Histone octamer assembly

CHROMATIN Chromatin Histone structure Nucleosome Chromatin fiber Histone acetylation Chromatin transcription Regulation of gene expression: -Cell cycle control (+/-) -Cell differentiation Genome integrity References: -Special issue on Epigenetic and Chromatin Organization. Cell 2007 vol. 128 issue 4 -Special issue Focus on Chromatin. Nat Struct Mol Biol 2007 vol. 14 issue 11 Contact: jacques.cote@crhdq.ulaval.ca 1

Chromatin Domains 2

H1 Linker histone H2A H2B Core histones H3 H4 N helix variable conserved HISTONES are highly conserved, small, basic proteins Histone acetylation is a reversible modification of lysines in the N-termini of the core histones. Result: reduced binding to DNA destabilization of chromatin Histone octamer assembly H3-H4 tetramer Histone octamer H2A-H2B dimer 3

Where are the N-termini of the core histones? Luger, Mader, Richmond, Sargent & Richmond Nature 389, 251-260 (1997) Question 1: What is the function of histone N-termini? Question 2: Are all N-termini functionally equivalent? 4

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Factors regulating local chromatin/ accessibility to DNA -Histone modifying enzymes (HAT, HDAC, HMT, HDM, kinase) -ATP-dependent chromatin remodelers (SWI/SNF, BRG1/BRM, NuRD, NURF) -Histone variants (H2A.Z, H2A.X, CENP-A, macro-h2a) -Histone chaperones (ASF1, NAP1, FACT, CAF1) Acetylation of conserved lysines The N-termini of histones H4 and H3, and their acetylation patterns, are absolutely conserved. H4 N-terminus H3 N-terminus 5 8 12 16 20 Ac Ac Ac Ac Ac or Me Ac-S-G-R-G-K-G-G-K-G-L-G-K-G-G-A-K-R-H-R-K-V-L-R-D- + + + + + + + + + + 4 9 14 18 23 27 Ac or Me Me Ac Ac Ac Ac A-R-T-K-Q-T-A-R-K-S-T-G-G-K-A-P-R-K-Q-L-A-T-K-A-A-R-K-S-A-P- + + + + + + + + + Acetyl-CoA Lysine HAT (Histone Acetyl-Transferase) CoA ε-n-acetyl-lysine O O N C C γ C C α β δ ε O O C C N+ - P P O O reversible reactions N C C C C C ε C N C O Histone Deacetylase C DNA backbone binding no DNA binding - - - - - - - P - - - - - - - - - - - - - - - 6

Chromatin fibers 30 nm chromatin fiber 11 nm (beads) + charged N termini (bind DNA and neigboring nucleosomes) highly acetylated core histones (especially H3 and H4) HIGH level of histone H1 NO gene transcription Reduced level of histone H1 Gene transcription possible Acetylation of Chromatin Domains Example: the chicken β-globin gene domain domain boundary DNA remaining 0 10 20 30 kb β ρ β H β A β ε domain boundary α(ac-lys) antibody nucleosome ppt DNA loop domain β = β-globin genes: DNase I hyper-sensitive site General DNase I sensitivity Control: inactive gene chicken ovalbumin DNase I High levels of chromatin acetylation, across complete chromatin domains (DNA loops), induces chromatin changes detected as general DNase I sensitivity Within these chromatin domains, at functional genes or transcription factors, the chromatin structure is interrupted by small DNase I hypersensitive sites 0 1 2 DNase I (U/ml) Hebbes, Clayton, Thorne, Crane-Robinson EMBO J. 13, 1823 (1994) 7

DOSAGE COMPENSATION MECHANISMS SPECIFIC ACETYLATION VS TRANSCRIPTION Drosophila Human Both X chromosomes are normaly expressed. The X chromosome is two-fold hypertranscribed K16 of histone H4 is hyperacetylated on the X chromosome. Only one X chromosome is transcribed The other one is hypoacetylated on all 4 sites of H4 The X chromosome is normaly expressed and acetylated. Acetylation at Promoters Transcriptional ACTIVATION TAF 250 II TBP HAT TATA ACGGTC ADA2 ADA3 GCN5 HAT pol.ii TH TH TACCCG p300 CPB HAT P/CAF + hormone HAT TR/RXR Thyroid Hormone Receptor example of DNA-binding Transcription Factor co-activator Histone Acetyl Transferases TAF 250 II TBP HAT Transcriptional REPRESSION Without Thyroid Hormone co-repressor TATA Histone Deacetylase TACCCG N-CoR Sin3 RPD3 Adapted from Wolffe Nature 387, 16 (1997) 8

