Download Chromatin Modifications

Chromatin
Modifications
Vered Fishbain
Reading Group in Computational Molecular Biology
21/12/2006
Some Definitions…
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Chromatin is the complex of DNA and proteins found
inside the nuclei of eukaryotic cells.
Nucleosomes are the fundamental repeating subunits of
all eukaryotic chromatin. They are made up of DNA and
protein core, which is the histone core.
The histone core is composed by two copies of the
following set of proteins, called histones:
H2A, H2B, H3 and H4.
147 bp in each nucleosome.
Heterochromatin is condensed chromatin, includes
inactive genes and untranscribed regions (like the
centromer).
Euchromatin is non-condensed chromatin, includes active
and repressed genes.
The Histone Core
Chromatin Modifications
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Chromatin modifications are covalent
modifications that can effect transcription.
Acetylation
Methylation
Phosphorylation
Ubiquitination
Sumoylation
Adenosine-diphosphate ribosylation
Histone Acetylation
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2.
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Associated with transcription activation.
Influence gene expression in (at least) two ways:
Neutralize Lysine’s positive charge, which can
weaken DNA-histone contacts, or histone-histone
contacts.
Acetyl-Lysine is bound by a specific protein
domain that is found in many transcription
factors and calls bromodomain.
Rapidly reversible, and can turn over rapidly in
vivo.
Histone Methylation
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Characterized mainly for histone 3-lysin 4 (H3K4).
The Lysine can be mono-, di- or tri-methylated.
Doesn’t change the Lysine charge (naturally
positive).
methyl-Lysine can be bound by a methyl-lysin
binding domain, such as chromodomain, WD40
domain, Tudor domain, etc.
Long-lived.
Research Challenges
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Absence of sufficient verified data.
Contradictory evidences.
The available data is in a low resolution.
Outline
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TAF1 as an acetyltransferase (HAT).
TAF1 and Gcn5 – is there a redundancy?
TAF1 and other HATs in yeast (Durant and Pugh).
Acetylation and methylation across promoters and
ORFs (Pokholok et al.)
High resolution mapping of acetylation and
methylation (Liu et al.)
Identifying two major groups with similar modification
patterns within.
Summary (Millar and Grunstein)
Genome-Wide Relationships
between TAF1 and Histone
Acetyltransferases in
Saccharomyces cerevisiae
Melissa Durant and B. Franklin Pugh
Molecular and Cellular Biology,
April 2006
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The transcription machinery assembles at
promoters via two complexes, TFIID and
SAGA, which have a compensatory function
(Inna’s lecture…).
Both complexes contain subunits (TAF1 and
Gcn5) that harbor bromodomain and
acetyltransferase (HAT) activity.
In Saccharomyces cerevisiae, the bromodomains
appear on the TFIID-interacting protein Bdf1.
Do TAF1 and Gcn5 play redundant
role in yeast?
H3
Lysines:
Gcn5, and not TAF1,
is important for bulk
H3 acetylation levels.
Promoter vs. Non-promoters regions
• TAF1 is not a major H3K9, H3K14 acetyltransferase (HAT).
• Gcn5 is a HAT at most yeast promoters.
Acetylation and Transcription
A strong correlation
between H3 K9, K14 in
W.T and without
transcription (without
PolII).
A little REAL biology…
Acetylation of H4K8 is
dependant on Elp3, a HAT that
is associated with PolII during
elongation, while acetylation in
other sites in H4 might be less
PolII dependent.
Same
Acetylation
Decrease in
levelK8in
mutant
acetylation.
and WT.
Gcn5 and TAF1 contribution to Gene
Expression
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Recent studies: changes in gene expression for about
25% were observed only when both Gcn5 and TAF1
are eliminated.
If Gcn5 and TAF1 each make independent
contributions to transcription, the loss of both should
be equivalent to the multiplicative result (additive on a
log scale) of losing each individually.
If the two are functionally redundant, the double
mutant should result in an effect that is substantially
greater than the multiplicative effects of the individual
mutants.
