Chromatin Assembly

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Chromatin Assembly. Nucleosomal DNA Primary chromatin structure beads-on-a-string. ac. ac. ac. ac. DNA methylation. ac. Nucleosome. from Horn and Peterson Science , 2002. Visualizing this primary structure nucleosomal arrays. - PowerPoint PPT Presentation

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  • Chromatin AssemblyNucleosomal DNA Primary chromatin structurebeads-on-a-string

  • Nucleosomefrom Horn and Peterson Science, 2002

  • Visualizing this primary structure nucleosomal arraysPartial nuclease digestion generates mono, di, trinucleosomes

  • How is chromatin assembly achieved?

  • Irregular assemblyDynamics of ATP-dependent chromatin assembly by ACFDmitry V. Fyodorov & James T. KadonagaNATURE | VOL 418 | 22 AUGUST 2002 |Assembly

    in vitroHistones + DNA + saltassemble into aggregatesHistones + DNA + negatively charged proteins= mononucleosomes andpoor nucleosmal array (irregularly spaced; smear)

  • 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)

  • What is in the extract?

    Histone chaperones

  • Histone chaperonesnegatively charged proteinshelp assembly; prevent aggregation

    NAP1 H2A/H2BNuclear Import, nucleosomedisruption, histone variant incorporation

    nucleoplasmin H2A/H2BStorage, sperm histone exchange

    N1/N2 H3/H4Storage

  • CAF-1Chromatin assembly factor 1More histone chaperones

  • 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 bindingin many complexesRolesnucleosome deposition after replicationheterochromatin maintenance

  • Ransom et al., Cell, 2010Smallest CAF subunit bound to histone H4

  • Replication

  • Cell, Vol. 96, 575585, February 19, 1999, Replication-Dependent Marking of DNA by PCNAFacilitates CAF-1-Coupled Inheritance of ChromatinKei-ichi Shibahara and Bruce Stillman

  • Cac2 interacts with PCNA when Cac1 is presentPCNA connects DNA replication to epigenetic inheritance in yeastZhiguo Zhang, Kei-ichi Shibahara & Bruce StillmanNATURE | VOL 408 | 9 NOVEMBER 2000PCNA

  • 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 coloniesActivation of Ade in telomeric heterochromatin in PCNA mutantsPCNA connects DNA replication to epigenetic inheritance in yeastZhiguo Zhang, Kei-ichi Shibahara* & Bruce Stillman*NATURE | VOL 408 | 9 NOVEMBER 2000 |Ade -Ade +PCNA wtPCNA mutant

  • PCNA coordinates: DNA synthesis (processivity)Chromatin assemblyRepairMethylation Epigenetic InheritanceCell-cycle control

  • Parental nucleosome segregation (hatched)

    De novo nucleosome assembly (unhatched)

  • 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 G2Histone synthesis occurs at G1/S

  • 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)

  • Corpet and Almouzni TICB 2008H3K56acH3K9/14acGCN5Rtt109

  • These modifications are important reduced viability in yeast if mutatedRole:Nuclear importBinding/Deposition by histone chaperoneChromatin maturation

    Mark the histone as newly synthesized for chromatinassembly

    Modifications are rapidly removed after assembly

  • 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 Genevive Almouznior tetramer

  • ASF1

    acts together with CAF1, interacts with CAC2 subunit

    interacts with HAT1 complex

    interacts with MCM helicase

    Another histone chaperone

  • Trujillo and Osley Nature Structural & Molecular Biology 2008

  • The ins and outs of nucleosome assemblyJill A Mello* and Genevive Almouzni Current Opinion in Genetics & Development 2001, 11:136141

  • Role of ASF1:generates H3 H4 dimer

    prevents tetramer formationbreaks up tetramerspresents H3/H4 dimer to CAF1

    also role in H3K56ac by Rtt109

  • Ransom et al., Cell, 2010

  • Another histone chaperoneRtt106

    interacts with CAF1 complex

    involved in H3/H4 assembly during replication

    high affinity for H3K56ac (as does CAF1)

  • Modified from Fillingham and Greenblatt, Cell 2008

  • Ransom et al., Cell, 2010H2A/H2B chaperone: FACT?

