chromatin modifications
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Chromatin Chromatin ModificationsModifications
Vered FishbainVered Fishbain
Reading Group in Computational Molecular Reading Group in Computational Molecular BiologyBiology
21/12/200621/12/2006
Some Definitions…Some Definitions… ChromatinChromatin is the complex of DNA and proteins is the complex of DNA and proteins
found inside the nuclei of eukaryotic cells. found inside the nuclei of eukaryotic cells. NucleosomesNucleosomes are the fundamental repeating are the fundamental repeating
subunits of all eukaryotic chromatin. They are made subunits of all eukaryotic chromatin. They are made up of DNA and protein core, which is the histone up of DNA and protein core, which is the histone core.core.
The The histone corehistone core is composed by two copies of the is composed by two copies of the following set of proteins, called following set of proteins, called histoneshistones::H2A, H2B, H3 and H4.H2A, H2B, H3 and H4.
147 bp in each nucleosome.147 bp in each nucleosome. HeterochromatinHeterochromatin is condensed chromatin, is condensed chromatin,
includes inactive genes and untranscribed regions includes inactive genes and untranscribed regions (like the centromer).(like the centromer).
EuchromatinEuchromatin is non-condensed chromatin, includes is non-condensed chromatin, includes active and repressed genes.active and repressed genes.
The Histone CoreThe Histone Core
Chromatin ModificationsChromatin Modifications
Chromatin modificationsChromatin modifications are are covalent modifications that can covalent modifications that can effect transcription.effect transcription.
AcetylationAcetylation MethylationMethylation PhosphorylationPhosphorylation UbiquitinationUbiquitination SumoylationSumoylation Adenosine-diphosphate ribosylationAdenosine-diphosphate ribosylation
Histone AcetylationHistone Acetylation
Associated with transcription activation.Associated with transcription activation. Influence gene expression in (at least) two Influence gene expression in (at least) two
ways:ways:
1.1. Neutralize Lysine’s positive charge, which Neutralize Lysine’s positive charge, which can weaken DNA-histone contacts, or histone-can weaken DNA-histone contacts, or histone-histone contacts.histone contacts.
2.2. Acetyl-Lysine is bound by a specific protein Acetyl-Lysine is bound by a specific protein domain that is found in many transcription domain that is found in many transcription factors and calls factors and calls bromodomainbromodomain..
Rapidly reversible, and can turn over rapidly Rapidly reversible, and can turn over rapidly in vivoin vivo..
Histone MethylationHistone Methylation Characterized mainly for histone 3-lysin 4 Characterized mainly for histone 3-lysin 4
(H3K4).(H3K4). The Lysine can be mono-, di- or tri-The Lysine can be mono-, di- or tri-
methylated.methylated. Doesn’t change the Lysine charge Doesn’t change the Lysine charge
(naturally positive). (naturally positive). methyl-Lysine can be bound by a methyl-methyl-Lysine can be bound by a methyl-
lysin binding domain, such as lysin binding domain, such as chromodomainchromodomain, , WD40WD40 domaindomain, , Tudor Tudor domaindomain, etc., etc.
Long-lived.Long-lived.
Research ChallengesResearch Challenges
Absence of sufficient verified data.Absence of sufficient verified data. Contradictory evidences.Contradictory evidences. The available data is in a low The available data is in a low
resolution.resolution.
OutlineOutline TAF1 as an acetyltransferase (HAT).TAF1 as an acetyltransferase (HAT).- TAF1 and Gcn5 – is there a redundancy?TAF1 and Gcn5 – is there a redundancy?- TAF1 and other HATs in yeast (Durant and TAF1 and other HATs in yeast (Durant and
Pugh)Pugh).. Acetylation and methylation across Acetylation and methylation across
promoters and ORFs (Pokholokpromoters and ORFs (Pokholok et al.) et al.) High resolution mapping of acetylation and High resolution mapping of acetylation and
methylation (Liu methylation (Liu et al.et al.))- Identifying two major groups with similar Identifying two major groups with similar
modification patterns within.modification patterns within. Summary (Millar and Grunstein)Summary (Millar and Grunstein)
Genome-Wide Genome-Wide Relationships between Relationships between
TAF1 and Histone TAF1 and Histone Acetyltransferases in Acetyltransferases in
Saccharomyces cerevisiaeSaccharomyces cerevisiae
Melissa Durant and B. Franklin Melissa Durant and B. Franklin Pugh Pugh
Molecular and Cellular Biology, Molecular and Cellular Biology,
April 2006April 2006
The transcription machinery assembles The transcription machinery assembles at promoters via two complexes, TFIID at promoters via two complexes, TFIID and SAGA, which have a compensatory and SAGA, which have a compensatory function (Inna’s lecture…).function (Inna’s lecture…).
