Download - Chromatin Assembly
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Chromatin Assembly
Nucleosomal DNA Primary chromatin structure
beads-on-a-string
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Nucleosomefrom Horn and Peterson Science, 2002
acacac
ac
acDNA
methylation
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Visualizing this primary structure
nucleosomal arrays
Partial nuclease digestion generates mono, di, trinucleosomes…
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How is chromatin assembly achieved?
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Irregular assembly
Dynamics of ATP-dependent chromatin assembly by ACFDmitry V. Fyodorov & James T. KadonagaNATURE | VOL 418 | 22 AUGUST 2002 |
Assembly
in vitroHistones + DNA + saltassemble into aggregates
Histones + DNA + negatively charged proteins= mononucleosomes andpoor nucleosmal array (irregularly spaced; “smear”)
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Dynamics of ATP-dependent chromatin assembly by ACFDmitry V. Fyodorov & James T. KadonagaNATURE | VOL 418 | 22 AUGUST 2002 |
in vitroXenopus/Drosophila extracts=chromatin (±regularly spaced)
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What is in the extract?
Histone chaperones
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Histone chaperonesnegatively charged proteinshelp assembly; prevent aggregation
NAP1 H2A/H2B Nuclear Import, nucleosomedisruption, histone variant incorporation
nucleoplasmin H2A/H2B Storage, sperm histone exchange
N1/N2 H3/H4 Storage
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CAF-1
Chromatin assembly factor 1
More histone chaperones
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Replication-coupled chromatin assembly factorCAF-1 (identified in yeast; Bruce Stillman lab)
brings H3/H4 onto newly synthesized DNA
CAF-13 subunits: p150 (Cac1) binds PCNA
p60 (Cac2) binds ASF1p48 (Cac3) binds histones histone binding
in many complexesRoles
nucleosome deposition after replicationheterochromatin maintenance
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Ransom et al., Cell, 2010
Smallest CAF subunit bound to histone H4
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Replication
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Cell, Vol. 96, 575–585, February 19, 1999, Replication-Dependent Marking of DNA by PCNAFacilitates CAF-1-Coupled Inheritance of ChromatinKei-ichi Shibahara and Bruce Stillman
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Cac2 interacts with PCNA when Cac1 is present
PCNA connects DNA replication to epigenetic inheritance in yeastZhiguo Zhang, Kei-ichi Shibahara & Bruce StillmanNATURE | VOL 408 | 9 NOVEMBER 2000
PCNA
- - -
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Role of PCNA in chromatin assembly?1. Adenine deficient yeast are red2. Move Adenine biosynthesis gene into heterochromatin ± inactive; red colonies3. Mutagenize strain4. improper heterochromatin assembly in PCNA mutants causes
Adenine biosynthesis gene reactivation; more whitish colonies
Activation of Ade in telomeric heterochromatin in PCNA mutants
PCNA connects DNA replication to epigenetic inheritance in yeastZhiguo Zhang, Kei-ichi Shibahara* & Bruce Stillman*NATURE | VOL 408 | 9 NOVEMBER 2000 |
Ade - Ade +
PCNA wt PCNA mutant
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PCNA coordinates: DNA synthesis (processivity)Chromatin assemblyRepairMethylation Epigenetic InheritanceCell-cycle control
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Parental nucleosome segregation (hatched)
De novo nucleosome assembly (unhatched)
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New nucleosome formationafter replication1 H3/H4 tetramer OR 2 H3/H4 dimers bind DNA2 dimers H2A/H2B bind H3/H4 tetramer
DNA first very reactive with histones, then matures
Nucleosome deposition is essentialif repressed, DNA will be duplicated but cell cycle arrest in G2
Histone synthesis occurs at G1/S
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Newly synthesized histones have acetylated tails cytoplasmic (B-type) HAT: HAT1 HAT: histone acetyltransferase
pattern of acetylation differs from acetylation involved in gene regulation
Modifications on H4: H4K5ac, H4K12ac
modifications lost after incorporation (HDAC, histone deacetylase)
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Corpet and Almouzni TICB 2008
H3K56ac
H3K9/14acGCN5
Rtt109
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These modifications are important reduced viability in yeast if mutated
Role: Nuclear importBinding/Deposition by histone chaperoneChromatin maturation
Mark the histone as newly synthesized for chromatinassembly
Modifications are rapidly removed after assembly
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Journal of Cell Science 113, 2647-2658 (2000)CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repairPatricia Ridgway and Geneviève Almouzni
or tetramer
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ASF1
acts together with CAF1, interacts with CAC2 subunit
interacts with HAT1 complex
interacts with MCM helicase
Another histone chaperone
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Trujillo and Osley Nature Structural & Molecular Biology 2008
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The ins and outs of nucleosome assemblyJill A Mello* and Geneviève Almouzni †Current Opinion in Genetics & Development 2001, 11:136–141
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Role of ASF1:
generates H3 H4 dimer
prevents tetramer formationbreaks up tetramers
presents H3/H4 dimer to CAF1
also role in H3K56ac by Rtt109
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Ransom et al., Cell, 2010
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Another histone chaperone
Rtt106
interacts with CAF1 complex
involved in H3/H4 assembly during replication
high affinity for H3K56ac (as does CAF1)
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Modified from Fillingham and Greenblatt, Cell 2008
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Ransom et al., Cell, 2010
H2A/H2B chaperone: FACT?