S phase Gene Activation co-activator CPB HAT HAT TAF 250 II Rb co-repressor HDAC1 Histone Deacetylase Rb G2 phase Mitosis S phase G1 phase Cell cycle control: the G1-S transition (Restriction point) R cyclinecdk2 kinase Low activity HAT P + P Rb Weakly active Adapted from Ait-Si-Ali et al. Nature 396, 184 (1998) TBP TATA TAF 250 II TBP HAT S phase gene: OFF TATA Histone Acetyl Transferase CPB P HAT pol.ii Active HAT S phase gene: ON 9

Deacetylation of Chromatin Transcriptional Silencing X-chromosome Inactivation me 5 5..pCpGp..3 transcriptional repressor MeCP2 co-repressor Histone Deacetylase 5 me 3..pGpCp..5 º º Sin3 RPD3 l l l l 5meC CpG DNA modification is observed in repressed genes and inactivated X chromosomes 5meC CpG-methylation is maintained after DNA replication by Maintenance Methylase action on hemi-methylated DNA 5meC binds transcriptional repressor MeCP2 (MethylC-binding Protein-2) MeCP2 binds Sin3 with RPD3 histone deacetylase + + CpG methylation Hypo-acetylated repressed chromatin fiber Nan et al. Mol.Cell.Biol. 16, 414 (1996); Cell 88, 1 (1997); Jones et al. Nat.Genet. 19, 187 (1998) Sas2 Sir4 Sir4 Sir4 Sir4 Sir4 Sir4 Sir4 Sir4 Sir4 Sir2 Sir3 Sir3 Sir3 Sir3 Sir2 Sir2 Sir2 Sir2 Rap1 Heterochromatin Gene activity Euchromatin Sas2 Sir4 Sir4 Sir4 Rap1 Sir4 Sir4 Sir2 Sir2 Sir2 Sir2 Sir3 Sir3 Sir3 Sir3 Sir4 Sir4 Sir4 Sir4 Sir2 Sir2 Sir2 Sir4 Sir4 Sir4 Sir4 Sir3 Sir3 Sir3 Sir3 Sir2 Sir2 Gene repression Dot1 H3 MeK79 Heterochromatin Sir2 Sir4 Sir4 Sir4 Sir4 Sir4 Sir4 Sir3 Rap1 Gene activity Heterochromatin Euchromatin 10

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ATP-dependent Chromatin Remodelers 12

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G&D 13:2339(1999) 15

Histone Acetyltransferases 16

Recruitment of NuA4 in transcription activation 17

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Phosphate rich (+Pi) -4-3 ClaI -2 UAS2-1 TATA UAS1 yng2 WT WT esa1 ts WT esa1 ts +Pi -Pi +Pi -Pi +Pi -Pi +Pi -Pi +Pi -Pi -Pi DNaseI Non Digested Digested RT 37 o C RT 37 o C RT -1-2 -3-4 NuA4 is required for chromatin remodeling at the PHO5 promoter p53 Element WT or Δyng2 p21-his3 + p53 WT p53 Δyng2 p53 Galactose WT Δyng2 + + p21-his3 GAL1 ACT1 25S rrna α p53 YNG2 is required for the transactivation function of p53 in yeast 19

WT+p21 WT+p21/p53 Δyng2+p21/p53 input αach4 αhyperach4 αach3 p53 targets NuA4 dependent histone H4 hyperacetylation to p21 promoter in yeast Cooperativity between Histone Acetylation and Chromatin Remodeling 20

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HO URS 1 URS 2 Swi5 Swi5 URS 1 URS 2 HO Swi5 URS 1 URS 2 HO Swi/Snf Ash1 Swi5 URS 1 Swi/Snf URS 2 HO Swi5Ash1 URS 1 URS 2 HO SAGA HO URS 1 URS 2 Swi/Snf SAGA HO URS 1 URS 2 Swi6 SBF Swi4 Swi/Snf SAGA Swi6 Swi4 URS 1 URS 2 HO Transcription de HO 22

Bromodomain: an acetylated histone binding module 23

Bromodomains bind acetylated histone tails; mechanism of retention? Bromodomains bind acetylated histone tails; mechanism of retention? 24

A code formed by histone modifications 25

La modification des histones affecte la fonction de la chromatine Empreinte Épigénétique 26

Specific protein domains recognize different epigenetic marks (read a local code/signature?) 27

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Mécanisme 29

Le code épigénétique des histones La théorie dit que différentes modifications à différents endroits et en différentes combinaisons sur les extrémités N terminales des histones formeraient des marques pour la formation d un état chromatinien transmissible particulier et pour des protéines qui les reconnaîtraient spécifiquement. 30

L héritage des histones parentales permet la transmission de l état chromatinien de génération en génération Tout comme pour la réplication de l ADN, la distribution des constituants des nucléosomes se fait selon un modèle SEMI-CONSERVATIF 31

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Modulation of chromatin modifications during the cell cycle and in response to signaling pathways 33

