REGULATION

REGULATION OF
OF GENE
GENE EXPRESSION
EXPRESSION
Assistant Professor Dr. Chatchawan Srisawat

 (Gene Expression)

Gene expression =
( RNA)

RNA

Protein

Gene expression

Gene (DNA)

 (Gene Expression)

Human genome ~ 20,000 25,000

~ 5,000

Regulation
Regulationof
ofgene
geneexpression
expression
Developmental and differentiation cues.

myocyte

adipocyte

neuron

erythrocyte

Adaptation (nutrients, osmotic pressure, temperature, hormones, etc)

REGULATION OF GENE EXPRESSION

Nucleus

1. Gene
2. Transcription
3. RNA processing
4. RNA export
5. Translation
6. RNA stability

cytosol

7. Post-translational
modifications

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION

Nucleus

1. Gene

cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - GENE LEVEL

1. Gene amplification
2. Gene rearrangement

REGULATION OF GENE EXPRESSION - GENE LEVEL

1. Gene amplification
copy number ribosomal RNA gene
(X. laevis) ribosome zygote
fertilization

Xenopus laevis

Oocyte

REGULATION OF GENE EXPRESSION - GENE LEVEL

1. Gene amplification
copy number ribosomal RNA gene
(X. laevis) ribosome zygote
fertilization

REGULATION OF GENE EXPRESSION - GENE LEVEL

2. Gene rearrangement
Immunoglobulin (antibody) or T-cell receptor genes
Antigen-binding site

Antigen (bacteria,
viruses, cancer)

Light chain
Heavy chain

immunoglobulin (antibody)
antigen
B-lymphocyte

REGULATION OF GENE EXPRESSION - GENE LEVEL

Antibody antigen antibody
antibody
antigen ( bacteria,
viruses, cancer)

bacteria

viruses

cancer cells

REGULATION OF GENE EXPRESSION - GENE LEVEL

Problem
Problem
1 1
Gene (DNA)

RNA

Protein (e.g. antibody)

antibody
20,000 25,000

antibody ?

REGULATION OF GENE EXPRESSION - GENE LEVEL

Solution
Solution

GENE REARRANGEMENT

Susumu
SusumuTonegawa
Tonegawa
Nobel 1987
gene rearrangement
antibody

REGULATION OF GENE EXPRESSION - GENE LEVEL

GENE REARRANGEMENT

Solution
Solution

heavy chain antibody

V
1 2 3

300

1 23 4 20

1 23 4 5

Germ
Germcells
cells

~ 300 V segments (V1, V2, V3, ..., V300)

~ 20 D segments (D1, D2, D3, , D20)
~ 4-5 J segments (J1, J2, J3, J4, J5)
segment coding sequence

REGULATION OF GENE EXPRESSION - GENE LEVEL

differentiation lymphocyte
antibody rearrangement V, D,
J segment
D

1 23 4 20

1 23 4 5

V
1 2 3

300

Germ cell DNA

DJ joining ( D2+J3)

1 2 3

DJ
23

300

Rearranged DNA

VD J
223

VDJ joining (V2 +D2J3)

C

Rearranged DNA
VD J
223

Transcription and splicing

C

An

mRNA

REGULATION OF GENE EXPRESSION - GENE LEVEL

V
1 2 3

300

1 23 4 20

1 23 4 5

heavy chain gene

rearrangement V, D, J segment random
lymphocyte
V53D19J1

V6D3J4

V217D8J3 ...

Rearranged gene heavy chain

= 300 x 20 x 5 30,000

DNA V, D, J segment
antibody rearranged gene (various combinations of
V,D,J)

REGULATION OF GENE EXPRESSION - GENE LEVEL

light chain antibody heavy chain
V J segments ~100 4 segments
rearrangement

V
1 2 3

100

J
1 23 4

light chain gene

Rearranged gene light chain

= 100 x 4 400

antibody heavy light chain

= 30,000 x 400 > 10,000,000
Gene
Generearrangement
rearrangement

antibody
antibody

REGULATION OF GENE EXPRESSION - GENE LEVEL

**
rearrangement
rearrangement

DNA
DNA
(
(
splicing
splicing

RNA)
RNA)

rearrange
rearrange

germ
germcell
cell

REGULATION OF GENE EXPRESSION

Nucleus

2. Transcription

cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

transcription
active ( mRNA
) inactive
transcription transcription
initiation

Termination
Transcription initiation step

Elongation

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

transcription 3

1. transcription factors
2. chromatin
3. methylation DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

1. transcription factors
upstream

-1 +1

downstream

transcription start site

exon1

intron

promoter
(TATA box)

eukaryote

exon2

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

1. transcription factors
upstream

RNA polymerase II

-1 +1

downstream

transcription start site

exon1

intron

exon2

promoter
(TATA box)
transcription RNA polymerase II complex
promoter TATA box (transcription initiation step)
Transcription elongation
Transcription termination

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

RNA polymerase II complex promoter TATA box
(inefficient trancription initiation)
transcription

RNA polymerase II

transcription start site

exon1

intron

exon2

promoter
(TATA box)

transcription
transcription

transcription
transcription
factors
factors

transcription
transcriptioninitiation
initiation

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

What are transcription factors?

