Transcript Document 7111559

Epigenetics
• Epigenetics - Heritable changes in
gene expression that operate outside
of changes in DNA itself
- stable changes in gene expression
caused by changes in chromatin
structure
Epigenetics
• DNA methylation
• Histone tail post-translational
modifications
• microRNA
Mechanisms of Epigenetics:
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Nature 441, 143-145 May 2006.
The five nucleotides that make
up DNA
Cytosine methylation occurs
predominantly at CpG dinucleotides
which are palindromic
5’ CpG 3’
3’ GpC 5’
Roles of DNA methylation
• Transcriptional silencing
• Protecting the genome from
transposition
• Genomic imprinting
• X inactivation
• Tissue specific gene expression
Model for Role of Methylation in
Heterochromatin Formation
•
HP1 (heterochromatin protein 1) binds
to H3K9-Me3
•
HP1 oligomerization
•
Spreads along chromatin
•
Condenses chromatin into
heterochromatin
DNA methylation and Transcription:
• only known covalent modification of DNA
in mammals
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• occurs in context of CpGs
CpG islands:
• regions of the genome rich in CpG dinucleotide
• comprise 60% of promoters
• hypomethylated in differentiated tissue but, variable methylation patterns found in tumors
Non-CpG island promoters:
• 40% of promoters
• often methylated
• tissue-specific differentially methylated regions exist
• primary or secondary event?
CpG Islands:
- Regions of the genome in which the CpG dinucleotide occurs
at the EXPECTED frequency
- Usually located in 5’ flanking sequence, around the proximal
promoter, and/or within the first exon and intron
- Refractory to methylation in somatic cells
- Frequently methylated in tumor cells
Exon 1
Exon 2
How does one study DNA methylation ?
•Bisulphite sequencing
•Methylation-specific restriction endonucleases
e.g., HspII/MSPI
•Methylation specific assays, e.g. MethylLight
Epigenetics
• DNA methylation
• Histone tail post-translational
modifications
• microRNA
Bednar et al.,
PNAS, 95, 1998
Wolffe and Hayes,
NAR 27, 1999
Post-translational Modification of
Histone N-terminal Tails
Wide Range of Histone Modifications to Regulate the “STATE” of Chromatin
Proteins with bromodomains (acetate groups) or chromodomains (CH3)
DNA methylation
In establishing heterochromatin, which is the driving force
– DNA methylation?
- Histone modification?
Variety of Histone modifications:
Landmarks for Chromatin-binding Proteins
Chromodomain
•CH3(Methyl)- recognition domain
•HP1 has a chromodomain
•Targets to Me-lys or H3K9me
•Promote packed “closed” chromatin
Demethylation of Lys 9 in H3 tail
facilitates phosphorylation (P) of Ser 10
Acetylation (Ac) of Lys 9 and 14
leads to “OPEN” chromatin
Bromodomain
Binds to acetylated lysines “OPEN”
Wide range of histone modifications ->
Repressors Regulate Gene Expression by
Modulating Chromatin Structure to be Closed
Fig. 7-38
-The DBD of repressors (like Ume6) bind a DNA element (URS1) and the Repression Domain (RD)
recruits a protein complex containing a histone deacetylase like Rpd 3.
-The subsequent deacetylation of histone N-terminal tails results in chromatin condensation which
promotes gene repression.
Activators Regulate Gene Expression by
Modulating Chromatin Structure to be Open
Fig. 7-38
-The DBD of Activators like Gcn4 bind their Upstream Activating Sequence (UAS).
-Activation Domain (AD) attracts protein complexes containing histone acetylases (Gcn5)
-Subsequent acetylation of histone tails serve to open up chromatin.
-Thus HDACs and HATs are important global regulators of transcription
Model for Heterochromatin Formation
Condensation assisted by recruitment of HMT (histone methyltransferase),
which methylates adjacent H3K9
Chromatin condensed until a boundary element is reached.
