Drug Discovery in Cancer Epigenetics

USA

Preface

Gerda Egger and Paola Arimondo

The causal involvement of epigenetic pathways in tumor development and progression has been widely accepted and recent findings have nourished our knowledge of tumor biology and opened potential clinical applications dependent on epigenetic aberrations. Deregulation and mutation of epigenetic enzymes as well as global and local changes of epigenetic chromatin modifications are implicated in a variety of malignancies and provide novel therapeutic and diagnostic targets for oncology. There is great interest in developing novel epigenetic drugs targeting chromatin modifiers as well as chromatin reader proteins and some successful recent studies have confirmed the validity of these drugs for cancer therapy.

We were fortunate to gather many experts and renowned authors to contribute to this book and are very thankful for their efforts and valuable contributions. This book intends to provide (i) an introduction into cancer epigenetics and to give a comprehensive overview on, (ii) methods and tools for epigenetic drug development, (iii) classes of epigenetic drugs, (iv) development of diagnostic tools, and (v) clinical implications of epigenetic therapy. Emerging concepts, such as episensitization, are also presented alongside with indications beyond cancer.

The intended audience includes both basic scientists as well as clinicians. The book is directed toward scientists of the academic and industrial sector, who are aiming to test and develop epigenetic cancer drugs. Importantly, the book will increase the awareness level of epigenetic drugs for oncologists. We envision that this might foster increased and more rapid translation of epigenetic drugs into the clinics.

Part I

Introduction

Outline

Chapter 1 Basic Epigenetic Mechanisms and Phenomena

Chapter 2 Cancer Epigenetics

Chapter 1

Basic Epigenetic Mechanisms and Phenomena

Melanie R. Hassler¹, Elisa Redl¹, Quanah J. Hudson², Wolfgang J. Miller³ and Gerda Egger¹,    ¹Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria,    ²CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria,    ³Laboratories of Genome Dynamics, Department of Cell and Developmental Biology, Center of Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria

Abstract

The term epigenetics literally defines mechanisms that act above genetics and was originally coined to describe processes that are essential to guide development from the fertilized oozyte to the mature offspring. Today, epigenetics is understood as mitotically/meiotically inherited information that is not dependent on DNA sequence. Epigenetic mechanisms are initiated in response to a factor or environmental signal and are stable even in the absence of the initial signal. Within the last two decades much progress has been made in the field owing to the discovery of proteins that are involved in setting and interpreting epigenetic marks. We can now appreciate epigenetic mechanisms that act on different layers of chromatin and their impact on development, differentiation, homeostasis, and also their causal involvement in a variety of diseases including cancer.

This chapter will provide an introduction to basic epigenetic mechanisms and phenomena and discuss the role of epigenetics for different disease.

Keywords

DNA methylation; histone modification; chromatin remodeling; noncoding RNA; X chromosome inactivation; imprinting; position effect variegation; transgenerational epigenetic inheritance

Chapter Outline

1.1 Introduction 4

1.2 Basic Epigenetic Mechanisms 6

1.2.1 DNA Methylation 7

1.2.2 DNA Demethylation 8

1.2.3 Histone Modifications 8

1.2.3.1 Histone Acetylation and Deacetylation 8

1.2.3.2 Histone Phosphorylation 10

1.2.3.3 Histone Methylation and Demethylation 10

1.2.3.4 Chromatin-Remodeling Complexes and Histone Variants 11

1.2.4 Noncoding RNAs 11

1.3 Epigenetic (Re)Programming 12

1.3.1 Epigenetic Asymmetry in the Zygote 12

1.3.2 Reprogramming in the Germline 14

1.3.3 Induced Pluripotency 14

1.4 Genomic Imprinting as a Model of Epigenetic Silencing 15

1.5 Dosage Compensation in Mammals 17

1.6 PEV in Drosophila 19

1.7 Transgenerational and Intergenerational Epigenetic Inheritance 20

1.8 Epigenetics and Disease 23

1.8.1 Selected Monogenetic Diseases 23

1.8.2 Selected Neurodegenerative Diseases 26

1.8.3 Selected Autoimmune Diseases 27

References 28

1.1 Introduction

The history of epigenetics dates back to the early Greek philosopher Aristotle, who considered development as a process that provides form from unformed material within male and female germ cells and involves a dynamic course based on intrinsic factors [1]. During the last few centuries vivid debates were fueled by rather extreme scientific views as to the development of an organism. Preformation as a predictable predetermined and stable process was opposed to epigenesis, which involves morphogenesis and differentiation based on regulatory response to the environment, cellular communication, and dynamic processes [2]. The term epigenetics was coined by Conrad Hal Waddington to describe causal mechanisms by which the genes of a genotype bring about phenotypic effects during development [3]. He introduced the concept of epigenotype to indicate the complex processes and networks of genetic control linking the genotype and the phenotype [4]. Thus, disruption of genes at early stages of development could affect this epigenotype and have far-reaching consequences on different organs and tissues. He modified this name from Valentin Haecker, who created the term phenogenetics to describe visible stages of ontogeny [5]. Being both an embryologist and geneticist he felt there was a need to combine genetics and experimental embryology and the expression epigenetics for him was in accordance with the classical concept of epigenesis. In the years and decades to follow, the term epigenetics was used to describe a variety of phenomena and mechanisms [6] and up to now no clear definition as to the use of the term has been adopted. The persistence of epigenetic systems to implement different phenotypes and a potential heredity or cellular inheritance was tied to epigenetics by Nanney and Ephrussi, respectively [7,8]. With his new concept of carcinogenesis, Holliday [9] proposed that malignant transformation not only results from genetic mutations but also from epigenetic changes in DNA methylation resulting in heritable alterations in gene expression. This put DNA methylation at center stage as an epigenetic mechanism. The discovery of the pivotal role of DNA methylation for imprinting and X chromosome inactivation (XCI), plus the discovery of histone-modifying enzymes provided a revival of the term epigenetic in the 1990s [6,10–12]. Histone modifications were suggested to provide an epigenetic code allowing for both transient regulation of gene expression and long-term epigenetic memory [13,14]. Among epigenetic mechanisms, RNAi was shown to induce not only posttranscriptional gene silencing but was also found to confer chromatin alterations including DNA methylation and histone modifications in plants and in yeast [15]. Another noncoding RNA component, long noncoding RNA (lncRNA), was shown to bridge long-range chromatin silencing and chromatin modification by recruitment of histone-modifying complexes both at the inactive X (Xi)-chromosome or gene-specific loci to promote stable chromatin states [16,17]. Thus, epigenetics, which is in fact a transdisciplinary field, has moved from observing phenomena to elucidating its underlying mechanisms, which include DNA methylation, chromatin modification/remodeling, and noncoding RNA. All three are interrelated and cooperate to maintain the stable epigenome of a given cell lineage in response to internal or external stimuli (Figure 1.1).

