Chromatin Modifications

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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

Text of Chromatin Modifications

  • Chromatin ModificationsVered Fishbain

    Reading Group in Computational Molecular Biology

    21/12/2006

  • Some DefinitionsChromatin 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 Chromatin modifications are covalent modifications that can effect transcription.AcetylationMethylationPhosphorylationUbiquitinationSumoylationAdenosine-diphosphate ribosylation

  • Histone AcetylationAssociated 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.

  • Histone MethylationCharacterized 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.

  • Research Challenges

    Absence of sufficientverified data.Contradictory evidences.The available data is in a low resolution.

  • About immunoprecipitation.

  • OutlineTAF1 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

  • 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.

  • Do TAF1 and Gcn5 play redundant role in yeast?Gcn5, and not TAF1, is important for bulk H3 acetylation levels.H3 Lysines:

  • Promoter vs. Non-promoters regionsTAF1 is not a major H3K9, H3K14 acetyltransferase (HAT).Gcn5 is a HAT at most yeast promoters.

  • Acetylation and TranscriptionA strong correlation between H3 K9, K14 in W.T and without transcription (without PolII).A little REAL biologySame 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 contribution to Gene ExpressionRecent 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 ExpressionTAF1 and Gcn5 make independent contribution to gene expression - No redundancy in TAF1 and Gcn5 function.

  • TAF1 redundancy with other HATsSas3Elp3Hpa2Hat1Esa1Their is no (or a very little) redundancy between TAF1 and each of the 5 tested HATs.

  • Some Other HATs and AcetylationWhy 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 Esa1Esa1 is the main HAT for H4 acetylation of K5, K8, K12.

  • 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?

  • The current model

  • Acetylated H4 tails primarily bind Bdf1 which generally (but not always) bind TFIID.

  • ConclusionsTaf1 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.

  • ConclusionsTaf1 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.

  • 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

  • Global Nucleosome OccupancyNucleosome 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 enrichmentof 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 gene expression and nucleosome occupancy?The genes were divided into five classes of transcription level.Before NormalizationAfter NormalizationNucleosome occupancy is reduced maximally at the promoters of active genes.

  • Histone AcetylationTwo 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.

  • Histone Acetylation results:

  • 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.

  • ?

  • Three interesting trimethylation patterns were observed(Will be discusses later to details) 1

  • 2 3

  • Histone Methylation - conclusionsThere 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.

  • 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.

  • Acetylation of H4K16Transcription start siteGenes

  • Methylation of H3K4:Gradient from tri-methylion in 5, to di-methylation, and then to mono-metylation on the 3.Transcription-independent modificationsTranscription-dependent modifications

  • Nucleosomes

  • Correlation between modificationthe 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.

  • Principal Component Analysis -PCA81% 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 -PCAComponent #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 Tr