Transcription factors (TF)
DNA transcription
example

CTF

SP1

Oct1

etc.

* eukaryote transcription factors

transcription factor 2
1. DNA (DNA-binding domain)
2. transcription
(Transcription-regulatory domain)

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA-binding domain

Transcription-regulatory domain

DNA
transcription element
1. DNA (DNA-binding domain)
DNA TF
(transcription element)

rasmol

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA-binding domain

Transcription-regulatory domain

DNA
transcription element
1. DNA (DNA-binding domain)
DNA TF
(transcription element)
examples

Transcription
factors

DNA sequence
(transcription element)

CTF

GGCCAATCT

SP1

GGGCGG

Oct-1

ATTTGCAT

5 CAGGGCGGATCTAAGAGGCCAATCTTATAGACTACTTATAAATCTCTCTAGACC 3
3 TGCCCGCCTAGATTCTCCGGTTAGAATATCTGATGAATATTTAGAGAGATCTGG 5
SP1

CTF
rasmol

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

SP1

CTF

5 CAGGGCGGATCTAAGAGGCCAATCTTATAGACTACTTATAAATCTCTCTAGACC 3
3 TGCCCGCCTAGATTCTCCGGTTAGAATATCTGATGAATATTTAGAGAGATCTGG 5
SP1

CTF

2.
transcription (Transcriptionregulatory domain) transcription
- interact RNA polymerase II enzyme complex
promoter

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

RNA polymerase II

SP1
CTF
5 CAGGGCGGATCTAAGAGGCCAATCTTATAGACTACTTATAAATCTCTCTAGACC 3
3 TGCCCGCCTAGATTCTCCGGTTAGAATATCTGATGAATATTTAGAGAGATCTGG 5
SP1

CTF

promoter
(TATA box)
2.
transcription (Transcriptionregulatory domain) transcription
- interact RNA polymerase II enzyme complex
promoter
- modulating enzyme complex activity
transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

Transcription factor transcription
transcription factor transcription
TF transcription = activator

Transcription element = enhancer

TF transcription = repressor

Transcription element = silencer

activator

repressor
activator

RNA polymerase II

5 CAGGGCGGATCTAAGCCATGGATCATAGGCCAATCTTATAGACTACTTATAAATCTCTCTAGACC 3
3 TGCCCGCCTAGATTCGGTACCTAGTATCCGGTTAGAATATCTGATGAATATTTAGAGAGATCTGG 5

SP1

repressor

CTF

promoter (TATA box)

- inhibit activation domain activator

- activator DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

: transcription factor
+

RNA polymerase II

+
+

+ start site
exon1

up to 10 kilobases
from the start site

intron

exon2

core promoter
(TATA box)
upstream

enhancer
silencer
downstream

transcription factor
activator DNA enhancer

transcription

repressor DNA silencer

transcription

* enhancer silencer (
upstream downstream ), promoter,

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

Transcription factor (activator/repressor)

transcription cooperative regulation transcription activity

(switch on/off).

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

TF
TF

Transcription factors

Tissue-specific gene expression

Differentiation-specific gene expression
example

albumin gene

C/EBP HNF-1 (hepatic nuclear factor-1)

albumin

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

Inducible transcription factor
activity transcription factors
ligand protein modification ( phosphorylation
dephosphorylation) TF
steroid hormones (e.g.
glucocorticoid, estrogen)

Examples
inhibitor

steroid receptor

GRE
glucocorticoid response element
cytosol

nucleus

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

Inducible transcription factor
activity transcription factors
ligand protein modification ( phosphorylation
dephosphorylation) TF
Interferon

Examples

inteferon
receptor
JAK kinase (transducer)
phosphorylation

unphosphorylated
STAT1 monomer

Response element
cytosol

nucleus

phosphorylated
STAT1 dimer

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

transcription 3

1. transcription factors
2. chromatin
3. methylation DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

2. chromatin

(chromatin remodeling)
DNA

DNA eukaryote
histone
nucleosome
Histone
( lysine, arginine)

DNA
(negatively-charged molecule)
histone

free DNA
10 nm chromatin fibril

30 nm chromatin fiber
Nucleosome

chromatin fiber chromosome

chromosome

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA histone nucleosome
transcription RNA polymerase
transcription factor DNA