Figure 1.1 Epigenetic mechanisms and their role in different processes. Epigenetic mechanisms include DNA methylation, histone modification, and remodeling, as well as noncoding RNA. All three are interrelated and are involved in different phenomena and developmental processes, and also in a variety of diseases. Changes in epigenomic signatures can be due to genetic alterations in epigenetic pathway proteins and to exposure to different environmental stimuli.

A generally well-accepted definition of modern epigenetics was coined by Arthur D. Riggs describing epigenetics as The study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence [18]. Currently, the term epigenetics is en vogue and used in very different scientific communities, including ethics and social sciences or religious studies and the debate on nature versus nurture has been newly sparked by discussions of whether environmental factors can alter the epigenome and be transmitted to subsequent generations. Such discussions and studies are usually based on unresolved questions and often lack biological evidence. Clearly, epigenetic mechanisms play a central role for development, cellular differentiation, and homeostasis and their deregulation is associated with diverse disease states including cancer. As discussed in more detail in Chapter 2, epigenetic alterations in cancer can be the result of mutations in genes involved in epigenetic regulation and very often occur as a consequence of exposure to harmful environmental factors.

One prime aspect of epigenetic regulation is the reversibility of epigenetic marks that can be modulated by chemical compounds, which makes it an important target for developing new therapeutic drugs, which will be addressed in great detail in the following chapters of this book. Starting in the 1960s, antineoplastic activity of 5-azacytidine (5-azaCR) and 5-aza-2′deoxycytidine (5-azaCdR) was shown against leukemia in mice and the first clinical studies were initiated in 1967 in Europe [19–21]. Initially, these compounds were used as cytotoxic agents as they were incorporated into RNA (5-azaCR) and DNA (5-azaCR, 5-azaCdR) and inhibit DNA synthesis. Jones and Taylor then showed that cytidine analogs induced (trans)differentiation of mouse embryonic and fibroblast cells into myotubes, striated muscle cells, adipocytes, and chondrocytes, and that this was due to inhibition of DNA methylation of newly synthesized DNA [22–24]. These two inhibitors were also the first epigenetic drugs to be approved by the FDA and are currently in clinical use for patients with myelodysplastic syndromes (MDSs), acute myeloid leukemia, and chronic myelomonocytic leukemia [25,26]. At approximately the same time, N-butyrate was identified to alter histone acetylation [27–29], and interestingly, HDAC inhibitors were the second class of epigenetic drugs to be approved by the FDA for the treatment of cutaneous T-cell lymphoma [30]. The list of inhibitors targeting epigenetic machineries is ever-growing and has recently attracted the interest of the pharmaceutical industry to develop programs for epigenetic drug discovery.

This chapter will give an overview on basic epigenetic mechanisms including DNA methylation, histone modification, and remodeling as well as noncoding RNA-based mechanisms. Furthermore, we will address some classic epigenetic phenomena such as dosage compensation, imprinting, and position effect variegation (PEV). Although epigenetic mechanisms were suggested to maintain gene expression patterns persistently through subsequent cell generations it was also suggested that environmental factors could cause dynamic epigenetic alterations and result in diseases. Epidemiological studies have suggested that the environment can cause phenotypic effects and directly impact on epigenetics, and that these changes can be heritable not only during mitotic cell divisions but through subsequent generations. We will discuss some of the current data available analyzing potential inter- and transgenerational epigenetic effects and disease.

1.2 Basic Epigenetic Mechanisms

In eukaryotic cells DNA is not naked but exists as an intimate complex with specialized proteins called histones, which together with DNA comprise chromatin. Nuclear DNA is spooled around nucleosomal units consisting of small histone proteins in order to fit into the nucleus [31]. Chromatin exists in two forms: the less condensed euchromatin that is associated with transcriptionally active regions and the highly condensed, usually transcriptionally inactive heterochromatin. Chromatin can be modified by posttranslational histone modifications, histone variants, energy-dependent chromatin-remodeling steps that mobilize or alter nucleosome structures, and noncoding RNA that can influence chromatin structure. Furthermore, DNA itself can be modified covalently by methylation of cytosines, usually of CpG dinucleotides.

1.2.1 DNA Methylation

DNA methylation is an epigenetic modification that is correlated with gene repression and is known to play an important role in gene regulation, development, and tumorigenesis. It consists of the addition of a methyl group to the carbon at position 5 of cytosine residues in the DNA template. In mammals it usually occurs at CpG dinucleotides but symmetric, asymmetric and non-CpG methylation is also known in embryonic stem cells (ESC), Neurospora crassa, and plants [32–34]. In mammalian somatic tissues approximately 70% of all CpG sites within the DNA are methylated and DNA methylation distribution shows enrichment in noncoding regions and interspersed repetitive elements [35] but not in CpG islands of active genes [36]. CpG islands were first defined in 1987 by Gardiner-Garden and Frommer as 200 bp stretches of DNA with a G+C content of at least 50% and an observed CpG/expected CpG excess of 0.6 [37]. CpG islands are often associated with promoters and usually lack DNA methylation, thus allowing gene expression. Approximately 60% of human genes have CpG island promoters. However, methylation of CpG islands can reinforce silencing of genes, for example, for genes on the Xi chromosome or for some imprinted genes. Additionally, in cancer cells, genes are often aberrantly silenced by CpG island methylation.

