Eliza Hall Institute of Experience-dependent adaptation of Epigenetic control of transcriptional
Medical Research synapses: hebb and homeostasis memory
Melbourne, Australia
Identification of genes and biological S. Nakanishi R. Paro
processes regulated by the transcription Emory University ZMBH
factor RUNX1/AML1 Atlanta, USA Heidelberg, Germany
Activity-dependent signaling in the Epigenetics of tissue regeneration
O. Mittelsten-Scheid regulation of neuronal excitability and and remodeling
Gregor Mendel Institute of Molecular synaptic efficacy
Plant Biology U. Paszkowski
Vienna, Austria P. Norio University of Geneva
A short history of mute genes and Albert Einstein College of Medicine Geneva, Switzerland
expressive mutations New York, USA Radical underground associations:
Developmental regulation of DNA genetics and genomics of an ancient
G. Montoya replication initiation in the mouse plant symbiosis
Spanish National Cancer Center immunoglobulin heavy chain locus;
Madrid, Spain implications for the functional L. Pearl
Molecular and structural studies on organization of the locus ICR
Polo-like kinase1 substrate recognition London, UK
B. Olveczky Chaperoning ubiquitylation structure
N. Mori Harvard University and mechanism of the CHIP E3
Nagasaki University School of Medicine Cambridge, USA ubiquitin ligase
Nagasaki, Japan Using behaviour as a guide for studying
Role of neural specific phosphotyrosine neural circuit function: insights from G. Pendyala
signal adaptor N-Shc/ShcC in neural salamanders and songbirds University of Fribourg
development, plasticity and brain aging Fribourg, Switzerland
C. Oste Circadian rhythms and glutamatergic
P. Morin NimbleGen Systems Inc. neurotransmission: implications on
Carleton University Madison, USA alcoholism
Ottawa, Canada High-definition genomics: ChIP chip,
Transcriptional control during array CGH, DNA methylation and gene O. Pertz
hibernation; tales of HIF and NRF expression using tiling arrays The Scripps Research Institute
La Jolla, USA
O. Mhlemann F. Palladino Spatio-temporal analysis of RhoA
University of Berne Ecole Normale Suprieure de Lyon activity in single living cells using a
Berne, Switzerland Lyon, France FRET-based probe
Nonsense-mediated transcriptional Unique and redundant functions
silencing of immunoglobulin mu genes, of C. elegans HP1 proteins in M. Peter
a novel quality control mechanism post-embryonic development ETH
of gene expression Zurich, Switzerland
D. Panne Function and regulation of cullin-
A. Mueller Harvard Medical School based ubiquitin-ligases
University of Arizona Boston, USA
Tuscon, Arizona Structure of the interferon-beta T. Petrova
Functional genomics of chromatin enhanceosome University of Helsinki
and gene regulation in Arabidopsis Helsinki, Finland
thaliana an RNAi approach A. Parcellier Transcriptional regulation of
INSERM U517 lymphangiogenesis
A. Mller Dijon, France
University of Wrzburg HSP27 and regulation of proteasome B. Pfander
Wrzburg, Germany function Max Planck Institute of Biochemistry
Neural and hematopoietic stem cell Martinsried, Germany
activities are increased by epigenetic M. Park Control of replicative bypass of
modifications McGill University DNA lesions through ubiquitin and
Montreal, Canada SUMO-modification of PCNA
H. Mller CrkI and CrkII function as key
DKBW, University of Basel signaling integrators for migration S. Pfeffer
Basel, Switzerland and invasion of cancer cells The Rockefeller University
Prevention of colorectal cancer New York, USA
through genetics Virus-encoded microRNAs