Both complexes contain subunits (TAF1 Both complexes contain subunits (TAF1 and Gcn5) that harbor bromodomain and Gcn5) that harbor bromodomain and acetyltransferase (HAT) activity.and acetyltransferase (HAT) activity.
In In Saccharomyces cerevisiaeSaccharomyces cerevisiae, the , the bromodomains appear on the TFIID-bromodomains appear on the TFIID-interacting protein Bdf1.interacting protein Bdf1.
Do TAF1 and Gcn5 play Do TAF1 and Gcn5 play redundant role in yeast?redundant role in yeast?
Gcn5, and not TAF1, is important for bulk H3 acetylation levels.
H3 Lysines:
Promoter vs. Non-Promoter vs. Non-promoters regionspromoters regions
• TAF1 is not a major H3K9, H3K14 acetyltransferase (HAT).
• Gcn5 is a HAT at most yeast promoters.
Acetylation and Acetylation and TranscriptionTranscription
A strong correlation between H3 K9, K14 in W.T and without transcription (without PolII).
A little REAL biology…
Same Acetylation level
in mutant and WT.
Decrease in K8
acetylation.
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.
Gcn5 and TAF1 Gcn5 and TAF1 contribution to Gene contribution to Gene
ExpressionExpression Recent studies: changes in gene expression Recent studies: changes in gene expression
for about 25% were observed only when for about 25% were observed only when bothboth Gcn5 and TAF1 are eliminated.Gcn5 and TAF1 are eliminated.
If Gcn5 and TAF1 each make independent If Gcn5 and TAF1 each make independent contributions to transcription, the loss of both contributions to transcription, the loss of both should be equivalent to the multiplicative should be equivalent to the multiplicative result (additive on a log scale) of losing each result (additive on a log scale) of losing each individually.individually.
If the two are functionally redundant, the If the two are functionally redundant, the double mutant should result in an effect that double mutant should result in an effect that is substantially greater than the multiplicative is substantially greater than the multiplicative effects of the individual mutants.effects of the individual mutants.
Gcn5 and TAF1 Gcn5 and TAF1 contribution to Gene contribution to Gene
ExpressionExpressionTAF1 and Gcn5 make independent contribution to gene expression - No redundancy in TAF1 and Gcn5 function.
TAF1 redundancy with TAF1 redundancy with other HATsother HATs
Sas3Elp3
Hpa2Hat1
Esa1
Their is no (or a very little) redundancy between TAF1
and each of the 5 tested HATs.
Some Other HATs and Some Other HATs and AcetylationAcetylation
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 Esa1TAF1 and Esa1Esa1 is the main HAT for H4 acetylation of K5, K8, K12.
ConclusionsConclusions
Taf1 and Gcn5 have no redundancy. Taf1 and Gcn5 have no redundancy. In fact, Taf1 may not be a HAT in In fact, Taf1 may not be a HAT in yeast.yeast.
Transcription depends upon Transcription depends upon acetylation, but acetylation doesn’t acetylation, but acetylation doesn’t depend upon transcription.depend upon transcription.
Gcn5 and Esa1 have a major gene Gcn5 and Esa1 have a major gene regulatory HATs, but not Hat1, Elp3, regulatory HATs, but not Hat1, Elp3, Hpa2 and Sas3.Hpa2 and Sas3.
Genome-wide Map of Genome-wide Map of NucleosomeNucleosome
Acetylation and Acetylation and Methylation in YeastMethylation in Yeast
Dmitry K. Pokholok, Christopher T. Dmitry K. Pokholok, Christopher T. Harbison, Stuart Levine, Megan Cole, Harbison, Stuart Levine, Megan Cole,
Nancy M. Hannett, Tong Ihn Lee, George W. Nancy M. Hannett, Tong Ihn Lee, George W. Bell, Kimberly Walker, P. Alex Rolfe, Bell, Kimberly Walker, P. Alex Rolfe,
Elizabeth Herbolsheimer, Julia Zeitlinger, Elizabeth Herbolsheimer, Julia Zeitlinger, Fran Lewitter, David K. Gifford, and Fran Lewitter, David K. Gifford, and
Richard A. YoungRichard A. YoungCell, August 2005Cell, August 2005
Global Nucleosome Global Nucleosome OccupancyOccupancy
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.