DNA methylation is catalyzed by DNA methyltransferases (DNMTs), which can either catalyze de novo methylation (DNMT3A/B) at novel sites or maintenance methylation (DNMT1) following DNA replication [38]. DNMT1 is specific for CpG and its preferred substrate is hemimethylated DNA, DNA methylated at CpG on one strand. Inactivation of Dnmt1 in mouse ESC results in genome-wide loss of CpG methylation, indicating that this DNMT is necessary for stable maintenance of DNA methylation [39]. However, Lei et al. [40] showed that de novo methylation of proviral DNA introduced into ES cells was not catalyzed by DNMT1 but another unknown DNMT. Besides DNMT1, three candidate proteins that could potentially encode additional DNMTs were found. DNMT2 had minimal DNMT activity in vitro and deletion of Dnmt2 did not alter the level of DNA methylation [41]. However, DNMT2 acts as an RNA methyltransferase that methylates tRNAs [42]. In contrast to DNMT1, DNMT3A and DNMT3B did not show preference for hemimethylated DNA in vitro [43] and both genes were necessary for de novo methylation of proviral genomes and repetitive elements in embryos and ESC [44]. In mice, inactivation of Dnmt3a and Dnmt3b results in early embryonic lethality and loss of one gene causes postnatal or embryonic lethality. Furthermore, in humans, mutations of DNMT3B are associated with the ICF syndrome, a rare condition characterized by immunodeficiency, centromeric instability, and facial abnormalities [45].

But how does DNA methylation interfere with gene expression? First, the presence of methyl groups interferes with the binding ability of transcription factors that are crucial for transcriptional activation. Transcription factors often recognize CG-rich motifs and several of these are unable to bind to methylated CpG sequences [46]. Second, proteins that bind to methyl-CpGs can also repress gene expression at methylated sites. These proteins were found by performing band-shift assays using random methylated DNA sequences as probes [47]. MeCP1, a DNA–protein complex that is specific for methylated DNA, was discovered in a variety of mammalian cell types. However, the first individual methyl-CpG-binding protein to be purified and cloned was MeCP2. Upon the presence of a methyl-CpG-binding domain four other members of this family, MBD1, MBD2, MBD3, and MBD4, could be identified [48]. Furthermore, two other structural domains are currently known to bind methylated DNA: the SET and RING finger-associated domain, found in UHRF1 and UHRF2 and zinc fingers, found in Kaiso and Kaiso-like proteins [49]. Methyl-CpG-binding proteins recruit a variety of proteins including histone deacetylases (HDACs) and chromatin-remodeling factors that are responsible for transcriptional repression [50] and can also interact with transcription factors, such as MeCP2 with TFIIB [51].

1.2.2 DNA Demethylation

Studies of cellular reprogramming have demonstrated that differentiated cellular states can be radically altered, suggesting that DNA methylation may be reversible in mammalian cells. Today it is known that not only passive DNA demethylation due to reduction in activity or absence of DNMTs, but also active demethylation catalyzed by special enzymes takes place within a cell. Active DNA demethylation plays an important role in early mammalian development as well as in tissue-specific differentiation but is also observed in adult cells. One of the most prominent processes that involve rapid active DNA demethylation in adult cells is the activity-dependent DNA demethylation of brain-derived neurotrophic factor and fibroblast growth factor 1 promoters in postmitotic neurons [52]. 5-Methylcytosine (5mC) can be hydroxylated to 5-hydroxymethylcytosine (5hmC) by members of the ten–eleven translocation (TET) enzyme family. 5hmC can further be oxidized to 5-formylcytosine or 5-carboxylcytosine. Furthermore, 5mC or 5hmC can be deaminated to 5-methyluracil or 5-hydroxymethyluracil by members of the AID/APOBEC enzyme family. Finally, the intermediates produced by TET and AID/APOBEC are replaced by the uracil DNA glycosylase [53] family of base excision repair glycosylases, that mediate DNA repair [54].

1.2.3 Histone Modifications

The basic chromatin unit, or nucleosome, consists of a protein octamer containing two molecules of each canonical (or core) histone (H2A, H2B, H3, H4), around which 147 bp of DNA are wrapped. The core histones consist of a globular domain and flexible aminoterminal histone tails [55]. These tails, particularly, those of H3 and H4, are accessible for modifications, such as phosphorylation, acetylation, methylation, and ubiquitylation, which can be correlated to both transcriptional activation or repression. In general, active marks include acetylation, arginine methylation, and lysine methylation, such as H3K4 and H3K36, while repressive marks include methylation of H3K9, H3K27, and H4K20. However, the globular domains of core histones can also be modified [56] (Figure 1.2).

Figure 1.2 Histone modification and nucleosome occupancy determine different genomic regions. Dependent on their activity state, genes are marked by characteristic chromatin modifications, nucleosome density, and histone variants, which together reflect the on/off state of a gene (top). Note that only selected histone modification marks are illustrated. NDR indicates nucleosome-depleted regions in enhancers, promoters and 3′ regions of active genes. BLOCs and LOCKs designate large silent gene-rich regions marked by repressive histone marks H3K27me3 and H3K9me2, respectively [57,58]. (Bottom) Chromatin composition including histone modifications, grade of compaction, and histone variants is indicated for intergenic regions and heterochromatin. Inverted arrows on top indicate inverted repeats. These regions show generally high levels of DNA methylation and are associated with repressive histone marks. Red, nucleosomes of active genes; blue, nucleosomes found in repressed genes, intergenic regions or heterochromatin; DRE, distal response element; TSS, transcription start site; E, exon.

But how do histone modifications work in general? First, histone modifications can act in cis by directly altering the chromatin structure by disrupting the contact between adjacent nucleosomes or between histones and DNA, for example, by charge changes [59]. Second, histone modifications can also act in trans and can be recognized by specific binding proteins, so-called reader proteins, which can further recruit chromatin-modifying complexes and modulate chromatin structure [60].

1.2.3.1 Histone Acetylation and Deacetylation

Histone acetylation, catalyzed by histone acetyltransferases (HATs), can directly alter chromatin structure through the loss of positive charges within histones, resulting in reduced interactions of histones with negatively charged DNA and increased accessibility of DNA-binding sites [61]. Reader proteins with bromo-like domains recognize and bind acetylated histones. These proteins can either be basal transcription factors, HATs, such as p300 or GCN5 or part of large chromatin-associating/altering complexes such as the ATP-dependent remodeling complex SWI/SNF, which further alter the chromatin structure [62]. The same principle applies to histone methylation, which is recognized by chromo-like domains, and phosphorylation, which is recognized by 14-3-3 proteins [63,64].