FMI Report 2005/2006 93

Lectures and seminars

K. Plath G. Reuter L. Schmitz

Whitehead Institute University of Halle University of Berne
Cambridge, USA Halle, Germany Berne, Switzerland
Tails of histone methylation The control of gene silencing in HIPK2, a kinase with many talents
X-inactivation and polycomb group Drosophila
proteins H.R. Schler
D. Rhodes Max Planck Institute
J. Pleiss MRC Laboratory of Molecular Biology Mnster, Germany
ITB Cambridge, UK Pluripotency in mouse preimplantation
Stuttgart, Germany The structure of the 30-nm chromatin embryos
Bioinfo@FMI: from sequence to function: fibre
in silico methods for optimising G. Schratt
biocatalysts E. Rial-Verde Harvard Medical School
Cold Spring Harbor Laboratory Boston, USA
I. Potrykus Cold Spring Harbor, USA A role for microRNAs in the regulation
ETH The role of the immediate-early gene of dendritic protein synthesis
Zurich, Switzerland Arc in synaptic transmission and synapse structure in neurons
From basic research toward a deregulated
product not a concept for the public L. Ringrose M.J. Schroeder
domain! ZMBH, University of Heidelberg University of Virginia
Heidelberg, Germany Charlottesville, USA
T. Preiss Negotiating silence: epigenetic dialogue Differential analysis of phosphopeptides
Victor Chang Cardiac Research Institute at polycomb/trithorax response elements and characterization by electron
Sydney, Australia transfer dissociation
MicroRNAs control translation initiation P. Robinson
by inhibiting eIF4E/cap and poly(A) MRC W. Schultz
tail function Cambridge, UK University of Cambridge
Structural and biochemical analysis Cambridge, UK
S.B. Prusiner of reconstituted 30-nm chromatin fibres Management of uncertainty by reward
University of California neurons
San Francisco, USA P. Sah
Synthetic prions University of Queensland J. Schwaller
Brisbane, Australia University of Basel
A. Quatrone Interneuronal networks in the basolateral Basel, Switzerland
University of Florence amygdala Understanding critical genetic
Florence, Italy alterations leading to acute leukemia
A systems perspective on sequence- R. Santoro
dependent translational control: German Cancer Research N. Seeds
from neural plasticity to colorectal Heidelberg, Germany University of Colorado
cancer progression Silencing of ribosomal gene transcription: Denver, USA
epigenetic interplay of chromatin Induction of plasminogen activator
M. Radman remodeling, histone modifications and concomitant with synaptic plasticity in
Universit Paris DNA methylation cerebellum and spinal cord
Paris, France
The fidelity of DNA replication and P. Sassone-Corsi H. Seitz
recombination processes IGBMC University Paul Sabatier
Strasbourg, France Toulouse, France
F. Radtke Unique chromatin remodeling and gene Imprinted small RNA (microRNA) genes
Ludwig Institute expression in male germ cells
Lausanne, Switzerland W. Senn
Notch: oncogene, tumor suppressor F. Savarese University of Berne
and progenitor gate keeper Research Institute of Molecular Pathology Berne, Switzerland
Vienna, Austria The cortical representation of time:
F. Radvanyi Dynamic X-inactivation during B-cell climbing activity, synaptic plasticity
Institute Curie development and Weber's law
Paris, France
Exploring tumour transcriptome: F. Schatzmann H.-S. Shin
identification of genes involved in University of Manchester Korea Institutes of Science & Technology
tumour progression Manchester, UK Seoul, Korea
A key role for focal adhesion kinase in Effect of altered Ca 2+ regulation on
M. Rape mammary epithelia brain functions
Harvard Medical School
Boston, USA M. Schmidt P. Singh
At the core of the cycle: regulation Harvard Medical School Research Center Borstel
of the anaphase-promoting complex Boston, USA Borstel, Germany
in mitosis and G1 The proteasome activator Blm10 links HP1 and epigenetic inheritance
proteasome function to metabolic
U. Rapp signaling pathways R. Skoda
University of Wrzburg University of Basel
Wrzburg, Germany S. Schmidt Basel, Switzerland
Role of Raf kinases in normal- and Harvard Medical School An activating Jak2 mutation
cancer-cell signaling Boston, USA in myeloproliferative disorders
Structure-function relationships:
homology is not destiny