…Surprise! Differential enrichment
of intergenic and genic regions also occurred in controlexperiments 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 Is there a correlation between gene expression and gene expression and
nucleosome occupancy?nucleosome occupancy?The genes were divided into five The genes were divided into five classes of transcription level.classes of transcription level.
Before Normalization After Normalization
Nucleosome occupancy is reduced maximally at the promoters of active
genes.
Histone AcetylationHistone Acetylation
Two HATs were checked: Gcn5, Two HATs were checked: Gcn5, which acetylates H3K9 and H3K14, which acetylates H3K9 and H3K14, and Esa1, which acetylates the four and Esa1, which acetylates the four residues of H4.residues of H4.
The acetylation level were measured The acetylation level were measured relative to the histones level.relative to the histones level.
Histone Acetylation – Histone Acetylation – results:results:
Histone Acetylation – Histone Acetylation – Conclusion:Conclusion:
There is a positive association between There is a positive association between Gcn5, the modifications known to be Gcn5, the modifications known to be catalyzed by Gcn5, and transcriptional catalyzed by Gcn5, and transcriptional activity.activity.
There is also a positive association There is also a positive association between Esa1, the modifications known between Esa1, the modifications known to be catalyzed by Esa1, and to be catalyzed by Esa1, and transcriptional activity, although the transcriptional activity, although the association is not as strong as that association is not as strong as that observed for Gcn5.observed for Gcn5.
Three interesting Three interesting trimethylation patterns trimethylation patterns
were observedwere observed
(Will be discusses later to details…)
11
22
33
Histone Methylation - Histone Methylation - conclusionsconclusions
There is a positive correlation between There is a positive correlation between H3K4 trimethylation near the 5’ end of H3K4 trimethylation near the 5’ end of transcribed gene and transcription rate.transcribed gene and transcription rate.
There is also a positive correlation There is also a positive correlation between H3K36 trimethylation near the between H3K36 trimethylation near the 3’ end of transcribed gene, and 3’ end of transcribed gene, and transcription rate.transcription rate.
Somewhat correlation exists between Somewhat correlation exists between H3K79 trimethylation and transcription H3K79 trimethylation and transcription rate.rate.
http://web.wi.mit.edu/young/http://web.wi.mit.edu/young/nucleosome/nucleosome/
Single-Nucleosome Single-Nucleosome Mapping of Histone Mapping of Histone
Modifications Modifications in in S. cerevisiaeS. cerevisiae
Chih Long Liu, Tommy Kaplan, Minkyu Chih Long Liu, Tommy Kaplan, Minkyu Kim, Stephen Buratowski, Stuart L. Kim, Stephen Buratowski, Stuart L.
Schreiber, Nir Friedman, Oliver J. RandoSchreiber, Nir Friedman, Oliver J. Rando
PLoS Biology, October 2005PLoS Biology, October 2005
For the first time, high-resolution For the first time, high-resolution measurement of histone measurement of histone
modifications.modifications.
Acetylation of H4K16Acetylation of H4K16
Transcription start site
Genes
Methylation of H3K4:
Gradient from tri-methylion in
5’, to di-methylation, and
then to mono-metylation on
the 3’.
Transcription-independent modifications
Transcription-dependent
modifications
Nucleosomes
Correlation between Correlation between modificationmodification
the matrix of correlations between the matrix of correlations between the 12 modifications shows that the 12 modifications shows that there are two groups of strongly there are two groups of strongly correlated acetylations:correlated acetylations:
Tri-methylation Tri-methylation of H3K4 of H3K4 correlates with correlates with the larger the larger group.group.Mono- and di-Mono- and di-methylation methylation orrelates with orrelates with the smaller the smaller group.group.
Principal Component Principal Component Analysis -Analysis -
PCAPCA81% 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 Principal Component Analysis -Analysis -
PCAPCA Component #1: Overall level of histone Component #1: Overall level of histone
modification.modification. Component #2: Relative levels of two Component #2: Relative levels of two
groups of histone modification - the groups of histone modification - the “Transcription -dependent “Transcription -dependent modifications” that occur in 5’ to 3’ modifications” that occur in 5’ to 3’ gradients over coding regions, and the gradients over coding regions, and the “Transcription - independent “Transcription - independent modifications” that characterized by modifications” that characterized by short hypo-acetyl domains surrounding short hypo-acetyl domains surrounding TSS.TSS.
Association Between Association Between Chromosomal Location and Chromosomal Location and
Histone ModificationHistone Modification
In the PCA plot, it is easy to distinguish between the
promoters nucleosomes and the genic nucleosomes.
Promoter
Coding region
Moreover, it is possible to distinguish between the
promoters nucleosomes and different coding regions (5’,
middle and 3’).
