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Shelley Berger The Wistar Institute University of Pennsylvania Overview of Histone Variants Bio 483 October 9, 2012 Shelley Berger Biology; CDB; Genetics Penn Epigenetics Program bergers@upenn.edu Slide 2 Lecture Outline What are histone variants? Review nuclesome structure wrt replacement Consideration of following issues: - sequence variation - nucleosome structure - genome function - location within the genome - physiological function DiscussH2A: macroH2A, H2A.Bbd, H2Ax, H2AZ H3: CENPA, H3.3 testes histone variants Slide 3 Core vs. variant histones H3 H4 H2A H2B globular NC Are there additional histones? Slide 4 H2A H2B H3 H4 H1 Slide 5 Luger and Richmond, Nature 1997 H3 H4 H2A H2B H3 tail Slide 6 New nucleosome formation: after replication H3/H4 tetramer binds to DNA 2 dimers H2A/H2B bind H3/H4 tetramer Slide 7 How to replace? 1.During replication assemble with variant histones 2.H2A/H2B dimer exchange 3.Disassembly of nucleosome during DNA damage repair, transcription Slide 8 H2A H2B H3 H4 DNA repair Gene repression X chrom inact Gene activation Centromere identity Gene activation Slide 9 H2A Slide 10 Chromatin compaction by macroH2A Slide 11 Chromatin destabilization by H2A.Bbd Slide 12 H2Ax H2A Histone H2Ax in DNA damage pathway Slide 13 DNA damage leads to complex repair pathways Slide 14 Molecular details of DNA repair Slide 15 H2Ax evolutionary conservation Slide 16 H2Ax phosphorylation is an early step in the repair process Slide 17 Histone H2AZ (= HTZ1): How does it affect NCP structure and positioning? What is its localization in the genome? What genomic process does it regulate? Histone H2AZ in gene regulation Slide 18 Genome-wide view of H2AZ at promotors TSS Nuc Depleted Region Gene ORF Promoter TSS H2AZ tends to occur in one nuc up- and one nuc down-stream of the TSS Does it regulate transcription? Slide 19 H2AZ is released as genes are activated Its release is required for full activation Slide 20 Association of H2A/H2AZ with chromatin H2AZ is more loosely bound to chromatin (although NCP structure very similar) Is there a difference in nucleosome stability? Slide 21 What is the localization of H2AZ around active/inactive genes? Active genes less enriched than inactive Slide 22 H2AZ How does it affect NCP structure and positioning? What is its localization in the genome? What genomic process does it regulate? Inactive promoters Poises genes for activation Destablizes NCP: correlates with depletion of one NCP over the TSS How is it incorporated? a digression Slide 23 Nucleosomes can be changed in detailed association with DNA, position, presence ATP-dependent Nucleosome Remodeling Slide 24 Activities of ATP-dependent chromatin remodeling enzymes ATP ADP + P i ATP Remodeler Slide 25 Evolutionary Tree of SWI2/SNF2 Remodelers BRM-Hs BRG-1-Hs Brahma-Dm SWI2-I STH1-Sc CHD-Dm CHD1-Hs CHD2-Hs CHD1-Sc MI2-Dm CHD3-Hs CHD4-Hs CHD5-Hs ISWI-Dm SNF2H-Hs SNF2L-Hs ISW2-Sc ISW1-Sc ino80-sc SWI/SNF ISWI CHD INO80 Slide 26 Start with H2A NCPs ATP dependent remodelers and Histone Variant deposition Swr1 H2AZ Slide 27 What do ATP-dependent remodeling complexes do? 2. Exchange histones and histone variants: New paradigm! 1. Directly alter NCP position Slide 28 Biochem. Cell Biol vol 84 2006 Model for H2AZ Slide 29 Instability of H2AZ NCPs allows high temp to turn on/off genes in plants Slide 30 Histone H3 and its Variants CenpA Replication Coupled deposition (only S phase) Replication independent Deposition (not strictly during S phase) Slide 31 H3/H3.1 H4 H3.3 Histone H3.1 and H3.3 Slide 32 H3.3 is very similar to H3.1 H3.3 is more ancient Slide 33 What genomic processes do H3.1/H3.3 regulate? Replication Dependent: Either H3.1 or H3.3 Replication Independent: Only H3.3 Slide 34 H3.3 localizes with RNAPII and K4me genes RNAP K4me2 H3.1 H3.3 Slide 35 H3.1 H3.3 How are H3.1 and H3.3 deposited in RC and RI pathways? HIRA IP-Western Two different chaperone/ assembly factors p150 p60 Slide 36 Nucleosome assembly RC or RI Slide 37 CENPA H3 H4 Histone CENPA Slide 38 What are centromeres? Slide 39 Sequence complexity CENPA, an H3 variant, is in a specialized NCP at centromeres in all eukaryotes Slide 40 Targeting of CENPA to centromeres Mapping in vitro and in vivo defined a region in the histone fold as CATD Slide 41 Chaperone for Centromeric Localization Cell line expressing CENPA-TAP Slide 42 HJURP required for CENPA targeting to centromeres HJURP Knock Down cell line No targeting of CENPA in HJURP KD 1. OE CENPA did not restore targeting 2. H3-CATD recruited by HJURP Slide 43 Controversy regarding structure of the centromeric CENPA NCP Proposed structures: Slide 44 Is there a distinct structure? (CENPA-H4)2 is more compact Slide 45 Structure of CENPA NCP Structural features map to CATD! It forms a well behaved octamer Slide 46 Cenp A HJURP HIRA CAF1 Summary: H3 variants and their functions Slide 47 Spermatogenesis and chromatin compaction: key role for histone variants Slide 48 Testes-specific histone variants replace core histones following meiosis Slide 49 All histones (except H4) have testes variants Slide 50 H2A H2B H3 H4 H1 A remarkable variety of histone variants to regulate the genome via location-specific nuclesome funtion Slide 51 Penn Epigenetics Monthly Meeting Every month Thursday 4:00 pm Check website Website: Penn Epigenetics