94 FMI Report 2005/2006

J. Skok R. Terranova
University College London Lymphocyte Development Group, MRC
London, UK London, UK
Regulation of immunoglobulin gene Switching programs: from lymphocyte
recombination: position matters to muscle
N. Sonenberg N. Thom
McGill University Memorial Sloan Kettering Cancer Center
Montreal, Canada New York, USA
Translational control: links to cancer Damage patrol: structural and
and learning and memory mechanistic studies of DNA damage
detection and repair
M. Stalder
Massachusetts Institute of Technology A. Tomilin
Cambridge, USA Max Planck Institute of Immunobiology
Prediction of regulatory sequences Freiburg, Germany
for pre-mRNA splicing Pluripotent cells derived from mouse
trophoblast
P. Stein
University of Pennsylvania N. Toni
Philadelphia, USA The Salk Institute
RNAi in the oocyte: a powerful La Jolla, USA
tool to study gene function in oocyte Synapse formation on new neurons
maturation and fertilization in the adult hippocampus
R. Sternglanz M.E. Torres Padilla
Stony Brook University University of Cambridge
New York, USA Cambridge, UK
Transcriptional silencing in yeast Transcription in the life of an embryo:
how does the mouse activate its genome?
J. Stetefeld
Biozentrum L. Trotman
Basel, Switzerland Sloan Kettering Cancer Center
The extracellular matrix: model New York, USA
systems to study their structure-function Emerging principles of tumor suppression
relationships learning from PTEN deficiency
O. Stork D. van Aalten
University of Magdeburg University of Dundee
Magdeburg, Germany Dundee, UK
Neuronal functions of the novel Towards a structural understanding
serine/threonine kinase NDR2 of the heterotrimeric LKB1 tumor
suppressor complex
H. Stunnenberg
University of Nijmegen G. Van der Goot
Nijmegen, The Netherlands University of Geneva
CHIP-chip: global deconvolution of Geneva, Switzerland
transcriptional pathways Making your way into mammalian
cells: the route of the anthrax toxin
E. Sum
Walter and Eliza Hall Institute of Medical B. Vanhaesebroeck
Research Ludwig Institute for Cancer Research
Parkville, Australia London, UK
The role of the LIM domain protein Signaling by PI3-kinase isoforms
LMO4 in breast tumorigenesis
and mammary development F. Vasguez
University of Lille
K. Svoboda Lille, France
Cold Spring Harbor Laboratory A third class of small RNAs in plants
Cold Spring Harbor, USA
Imaging synaptic plasticity in the A. Vichalkovski
neocortex in vivo University of Berne
Berne, Switzerland
J. Tazi Interaction of G-protein coupled
CNRS receptors with tyrosine kinases during
Montpellier, France growth and differentiation of human
Control of foetal growth and neonatal hematopoietic progenitor cells
survival by the RasGAP associated
endoribonuclease G3BP D. Vivien
University of Caen Lower-Normandy
Caen, France
Is tissue type plasminogen activator
(tPA) a neuromodulator?
S. Volarevic
University of Rijeka
Rijeka, Croatia
The response of developing and mature
cells to a defect in ribosome biogenesis
D.G. Wansink
Nijmegen Center for Molecular
Life Sciences
Nijmegen, The Netherlands
Myotic dystrophy protein kinase isoforms:
role of alternative splicing in protein
structure, enzymatic activity, and
ER-mitochondrial membrane anchoring
H. Wohlgemuth
LI-COR Biosciences GmbH
Bad Homburg, Germany
TILLING high throughput functional
genomics: screening for genetic variation
J. Woodgett
Ontario Cancer Institute
Toronto, Canada
Genetic analysis of GSK-3: a key
regulator of cellular fate
J. Workman
Stowers Institute for Medical Research
Kansas City, USA
Protein complexes that modify chromatin
J. Yang
Whitehead Institute for Biomedical
Research
Cambridge, USA
Molecular dissection of multi-step
tumor metastasis
W. Yang
National Institutes of Health
Bethesda, USA
Three ATPases in DNA mismatch
repair: fidelity check, matchmaking
and strand separation
S. Yokobayashi
University of Tokyo