Methylation of histone tails long lasting compared to acetylation
Can be Inherited by daughter cells: Responsible for X-inactivation
Epigenetics: chromatin structure controls gene expression rather than nt. sequence
Histone Post-translational Modifications (PTMs) involved in transcription:
• Histone modifying enzymes
generate “the histone code”
•PTMs dictate chromatin structure
and serve as a scaffold for additional
regulatory proteins
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• acetylation, methylation, ubiquitination
sumolyation and phosphorylation
• H3KAc, H4KAc, H3K4me, H3K36me
H3K27me, H3K9me
We examined histone PTMs
across the fpgs to study their
role in promoter choice and silencing.
Workman et. al 2007, review
Current thoughts on the role of chromatin in transcriptional initiation:
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are needed to see this picture.
Workman et. al 2007, review
Does this model apply to most tissue-specific promoters?
How does one detect histone PTMS ?
•Chromatin Immunoprecipitation (ChIP)
-variations, i.e., ChIP walking, ChIP on chip
Or
•Mass Spectrometry
Chromatin Immunoprecipitation
Identify proteins that interact with DNA
Cross Link
Sonicate
Primary Ab & Protein G Beads
Supe
after IgG
IP
IP
Ab Enriched DNA
Quantitate by
Real Time PCR
INPUT
Histone H3 lysine 4 tri-methylation across the fpgs gene:
H3k4
Workman et. al 2007
Vakoc et. al 2006
Histone H3K4me3 decorates P2 in all tissues, but appears limited in liver
and P1 K4me3 correlates with loss of DNA methylation and promoter activation.
Histone H3 lysine 4 tri-methylation across the fpgs gene:
H3k4
L1210
P1
P2
Liver
P1
P2
Brain
P1
P2
Histone H3K4me3 decorates P2 in all
tissues, but appears limited in liver
and P1 K4me3 correlates with loss of
DNA methylation and promoter
activation.
Histone H3 and H4 acetylation across the fpgs gene:
QuickTime™ and a
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Vakoc et. al, G and D, 2006
H3KAc and H4KAc
H3K36me3
TFIIH
a.)
\
TFIIE
TFIID
TFIIA
TBP
(Y
SP
TS
PS
)5
2
H3K4me3
TFIIBTFIIF
RNA
polymerase
TATA
SP
TS
PS
)5
2
Histone H3 and H4 acetylation
decorates
HMT
P
P2 in all tissues, and P1 acetylation
correlates
Memethylation and promoter activation.
with b.)
loss of DNA
TFIIH
Racanelli et al, MCB, 28, 836-48, 2008
Histone Post-Translational Modifications
Ac
Ac
3Me
3Me
3Me
2Me
2Me
Ac
2Me
P 1Me
1Me P
Ac
P
1Me Ac
2Me
1Me 1Me P
1Me
H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH- 39
P
Ac
1Me P
ARTKQTARKSTGGKAPRKQL
2Me
Ac
Ac
ARTKQTARKSTGGKAPRKQL
Ac
P
ATKAARKSAPATGGVKKPH
Ac
1Me
2Me
ATKAARKSAPATGGVKKPH
Summary: Layers of Gene Regulation
Chromatin Remodeling Factors (ATPase)
Histone Modifications
repress
DeAcetylation/Acetylation
Phosphorylation, methylation, Ub
activate
Regulatory Factors-gene specific
Also subject to post-translational modifications
HDATs
Co-repressors
General Transcription Factors
Pol II
HATs
Co-activators
Once Transcription Complex Assembled, what starts transcription?
FACT: CTD-becomes hyperphosphorylated
The dynamic epigenome and its
implications for modulating gene
expression in the cell
Chromatin modifications with
impact on gene expression
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•
•
•
•
•
K9 histone H3 and H4 acetylation
K9 H3 methylation
K4 H3 methylation
Ser 10 phosphorylation
Ubiquitination
Association of chromatin remodeling
complexes