Accepted Manuscript Title Distinct factors control histone variant H3 3 localization at specific genomic regions Authors Aaron D Goldberg Laura A Banaszynski Kyung Min Noh Peter W Lewis Simon J Elsaesser Sonja Stadler Scott Dewell Martin Law Xingyi Guo Xuan Li Duancheng Wen Ariane Chapgier Russell C DeKelver Jeffrey C Miller Ya Li Lee Elizabeth A Boydston Michael C Holmes Philip D Gregory John M Greally Shahin Rafii Chingwen Yang Peter J Scambler David Garrick Richard J Gibbons Douglas R Higgs Ileana M Cristea Fyodor D Urnov Deyou Zheng C David Allis PII DOI Reference S0092 8674 10 00004 8 10 1016 j cell 2010 01 003 CELL 4956 Published in Cell Received date 9 September 2009 Revised date 23 November 2009 Accepted date 31 December 2009 Cite this article as Goldberg AD Banaszynski LA Noh K M Lewis PW Elsaesser SJ Stadler S Dewell S Law M Guo X Li X Wen D Chapgier A DeKelver RC Miller JC Lee Y L Boydston EA Holmes MC Gregory PD Greally JM Rafii S Yang C Scambler PJ Garrick D Gibbons RJ Higgs DR Cristea IM Urnov FD Zheng D Allis C D Distinct factors control histone variant H3 3 localization at specific genomic regions Cell doi 10 1016 j cell 2010 01 003 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting typesetting and review of the resulting proof before it is published in its final citable form Please note that during the production process errors may be discovered which could affect the content and all legal disclaimers that apply to the journal pertain 2010 Elsevier Inc All rights reserved Distinct factors control histone variant H3 3 localization at specific genomic regions Aaron D Goldberg1 Laura A Banaszynski1 Kyung Min Noh1 Peter W Lewis1 Simon J Elsaesser1 Sonja Stadler1 Scott Dewell2 Martin Law4 Xingyi Guo11 Xuan Li3 Duancheng Wen5 6 7 Ariane Chapgier8 Russell C DeKelver9 Jeffrey C Miller9 Ya Li Lee9 Elizabeth A Boydston9 Michael C Holmes9 Philip D Gregory9 John M Greally12 13 Shahin Rafii5 6 7 Chingwen Yang3 Peter J Scambler8 David Garrick4 Richard J Gibbons4 Douglas R Higgs4 Ileana M Cristea10 Fyodor D Urnov9 Deyou Zheng11 13 14 C David Allis1 1 Laboratory of Chromatin Biology 2Genomics Resource Center 3Gene Targeting Resource Center The Rockefeller University 1230 York Avenue New York NY 10065 USA 4 MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine John Radcliffe Hospital Headington Oxford OX3 9DS UK 5 Howard Hughes Medical Institute 6Ansary Stem Cell Institute 7Department of Genetic Medicine Weill Cornell Medical College New York NY 10065 USA 8 Molecular Medicine Unit Institute of Child Health 30 Guilford Street London WC1N 1EH UK 9 Sangamo BioSciences Inc Pt Richmond Tech Center 501 Canal Blvd Suite A100 Richmond CA 94804 USA 10 Department of Molecular Biology Princeton University Princeton NJ 08544 USA 11 Department of Neurology 12Department of Medicine 13Department of Genetics and 14 Department of Neuroscience Albert Einstein College of Medicine Bronx NY 10461 USA These authors contributed equally Contact alliscd rockefeller edu deyou zheng einstein yu edu 1 Summary 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 ES cells and neuronal precursor cells Genome wide 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 Introduction 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 non centromeric 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 4 or 5 amino acids Figure S1A H3 3 is specifically enriched at transcriptionally active genes and regulatory elements in non pluripotent cells Ahmad and Henikoff 2002 Jin et al 2009 Mito et al 2005 2007 2 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 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 Hodl 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
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