1. Histone Modifications

2. Outline • Overview of histone modifications: • Types of modifications and modifiers • General roles of modifications • Techniques used to study histone modifications • Specific modifications (acetylation, methylation, etc): • Residues/positions that are frequently modified • Enzymes that add/remove the modification • Biological roles • Cross-talk between histone modifications • Summary • A histonecode?

3. Quick Overview Modifications and modifiers General roles Techniques

4. Types of Histone Modifications Bhaumik, Smith, and Shilatifard, 2007.

5. Features of Histone Modifications SUMO Methyl Acetyl Phospho Ubiquitin Cell, 111:285-91, Nov. 1, 2002 • Covalently attached groups (usually to histone tails) • Reversible and Dynamic • Enzymes that add/remove modification • Signals • Have diverse biological functions

6. Features of Histone Modifications • Small vs. Large groups • One or up to three groups per residue Ub = ~8.5 kDa H4 = 14 kDa Jason L J M et al. J. Biol. Chem. 2005

7. Histone Modifications and Modifers • Writers: enzymes that add a mark • Readers: proteins that bind to and “interpret” the mark • Erasers: enzymes that remove a mark Tarakhovsky, A., Nature Immunology, 2010.

8. Histone Modifications and Modifers • Others: Sumoylation (Lysine), ADP Ribosylation (Glutamate)

9. Histone Modifiers • Do not bind to DNA themselves • Can be recruited by: • Histone modifications (through chromodomains, bromodomains, etc.) • Transcription factors • RNA (fission yeast, mammals, plants) • DNA damage • Act as transcriptional co-regulators • Enhance activities of transcriptional repressors or activators • Co-repressor: ex. HDACs • Co-activator: ex. HATs

10. General Roles of Histone Modifications • Intrinsic • Single nucleosome changes • Example: histone variant specific modifications (H2AX) • Extrinsic • Chromatin organization: nucleosome/nucleosome interactions • Alter chromatin packaging, electrostatic charge • Example: H4 acetylation • Effector-mediated • Recruitment of other proteins to the chromatinvia • Bromodomains bind acetylation • Chromo-like royal domains (chromo, tudor, MBT) and PHD bind methylation • 14-3-3 proteins bind phosphorylation • Prevent binding: H3S10P prevents Heterochromatin • Protein 1 binding Kouzarides, Cell 2007

11. General Roles of Histone Modifications Gene Regulation DNA Damage Moggs and Orphanides, Toxicological Sciences, 2004. Wade P A Hum. Mol. Genet. 2001.

12. General Roles of Histone Modifications Chromatin Condensation Spermatogenesis Kruhlak M J et al. J. Biol. Chem. 2001.

13. Techniques to Study Histone Modifications • Chromatin immunoprecipitation (ChIP): Strategy for localizing histone marks • ChIP-qPCR: if you know the target gene • ChIP-chip, ChIP-seq, or other genome-wide techniques: unbiased • Limitations: • Antibody specificity • Inherent biases of localization methods • Mass Spectrometry: • Requires digestion of histones

14. ChIP-chip

15. ChIP-seq Local amplification Massively parallel sequencing

16. ChIP-seq “Depth”: the number of mapped sequence tags Genome “Coverage”: how much of the genome the reads can be mapped to

17. ChIP-seq vs. ChIP-chip Mikkelsen et al., 2007

18. Outline • Overview of histone modifications: • Types of modifications and modifiers • General roles of modifications • Techniques used to study histone modifications • Specific modifications (acetylation, methylation, etc): • Residues/positions that are frequently modified • Enzymes that add/remove the modification • Biological roles • Cross-talk between histone modifications • Summary • A histonecode?

19. Specific Histone Modifications Positions that are modified Modifying enzymes Functions of the modification

20. Lysine Acetylation Bhaumik, Smith, and Shilatifard, 2007.

21. Acetylation • Many lysine residues can be acetylated • mainly on histone tails (sometimes in core) • Can be part of large acetylation domains • Modifying enzymes: • often multi-enzyme complexes • can modify multiple residues • Well correlated with transcriptional activation • Other roles (chromatin assembly, DNA repair, etc.)

22. Histone Acetyl Transferases (HATs/KATs) • Two general types: • Type B: cytoplasmic (newly synthesized histones) • HAT1 Type B Kouzarides, Cell, 2007.

23. HAT Superfamilies Type A: nuclear 1. GNAT 2. MYST Other HATs exist as well: e.g. associated with nuclear receptors or Pol III 3. P300/CBP (metazoan) 4. Rtt109 (fungal)

24. HAT Superfamilies • Similarities • Structurally conserved central core • Can acetylate non-histone proteins • Differences • Sequence divergence • Catalytic mechanisms • Interacting proteins (regulate specificity) GNAT MYST P300/CBP Rtt109 Marmorstein and Trievel, 2009

25. Histone Deacetylases (HDACs) • Multi-enzyme complexes • Targeted by transcriptional repressors • Deactylate histone tails

26. HDAC Superfamilies • Class I HDACs • RPD3-like (HDAC 1, 2, 3) • most cell types • in nucleus • Class II HDACs • HDA1- like (HDAC 4, 5, 6, 7, 9, 10) • HDAC and N-term repressor motif • restricted expression • shuttle in/out nucleus • Class III HDACs • Sir-2 (NAD-dependent) • sirtuins

27. HDACs are in Complexes Butler and Bates, Nature Reviews Neuroscience, 2006.

28. Roles of Acetylation 1. Opens up chromatin: • Reduces charge interactions of histones with DNA (K has a positive charge) • Prevents chromatin compaction (H4K16ac prevents 30nm fiber formation) 2. Recruits chromatin proteins with bromodomains (SWI/SNF, HATs: GCN5, p300) 3. May occur at same residues as methylation with repressive effect (competitive antagonism) Robinson et al., J. Mol. Biol., 2008. PCAF H3K27ac H3K27me3 Mujtaba et al., Oncogene, 2007. Yang and Chen, Cell Research, 2011.