5’ end
Middle
3’ end
ConclusionConclusion
Specific genomic regions are characterized Specific genomic regions are characterized by distinct modification patterns, with little by distinct modification patterns, with little overlap in modification types between the overlap in modification types between the different regions. different regions.
But…But…
This correlation is imperfect, and it might This correlation is imperfect, and it might be due to the different expression level of be due to the different expression level of the genes. the genes.
Is there a better correlation while separate Is there a better correlation while separate genes according to the PolII activity level?genes according to the PolII activity level?
Highly Transcribed Genes
Poorly Transcribed Genes
High PolII activity
level
Medium PolII
activity level
Low PolII activity
level
5’ coding region
nucleosomes
Correct classification: 75.4%
Is there a difference Is there a difference between TSS proximal between TSS proximal
nucleosomes and TSS distal nucleosomes and TSS distal nucleosomes? nucleosomes?
TSS proximal nucleosomes
TSS distal nucleosomes
Modifications occur proximal to
transcribed gene contain data about transcription level.
Modifications occur distal to transcribed
gene can’t help predict transcription
level.
Correct classification: 72.8%
Correct classification: 58.4%
Association Between Association Between Modifications and Modifications and
Transcription Factor Transcription Factor DomainsDomains
Modification BoundariesModification Boundaries
Tri-methylation for nucleosome N
Tri-methylation for nucleosome N-1
Example of “punctate” Example of “punctate” nucleosomenucleosome
ConclusionsConclusions
For the first time, modifications For the first time, modifications mapping in a single-nucleosome mapping in a single-nucleosome resolution.resolution.
Two distinct groups of acetylation Two distinct groups of acetylation modifications. modifications.
The modification patterns can be The modification patterns can be explained by only two principle explained by only two principle components.components.
There is no “Histone Code”.There is no “Histone Code”.
Genome-wide Genome-wide patterns of histone patterns of histone
modifications modifications in yeastin yeast
Catherine B. Millar and Catherine B. Millar and Michael GrunsteinMichael Grunstein
Nature, September 2006Nature, September 2006
Histone Modification Histone Modification EnzymesEnzymes
Substrate preference:Substrate preference: In yeast, all known HMT methylate only one In yeast, all known HMT methylate only one
substrate.substrate. HATs and HDACs act on several sites, but have HATs and HDACs act on several sites, but have
distinct preferences.distinct preferences.Enzyme targeting:Enzyme targeting: Specific targeting – recruitment by a Specific targeting – recruitment by a
transcription factor/repressor. This can result in a transcription factor/repressor. This can result in a class-specific modification.class-specific modification.
Global – function over large regions, irrespective Global – function over large regions, irrespective of promoters and coding regions, and without TFs. of promoters and coding regions, and without TFs. Global targeting thought to be independent on Global targeting thought to be independent on transcription status.transcription status.
Histone Modification Histone Modification Enzymes – cont.Enzymes – cont.
Some HATs function as subunits in a few Some HATs function as subunits in a few complexes, one of them has a speciofic complexes, one of them has a speciofic targeting and the other has a global targeting and the other has a global targeting.targeting.
Some HATs have a large but limited Some HATs have a large but limited region – usually enzymes that are region – usually enzymes that are involved in heterochromation formation.involved in heterochromation formation.
No specific HMTs are known to interact No specific HMTs are known to interact with TFs, but some do recruit with TFs, but some do recruit specifically to coding regions.specifically to coding regions.
Histone Modification Histone Modification EnzymesEnzymes
HAT
HDAC
HMT
HDM
Gradient of histone Gradient of histone modifications in Active modifications in Active
GenesGenes
Patterns of multiple histone Patterns of multiple histone modificationmodification
K-means clustering – identified groups of K-means clustering – identified groups of at least 20 promoters that have a similar at least 20 promoters that have a similar acetylation state at 11 different sites, 53 acetylation state at 11 different sites, 53 clusters were defined (kurdistany clusters were defined (kurdistany et alet al.). .).
The promoters within 55% of these The promoters within 55% of these clusters share DNA-sequence motifs, clusters share DNA-sequence motifs, whereas 26% bind similar transcription whereas 26% bind similar transcription factors, and 23% of clusters contain factors, and 23% of clusters contain promoters that lie upstream of genes promoters that lie upstream of genes that belong to the same functional that belong to the same functional category.category.
Histone modifications in Histone modifications in two different clusterstwo different clusters
Thanks for your listening,Thanks for your listening,andand
!!!!!!חנוכה שמחחנוכה שמח
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