Tokyo, Japan
The kinetochore protein Moa1
enables cohesion-mediated monopolar
attachment at meiosis I
M. Zavolan
Biozentrum
Basel, Switzerland
The bioinformatics of non-coding RNAs
Y. Zhou
German Cancer Research Center
Heidelberg, Germany
Transcription regulation of ribosomal
genes on chromatin
A. Zuniga
DKBW, University of Basel
Basel, Switzerland
Epithelial-mesenchymal interactions
during vertebrate organogenesis
FMI Report 2005/2006 95
 Teaching
FMI laboratories are home to approximately
90 students. These are PhD or MSc students
registered at local universities and carrying
out their dissertation studies under the super-
vision of FMI group leaders, or they are stu-
dents visiting from abroad, for example under
the IAESTE scheme. In addition, FMI group
leaders were involved in lecture and labo-
ratory courses presented at universities in
Switzerland and other countries. Unless indi-
cated otherwise, the courses were delivered at
the University of Basel:
96 FMI Report 2005/2006
Professor
Silvia Arber
Neurobiology foundation course
Developmental neuroscience
Cell biology foundation course
Genes, brain and behaviour
Neurobiology
Ruth Chiquet-Ehrismann
Extracellular matrix and cell adhesion
in development and disease
New literature in cell-cell and
cell-extracellular matrix adhesion
Seminars in growth control
Witold Filipowicz
Seminars on recent literature in
epigenetics
Advanced seminars in epigenetics
Function, structure and processing
of RNA
Biogenesis and function of miRNAs
(University of Bern)
Susan Gasser
Advanced seminars in epigenetics
Masters in molecular biology
Function and analysis of post-
translational protein modifications
Integrated biological systems
Molecular biology of yeast and
filamentous fungi
Progress on supramolecular cellular
assemblies
Nancy Hynes
Experimental cancer research
Experimental cancer research II
Cell and molecular biology of cancer
Andreas Lthi
Signalling in the nervous system
Genes, brain and behaviour
Neurobiology foundation course
Neurophysiology
The nervous system
Seminars on new literature in
neurobiology
Postgraduate course in synaptic
plasticity (University of Zurich)
Neuroscience (NCBS, Bangalore, India)
Patrick Matthias
Advanced immunology
Transcription regulation and gene
expression in eukaryotes
Andrew Matus
Graduate course on neurobiology:
Cortical maps
Graduate course on neurobiology:
Consciousness
Evolution of brain and behaviour
Cell Biology of the neuronal
cytoskeleton (University of Stockholm)
Frederick Meins Jr
Block course in plant biology
Research seminar in plant cell and
molecular biology
Research seminar on epigenetics
and post-transcriptional
Current literature in epigenetics
Lecture course in gene silencing
Plant developmental biology
Advanced seminar in epigenetics
Spectrum in plant sciences
(Zurich-Basel Plant Science Centre)
Intensive course on epigenetic
regulation in plants
(Zurich-Basel Plant Science Centre)
Denis Monard
New literature in neurobiology
Basel neurobiology lectures
Faculty member responsible for
FMI PhD students
Member MD-PhD commission,
University of Basel
Privatdozent
Pico Caroni
Systems neuroscience
Signalling in neuroscience
Developmental neuroscience
Jan Hofsteenge
Protein modifications
Non-faculty
Joy Alcedo
Developmental neuroscience
Helge Grosshans
Seminars on recent literature
in epigenetics
Brian Hemmings
Seminars in growth control
SS2005/2006
Seminars in growth control
WS2005/2006
Thomas Oertner
Signalling in the nervous system
(Woods Hole, USA)
Cell biology und neurobiology
Antoine Peters
Protein modifications
Advanced seminars in epigenetics
Seminars on recent literature
in epigenetics
Dirk Schbeler
Current literature in epigenetics
Protein modifications
Gene silencing
Technology platforms The FMI
maintains a range of central
technical services that are
continually updated to include
the latest developments.