Gcn5 and TAF1 contribution to Gene
Expression
TAF1 and Gcn5
make independent
contribution to
gene expression No redundancy
in TAF1 and
Gcn5 function.
TAF1 redundancy with other HATs
Sas3
Elp3
Hpa2
Their is no (or a very little)
Hat1
redundancy between TAF1 and
each
Esa1
of the 5 tested HATs.
Some Other HATs and Acetylation
Why there is no effect of any HAT
mutant on acetylation?
(i) Having highly selective gene
targets.
(ii) Having Lysine specificities other
than those tested.
(iii) Making transient contributions.
(iv) Being highly redundant with other
HATs.
TAF1 and Esa1
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Esa1 is the main
HAT for H4
acetylation of K5,
K8, K12.
Conclusions
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Taf1 and Gcn5 have no redundancy. In fact,
Taf1 may not be a HAT in yeast.
Transcription depends upon acetylation, but
acetylation doesn’t depend upon transcription.
Gcn5 and Esa1 have a major gene regulatory
HATs, but not Hat1, Elp3, Hpa2 and Sas3.
Genome-wide Map of Nucleosome
Acetylation and Methylation in Yeast
Dmitry K. Pokholok, Christopher T. Harbison, Stuart Levine,
Megan Cole, Nancy M. Hannett, Tong Ihn Lee, George W. Bell,
Kimberly Walker, P. Alex Rolfe, Elizabeth Herbolsheimer, Julia
Zeitlinger, Fran Lewitter, David K. Gifford, and Richard A.
Young
Cell, August 2005
Global Nucleosome Occupancy
Nucleosome occupancy
at the promoter of
CPA1, a gene encoding
an amino acidbiosynthetic enzyme.
A composite profile of
histone occupancy at
5,324 genes.
…Surprise!
Differential enrichment
of intergenic and genic
regions also occurred in
control
experiments lacking antibody.
After normalization to the
control:
No substantial differences in the
relative levels of intergenic vs. genic
DNA at the average gene, but 40% of
the promoters have lower level of
histones than their transcribed genes.
Is there a correlation between gene
expression and nucleosome occupancy?
The genes were divided into five classes of
transcription level.
Before Normalization
After Normalization
Nucleosome occupancy is reduced
maximally at the promoters of active
genes.
Histone Acetylation
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Two HATs were checked: Gcn5, which
acetylates H3K9 and H3K14, and Esa1, which
acetylates the four residues of H4.
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The acetylation level were measured relative to
the histones level.
Histone Acetylation – results:
Histone Acetylation – Conclusion:
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There is a positive association between Gcn5,
the modifications known to be catalyzed by
Gcn5, and transcriptional activity.
There is also a positive association between
Esa1, the modifications known to be catalyzed
by Esa1, and transcriptional activity, although
the association is not as strong as that observed
for Gcn5.
Three interesting trimethylation
patterns were observed
1
(Will be discusses later to details…)
2
3
Histone Methylation - conclusions
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There is a positive correlation between H3K4
trimethylation near the 5’ end of transcribed
gene and transcription rate.
There is also a positive correlation between
H3K36 trimethylation near the 3’ end of
transcribed gene, and transcription rate.
Somewhat correlation exists between H3K79
trimethylation and transcription rate.
http://web.wi.mit.edu/young/nucleosome/
Single-Nucleosome Mapping of
Histone Modifications
in S. cerevisiae
Chih Long Liu, Tommy Kaplan, Minkyu Kim, Stephen
Buratowski, Stuart L. Schreiber, Nir Friedman, Oliver J. Rando
PLoS Biology, October 2005
For the first time, high-resolution measurement
of histone modifications.
Acetylation of H4K16
Transcription start
site
Genes
Methylation of H3K4:
Gradient from trimethylion in 5’, to dimethylation, and then
to mono-metylation
on the 3’.