29. Roles of Acetylation Roles of Acetylation -- Continued 4. Highly correlated with active transcription i.e. enriched at TSS of actively transcribed genes H4Ac H3Ac H3 RNAPII Expression: P1<P2<P3<P4 208 TSS investigated Heintzman N et al., Nature Genetics, 2007.

30. Roles of Acetylation 5. Correlated with binding of activating transcription factors i.e. enriched at promoters and enhancers H4Ac H3Ac p300 Expression: E1<E2<E3 74 enhancers (distal p300 binding sites) Heintzman N et al., Nature Genetics, 2007.

31. Lysine Methylation Bhaumik, Smith, and Shilatifard, 2007.

32. Lysine Methylation • Many lysine residues can be methylated • Mainly on histone tails (sometimes in core) • Can be mono-, di-, or tri-methylated • Depending on residue and number of methyl groups, can be associated with active or repressive transcription • Other roles • Transcriptional elongation • Pericentromeric heterochromatin • X chromosome inactivation Liu et. al, Annu. Rev. Plant Biol., 2010.

33. Lysine Methyltransferases: KMTs • Enzymes very specific • Target a certain lysine on a certain histone • Put on mono, di, and/or tri methyl (me, me2, me3) • Many contain SET domains (me-transferase) • ‘Readout’ is very specific • Ex. H3K4me1 vs. H3K4me3 Only room for one methyl group Set7/9 Xiao et al., Nature, 2003.

34. KMTs H3K4me: trithorax/Set1, KMT2 H3K9me: Suv3-9,KMT1 H3K27me: polycomb Group (E(z)),KMT6 H3K36me: Set2, KMT3 H3K79me: Dot1 (nonSET) KMT4 H4K20me: Suv4-20, KMT5 ***Most contain SET Domains

36. Roles of Lysine Methylation Roles of Lysine methylation 1. Recruitment of other chromatin proteins through specific domains: Chromodomain (CHD ATPases, HP1, PC) Tudor (some histone demethylases) PhD (many chromatin regulators BPTF, ING2) MBT (in some polycomb proteins) WD-40 (WDR5) Chromo-like (Royal)

37. Roles of Lysine Methylation Bannister and Kouzarides Cell Research 2011

38. Roles of Lysine Methylation 2. Different readout depending on level of methylation Liu et. al, Annu. Rev. Plant Biol., 2010.

39. Roles of Lysine Methylation 2. Different readout depending on level of methylation • Methylation status of H3K4 is recognized by different domains • H3K4me1: chromodomain in CHD1 (ATPase) = transcription activation • H3K4me2: WD-40 domain in WDR5 in MLL (Trx) = transcription activation • H3K4me3: PhD domain of BPTF in NURF (ISWI) = transcription activation • H3K4me3: PhD ING2 recognizes during DNA damage and shuts down active transcription through mSin3a-HDAC1 Promoters Enhancers Heintzman N et al., Nature Genetics, 2007.

40. Roles of Lysine Methylation 3. Transcriptional activation • H3K4me3: euchromatin promoter, 5’ end activates transcription (Set1) • H3K36me3: in gene (ORF), transcriptional elongation (Set2) Li et al., Cell, 2007.

41. Roles of Lysine Methylation 3. Transcriptional activation • H3K36me3 marks actively transcribed genes PolII H3K36me3 Guenther et al., Cell 2007

42. Roles of Lysine Methylation 4. Transcriptional repression • H3K9me (Suv39H1) • In promoter, represses transcription • In larger domains, heterochromatin formation • H3K27me (EZH2) • Repression of transcription • Polycombgroup silencing • H4K20me (Suv420H1) • Some forms of silencing and repression of gene expression • In repetitive elements, similar localization as H3K9me3 in ES cells Li et al., Cell, 2007.

43. Co-occurrence of activating and repressive lysine methylation Bivalency marks “poised” genes Mikkelsen et al., Nature 2007

44. Position of histone modifications H3K9me3 and H4K20me3 can occur in actively transcribed genes Thought to prevent access to cryptic transcription initiation Mikkelsen et al., Nature, 2007.

45. Lysine Demethylation LSD1: H3K4 Jumonji family: H3K9 (JHDM2A:me1 and me2) JHDM1A demethylase: H3K36 me1 and me2 Wysocka et al., Cell, 2005.

46. Lysine Demethylation • LSD1 • first histone demethylase • amine oxidase • only me1 and me2 can serve as substrates • Different domain structure from other demethylase • Complex determines specificity (H3K4me vs. H3K9me) Stavropoulos et al., NSMB, 2006.

47. Lysine Demethylation • JmjC • JmjC-domain containing oxygenases • 27 family members • Catalytic JmjC domain can accommodate me3 as substrate Wolf et al., EMBO Reports, 2007.

48. Lysine Methylation/Demethylation Individual lysines can be targeted by different methyltransferases/demethylases Shi, Nature Reviews, 2007.

49. Other Histone Modifications Kouzarides, Cell, 2007.

50. Arginine Methylation Bhaumik et al., NSMB, 2007.