Histone deacetylation is catalyzed by HDACs that remove acetyl groups [65]. HDACs are grouped into four classes according to their homology with yeast proteins: type I, type II, and type IV HDACs are referred to as classical HDACs and are zinc-dependent enzymes, whereas type III or SIR2-related HDACs require the cofactor NAD. HDACs are frequently part of large multisubunit complexes, which target the enzyme to genes. This targeting often requires other histone interacting proteins, for example, the Sin3 corepressor, which is targeted by Rpd3 to H3K36me sites and suppresses transcription in yeast [66,67].

1.2.3.2 Histone Phosphorylation

Histone phosphorylation was the first characterized histone modification since it has long been understood that kinases regulate signal transduction pathways in the cell. In 1991, Mahadevan et al. were able to show that stimulation of proliferation and transcription of the so-called immediate-early genes in cells correlate with histone H3 phosphorylation. Phosphorylation of H3S10 has been shown to be required for chromosome condensation and segregation during mitosis [68]. H3S10 phosphorylation occurs at the onset of mitosis, interferes with HP1-H3K9me3 binding and leads to release of heterochromatin protein 1 (HP1), which might be necessary for full mitotic chromatin condensation [69]. Furthermore, phosphorylation of the histone variant H2AX has been shown to be associated with double-strand DNA breaks, where it helps to recruit repair proteins to the site of the break [70].

1.2.3.3 Histone Methylation and Demethylation

Histone methylation is the most complex histone modification since it can occur on either lysines or arginines and is associated with either transcriptional activation or repression. Furthermore, lysines can be mono- (me1), di- (me2), or tri- (me3) methylated and arginines can be monomethylated or symmetrically or asymmetrically dimethylated.

Enzymes of three distinct families can catalyze lysine methylation of histones: the PRMT1 family, whose substrate is arginine, the SET domain containing protein family, and the non-SET domain proteins DOT1/DOT1L, which methylate lysine residues [71,72].

In general, methylation of H3K4, H3K36, and H3K79 is linked to transcriptional activation, however, depending on the methylation state and the genomic location, the same modification may lead to different effects [59,73]. In contrast, methylation of H3K9, H3K27, H3K64, and H4K20, as well as methylation of the linker histone H1, has been implicated in transcriptional repression [73]. Specific reader proteins bind to the methylation sites and alter gene expression. These proteins contain one of three distinct methyl lysine recognition domains: the so-called chromo, tudor or PHD repeat domains [74]. Furthermore, H3K79me3 and H4K20me are associated with DNA repair [75].

For a long time it was not clear whether histone demethylation takes place in the cell or not. Shi et al. [76] discovered the protein LSD1, an enzyme that removes methyl groups specifically from H3K4. It can associate with different complexes and depending on the complex it can also change its demethylation specificity [77]. Today several families of histone demethylases are known, which act on various substrates and therefore play different roles in gene activation and repression [78].

1.2.3.4 Chromatin-Remodeling Complexes and Histone Variants

Another major mechanism to alter chromatin and nucleosome composition in a noncovalent manner is the recruitment of ATP-dependent chromatin-remodeling complexes. These protein complexes can be generally categorized into two families: the SNF2H or ISWI family, which mobilizes nucleosomes and the Brahma or SWI/SNF family, which alters the structure of nucleosomes [79].

Furthermore, core histones can be exchanged in an ATP-dependent manner by specialized histone variants. Histone variants are associated with specific expression states, genomic localization, and species-distribution patterns. They change the structural and functional properties of nucleosomes by affecting chromatin remodeling and histone modifications. There are two important histone variants of H3: H3.3, which marks transcriptionally active genes [80] and CenpA, which is found in centromeric chromatin and is essential for centromeric function and chromosome segregation [81].

Histone H2A shows three histone variants. The presence of H2A.Z correlates with transcriptional activity, while H2A.X senses DNA damage and is therefore crucial for DNA repair [82]. MacroH2A specifically associates with the Xi chromosomes in mammals and is an epigenetic regulator of key developmental genes.

1.2.4 Noncoding RNAs

Within the last few years different classes of noncoding RNAs have been identified, which can either cause transcriptional or posttranscriptional gene silencing or directly impact on chromatin structure by recruiting chromatin-modifying complexes.

RNA interference was originally discovered in Caenorhabditis elegans, where exogenously introduced double-stranded RNA molecules are able to silence the expression of homologous sequences within the genome [83]. RNAi and RNAi-mediated transcriptional and post-transcriptional silencing is best understood in Schizosaccharomyces pombe, where deletion of one of the genes encoding for Argonaute, Dicer or RNA-dependent RNA polymerase proteins leads to loss of heterochromatic gene silencing, reduced H3K9 methylation at centromeric repeats and accumulation of noncoding RNAs [84–86]. Furthermore, RNAi has also been shown to be required for heterochromatin assembly in Schizosaccharomyces pombe and it was shown that pericentromeric siRNA accumulation requires the H3K9 methyltransferase Clr4 [87]. In higher organisms, for example, Drosophila melanogaster mutations of Argonaute proteins lead to a decrease of H3K9me as well as HP1 binding [88] and mouse cell lines with mutations in Dicer show loss of silencing at centromeres [89]. Furthermore, gene silencing triggered by RNAi can be long term and heritable, as demonstrated in Caenorhabditis elegans, where four chromatin-remodeling factors are necessary for maintaining the silent state of genes [90].

In higher organisms microRNAs (miRNA), a class of small noncoding RNAs, are essential for the regulation of early differentiation programs. For example, HOX genes encode a family of transcriptional regulators important for patterning along body axes. Expression of HOX genes is regulated by different molecular mechanisms including nuclear dynamics, RNA processing, translational regulation, and miRNAs [91]. Most of the miRNAs that regulate HOX gene expression are encoded within the HOX cluster [92]. In mammals there are four HOX gene clusters, which contain five genes encoding miRNA-10 and miRNA-96 [93].