Transcriptionindependent
modifications
Transcriptiondependent
modifications
Nucleosomes
Correlation between modification
the matrix of correlations between the 12
modifications shows that there are two groups
of strongly correlated acetylations:
Tri-methylation of
H3K4 correlates
with the larger
group.
Mono- and dimethylation
orrelates with the
smaller group.
Principal Component Analysis PCA
81% of the variance in
histone modification
patterns is captured by
these two principal
components.
Nucleosomes have
continuous variation, both in
the total level of acetylation,
and in the relative ratio of
the two groups of
modifications, but they do
not show much complexity
beyond these two axes.
Principal Component Analysis PCA
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Component #1: Overall level of histone
modification.
Component #2: Relative levels of two groups
of histone modification - the “Transcription dependent modifications” that occur in 5’ to 3’
gradients over coding regions, and the
“Transcription - independent modifications”
that characterized by short hypo-acetyl domains
surrounding TSS.
Association Between Chromosomal
Location and Histone Modification
Promoter
Coding region
In the PCA plot, it is easy to distinguish
between the promoters nucleosomes and the
genic nucleosomes.
5’ end
Middle
3’ end
Moreover, it is possible to distinguish
between the promoters nucleosomes and
different coding regions (5’, middle and 3’).
Conclusion
Specific genomic regions are characterized by
distinct modification patterns, with little overlap in
modification types between the different regions.
But…
This correlation is imperfect, and it might be due to
the different expression level of the genes.
Is there a better correlation while separate genes
according to the PolII activity level?
Highly
Transcribed
Genes
Poorly
Transcribed
Genes
5’ coding region
nucleosomes
High PolII
activity level
Correct
classification:
75.4%
Medium
PolII activity
level
Low PolII
activity level
Is there a difference between TSS
proximal nucleosomes and TSS
distal nucleosomes?
TSS proximal Correct
nucleosomes classification:
72.8%
Modifications occur proximal
to transcribed gene contain
data about transcription level.
TSS distal Correct
nucleosomes classification:
58.4%
Modifications occur distal to
transcribed gene can’t help
predict transcription level.
Association Between Modifications
and Transcription Factor Domains
Modification Boundaries
Tri-methylation for
nucleosome N-1
Tri-methylation
for nucleosome N
Example of “punctate” nucleosome
Conclusions
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For the first time, modifications mapping in a
single-nucleosome resolution.
Two distinct groups of acetylation
modifications.
The modification patterns can be explained by
only two principle components.
There is no “Histone Code”.
Genome-wide patterns of
histone modifications
in yeast
Catherine B. Millar and Michael Grunstein
Nature, September 2006
Histone Modification Enzymes
Substrate preference:
 In yeast, all known HMT methylate only one substrate.
 HATs and HDACs act on several sites, but have
distinct preferences.
Enzyme targeting:
 Specific targeting – recruitment by a transcription
factor/repressor. This can result in a class-specific
modification.
 Global – function over large regions, irrespective of
promoters and coding regions, and without TFs. Global
targeting thought to be independent on transcription
status.
Histone Modification Enzymes –
cont.
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Some HATs function as subunits in a few
complexes, one of them has a speciofic targeting
and the other has a global targeting.
Some HATs have a large but limited region –
usually enzymes that are involved in
heterochromation formation.
No specific HMTs are known to interact with
TFs, but some do recruit specifically to coding
regions.
Histone Modification Enzymes
HAT
HDAC
HMT
HDM
Gradient of histone modifications in
Active Genes
Patterns of multiple histone
modification
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K-means clustering – identified groups of at
least 20 promoters that have a similar acetylation
state at 11 different sites, 53 clusters were
defined (kurdistany et al.).
The promoters within 55% of these clusters
share DNA-sequence motifs, whereas 26% bind
similar transcription factors, and 23% of clusters
contain promoters that lie upstream of genes
that belong to the same functional category.
Histone modifications in two
different clusters
Thanks for your listening,
and
!!!‫חנוכה שמח‬