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

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

Core vs. variant histones H3 H4 H2A H2B globular NC Are there additional histones?

H2A H2B H3 H4 H1

Luger and Richmond, Nature 1997 H3 H4 H2A H2B H3 ‘tail’

New nucleosome formation: after replication H3/H4 tetramer binds to DNA 2 dimers H2A/H2B bind H3/H4 tetramer

How to replace? 1.During replication assemble with variant histones 2.H2A/H2B dimer exchange 3.Disassembly of nucleosome during DNA damage repair, transcription

H2A H2B H3 H4 DNA repair Gene repression X chrom inact Gene activation Centromere identity Gene activation

H2A

Chromatin compaction by macroH2A

Chromatin destabilization by H2A.Bbd

H2Ax H2A Histone H2Ax in DNA damage pathway

DNA damage leads to complex repair pathways

Molecular details of DNA repair

H2Ax evolutionary conservation

H2Ax phosphorylation is an early step in the repair process

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

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?

H2AZ is released as genes are activated Its release is required for full activation

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?

What is the localization of H2AZ around active/inactive genes? Active genes less enriched than inactive

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

Nucleosomes can be changed in detailed association with DNA, position, presence ATP-dependent Nucleosome Remodeling

Activities of ATP-dependent chromatin remodeling enzymes ATP ADP + P i ATP Remodeler

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

Start with H2A NCPs ATP dependent remodelers and Histone Variant deposition Swr1 H2AZ

What do ATP-dependent remodeling complexes do? 2. Exchange histones and histone variants: New paradigm! 1. Directly alter NCP position

Biochem. Cell Biol vol Model for H2AZ

Instability of H2AZ NCPs allows high temp to turn on/off genes in plants

Histone H3 and its Variants CenpA Replication Coupled deposition (only S phase) Replication independent Deposition (not strictly during S phase)

H3/H3.1 H4 H3.3 Histone H3.1 and H3.3

H3.3 is very similar to H3.1 H3.3 is more ancient

What genomic processes do H3.1/H3.3 regulate? Replication Dependent: Either H3.1 or H3.3 Replication Independent: Only H3.3

H3.3 localizes with RNAPII and K4me genes RNAP K4me2 H3.1 H3.3

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

Nucleosome assembly RC or RI

CENPA H3 H4 Histone CENPA

What are centromeres?

Sequence complexity CENPA, an H3 variant, is in a specialized NCP at centromeres in all eukaryotes

Targeting of CENPA to centromeres Mapping in vitro and in vivo defined a region in the histone fold as “CATD”

Chaperone for Centromeric Localization Cell line expressing CENPA-TAP

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

Controversy regarding structure of the centromeric CENPA NCP Proposed structures:

Is there a distinct structure? (CENPA-H4)2 is more compact

Structure of CENPA NCP Structural features map to CATD! It forms a well behaved octamer

Cenp A HJURP HIRA CAF1 Summary: H3 variants and their functions

Spermatogenesis and chromatin compaction: key role for histone variants

Testes-specific histone variants replace core histones following meiosis

All histones (except H4) have testes variants

H2A H2B H3 H4 H1 A remarkable variety of histone variants to regulate the genome via location-specific nuclesome funtion

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