RNA polymerase

promoter

transcription

transcription

 heterochromatin = denselypacked chromatin

nucleus

heterochromatin
active
transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

drumstick

Barr body

example Condensation of X chromosome in cells derived from females

 euchromatin =
loosely-packed chromatin
nucleus

euchromatin
active
transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

chromatin
chromatin(chromatin
(chromatin
remodeling)
remodeling)

suppress transcription

increase chromatin condensation

enhance transcription

decrease chromatin condensation

chromatin
chromatin

acetyl
acetylgroup
group
histone
histone
1. Histone acetyl transferase
2. Histone deacetylase

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

+NH

3
CH 2
CH 2
O
CH 2 O
CH 2
... NH CH C NH CH C ...
2
Gly
Lys

Histone acetyl
AcCoA

CH 3
C
NH
transferases
CH 2
CoA
CH 2
O
CH 2 O
CH 2
... NH CH C NH CH C ...
2
O

Ac

Histone deacetylases

Acetylated histone

chromatin condensation

transcription factors
DNA transcription

Deacetylated histone

chromatin condensation

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

transcription 3

1. transcription factors
2. chromatin
3. methylation DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

3. methylation DNA
2-7% cytosine DNA 5-methylcytosine

DNA methyltransferases

methylation cytosine DNA

5 CG 3 (CpG)
m

5 A T G C G A C C 3
3 T A C G C T G G 5
m

methylation
cytosine
DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

3. methylation DNA
1 CpG 100 bp CpG
(>10 CpG 100 bp)
CpG island

CpG island

transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

state of
methylation CpG activity gene

Methylated
y
g
genes

inactive genes

Non-methylated genes

active genes

DNA methyltransferase
Non-methylated genes

Methylated genes

5 methylcytosine DNA glycosylase / DNA demethylase

5-methylcytosine

state of methylation

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA methylation transcription
methylation transcription
a)
methylation
cytosine
transcription
factor DNA

activator

5 CAGGGCGGATCTAA 3
3 TGCCCGCCTAGATT 5

transcription

transcription factor
transcription
(repressor)
MeCP1, 2 DNA
5-methylcytosine

RNA pol II

repressor

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA methylation transcription
methylation transcription
a)
MeCP gene (transcribed region)
RNA polymerase transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

DNA methylation transcription
b)
methylation chromatin remodeling
increase chromatin condensation
suppress gene expression

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

Inheritance of Gene Methylation Patterns

hemimethylated DNA

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

activator
repressor
Transcription factors

Chromatin
remodeling

DNA methylation

chromatin condensation

DNA methylation

chromatin condensation

DNA methylation

3
transcription

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

REGULATION OF GENE EXPRESSION - TRANSCRIPTIONAL LEVEL

REGULATION OF GENE EXPRESSION

Nucleus

3. RNA processing

cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

1. RNA splicing
2. RNA editing

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

1. RNA splicing

Exon, Intron, 5 and 3 untranslated regions (UTR)

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

Various machanisms in alternative splicing

a) Alternative 5 splice sites

b) Alternative 3 splice sites

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

Various machanisms in alternative splicing

c) Exon inclusion or skipping

d) Mutually exclusive use of exons

e) Intron retention

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

Alternative splicing
Alternative splicing

Alternative splicing is often tightly regulated depending on cell

types or developmental stages.

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

Alternative splicing
Alternative splicing

> 90% human genes process alternative splicing

mRNAs

(E. coli)
~ 4000

~13000

~ 20000

REGULATION OF GENE EXPRESSION - RNA PROCESSING LEVEL

2. RNA editing
RNA editing
RNA transcripts
,
Example

apolipoprotein B pre-mRNA
H

H
N

cytosine

N
O

Ribose

deamination

O
H
O

N
N

Ribose

uracil

Deamination:
C to U

REGULATION OF GENE EXPRESSION

Nucleus

4. RNA export

cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - RNA EXPORT LEVEL

mRNAs processed
nucleus cytoplasm template
translation

Quality controls

mRNAs

- Abnormally-spliced RNAs
- RNAs containing nonsense mutations

REGULATION OF GENE EXPRESSION

Nucleus

5. Translation
cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - TRANSLATIONAL LEVEL

translation translation initiation
A) Global regulation
of protein synthesis

modification of eukaryotic initiation

factors (eIFs) phosphorylation/
dephosphorylation translation

protein synthesis

REGULATION OF GENE EXPRESSION - TRANSLATIONAL LEVEL

translation translation initiation
B) mRNA-specific
gene regulation

translation
mRNA
mRNA
preinitiation complex

preinitiation complex mRNA 5 AUG start

codon (scanning) initiation complex

REGULATION OF GENE EXPRESSION - TRANSLATIONAL LEVEL

mRNA hairpin 5 AUG

start codon scanning preinitiation
complex

translation

translation
iron metabolism slide

REGULATION OF GENE EXPRESSION

Nucleus

6. RNA stability

cytosol

-S-S-

degraded mRNA

protein

active protein

REGULATION OF GENE EXPRESSION - RNA STABILITY LEVEL

Functions of 5 cap

Functions of poly(A) tail

Transport to cytoplasm
Increase translational efficiency
Protection of mRNA from degradation

