Chromatin Modifications

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Chromatin Modifications

• Vered Fishbain
• Reading Group in Computational Molecular Biology
• 21/12/2006

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Some Definitions

• 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.

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The Histone Core
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Chromatin Modifications

• Chromatin modifications are covalent
  modifications that can effect transcription.
• Acetylation
• Methylation
• Phosphorylation
• Ubiquitination
• Sumoylation
• Adenosine-diphosphate ribosylation

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Histone Acetylation

• Associated with transcription activation.
• Influence gene expression in (at least) two ways
• Neutralize Lysines 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.

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Histone Methylation

• Characterized mainly for histone 3-lysin 4
  (H3K4).
• The Lysine can be mono-, di- or tri-methylated.
• Doesnt 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.

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Research Challenges

• Absence of sufficient verified data.
• Contradictory evidences.
• The available data is in a low resolution.

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• About immunoprecipitation.

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Outline

• 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)

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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 (Innas
  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.

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Do TAF1 and Gcn5 play redundant role in yeast?

H3 Lysines
Gcn5, and not TAF1, is important for bulk H3
acetylation levels.
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Promoter vs. Non-promoters regions

• TAF1 is not a major H3K9, H3K14 acetyltransferase
  (HAT).
• Gcn5 is a HAT at most yeast promoters.

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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 level in mutant and WT.
Decrease in K8 acetylation.
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Gcn5 and TAF1 contribution to Gene Expression

• 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.

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Gcn5 and TAF1 contribution to Gene Expression
TAF1 and Gcn5 make independent contribution to
gene expression - No redundancy in TAF1 and Gcn5
function.
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TAF1 redundancy with other HATs
Sas3
Elp3
Hpa2
Their is no (or a very little) redundancy between
TAF1 and each of the 5 tested HATs.
Hat1
Esa1
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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.
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TAF1 and Esa1
Esa1 is the main HAT for H4 acetylation of K5,
K8, K12.
?
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What is the relationship between TFIID, TAF1,
Bdf1 and Esa?

• Genes whose expression was significantly
  dependent on Esa1 or Taf1 were clustered by
  K-means into eight groups.

Can the current model explain this behavior?
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The current model
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Acetylated H4 tails primarily bind Bdf1 which
generally (but not always) bind TFIID.
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Conclusions

• Taf1 and Gcn5 have no redundancy. In fact, Taf1
  may not be a HAT in yeast.
• Transcription depends upon acetylation, but
  acetylation doesnt depend upon transcription.
• Gcn5 and Esa1 have a major gene regulatory HATs,
  but not Hat1, Elp3, Hpa2 and Sas3.
• A model is suggested to define the mechanism
  linking Esa1, H4 acetylation, Bdf1 binding and
  TFIID recruitment.

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Conclusions

• Taf1 and Gcn5 have no redundancy. In fact, Taf1
  may not be a HAT in yeast.
• Transcription depends upon acetylation, but
  acetylation doesnt depend upon transcription.
• Gcn5 and Esa1 have a major gene regulatory HATs,
  but not Hat1, Elp3, Hpa2 and Sas3.

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Genome-wide Map of NucleosomeAcetylation 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
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Global Nucleosome Occupancy
Nucleosome occupancy at the promoter of CPA1, a
gene encoding an amino acid-biosynthetic enzyme.
A composite profile of histone occupancy at 5,324
genes.
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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.
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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.
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Histone Acetylation

• Two HATs were checked Gcn5, which acetylates
  H3K9 and H3K14, and Esa1, which acetylates the
  four residues of H4.
• The acetylation level were measured relative to
  the histones level.

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Histone Acetylation results
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Histone Acetylation Conclusion

• 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.

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Three interesting trimethylation patterns were
observed
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(Will be discusses later to details)
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3
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Histone Methylation - conclusions

• 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.

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http//web.wi.mit.edu/young/nucleosome/
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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

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• For the first time, high-resolution measurement
  of histone modifications.

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We can already see that

• Histone modifications generally occur in broad
  range.
• Modifications were generally homogeneous for all
  the probes within a given nucleosome.
• Correlations could be observed between a
  nucleosomes position relative to coding regions
  and its modification pattern.
• Low acetylation on heterochromatic regions.

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Acetylation of H4K16
Transcription start site
Genes
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Methylation of H3K4 Gradient from tri-methylion
in 5, to di-methylation, and then to
mono-metylation on the 3.
Transcription-dependent modifications
Transcription-independent modifications
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Nucleosomes
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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 di-methylation orrelates
with the smaller group.
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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.
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Principal Component Analysis -PCA

• 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.

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Association Between Chromosomal Location and
Histone Modification
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Promoter Coding region
In the PCA plot, it is easy to distinguish
between the promoters nucleosomes and the genic
nucleosomes.
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5 end Middle 3 end
Moreover, it is possible to distinguish between
the promoters nucleosomes and different coding
regions (5, middle and 3).
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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?

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Highly Transcribed Genes
Poorly Transcribed Genes
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• An arbitrary gene

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5 coding region nucleosomes
High PolII activity level
Correct classification 75.4
Medium PolII activity level
Low PolII activity level
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Is there a difference between TSS proximal
nucleosomes and TSS distal nucleosomes?
TSS proximal nucleosomes
TSS distal nucleosomes
Correct classification 58.4
Correct classification 72.8
Modifications occur proximal to transcribed gene
contain data about transcription level.
Modifications occur distal to transcribed gene
cant help predict transcription level.
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Association Between Modifications and
Transcription Factor Domains
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Modification Boundaries
Tri-methylation for nucleosome N-1
Tri-methylation for nucleosome N
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Example of punctate nucleosome
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Conclusions

• 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.

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Genome-wide patterns of histone modifications in
yeast

• Catherine B. Millar and Michael Grunstein
• Nature, September 2006

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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.

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Histone Modification Enzymes cont.

• 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.

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Histone Modification Enzymes
HAT HDAC HMT HDM
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Distinct patterns in chromosomal locations

• Heterochromatin no acetylation, methylation.
• Centromeric locations - usually surrounded by
  heterochromatin, but have a distinct modification
  pattern and also distinct from euchromatin
  modification patterns dimethylation on H3K4 and
  hypoacetylation on h3K9, H4K5, H4K8, H4K12 and
  H4K16.
• H3 is replaced by the histone variant cenH3, with
  present no modifications on yeast.
• CEN sequence

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• HAST domains and HZADs
• Hda1-affected subtelomeric domain (HAST) - groups
  of contiguous genes that are deacetylated by the
  HDAC Hda1, and are located in the subtelomeric
  euchromatin.
• Htz1-activated domain (HZAD) - Adjacent
  subtelomeric genes, the expression of which is
  downregulated in the absence of the H2A variant
  Htz1.

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Gradient of histone modifications in Active Genes
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Patterns of multiple histone modification

• 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.

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Histone modifications in two different clusters
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Thanks for your listening,and????? ???!!!