An additional class of noncoding RNAs constitute lncRNAs, which are able to recruit histone-modifying enzymes using RNAi-independent strategies to particular genomic sites [94,95]. The most prominent lncRNA in mammals so far is XIST that mediates XCI and was shown to target the Polycomb repressive complex 2 (PRC2) including the H3K27 methyltransferase EZH2 to chromatin [96,97]. Other lncRNAs that recruit PRC2 and confer silencing include HOTAIR, RepA, and Kcnq1ot1 amongst others [94]. Generally, lncRNAs appear to work as scaffolds to enable complex formation and recruitment to distinct genomic loci. A group of lncRNAs transcribed from enhancers can act as transcriptional activators in cis via DNA looping and recruitment of coactivators. An example for this is the lncRNA HOTTIP, which recruits a H3K4 methyltransferase complex to activate transcription of HOXA homeobox genes [98].

1.3 Epigenetic (Re)Programming

Generating a broad range of different cell types in a highly ordered and reproducible manner is one of the most remarkable hallmarks of complex genomes [99]. In mammals ~25,000 genes contain the information for the development of ~200 different cell types. During development of multicellular organisms different cells and tissues acquire specific gene expression programs, which are mainly regulated by epigenetic modifications such as DNA methylation and histone modification [36,100,101]. These epigenetic mechanisms propagate appropriate patterns of gene expression and can be heritable but potentially reversible [102].

Cells of multicellular organisms can be functionally divided into two major groups: totipotent reproductive germ cells, necessary for the transmission of genetic information to the next generation, and differentiated somatic cells. For somatic cells epigenetic marks become fixed once the cell differentiates [100] and developmental processes in these cells are usually associated with a progressive loss of developmental potential. However, in cells undergoing dedifferentiation (e.g., cancer cells), or transdifferentiation, reprogramming can also take place in differentiated somatic cells [103].

Major reprogramming and global erasure of epigenetic marks take place at two time points in the mammalian life cycle: during early embryonic development after fertilization and in primordial germ cells (PGCs), where totipotency or pluripotency is restored and parental imprints are erased (Figure 1.3).

Figure 1.3 Epigenetic reprogramming. After fertilization, the paternal genome (bottom blue line) becomes actively demethylated rapidly in the zygote, while the maternal genome (bottom red line) is demethylated by a passive mechanism. Both genomes are remethylated at the time of implantation. During embryonic development, another reprogramming event is occurring in germ cell development in PGCs, were genome-wide DNA methylation is followed by remethylation in male germ cells (top blue line) in prospermatogonia and in female germ cells (top red line) after birth. Both reprogramming events are associated also with changes in repressive histone modification patterns as indicated by methylation changes in histone H3K27 and H3K9.

1.3.1 Epigenetic Asymmetry in the Zygote

At fertilization, the parental genomes are in different stages of the cell cycle and contain divergent epigenetic marks and chromatin composition [100]. The paternal genome is single copy (1C), packed densely with protamines instead of histones, whereas the maternal genome is arrested in metaphase II (2C) and contains histones. Upon fertilization and zygote formation protamines in the paternal genome are rapidly replaced with histones that lack H3K9me2 and H3K27me3, followed by an active, genome-wide loss of DNA methylation. In contrast, the maternal pronucleus contains histones with H3K9me2 and H3K27me3 marks that were acquired during oocyte growth and does not lose 5mC at this early stage, but rather by a passive mechanism during subsequent cell divisions. This asymmetry in paternal and maternal epigenomes can be detected up to the four-cell state. However, the functional importance of this asymmetry in the zygote is not clear [99]. Gill et al. [104] hypothesized that chromatin modifications established in the gametes may be required for proper embryonic development, and it could be shown that loss of early zygotic paternal demethylation perturbs the activation of several pluripotency-associated genes and impairs development [105].

During preimplantation development a passive loss of 5mC can be detected in the maternal genome until the blastocyst state when the inner cell mass acquires high levels of 5mC, H3K9me2, and H3K27me3. In contrast to the paternal active DNA demethylation catalyzed by TET enzymes, it has been hypothesized that DNA demethylation in the maternal genome occurs passively through exclusion of the maintenance methyltransferase DNMT1 from the nucleus [106]. During active paternal demethylation the maternal genome is protected by DPPA3 (also called STELLA) from TET3 oxidation [107]. DPPA3 is essential for embryonic development during preimplantation and loss of Dppa3 leads to embryonic lethality in mice [108]. Furthermore, although DNA demethylation is a global process during preimplantation development, some genomic sequences, like imprinted loci, centromeric heterochromatin, and intracisternal A particle (IAP) retrotransposons maintain DNA methylation during preimplantational development, which is also mediated by DPPA3.

All in all, an elaborate temporal and spatial epigenetic modification program seems to be necessary for zygote development and to exploit the entire potential of the genome. However totipotency is lost as cleavage progresses [99]. Histone modifications as well as DNA methylation are necessary for regulation of lineage induction and defects in DNA and histone methyltransferases can lead to impaired differentiation in ESC [109]. Polycomb repressive complexes are required for the bivalency of key regulator developmental genes in a yet transcriptionally poised state, where activating and repressing histone marks are present at the same time, ensuring lineage flexibility.

1.3.2 Reprogramming in the Germline

A second major epigenetic reprogramming event takes place during development of germ cells in the early embryo. PGCs are formed from epiblast cells by signaling molecules that are produced by extraembryonic ectoderm and primary endoderm [110]. This process is associated with a loss of 5mC and H3K9me2 and an increase of H3K27me3, a more flexible histone modification that allows rapid activation of poised developmental regulator genes [111]. Global demethylation of PCGs occurs between E11.5 and E12.5 of mouse development and leads to erasure of methylation in imprinted genes and single copy genes [112] and to reactivation of the X chromosome in females [113]. However, there is evidence that not all epigenetic marks are erased in imprinted genes in PGCs. The paternal H19 and the maternal Snrpn alleles are demethylated during PGC development but later during spermatogenesis and oogenesis they need to be methylated again. This de novo methylation of formerly methylated genes seems to happen at an earlier stage than de novo methylation of originally unmethylated genes [114,115], indicating that other epigenetic marks are not completely erased during PGC development and provide signals for de novo methylation [100]. To distinguish between somatic cells and PGCs several markers can be used, which reflect the different genetic programs of PGC and somatic cell specification. PGCs were found to express several pluripotency genes, including Sox2 and Oct4, which were not expressed in the neighboring somatic cells, indicating that PGCs exhibit pluripotency, which is lost in somatic cells. On the other hand some genes, including Hoxb1 and Hoxa1, which are important for cell differentiation and morphogenesis are significantly upregulated in somatic cells, but not expressed in PGCs, indicating that PGCs repress somatic cell fate [116].

