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Berkeley MCELLBI 140 - Distinct Factors Control Histone

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Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic RegionsIntroductionResultsGenome-wide Patterns of H3.3 Enrichment Are Dependent upon Endogenous Amino Acid SequenceH3.3 Is Enriched at TSS of Active and Repressed HCP Genes, and in the Bodies and TES of Transcribed SequencesThe Profile of H3.3 at Cell Type-Specific Genes and Regulatory Elements Changes with Cell DifferentiationHira Is Required for Enrichment of H3.3 at Active and Repressed GenesHira-Independent Enrichment of H3.3 at Transcription Factor Binding SitesHira-Independent Association of Atrx and Daxx with Histone H3.3Atrx Is Required to Maintain H3.3 Localization at Telomeres and for Repression of Telomeric Repeat-Containing RNA in ESCsDiscussionExperimental ProceduresESC Culture and DifferentiationZFN Design, Targeting, and VerificationCellular Extract PreparationChIP and ChIP-SeqChIP-Seq Data AnalysisGene Expression Analysis of Wild-Type and Hira-/- ESCsIsolation of Protein Complexes and Mass Spectrometric AnalysisImmunofluorescence and Telomere Fluorescence In Situ HybridizationTERRA AnalysisAccession NumbersSupplemental InformationAcknowledgmentsReferencesSupplemental InformationExtended Experimental ProceduresMouse ES CellsZinc Finger Nuclease Design and ValidationH3.3B EYFP and HA-IRES-EYFP Donor Plasmid ConstructionZFN Targeting and VerificationES Cell Culture and NPC DifferentiationRT-PCRFlow CytometryMetaphase Chromosome SpreadsIsolation of Protein Complexes and Mass Spectrometric AnalysisAntibodiesCrosslinking and Native ChIPValidation of ChIP-SeqChIP-SeqData AnalysisGenome-wide Density Maps of H3 Variants and Determination of Enriched RegionsThe Relative Enrichment of H3 Variants in Repetitive ElementsDistributions of H3 Variants in Transcription Start and End SitesThe Relative Enrichment of H3 Variants in TFBSImmuno-FISH and MicroscopySupplemental ReferencesDistinct Factors Control HistoneVariantH3.3Localizationat Specific Genomic RegionsAaron D. Goldberg,1Laura A. Banaszynski,1,15Kyung-Min Noh,1,15Peter W. Lewis,1Simon J. Elsaesser,1Sonja Stadler,1Scott Dewell,2Martin Law,4Xingyi Guo,11Xuan Li,3Duancheng Wen,5,6,7Ariane Chapgier,8Russell C. DeKelver,9Jeffrey C. Miller,9Ya-Li Lee,9Elizabeth A. Boydston,9Michael C. Holmes,9Philip D. Gregory,9John M. Greally,12,13Shahin Rafii,5,6,7Chingwen Yang,3Peter J. Scambler,8David Garrick,4Richard J. Gibbons,4Douglas R. Higgs,4Ileana M. Cristea,10Fyodor D. Urnov,9Deyou Zheng,11,13,14,*and C. David Allis1,*1Laboratory of Chromatin Biology and Epigenetics2Genomics Resource Center3Gene Targeting Resource CenterThe Rockefeller University, 1230 York Avenue, New York, NY 10065, USA4MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK5Howard Hughes Medical Institute6Ansary Stem Cell Institute7Department of Genetic MedicineWeill Cornell Medical College, New York, NY 10065, USA8Molecular Medicine Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK9Sangamo BioSciences, Point Richmond Tech Center, 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA10Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA11Department of Neurology12Department of Medicine13Department of Genetics14Department of NeuroscienceAlbert Einstein College of Medicine, Bronx, NY 10461, USA15These authors contributed equally to this work*Correspondence: [email protected] (D.Z.), [email protected] (C.D.A.)DOI 10.1016/j.cell.2010.01.003SUMMARYThe incorporation of histone H3 variants has beenimplicated in the epigenetic memory of cellular state.Using genome editing with zinc-finger nucleasesto tag endogenous H3.3, we report genome-wideprofiles of H3 variants in mammalian embryonicstem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acidsequence and change with cellular differentiation atdevelopmentally regulated loci. The H3.3 chaperoneHira is required for H3.3 enrichment at active andrepressed genes. Strikingly, Hira is not essential forlocalization of H3.3 at telomeres and many transcrip-tion factor binding sites. Immunoaffinity purificationand mass spectrometry reveal that the proteinsAtrx and Daxx associate with H3.3 in a Hira-indepen-dent manner. Atrx is required for Hira-independentlocalization of H3.3 at telomeres and for the repres-sion of telomeric RNA. Our data demonstrate thatmultiple and distinct factors are responsible forH3.3 localization at specific genomic locations inmammalian cells.INTRODUCTIONGenetic and biochemical evidence have recently convergedto connect epigenetic mechanisms at the level of chromatin(Bernstein et al., 2007; Goldberg et al., 2007; Henikoff, 2008).In addition to nucleosome remodeling and covalent modifi-cations, eukaryotic cells generate variation in chromatin bythe introduction of variant histone proteins (Henikoff, 2008).Mammalian cells express three major types of noncentromerichistone H3 variants, H3.1, H3.2, and H3.3 (Hake and Allis,2006; Hake et al., 2006). Although histone H3.3 differs fromH3.2 and H3.1 at only four or five amino acids (Figure S1A avail-able online), H3.3 is specifically enriched at transcriptionallyactive genes and regulatory elements in nonpluripotent cells(Ahmad and Henikoff, 2002; Jin et al., 2009; Mito et al., 2005,2007).Histone H3.3 is incorporated into chromatin in both a replica-tion-coupled (RC) and replication-independent (RI) manner,while the incorporation of H3.2 is coupled to replication (Ahmadand Henikoff, 2002; De Koning et al., 2007). The histone chap-erone CAF-1 is found in a complex with H3.1 and mediates RCnucleosome assembly (Smith and Stillman, 1989; Tagamiet al., 2004). In contrast, the histone chaperone Hira has beenfound in a complex with H3.3 and mediates RI nucleosomeassembly (Ray-Gallet et al., 2002; Tagami et al., 2004).678 Cell 140, 678–691, March 5, 2010 ª2010 Elsevier Inc.Hira has been implicated in H3.3-specific deposition and chro-matin assembly (Loyola and Almouzni, 2007). Although Hira isrequired for chromatin assembly and H3.3 deposition in themale pronucleus of Drosophila, Hira is not required for globalH3.3 deposition in Drosophila embryos or adult cells, suggestingthat alternate pathways may mediate H3.3 nucleosome assem-bly (Bonnefoy et al., 2007; Loppin et al., 2005). Indeed, the chro-matin remodeling factor CHD1 was shown to physically asso-ciate with Hira and has been suggested to work with Hira


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Berkeley MCELLBI 140 - Distinct Factors Control Histone

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