Increase translational efficiency

Protection of mRNA from degradation

mRNA stability

mRNA in closed-loop configuration RNA ribonuclease

REGULATION OF GENE EXPRESSION - RNA STABILITY LEVEL

mRNA (mRNA decay rate)

mRNA
transcription.
Transcription

mRNA
mRNAlevel
level

RNA decay

Half-life mRNA eukaryotic cells

RNA decay

REGULATION OF GENE EXPRESSION - RNA STABILITY LEVEL

Regulation of RNA decay:
mRNA sequence
stability mRNA

e.g.

AU-rich element (ARE) at 3 UTR

promotes poly-A shortening

Iron-responsive element (IRE) at 3UTR

makes mRNA susceptible to endonuclease attacks.

activity sequence

mRNA

iron metabolism slide

REGULATION OF GENE EXPRESSION

iron metabolism
translation RNA stability

metabolism iron
translation RNA stability
example Transferrin receptor gene ferritin gene
transferrin (iron-transport protein)

cell

transferrin receptor

Iron metabolism

Fe2+/Fe3+
ferritin
(iron-storage protein)
cell usage (e.g. cytochrome, heme)

 Transferrin receptor iron

Ferritin iron iron
gene expression
iron metabolism
transferrin (iron-transport protein)

cell

transferrin receptor

Iron metabolism

Fe2+/Fe3+
ferritin
(iron-storage protein)
cell usage (e.g. cytochrome, heme)

 gene expression
Transferrin receptor

Ferritin

Iron excess

Transferrin receptor

Ferritin

Iron depletion

Fenton reaction
and oxidative
damages to cells
Defective cellular
functions

transferrin (iron-transport protein)

cell

transferrin receptor

Iron metabolism

Fe2+/Fe3+
ferritin
(iron-storage protein)
cell usage (e.g. cytochrome, heme)

 iron-responsive elements (IREs) =

RNA sequence forming a hairpin structure.
IRE-binding proteins (IRPs) = IRE

IRP
IRE
5

RNA

IRP
iron IRP
(iron-bound IRP) IRE
Iron-bound IRP

no binding to IRE

iron
iron

transferrin
transferrinreceptor
receptor

iron
iron

ferritin
ferritin

free
freeiron
iron

Transferrin
Transferrinreceptor
receptormRNA
mRNA

contains IRE at 3 UTR

binding of IRP to IREs blocks
endonucleolytic cleavage of mRNA
mRNA stability
translation and level of
transferrin receptor

endonucleolytic cleavage

RNA
RNAstability
stabilitycontrol
control

iron
iron

transferrin
transferrinreceptor
receptor

iron
iron

ferritin
ferritin

free
freeiron
iron

Ferritin
FerritinmRNA
mRNA

contains an IRE at 5 UTR

binding of IRP to IRE stabilizes
the hairpin structure and blocks
translation initiation.
translation initiation
translation and
level of ferritin

translational
translationalcontrol
control

iron
iron

transferrin
transferrinreceptor
receptor

iron
iron

ferritin
ferritin

free
freeiron
iron

free
freeiron
iron

Transferrin
Transferrinreceptor
receptormRNA
mRNA

contains IRE at 3 UTR

binding of IRP with iron prevents it
from bindng to IREs.

IRP

Fe

no blocking from endonucleolytic cleavage

mRNA stability
translation and level of
transferrin receptor
endonucleolytic cleavage

iron
iron

transferrin
transferrinreceptor
receptor

iron
iron

ferritin
ferritin

free
freeiron
iron

free
freeiron
iron

Ferritin
FerritinmRNA
mRNA

IRP

Fe

contains an IRE at 5 UTR

binding of IRP with iron prevents it
from bindng to IREs.
no stabilization of IRE

translation initiation
translation and
level of ferritin

REGULATION OF GENE EXPRESSION

Transcriptional VS. Post-transcriptional control of gene expression

REGULATION OF GENE EXPRESSION

Nucleus

1. Gene
2. Transcription
3. RNA processing
4. RNA export
5. Translation
6. RNA stability

cytosol

7. Post-translational
modifications

-S-S-

degraded mRNA

protein

active protein