The broad-based approach at the FMI to the regu-

lation of the human genome and proteome, or-
ganised loosely into the areas of growth control,
epigenetics and neurobiology, has always been fer-
tile ground for comparative studies at different
structural levels, in different model systems, using
multiple techniques. What has now crystallised in
many institutes into "systems biology" is dependent
upon high-throughput and sensitive techniques to
both quantify changes in the genome and proteome
and to integrate and analyse large amounts of re-
sulting data in order to model the function of par-
ticular biological systems. Equally important is the
availability of dedicated, highly trained technical
staff.
The FMI maintains a wide range of central ser-
vices that are continually updated to include the
latest developments. The technology platforms
available at the FMI include analytical and prepar-
ative fluorescence activated cell sorting (FACS),
functional genomics and proteomics (gene se-
quence analysis and DNA microarray technology),
mass spectrographic protein analysis, tissue prepa-
ration and histology, monoclonal antibody pro-
duction and screening, peptide synthesis, confocal
and time-lapse microscopy, and quantitative fluo-
rescence image analysis.
An extensive animal facility produces and main-
tains new strains of transgenic mouse lines and of-
fers stem cell technology. Yeast, Drosophila and
Caenorhabditis elegans genetics are also well sup-
ported. Our information technology group main-
tains high speed networked computer services as
well as a dedicated facility that includes computer-
based imaging and printing. An extensive and ex-
panding online library provides rapid access to the
latest scientific literature.

Several of these platforms are featured below.

FMI Report 2005/2006 97

 HISTOLOGY
Sandrine Bichet

Histology and related techniques are used more and

more to complement molecular and biochemical
studies. Transgenic mouse models also involve phe-
notype analysis at the tissue level. The Histology
unit is equipped with Leica rotary microtomes for
paraffin sectioning, an automated tissue processing
unit, paraffin embedding unit, automated haema-
toxylin and eosin stainer, a microwave processing
labstation for antigen retrieval and an MMI laser
dissection microscope for the isolation of tissue for
genomic transcriptome analysis. Working closely
with the microscopy and imaging core facility and
using multiple deconvolution microscopes, con-
focal microscopy and automated plate readers, the
team provides immunohistochemistry, imaging
and morphometry expertise and assists in setting
up and optimising new immunohistochemical,
immunofluorescence and in situ hybridisation
approaches.

MONOCLONAL ANTIBODIES
Susanne Schenk Ernst

The production of monoclonal antibody molecules

(MABs) by hybridoma cells has been of immense
benefit to cutting-edge biomedical research. How-
ever, their generation is complex and time-con-
suming and interest in the technology stagnated for
several years. More recent appreciation of their di-
agnostic and therapeutic potential has revived ac-
tivity in the field. As they bind to only one defined
epitope of an antigen, MABs are excellent tools for
identifying particular antigen structures/regions or
distinguishing different forms of an antigen. Bar-
ring genetic alterations,the capacity of a hybridoma
to grow and secrete MABs is unlimited.
The Monoclonal Antibodies service assists in
the generation of MAB-producing B cell hybrido-
mas by immunization of mice, fusion of activated
B-lymphocytes with a myeloma cell line and ELISA
screening, subcloning of selected hybridoma cells
to stabilise the cell line, and subsequent expansion
and freezing of selected positive clones. The team
also advises in the preparation of antigens for
immunisation and in the development of assays
for re-evaluation of the ELISA-positive hybridoma
supernatants.

