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Chromatin Modifications

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.

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Chromatin Modifications

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

  2. 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.

  3. The Histone Core

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

  5. Histone Acetylation • 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.

  6. Histone Methylation • 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, WD40domain, Tudor domain, etc. • Long-lived.

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

  8. 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)

  9. Genome-Wide Relationships between TAF1 and Histone Acetyltransferases in Saccharomyces cerevisiae Melissa Durant and B. Franklin Pugh Molecular and Cellular Biology, April 2006

  10. 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.

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

  12. Promoter vs. Non-promoters regions • TAF1 is not a major H3K9, H3K14 acetyltransferase (HAT). • Gcn5 is a HAT at most yeast promoters.

  13. 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.

  14. 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.

  15. Gcn5 and TAF1 contribution to Gene Expression TAF1 and Gcn5 make independent contribution to gene expression - No redundancy in TAF1 and Gcn5 function.

  16. 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

  17. 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.

  18. TAF1 and Esa1 Esa1 is the main HAT for H4 acetylation of K5, K8, K12. 

  19. Conclusions • 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.

  20. 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

  21. 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.

  22. …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.

  23. 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.

  24. 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.

  25. Histone Acetylation – results:

  26. 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.

  27. Three interesting trimethylation patterns were observed 1 (Will be discusses later to details…)

  28. 2 3

  29. 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.

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

  31. 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

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

  33. Acetylation of H4K16 Transcription start site Genes

  34. 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

  35. Nucleosomes

  36. 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.

  37. 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.

  38. 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.

  39. Association Between Chromosomal Location and Histone Modification

  40. Promoter Coding region In the PCA plot, it is easy to distinguish between the promoters nucleosomes and the genic nucleosomes.

  41. 5’ end Middle 3’ end Moreover, it is possible to distinguish between the promoters nucleosomes and different coding regions (5’, middle and 3’).

  42. 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?

  43. Highly Transcribed Genes Poorly Transcribed Genes

  44. 5’ coding region nucleosomes High PolII activity level Correct classification: 75.4% Medium PolII activity level Low PolII activity level

  45. 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 can’t help predict transcription level.

  46. Association Between Modifications and Transcription Factor Domains

  47. Modification Boundaries Tri-methylation for nucleosome N-1 Tri-methylation for nucleosome N

  48. Example of “punctate” nucleosome

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