Chromatin and the Transcription Cycle 34

Factors regulating local chromatin/ accessibility to DNA -Histone modifying enzymes (HAT, HDAC, HMT, HDM, kinase) -ATP-dependent chromatin remodelers (SWI/SNF, BRG1/BRM, NuRD, NURF) -Histone variants (H2A.Z, H2A.X, CENP-A, macro-h2a) -Histone chaperones (ASF1, NAP1, FACT, CAF1) Workman G&D 2006 35

J. Mellor Mol. Cell 2005 Presetting of highly inducible genes by NuA4 The human NuA4/Tip60 complex is structurally equivalent to a physical merge of yeast NuA4 and SWR1 complexes Link between H4 acetylation and H2AZ-H2A exchange in chromatin? 36

NuA4-dependent chromatin acetylation influences H2AZ deposition on chromatin Chromatin domains along a transcription unit K4-trimethylated nucleosomes Lieb and Clarke, Cell 2005 37

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Figure 7. Model of the role of ubiquitylation and deubiquitylation at GAL1 Karl W. Henry et al. Genes Dev. 2003; 17: 2648-2663 39

Swr1-dependent K4-trimethylated nucleosomes (Lieb and Clarke, Cell 2005) H3 trimek36 (Set2) Rpd3 Sin3 H3 trimek36(set2) H4/H2A acetylation on promoter(nua4) H3 trimek4(set1) Histone deacetylation on coding region(rpd3) K4-trimethylated nucleosomes (Lieb and Clarke, Cell 2005) 2 HDAC complexes with opposite functions Repression at promoters Accurate transcription elongation 40

HBO1-JADE1 HAT complexes are enriched on the coding region of genes High resolution localization analysis (ChIP-chip) 41

Chromatin and Gene silencing 42

Modèle Modèle suggéré: La méthylation des histones induit la méthylation de l ADN. 1. Une séquence signal recrute un facteur x. 2. Facteur x recrute HDAC et HMT Induction d un état transcriptionnel inactif. 3. Facteurs y recruté sur les sites méthylés 4. DMTase recrutée par facteur y. Propagation de l état inactif 43

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Chromatin and Genome stability 46

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N H2A.X P C 48

Redon et al. JCB 1999 H2A.X is rapidly phosphorylated at sites of DNA damage A B, TIP60 (BAF53 + HAT) TIP60 (HAT + ruvb-like helicases) 49

Ordered recruitment of NuA4 and ATP-dependent remodelers at a DSB in vivo 50

Recruitment and role of the NuA4/TIP60 HAT complex at DNA double-strand breaks Chromatin and DNA Replication 51

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Epigenetic inheritance 53

S-phase chromatin contains the bulk of acetylated H4 ING tumor suppressor family of proteins are critical regulators of chromatin acetylation in eukaryotes 54

ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication Schematic representation of the assembly of the pre-replicative complex (Pre-RC) and its activation upon entry into S-phase to initiate replication fork progression. (from Takeda and Dutta, Oncogene 2005) 55

Essential role of ING complexes during DNA replication Larrieu and Pedeux Cell Cycle 2009 Modified from Ann E. Ehrenhofer-Murray EJB 2004 56

Chromatin and Cancer Cancer Biology vs Chromatin Remodeling Translocations in Leukemia: -PML-RAR (functions trough HDAC) -MOZ/MORF-CBP/p300 (AML1-HATs) -MLL-CBP (HMT+HAT) -MLL-ENL/AF9 (HMT,SWI,HAT) -AML1-ETO (functions through HDAC) Tumor suppressors: -Rb, functions through SWI/SNF, HDAC, HMT and DNMT recruitment -p53, functions through HAT recruitment (ING) (SWI/SNF?) -BRCA1, is associated with SWI/SNF in vivo -Tip60, HAT subunit of NuA4 complex -ING1-5, subunits of HATs and HDACs Oncogenes: -Myc, functions through SWI/SNF and HAT recruitment -E2F, functions through HAT recruitment -E1A, disrupts HAT and HDAC complexes Mutations associated with leukemia and diverse tumors: -Ini1/Snf5, subunit of SWI/SNF -Brg1, subunit of SWI/SNF -HBO1, HAT subunit Mutations associated with metastasis/invasive growth -MTA1-3, subunits of NuRD -ING4, subunit of HBO1 HAT complex 57

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The NuRD complex (MTA s) and Breast Cancer 59

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Six hallmarks of cancer. Epigenetic silencing of tumor suppressor genes (examples in parentheses) plays a significant role in the development of each of these six tumor traits. Epigenetic therapy can resensitize cancer cells to conventional therapies. In a heterogenous tumor cell population, a small percentage of cells may respond directly to epigenetic drugs. The remaining tumor cells, however, could then be resensitized (e.g., by tumor suppressor gene re-expression) to conventional therapies. 61

Complex and Conserved roles of Chromatin in regulating Nuclear Functions 62

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