1.3.3 Induced Pluripotency

Briggs and King [117] performed the first nuclear transfer experiments with cells isolated from late blastula stage frog embryos from Rana pipiens that developed into complete embryos when transplanted into enucleated oocytes. Subsequently, Gurdon and colleagues succeeded in producing sexually mature frogs from nuclear transplantation of adult nuclei in Xenopus laevis [118]. Meanwhile, it could be demonstrated that adult somatic cell nuclei can be reprogrammed by nuclear transfer from different species including mammals [119]. These studies indicated on one hand that developmental restrictions are due to reversible epigenetic modifications [119] and on the other hand that the oocyte must contain factors that mediate the reprogramming of adult cells into an embryonic state [103].

However, the big breakthrough was the discovery of transcription factors that can induce pluripotency in somatic cells to generate induced pluripotent stem (iPS) cells without the need for oocytes. Takahashi and Yamanaka [120] identified four transcription factors, OCT4 (O), SOX2 (S), KLF4 (K), and cMYC (M) that were able to reprogram adult mouse fibroblasts into ES-like iPS cells. The advantages of transcription-factor-induced reprogramming lies in its simplicity and robustness. By ectopic expression of OSKM a broad range of different cell types can easily be reprogrammed to pluripotency. In their ideal state iPS cells are functionally indistinguishable from ESC, can form chimeras and teratomas, and are able to differentiate into cells of all three germ layers [121]. However, the induction of pluripotency upon OSKM expression requires a latency period of 1–2 weeks and occurs only in 1% of the starting cells. Furthermore, it has been shown that the differentiation state of a somatic cell can influence the efficiency of iPS cell generation and that compounds that inhibit DNA methylation and histone modification can increase the efficiency [122].

1.4 Genomic Imprinting as a Model of Epigenetic Silencing

Monoallelic expression can occur randomly for specific clusters of genes, for example, for B- or T-cell receptor genes [123], and for up to 10–24% of individual genes per cell [123,124]. Genetic differences between alleles that affect the activity of promoters or cis-regulatory elements can also lead to monoallelic or strongly biased allelic expression that may affect many genes in human [125]. In contrast, parental-specific monoallelic or imprinted expression is relatively rare, with only approximately 0.5% of genes (125 in mouse) showing imprinted expression in mammals (http://igc.otago.ac.nz, [126]). In spite of their small number, many imprinted genes play important roles in development and growth, as was already demonstrated in the 1980s by experiments that showed early embryonic lethality of androgenic and gynogenic embryos, indicating that both the maternally and paternally inherited genomes together are required for normal development [127,128]. Following this, a number of developmental disorders have been shown to be caused by defects at specific imprinted gene loci, such as the chromosome 15q11-13 imprinted region, where paternal mutations can cause Prader–Willi syndrome and maternal mutations can cause Angelman syndrome [129]. In such diseases a nonmutated allele is present, but epigenetically silenced by the imprinting mechanism, opening the possibility for treatment by reactivation of the silent allele with epigenetic drugs.

Genomic imprinting is a classical epigenetic process in mammals controlled by DNA methylation that is deposited in genomic regions called imprint control elements (ICEs) in either the male or female germline by the de novo DNA methyltransferases DNMT3A and DNMT3L [130–132] (Figure 1.4). Differential DNA methylation is then maintained on the same parental allele in all somatic cells in the organism by DNMT1 [133]. The majority of reported imprinted genes in mice lie near to one of the 24 reported gametic differentially methylated regions (gDMRs) [134,135], seven of which have been demonstrated to be ICEs by genetic deletion that resulted in loss of imprinted expression of nearby genes [10]. This indicates that most imprinted genes are in co-regulated clusters controlled by an ICE, that ranges in size from 3 to 12 genes spread over 100–3700 kilobases (kb) [10]. DNA methylation is associated with gene silencing, but paradoxically many imprinted genes are expressed from the same allele as where the ICE is methylated. This is explained by the mechanism by which imprinted silencing is achieved. Different mechanisms of imprinted silencing via the ICE have been shown, but the most common mechanism described so far for four of the seven defined ICEs (the Igf2r, Kcnq1, Gnas, and Prader–Willi clusters) is by lncRNA-mediated silencing [136–139]. In these cases the ICE is associated with the promoter of the lncRNA and silenced on the maternal allele by DNA methylation. The lncRNA is then expressed and silences imprinted genes in cis on the paternal allele. Similarly, in the Igf2 cluster where imprinted silencing is not controlled by an lncRNA, the imprinted protein-coding genes Igf2 and Ins2 are expressed from the paternal allele where the ICE is methylated. In this cluster the insulator protein CTCF binds the unmethylated maternal ICE blocking Igf2 from accessing enhancers, while CTCF binding is prevented by methylation on the paternal allele allowing the enhancers to activate Igf2 expression [140,141]. Hence, in imprinted silencing DNA, methylation acts to repress the repressor allowing the expression of imprinted genes. For a limited number of imprinted genes DNA methylation can then play a secondary role to reinforce silencing directly on the silent promoter, forming a somatic DMR [10].

Figure 1.4 Regulation of imprinted expression. Imprinted silencing is controlled by differential DNA methylation on the ICE that is established during either oogenesis or spermatogenesis by the DNMT3A/3L de novo DNMTs complex and maintained on the same parental chromosome by DNMT1. The unmethylated ICE then acts to silence a cluster of imprinted genes in cis, in the most common mechanism by acting as a promoter for a lncRNA that then causes imprinted silencing. Although, differential DNA methylation of the ICE is present in all somatic cells, and the lncRNA is also expressed in most cell types, many imprinted genes are only susceptible to imprinted silencing in some cell types or developmental stages, and hence show tissue-specific imprinted expression.