  • BOTH long arrays AND properly spaced nucleosomes

    require other enzymes: ATP-dependent chromatin remodelersChromatin assemblyChromatin array formationATPasecomplexISWI ACF/ChRACCHD1-

    INO80INO80

  • ISWI: chromatin remodeling ATPase

    roles in chromatin assembly together with NAP1 or Asf1linker length, formation of long nucleosomal arrays

    transcriptional elongationsilencingchromosome organization

  • CHD1: chromatin remodeling ATPaseroles in chromatin assembly together with NAP1histone variant assembly

  • Enhanced assemblyLusser et al., Nature Struc Mol Biol 2005

  • Different periodicityLusser et al., Nature Struc Mol Biol 2005

  • Clapier and Cairns, Annu. Rev. Biochem., 2009

  • INO80: chromatin remodeling ATPaseroles in replication stress

  • Morrison and Shen, Nature reviews Mol Cell Biol, 2009

  • 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

  • Epigenetic Memory

  • 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

  • Copy modification by recruiting the enzyme that makes the modificationto this same modification(Intermolecular)Hypothesis 2

  • DNA methylation (DNMT1) interacts with PCNA

    KMT1C (H3K9me) interacts with DNMT1KMT1C also interacts with CAF1

    HP1 interacts with CAF1

    Late replicatingHETEROCHROMATINCharacterized by DNA methylation, HeK9me, and presence of HP1

  • Heterochromatin: new possibilities for the inheritance of structureShiv IS Grewal and Sarah CR ElginCurrent Opinion in Genetics & Development 2002, 12:178187HS-2: heterochromatin

    39C-X: euchromatin

  • Chromatin AssemblyDuring Replication TranscriptionRepair

  • Chromatin assembly comes in two flavors:

    Replication coupled (RC)

    Replication independent (RI)

  • CAF1H3/H4assembly, disassembly

    ASF1H3/H4H3/H4 dimerassembly, disassembly

    FACTH2A/H2Bassembly, disassembly

    ISWINAnucleosomal arrays, periodicityHeterochromatin replic.CHD1NAnucleosomal arrays

    INO80NAreplication stress

    Chromatin Assembly (RC)

  • HirAH3.3/H4deposition during transcription; ASF1H3.3/H4 interact with transcription elongation factor FACTFACTH2A/H2B disassembly and reassembly during transcription elongation

    CHD1H3.3interacts with HirA

    SWRH2A.Z/H2Bdeposition during transcription

    INO80H2A.X/H2Bdeposition during DNA repairChromatin Assembly (RI, replacement)

  • = CAC3=Htz1=CENP-A

  • DNA repair

  • Transcription

  • Dis-assembly and assembly

  • Histone turnover in genes

    Consequence of nucleosome dis-assembly during transcription elongation

    FACT elongation factor

  • Transcription elongation

  • Histone turnover at promoters/genes

    Consequence of

    disassembly/assemblytranscription

    nucleosome eviction

    chromatin remodeling

    histone modifications

  • Histone turnover at promoters/genes

    Consequence of

    disassembly/assemblytranscription

    nucleosome eviction

    chromatin remodeling

    histone modifications

  • promoterpromoter5 end geneH3.3 incorporation during transcriptionDrosophila

  • Histone turnover at promoters/genes

    Consequence of

    disassembly/assemblytranscription

    nucleosome eviction

    chromatin remodeling

    histone modifications

  • High histone turnover at promoters is high = red nucleosomes

  • GlossaryPCNAsliding clampPOLII processivityMCMhelicaseDNA unwinding/replicationNAP1histone chaperoneH2A/H2B nuclear importN1/N2histone chaperoneH3/H4 (storage)Nucleoplasminhistone chaperoneH2A/H2B (storage)Rtt106histone chaperoneH3K56ac/H4 RC assemblyCAF1histone chaperoneH3/H4 RC assemblyASF1histone chaperoneH3/H4 RC and H3.3/H4 RI assemblyHirAhistone chaperoneH3.3/H4 RI assemblyHAT1cytoplasmic KATacetylates newly synthesized H4Rtt109KATacetylates H3K56ISWIchromatin remodelingassembly, spacingATPaseCHD1chromatin remodelingassembly, H3.3 with HirAATPaseACScomplexes containingassemblyISWIINO80chromatin remodelingreplication stress, repair (H2AX)ATPase/complexSWRchromatin remodelingH2AZ exchange

    *To change in 2011:For INO80 get a repair data sildeAdd more on histone variantsFlesh out and later copying memory H3/H4 slides***********Polymerase delta: continuous extension on leading strand;Discon

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