98 FMI Report 2005/2006

CELL SORTING (FACS)
Hubertus Kohler
Flow cytometry is an extremely flexible method for
characterising cells in suspension. Given a choice
of fluorochromes, fluorescence activated cell sort-
ing (FACS) allows selection of cells based on e.g.
surface, cytoplasmic and nuclear antigens, GFP ex-
pression, DNA content, cell cycle phase, or apop-
tosis characters. The method can also measure in-
tracellular cytokines and the movement of Ca2+
ions, as well as discriminate alive and dead cells.
The Cell Sorting service operates an analytical
FACSCalibur equipped with a primary laser al-
lowing two light-scatter parameters and three
fluorescence channels to be measured and a
second laser for the excitation of dyes such as
APC or Cy5. A second MoFlo instrument is a high-
speed 4-way cell sorter with a 3-laser set-up, pro-
viding sophisticated sorting based on multiple
fluorescent dyes.
PROTEIN ANALYSIS
Daniel Hess
The Protein Analysis facility provides structural de-
tails of proteins exploiting the latest developments
in the fields of protein separation, quantification
and mass spectrometry. Expertise is provided in the
preparation of samples for analysis (protein pre-
cipitation, labelling, etc.), in the separation of com-
plex samples (2D gel electrophoresis and chroma-
tography), in the quantification of protein expres-
sion, and in high-sensitivity identification of pro-
teins and post-translational modifications by mass
spectrometry.
This approach profits from the exponential
growth of sequence databases with many fully se-
quenced genomes. New developments in mass
spectrometry and separation have led to large in-
creases in sensitivity and speed of analysis. In the
FMI, 2 D gel electrophoresis using two different
units for isoelectric focusing (Multiphor II and
IPGphor) and two SDS PAGE systems (Ettan DALT
12 and Protean II xi Cell) is coupled to appropriate
scanners and software for image analysis. High-
sensitivity identification of proteins is carried out
with three mass spectrometers: a TofSpec 2E for
rapid identification of single proteins by peptide
mass fingerprinting, an LCQ Deca XP used in com-
bination with capillary HPLC (fmol range), and an
API300 combined with HPLC and equipped with
a nanospray source.
MICROSCOPY AND IMAGING CORE FACILITY
Jens Rietdorf/Patrick Schwarb
Quantitative fluorescence microscopy is increas-
ingly valuable in modern biomedical research. New
imaging tools such as 2-Photon, FLIM, FRET and
live confocal imaging combined with powerful anal-
ysis software go up to and beyond the limits of op-
tical resolution. The Microscopy and Imaging Core
Facility provides support at all steps of data prepa-
ration, imaging, analysis and visualization, as well
as training in image acquisition and processing.
Nine high-end imaging systems are used for
investigating and quantifying sample volumes,
shapes and densities as well as the locations, inter-
actions and dynamics of molecules, organelles and
cells. Know-how and equipment are available for
multi-dimensional fluorescence microscopy, vol-
ume time-lapse, confocal microscopy and spectral
detection, multi-beam real-time confocal micros-
copy, fluorescence recovery after photobleaching,
fluorescence resonance energy transfer, photo-
uncaging, photoactivation, spectral unmixing of
fluorescent labels, deconvolution, and environ-
mental control for live specimen imaging. Total
internal reflection microscopy and fluorescence
correlation spectroscopy are offered in cooperation
with almf@embl-heidelberg.
As an image without annotation is of low long-
term value, an image database has been developed
to allow the storage of experimental information
and microscope settings along with the raw image.
Both commercial packages in the form of plug-ins
and completely new, FMI-developed tools are used
to analyse image data.
BIOINFORMATICS SUPPORT
Michael Rebhan
The number of bioinformatics approaches,tools and
databases essential for current biological research
increases almost daily. To exploit these tools ef-
ficiently, Bioinformatics Support provides a help-
desk and training. The focus is on function predic-
tion, in-depth analysis of selected genes, transcripts
and proteins, genomic data mining and understan-
ding sequence-structure-function relationships. A
comprehensive environment for computational
biology analysis is being established in collabora-
tion with the Biozentrum, University of Basel and
the Swiss Institute of Bioinformatics. The establish-
ment of a protein structure facility will expand our
capabilities by the end of 2006.
FMI Report 2005/2006 99
 Students and postdocs Our students and
postdoctoral fellows are exposed not only to
the latest molecular approaches, but are also
immersed in a culture that takes delight in
relating knowledge to biomedical application.