Genomic imprinting is a phenomenon with a clear phenotype, making it a useful model to investigate aspects of epigenetic gene regulation. With the advent of high-throughput sequencing technologies, thousands of lncRNAs have been recently identified [142], but imprinted lncRNAs remain among the few lncRNAs that have so far been demonstrated to be functional by genetic experiments. lncRNAs could act by their transcription alone or via their RNA product to regulate gene expression. Posttranscriptional knockdown by RNA interference-based approaches will not affect the function of lncRNAs that act via their transcription, while genetic deletions of lncRNAs could also remove cis-regulatory elements such as enhancers, making neither strategy ideal for investigating lncRNA function. Therefore, in the imprinting field lncRNAs have been truncated by introducing a polyadenylation cassette to investigate their function and mechanism of action [136–139]. Using this approach, the lncRNA Airn that silences Igf2r in mouse was truncated to various lengths, demonstrating that Airn need only be transcribed across the Igf2r promoter to cause silencing due to transcriptional interference [143]. In contrast, the Airn lncRNA product was shown to be necessary to silence the nonoverlapped gene Slc22a3 by recruiting the repressive EHMT H3K9 dimethyltransferase to its promoter [144]. Similarly, the Kcnq1ot1 lncRNA responsible for silencing genes in the Kcnq1 cluster is associated with EHMT2 and the PRC2 H3K27trimethyltransferase complex, and imprinted expression of some genes in the Kcnq1 cluster is lost when these complexes are disrupted, indicating that Kcnq1ot1 may also cause silencing by recruiting repressive histone-modifying complexes [145]. The experimental approaches developed by studying imprinted lncRNAs, and the silencing mechanisms uncovered, may be applied to investigate the function and mechanism of action of lncRNAs outside of imprinted regions.

The majority of imprinted genes show tissue-specific imprinted expression [146], indicating that they may have a dose-dependent function in specific tissues. This indicates that the imprinted silencing mechanism may act tissue-specifically, which has been hypothesized to occur, for example, by imprinted lncRNAs blocking tissue-specific enhancer activity [147]. Open questions in well-studied imprinted clusters, and the likely possibility that other imprinted clusters may achieve imprinted silencing in different ways, makes genomic imprinting a fertile field of study to further elucidate mechanisms of epigenetic gene silencing.

1.5 Dosage Compensation in Mammals

The difference in the distribution of the sex chromosomes in mammals requires a mechanism of gene dose balancing to adjust for the double amount of X-linked genes in females. H.J. Muller was the first to develop the concept of dosage compensation in Drosophila following his studies on X-linked eye pigment genes [148]. While in Drosophila the dosage difference is equaled by transcriptional upregulation of the genes on the single male X chromosome [149], mammals have adopted a silencing mechanism that affects one of the two female X chromosomes [150]. This process involves random selection of one of the two Xs for inactivation, which is remarkably illustrated by the fur color mosaicism of calico and tortoiseshell cats. In these cats the different colored patches are directly reflecting the expression pattern resulting from random XCI of two different alleles of fur color encoded on the X chromosome (http://www.bio.miami.edu/dana/dox/calico.html). XCI utilizes all major epigenetic silencing mechanisms including DNA methylation, histone modification, and incorporation of histone variants, changes in nuclear architecture and noncoding RNA. These mechanisms act together to generate the Xi chromosome, which exists as facultative heterochromatin, is late replicating and condensed during interphase, and is represented by the Barr body in female somatic cells. Together, these structural characteristics are associated with transcriptional silencing of about 1000 X-linked genes.

XCI involves several important steps including sensing (more than one X chromosome is present), counting (only one X chromosome per cell should be active) and choice (random selection of one X chromosome for inactivation) [11]. The onset of XCI is associated with the expression of a noncoding RNA called Xist, which covers and marks the X chromosome in cis and is essential for the initiation of silencing [151,152]. It is transcribed from the X inactivation center, a key locus for the initiation and regulation of XCI, next to other noncoding RNAs such as the Xist repressor Tsix. Xist expression is imprinted and only expressed from the paternal X in marsupials and in the early zygote and extraembryonic tissues in rodents, whereas it appears to be randomly expressed from either X chromosomes in other eutherian mammals including human. Xist expression and coating of the X chromosome in cis represents the first step in XCI. The exact mechanism of interaction between Xist and the X chromosome is not clear, but it is suggested to involve several interaction domains. XCI initiation events also include loss of active histone marks (H3K9Ac, H3K4me2, H3K4me3), global H4 hypoacetylation, loss of RNA polymerase II, and transcriptional silencing of X-linked genes. The Xist transcript might also play a role for the propagation of silencing by recruitment of histone modification complexes such as PRC2, that deposit repressive marks (H3K27me3, H3K9me3, H4K20me1, H2AK119ub1). Interestingly, H3K9me and H3K27me are regionally enriched at nonoverlapping regions [153] and the association of repressive histone modification complexes is found early during XCI but diminished later on, suggesting a role for these in establishment rather than maintenance of the silenced state. The chromodomain-containing reader protein Cdyl was recently identified as a factor binding to the Xi and was suggested to propagate the silencing via recruitment of the histone methyltransferase G9a [154]. Following these early chromatin changes, the Xi chromosome is decorated with the histone variant macroH2A and DNA methylation of X-linked CpG islands, which is essential for the maintenance of a stable silenced X chromosome [11].

The X chromosome includes genes that are important for sexual dimorphism and reproduction, but also for neuronal development, general intelligence, and social cognition [155]. Intriguingly, almost 30% of known genes that are linked to mental retardation are encoded on the X chromosome [155]. Generally, XCI has important implications for different diseases, because mosaic inactivation of X-linked genes can dampen the phenotype of mutations in female patients, while skewed X inactivation can be associated with worse disease outcomes of X-linked disorders [156]. On the other hand, escape from silencing or mutations in escape genes are involved in disease including cancer [157]. Depending on the cell types and tissues some genes regularly escape XCI in humans. Besides genes located within the pseudoautosomal (PAR) regions, which are homologous between the X and Y chromosomes, genes outside the PAR regions with Y homologs (e.g., KDM5c, UTX) but also genes without Y-linked homologs can escape inactivation resulting in higher expression levels in females, although their exact biological function has been unresolved [11]. Interestingly, the H3K27me3 demethylase UTX was recently identified as a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia in males [158].

