BCH 2021 2008

1. BCH 2021 2008 Histone Modifications Peter N Lewis

2. Papers to be discussed • Nonprocessive methylation by Dot1 leads to functional redundancy of histone H3K79 methylation states • Frederiks F, Tzouros M, Oudgenoeg G, van Welsem T, Fornerod M, Krijgsveld J, van Leeuwen F. Nat Struct Mol Biol. 2008 15, 550-7 • Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation • McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW. Nature. 2008 453, 812-6

3. Background Reviews • 1) Is there a code embedded in proteins that is based on post-translational modifications? Sims RJ 3rd, Reinberg D. Nat Rev Mol Cell Biol. 2008 9, 815-20 • 2) Simplifying a complex code. Turner BM, Nat Struct Mol Biol. 2008 15, 542-4 • 3) Defining an epigenetic code Turner BM, Nat Cell Biol. 2007, 9, 2-6 • 4) Translating the histone code Jenuwein T, Allis CD. Science 2001 293,1074-8

4. Cell Nucleus - Chromosomes Chromatin Fibre Nucleosome DNA 2x109 bp < 2% transcribed DNA CompactionDNA 1Nucleosome 630 nm fibre 36Interphase 1000Metaphase 100,000 Metaphase Chromosome

5. Histones Linker histone H1 Core histones H2A H2B helix N variable H3 conserved H4 Highly conserved, small, basic proteins subject to many post-translational modifications

6. Histone Post-translational Modifications acetyl phosphoryl methyl Strahl and Allis Nature 403, 41 (2000)

7. Histone Code Hypothesis (Allis) “distinct histone modifications, on one or more tails, act sequentially or in combination to form a “histone code” that is read by other proteins to bring about distinct downstream events”

8. Epigenetic Code (Turner) “describes the way in which the potential for expression of genes in a particular cell type is specified by chromatin modifications put in place at an earlier stage of differentiation”

9. Defining an epigenetic code Turner BM Nat Cell Biol. 2007 Jan;9(1):2-6.

10. H H H + H H + H +

11. Yeast Telomeric Silencing Requires Sir3 binding to nucleosomal basic patch around H4K16 Sir3 does not bind if H4K16 is acetylated or H3K79 is methylated Dot1 – Disruptor of telomeric silencing – methyltransferase methylates H3K79 up tp 90%, prevents Sir3 binding Dot1 binds to H4 Arg 17, His 18 and Arg 19, but is not affected by H4K16 acetylation Dot1 activity is enhanced by H2B ubiquitination at K123 Dot1 is not processive (distributive) unlike SET containing methyltransferases H4 H2B H3

12. Simplifying a complex code. Turner BMNat Struct Mol Biol. 2008 15, 542-4

13. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation.McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW. Nature. 2008 453, 812-6 Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the ‘histone code’ hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3, by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

14. Fig 1 Semi-Synthesis of ubiquitylated H2B 1) 117-125 2) ubiquitin 5) 1-116

15. Figure 2 | Semi-synthesis of uH2B and incorporation into chemicallydefined histone octamers and nucleosomes

16. Figure 3 | Effects of uH2B on intranucleosomal methylation of H3 K79 by hDot1L.

17. Figure 4 | Methyltransferase assays on dinucleosomes using full-lengthhDot1L

19. Figure 1 Dot1 is a nonprocessive methyltransferase in vitro.

20. Figure 2 Dot1 is a nonprocessive methyltransferase in vivo.

21. Figure 3 Dot1-G401A is an active site mutant with reduced catalytic activity.

22. Figure 4 The functions of Dot1 in telomeric silencing depend on the overall levels of H3K79 methylation

23. Figure 6 H2B ubiquitination regulates all H3K79 methylation states and does not act via the putative ubiquitin binding domain of Dot1

24. Conclusions • Dot1 introduces multiple methyl groups at H3K79 by a nonprocessive mechanism. • Does not depend on a single methylation state (mono, di, tri) • Leads to a simple binary code, limits regulation by other proteins • Cross talk with H4 K16 and H2B K123