OUR GOALS INTERNATIONAL PhD PROGRAMME

In 2005, the FMI celebrated 35 years as an inter-
nationally recognised centre for fundamental bio-
medical research. Why do scientists continue to
explore the basic molecular mechanisms that lead
to healthy growth or to dysfunctional degeneration?
Despite our ever-expanding understanding of cells
and their genetic information, we simply do not
know enough about the molecular mechanisms of
human disease to be able to design treatments for
those who suffer. Thus, enhancing our grasp on the
fundamental mechanisms at work within living
cells is a goal of vital interest both to society at large
and to the pharmaceutical industry. Now more
than ever, it is clear that basic biological research
efforts can and will have a major impact on the
quality of our lives.
RESEARCH AT THE FMI
The FMI is situated at the interface of academic
research and pharmaceutical application and pro-
vides an open, collegial environment that encour-
ages scientists to explore new research areas with
daring ideas. Our research focuses on cell growth
control, epigenetics and neurobiology. By under-
standing the control of cell division, the patterning
of gene expression during cell differentiation, or the
formation and maintenance of neuronal circuitry,
we hope to forge new means to combat cancer, to
correct degenerative states, and/or to suppress dis-
ease correlated with neuronal dysfunction. With an
array of cutting-edge techniques, including genetic
approaches in model organisms, as well as detailed
proteomic and genomic analyses and live imaging,
FMI scientists hope that their discoveries will help
explain human disease and thereby contribute to
novel treatments.
The FMI is also dedicated to the training of young
scientists and fosters a lively educational atmos-
phere, having trained hundreds of young scientists
at the postgraduate and postdoctoral levels. We
offer dedicated programmes for MSc and PhD
students as well as research experience to visiting
students from abroad. The FMI is affiliated with the
University of Basel, where we contribute to the
teaching programme. Nine FMI group leaders are
professors at the University and a further nine are
involved in diverse teaching activities. As an im-
portant component of our institute we currently
count about 100 students and 70 postdocs from
about 30 different countries pursuing graduate or
postgraduate training. For details of the Interna-
tional PhD Programme, consult www.fmi.ch/grad-
uate studies.
FINANCIAL SUPPORT
Students are selected competitively from countries
the world over. The FMI provides financial support
to graduate students in accordance with the scale
of the Swiss National Science Foundation. The in-
come is generous relative to international standards
for PhD students.

100 FMI Report 2005/2006

FMI Report 2005/2006 101
102 FMI Report 2005/2006
STUDENT ACTIVITIES POSTDOCTORAL FELLOWSHIPS
The students at the FMI are involved pro-actively A recent poll by The Scientist magazine placed the
in science education, career development and, of FMI as the best place to work as a postdoc out-
course, social life. These activities are coordinated side of the USA. This result reflects not only the
by the student representatives. quality of the research programmes and the com-
A growing feature of FMI student activities is the mitment to maintaining state-of-the-art technol-
series of Students Science Colloquia. Nobel Lau- ogy platforms but also the appropriate remunera-
reates Tim Hunt and Avram Hershko kick-started tion of high quality candidates. Furthermore, the
the latest series, followed by Mario Cappechi with FMI is located in the city of Basel, an international
a stimulating lecture on gene targeting in the 21st centre for research in biology, with major academic
century. Local hero Walter Gehring impressed with institutions and the research departments of leading
a colourful lecture on the evolution of the eye and life science companies. The city is also rich in culture
photoreception, and Kim Nasmyth journeyed into and is an excellent starting point to explore Switzer-
the structural aspects of chromatin segregation, land and neighbouring France and Germany.
while Semir Zeki captivated our young scientists The FMI postdoc community elects four repre-
with the unconventional idea of looking at the sentatives from the different research areas, who
beautiful products of the brain, e.g. visual art, in organise vocational training opportunities, such as
order to understand its function. The list of speak- workshops on scientific writing, presentation skills
ers also included Adriano Aguzzi, Tom Cech, An- and grant writing, and also liase with the FMI di-
thony Grace, Michael Grunstein, Bruce Lahn, Polly rector, Prof. Susan Gasser.The average duration of
Matzinger, Erwin Neher, Miroslav Radman, Ueli a postdoctoral term at the FMI is 3-4 years.
Schibler, Rene Schroeder and David Tollervey. A
lively public debate on human embryonic stem cell For further details of postdoc life at the FMI, consult
research featured Martin Raff and Hans-Werner www.fmi.ch/postdoc
Denker and, looking to the future of our students,
Jack Wood from the IMD Business School, Lau-
sanne analysed The nature of leadership.
A further student initiative in May 2005 was the
Career Guidance Conference in Life Sciences
(www.cgc2005.com) under the motto of aspire, ad-
vance, achieve. This was organised by FMI students,
together with students from the Biozentrum and
the Department of Clinical and Biological Sciences
(DKBW), University of Basel. A full house of young
scientists from Switzerland, Germany and France
heard an impressive array of speakers ranging
from the Head of Biomedical Research at Novartis
and the Managing Director of Nature Publishing
Group to a patent attorney and a managing part-
ner of McKinsey.