Somatic loss of the Barr body, the Xi chromosome, in cancer was already reported in early studies and was also found in aging tissues in mice [159,160]. A role for loss of the Xi in cancer was indicated by silencing of Xist in the blood compartment of mice, being correlated with a highly aggressive myeloproliferative neoplasm and MDS [161]. However, only recently a report by Chaligne et al. [162] demonstrated for the first time that loss of the Xi is due to epigenetic erosion rather than genetic loss in the context of breast cancer.

Taken together, these findings highlight the essential role for proper XCI for development and disease, which are often associated with gender-specific prevalence.

1.6 PEV in Drosophila

In addition to the concept of dosage compensation of X-chromosomal linked genes, Muller [163] made in the same publication a second seminal discovery named PEV. Using X-rays as a mutagen, Muller observed a group of unusual phenotypes, in which the eye of Drosophila flies was variegating, with some patches of red and some patches of white facets. In contrast to classic loss-of-function mutations that strictly follow Mendelian rules, the variegating nature of the mosaic, so-called "white-mottled" eye phenotype of PEV mutants suggested that the X-chromosomal white gene, encoding for the red pigment of wild-type flies, itself was not damaged at all, since some facets in the eye remained red, and flies with entirely red eyes could be recovered, again using X-rays as a mutagen. Muller suggested that in this kind of mutation the white gene stays intact (not mutated), but has been clonally silenced in some of the facet cells (white), whereas in others it was normally expressed (red). Subsequent studies on somatic giant chromosomes of larval salivary glands showed that the PEV eye phenotype is caused by an inversion or rearrangement with one breakpoint within the pericentric heterochromatin, and one breakpoint adjacent to the white gene. Hence this phenomenon is referred to as PEV since the variegating phenotype is caused by a change in the position of the gene within the chromosome and not by a mutation of the gene itself [163].

Today, virtually every Drosophila gene that has been examined in an appropriate chromosomal rearrangement has been shown to variegate, and rearrangements involving the pericentric heterochromatin of any chromosome can lead to PEV [164]. This phenomenon is not limited to Drosophila but PEV has subsequently also been observed in a variety of organisms, including yeasts (recently reviewed in Ref. [165]), flies, and mammals (recently reviewed in Refs [166,167]). Generally, the phenotype of PEV results whenever a euchromatic gene is juxtaposed with heterochromatin by any kind of rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it shuts off transcription of the affected reporter gene in a stochastic, that is, variegating, pattern. The strength of PEV, however, can be modified by a variety of extrinsic as well as intrinsic factors [168] (and recently reviewed in Ref. [169]). The first group shown to affect the extent of variegation extrinsically is environmental factors such as temperature acting during development, and the latter represent factors like the amount of heterochromatin available within the genome. High temperatures from 25 to 29 °C during larval development result in suppression of variegation (loss of silencing, red eyes), whereas lower temperatures (e.g., 18 °C) cause enhancement of variegation (increase in silencing, white eyes). The presence of additional Y chromosomes (XXY females and XYY males) that mainly consist of heterochromatin can titrate-off heterochromatic silencing factors from the PEV reporter site and hence act as strong suppressors of PEV, whereas flies without a Y chromosome (X0) act as strong enhancers [170].

In addition to these chromosomal modifiers of PEV mentioned above, hypersensitive reporter genes such as white-mottled were intensely studied in Drosophila in order to screen for dominant second-site mutations of modifier genes that act either as suppressors or enhancers of PEV. Since then many genetic dominant suppressors and enhancers of PEV have been isolated [171,172] (and recently reviewed in Ref. [169]). These second-site mutations were induced by chemical mutagens that cause point mutations or small insertions/deletions by transposons or irradiation, but that do not impact the chromosome rearrangement responsible for the PEV phenotype. By this genetic approach two groups of PEV modifier genes were extracted from the fly genome. The first class are genes that, when mutated, result in a loss of silencing (reversion to red eyes) and hence act as suppressors of the variegation, thereby named Su(var). The second class are genes that when mutated result in an increase in silencing (white), hence acting as enhancer of variegation, consequently named E(var). In Drosophila about 150 loci have been identified from such screens, and of these approximately 30 modifiers of PEV have been studied in detail (recently summarized in Ref. [169]).

In recent years further genetic screens for dominant PEV mutations as well as bioinformatic analysis for extracting homologs of E(VAR) and SU(VAR) proteins not only of Drosophila but also mammals, have identified many conserved epigenetic factors including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. As one of the many examples, the heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Thereby, genetic, molecular, and biochemical analyses of PEV that were initiated in the Drosophila model system have contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.

1.7 Transgenerational and Intergenerational Epigenetic Inheritance

Transgenerational epigenetic effects are defined as changes in the offspring’s phenotype that are propagated over several generations and that are not due to changes in the genomic DNA sequence [173]. This concept represents a significant alteration regarding the inheritance of phenotypic traits across generations when opposed to Mendelian inheritance, which strictly correlates phenotypes to genotypes. However, it revives Lamarckian ideas by stating that characteristics (i.e., phenotypes) that were acquired or learned due to external influences can be passed on to the next generation without having an impact on the genome sequence.

Before giving an overview of the most well-established examples, we want to point out some important discussion points in the field of transgenerational inheritance. Although it is now known that transgenerational epigenetic effects are common in plants and worms, the occurrence of these phenomena in mammals is still to some extent subject to debate. This is due to the fact that mammalian PGCs and the zygote undergo efficient reprogramming at distinct developmental steps, which leads to nearly complete erasure of all inherited epigenetic marks [174]. Through this mechanism, totipotency of the early embryo is achieved, but also epigenetic changes acquired by parents are erased. However, during pregnancy, epigenetic alterations not only affect the mother and the fetus, but also the fetus’s PGCs, which will eventually give rise to the grandchildren. Considering this concomitant exposure of three generations during pregnancy, it is important to note that many of the currently termed transgenerational effects in mammals are observed when pregnant mothers, their unborn offspring, and the latters’ PGCs are exposed to certain external stimuli, but that these effects often cease in the subsequent generation [175]. If the effect on the phenotype thus does not persist longer than in the grandchildren of the affected generation, the effect on the phenotype should rather be considered as an intergenerational and not a transgenerational