Distinct Factors Control Histone Variant H3 3 Localization at Specific Genomic Regions Aaron D Goldberg 1 Laura A Banaszynski 1 15 Kyung Min Noh 1 15 Peter W Lewis 1 Simon J Elsaesser 1 Sonja Stadler 1 Scott Dewell 2 Martin Law 4 Xingyi Guo 11 Xuan Li 3 Duancheng Wen 5 6 7 Ariane Chapgier 8 Russell C DeKelver 9 Jeffrey C Miller 9 Ya Li Lee 9 Elizabeth A Boydston 9 Michael C Holmes 9 Philip D Gregory 9 John M Greally 12 13 Shahin Rafii 5 6 7 Chingwen Yang 3 Peter J Scambler 8 David Garrick 4 Richard J Gibbons 4 Douglas R Higgs 4 Ileana M Cristea 10 Fyodor D Urnov 9 Deyou Zheng 11 13 14 and C David Allis1 1Laboratory of Chromatin Biology and Epigenetics Resource Center 3Gene Targeting Resource Center The Rockefeller University 1230 York Avenue New York NY 10065 USA 4MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine John Radcliffe Hospital Headington Oxford OX3 9DS UK 5Howard Hughes Medical Institute 6Ansary Stem Cell Institute 7Department of Genetic Medicine Weill Cornell Medical College New York NY 10065 USA 8Molecular Medicine Unit Institute of Child Health 30 Guilford Street London WC1N 1EH UK 9Sangamo BioSciences Point Richmond Tech Center 501 Canal Boulevard Suite A100 Richmond CA 94804 USA 10Department of Molecular Biology Princeton University Princeton NJ 08544 USA 11Department of Neurology 12Department of Medicine 13Department of Genetics 14Department of Neuroscience Albert Einstein College of Medicine Bronx NY 10461 USA 15These authors contributed equally to this work Correspondence deyou zheng einstein yu edu D Z alliscd rockefeller edu C D A DOI 10 1016 j cell 2010 01 003 2Genomics SUMMARY INTRODUCTION The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state Using genome editing with zinc finger nucleases to tag endogenous H3 3 we report genome wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells Genomewide patterns of H3 3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci The H3 3 chaperone Hira is required for H3 3 enrichment at active and repressed genes Strikingly Hira is not essential for localization of H3 3 at telomeres and many transcription factor binding sites Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3 3 in a Hira independent manner Atrx is required for Hira independent localization of H3 3 at telomeres and for the repression of telomeric RNA Our data demonstrate that multiple and distinct factors are responsible for H3 3 localization at specific genomic locations in mammalian cells Genetic and biochemical evidence have recently converged to 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 modifications eukaryotic cells generate variation in chromatin by the introduction of variant histone proteins Henikoff 2008 Mammalian cells express three major types of noncentromeric histone H3 variants H3 1 H3 2 and H3 3 Hake and Allis 2006 Hake et al 2006 Although histone H3 3 differs from H3 2 and H3 1 at only four or five amino acids Figure S1A available online H3 3 is specifically enriched at transcriptionally active 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 replication coupled RC and replication independent RI manner while the incorporation of H3 2 is coupled to replication Ahmad and Henikoff 2002 De Koning et al 2007 The histone chaperone CAF 1 is found in a complex with H3 1 and mediates RC nucleosome assembly Smith and Stillman 1989 Tagami et al 2004 In contrast the histone chaperone Hira has been found in a complex with H3 3 and mediates RI nucleosome assembly Ray Gallet et al 2002 Tagami et al 2004 678 Cell 140 678 691 March 5 2010 2010 Elsevier Inc Hira has been implicated in H3 3 specific deposition and chromatin assembly Loyola and Almouzni 2007 Although Hira is required for chromatin assembly and H3 3 deposition in the male pronucleus of Drosophila Hira is not required for global H3 3 deposition in Drosophila embryos or adult cells suggesting that alternate pathways may mediate H3 3 nucleosome assembly Bonnefoy et al 2007 Loppin et al 2005 Indeed the chromatin remodeling factor CHD1 was shown to physically associate with Hira and has been suggested to work with Hira to mediate H3 3 incorporation into chromatin in Drosophila Konev et al 2007 In Drosophila both Hira and H3 3 are required for fertility and for transcriptional regulation of specific genes but not for developmental viability Bonnefoy et al 2007 Ho dl and Basler 2009 Nakayama et al 2007 Sakai et al 2009 However in mice targeted mutagenesis of Hira results in a more severe phenotype with gastrulation defects leading to early embryonic lethality Roberts et al 2002 Given the conserved association between H3 3 and active chromatin H3 3 has been speculated to play an important role in mammalian embryonic stem cells ESCs Creyghton et al 2008 Gaspar Maia et al 2009 However no genome wide studies in pluripotent cells distinguish between H3 variants nor do they examine the genome wide role of Hira or other histone chaperones in specifying H3 3 localization at specific genomic regions Here we report genome wide profiles of histone H3 variant localization in mammalian ESCs and neuronal precursor cells NPCs and we establish the dependence and independence of these patterns on Hira We find that Hira is required for genome wide H3 3 enrichment at active and repressed genes in ESCs Surprisingly H3 3 enrichment at many transcription factor binding sites TFBS and telomeres is Hira independent To identify factors that might mediate specific Hira independent localization of H3 3 we use immunoprecipitation and mass spectrometry We identify Atrx and Daxx as proteins that specifically associate with H3 3 in both pluripotent and nonpluripotent cells both in the presence and in the absence of Hira Unlike Hira Atrx is not required for H3 3 localization at genes or TFBS However Atrx is specifically required for enrichment of H3 3 at telomeres in ESCs and for the repression of telomeric RNA RESULTS Genome wide Patterns of H3 3 Enrichment Are Dependent upon Endogenous Amino Acid Sequence To distinguish H3 variants in our study without altering endogenous
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