A complete overview of FMI student activities can be

found at www.fmi.ch/student.

Applications
Please consult www.fmi.ch for full details of the
graduate studies programme with application forms
and deadlines and of openings for postdoctoral
fellows.

FMI Report 2005/2006 103

 Visiting the FMI
The FMI is located close to the centre of Basel on the north bank of the river Rhine, just
a few minutes from the borders of France and Germany. It is easily accessible by public
transport from the two main railway stations and the airport.

Bad
. Ba
sse
rstra FMI

hnh
lbee
Mau Maulbeerstrasse 66

Sch

of
Building 1066

wa
rzw
ald
alle
Ma

e
tte
nst
ras
se

se
as
l str
nta
Car Entrance se
Ro
Gate 1047
sse
Mes

tra
ns
se B

e
R ieh
asel

Gewerbeschule

From the Swiss /French railway station SBB / SNCF
Trams stop in front of the station. Take tram #1
or #2 from platform #3 (direction Bad.Bahnhof).
Trams leave every 5 minutes. Get off at the stop
Gewerbeschule at Mattenstrasse. Cross the tram
tracks and follow Mattenstrasse for about 200 m,
then turn right into Maulbeerstrasse. You will find
the FMI about 150 m along Maulbeerstrasse on
the right. Journey time is 10 minutes.
200 m, then turn right into Maulbeerstrasse.
From the Swiss /German railway station
Basel Bad. Bahnhof
The FMI is a short distance from the front en-
trance of this railway station. Cross the main road
Schwarzwaldallee using the subway. Turn right,
and 150 m on your left turn into Maulbeerstrasse.
The FMI is about 100 m down the street on the
left. Journey time is 3 minutes.
FMI telephone number for enquiries:
+ 41 61 697 66 51
Street map
www.geo-bs.ch/stadtplan_stadtplan_karte.cfm
From the airport
(EuroAirport Basel / Mulhouse/ Freiburg)
Leave the baggage claim hall by the Swiss exit!
Bus: Take bus #30 to the Swiss/French railway
station SBB/SNCF. Buses leave every 15 minutes
(more frequent at peak times). Trams stop in
front of the railway station. Take tram #1 or #2
from platform #3 (direction Bad. Bahnhof).
Trams leave every 5 minutes. Get off at the stop
Gewerbeschule at Mattenstrasse. Cross the
tram tracks and follow Mattenstrasse for about
You will find the FMI about 150 m along
Maulbeerstrasse on the right. Total journey time
is 35 minutes.
Taxi:The FMI is a short ride from the airport by
taxi. Journey time about 15 minutes.
By car
There is no parking space at the FMI entrance
area on Maulbeerstrasse. Parking is possible on
the adjacent Syngenta site. Enter the Syngenta
site at gate 1047 on Mattenstrasse (see map).
Register at the gate office. You will receive an
ID badge and be allocated a parking space. Park
the car and walk to the FMI (building 1066).

104 FMI Report 2005/2006