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BOTH long arrays AND properly spaced nucleosomes
require other enzymes:
ATP-dependent chromatin remodelers
Chromatin assemblyChromatin array formation
ATPase complexISWI ACF/ChRAC
CHD1 -
INO80 INO80
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ISWI: chromatin remodeling ATPase
roles in chromatin assembly together with NAP1 or Asf1linker length, formation of long nucleosomal arrays
transcriptional elongationsilencingchromosome organization
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CHD1: chromatin remodeling ATPase
roles in chromatin assembly together with NAP1histone variant assembly
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Enhanced assembly
Lusser et al., Nature Struc Mol Biol 2005
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Different periodicity
Lusser et al., Nature Struc Mol Biol 2005
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Clapier and Cairns, Annu. Rev. Biochem., 2009
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INO80: chromatin remodeling ATPase
roles in replication stress
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Morrison and Shen, Nature reviews Mol Cell Biol, 2009
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Open questions
H3/H4 assembled as tetramer or as dimer?
H2A/H2B chaperone?
how are the parental histones incorporated?
how are the chromatin marks copied?
Epigenetic memory
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Epigenetic Memory
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Hypothesis 1: H3H4 dimers are incorporatedcan “copy” modifications, variantsintramolecular
Hypothesis 2A: H3 H4 tetramers are incorporatedrepressive marks are defaultactivating marks incorporated during RI assembly variants
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Copy modification by recruiting the enzyme that makes the modificationto this same modification(Intermolecular)
Hypothesis 2
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DNA methylation (DNMT1) interacts with PCNA
KMT1C (H3K9me) interacts with DNMT1KMT1C also interacts with CAF1
HP1 interacts with CAF1
Late replicating
HETEROCHROMATINCharacterized by DNA methylation, HeK9me, and presence of HP1
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Heterochromatin: new possibilities for the inheritance of structureShiv IS Grewal and Sarah CR ElginCurrent Opinion in Genetics & Development 2002, 12:178–187
HS-2: heterochromatin
39C-X: euchromatin
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Chromatin Assembly
During Replication
Transcription
Repair
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Chromatin assembly comes in two flavors:
1. Replication coupled (RC)
2. Replication independent (RI)
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CAF1 H3/H4 assembly, disassembly
ASF1 H3/H4 H3/H4 dimerassembly, disassembly
FACT H2A/H2B assembly, disassembly
ISWI NA nucleosomal arrays, periodicityHeterochromatin replic.
CHD1 NA nucleosomal arrays
INO80 NA replication stress
Chromatin Assembly (RC)
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HirA H3.3/H4 deposition during transcription;
ASF1 H3.3/H4 interact with transcription elongation factor FACT
FACT H2A/H2B disassembly and reassembly during transcription elongation
CHD1 H3.3 interacts with HirA
SWR H2A.Z/H2B deposition during transcription
INO80 H2A.X/H2B deposition during DNA repair
Chromatin Assembly (RI, replacement)
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= CAC3
=Htz1
=CENP-A
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DNA repair
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Transcription
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Dis-assembly and assembly
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Histone turnover in genes
Consequence of nucleosome dis-assembly during transcription elongation
FACT elongation factor
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Transcription elongation
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Histone turnover at promoters/genes
Consequence of
disassembly/assembly
transcription
nucleosome eviction
chromatin remodeling
histone modifications
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Histone turnover at promoters/genes
Consequence of
disassembly/assembly
transcription
nucleosome eviction
chromatin remodeling
histone modifications
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promoter
promoter 5’ end gene
H3.3 incorporation during transcription
Drosophila
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Histone turnover at promoters/genes
Consequence of
disassembly/assembly
transcription
nucleosome eviction
chromatin remodeling
histone modifications
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High histone turnover at promoters is high = red nucleosomes
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GlossaryPCNA sliding clamp POLII processivityMCM helicase DNA unwinding/replicationNAP1 histone chaperone H2A/H2B nuclear importN1/N2 histone chaperone H3/H4 (storage)Nucleoplasmin histone chaperone H2A/H2B (storage)Rtt106 histone chaperone H3K56ac/H4 RC assemblyCAF1 histone chaperone H3/H4 RC assemblyASF1 histone chaperone H3/H4 RC and H3.3/H4 RI
assemblyHirA histone chaperone H3.3/H4 RI assemblyHAT1 cytoplasmic KATacetylates newly synthesized H4Rtt109 KAT acetylates H3K56ISWI chromatin remodeling assembly, spacing
ATPaseCHD1 chromatin remodeling assembly, H3.3 with HirA
ATPaseACS complexes containing assembly
ISWIINO80 chromatin remodeling replication stress, repair (H2AX)
ATPase/complexSWR chromatin remodeling H2AZ exchange