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

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Chromatin Modifications. Vered Fishbain Reading Group in Computational Molecular Biology 21/12/2006. Some Definitions…. Chromatin is the complex of DNA and proteins found inside the nuclei of eukaryotic cells. - PowerPoint PPT Presentation

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Page 1: Chromatin Modifications

Chromatin Chromatin ModificationsModifications

Vered FishbainVered Fishbain

Reading Group in Computational Molecular Reading Group in Computational Molecular BiologyBiology

21/12/200621/12/2006

Page 2: Chromatin Modifications

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.

Page 3: Chromatin Modifications
Page 4: Chromatin Modifications

The Histone CoreThe Histone Core

Page 5: Chromatin Modifications

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

Page 6: Chromatin Modifications

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..

Page 7: Chromatin Modifications

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.

Page 8: Chromatin Modifications

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.

Page 9: Chromatin Modifications

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)

Page 10: Chromatin Modifications

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

Page 11: Chromatin Modifications

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.

Page 12: Chromatin Modifications
Page 13: Chromatin Modifications

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:

Page 14: Chromatin Modifications

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.

Page 15: Chromatin Modifications

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.

Page 16: Chromatin Modifications

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.

Page 17: Chromatin Modifications

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.

Page 18: Chromatin Modifications

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.

Page 19: Chromatin Modifications

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.

Page 20: Chromatin Modifications

TAF1 and Esa1TAF1 and Esa1Esa1 is the main HAT for H4 acetylation of K5, K8, K12.

Page 21: Chromatin Modifications

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.

Page 22: Chromatin Modifications

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

Page 23: Chromatin Modifications

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.

Page 24: Chromatin Modifications

…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.

Page 25: Chromatin Modifications

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.

Page 26: Chromatin Modifications

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.

Page 27: Chromatin Modifications

Histone Acetylation – Histone Acetylation – results:results:

Page 28: Chromatin Modifications

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.

Page 29: Chromatin Modifications

Three interesting Three interesting trimethylation patterns trimethylation patterns

were observedwere observed

(Will be discusses later to details…)

11

Page 30: Chromatin Modifications

22

33

Page 31: Chromatin Modifications

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.

Page 32: Chromatin Modifications

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

Page 33: Chromatin Modifications

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

Page 34: Chromatin Modifications

For the first time, high-resolution For the first time, high-resolution measurement of histone measurement of histone

modifications.modifications.

Page 35: Chromatin Modifications

Acetylation of H4K16Acetylation of H4K16

Transcription start site

Genes

Page 36: Chromatin Modifications

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

Page 37: Chromatin Modifications

Nucleosomes

Page 38: Chromatin Modifications

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.

Page 39: Chromatin Modifications

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.

Page 40: Chromatin Modifications

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.

Page 41: Chromatin Modifications

Association Between Association Between Chromosomal Location and Chromosomal Location and

Histone ModificationHistone Modification

Page 42: Chromatin Modifications

In the PCA plot, it is easy to distinguish between the

promoters nucleosomes and the genic nucleosomes.

Promoter

Coding region

Page 43: Chromatin Modifications

Moreover, it is possible to distinguish between the

promoters nucleosomes and different coding regions (5’,

middle and 3’).

5’ end

Middle

3’ end

Page 44: Chromatin Modifications

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?

Page 45: Chromatin Modifications

Highly Transcribed Genes

Poorly Transcribed Genes

Page 46: Chromatin Modifications

High PolII activity

level

Medium PolII

activity level

Low PolII activity

level

5’ coding region

nucleosomes

Correct classification: 75.4%

Page 47: Chromatin Modifications

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%

Page 48: Chromatin Modifications

Association Between Association Between Modifications and Modifications and

Transcription Factor Transcription Factor DomainsDomains

Page 49: Chromatin Modifications

Modification BoundariesModification Boundaries

Tri-methylation for nucleosome N

Tri-methylation for nucleosome N-1

Page 50: Chromatin Modifications

Example of “punctate” Example of “punctate” nucleosomenucleosome

Page 51: Chromatin Modifications

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”.

Page 52: Chromatin Modifications

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

Page 53: Chromatin Modifications

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.

Page 54: Chromatin Modifications

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.

Page 55: Chromatin Modifications

Histone Modification Histone Modification EnzymesEnzymes

HAT

HDAC

HMT

HDM

Page 56: Chromatin Modifications

Gradient of histone Gradient of histone modifications in Active modifications in Active

GenesGenes

Page 57: Chromatin Modifications

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.

Page 58: Chromatin Modifications

Histone modifications in Histone modifications in two different clusterstwo different clusters

Page 59: Chromatin Modifications
Page 60: Chromatin Modifications

Thanks for your listening,Thanks for your listening,andand

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