title: Characterisation of histone variant distribution in human embryonic stem cells by transfection of in vitro transcribed mRNA.
abstract: Recent studies, primarily in mouse embryonic stem cells, have highlighted the unique chromatin state of pluripotent stem cells, including the incorporation of histone variants into specific genomic locations, and its role in facilitating faithful expression of genes during development. However, there is little information available on the expression and subcellular localisation of histone variants in human embryonic stem cells (hESCs). In this study, we confirmed the expression of a panel of histone variant genes in several hESC lines and demonstrated the utility of transfection of in vitro transcribed, epitope-tagged mRNAs to characterise the subcellular localisation of these proteins. The subcellular localisations of variant histone H3 (CENP-A, H3.3), H2A (MACROH2A, H2AX, H2AZ, H2ABBD) and H1 (H1A, HB, H1C, H1D) were examined, revealing distinct nuclear localisation profiles for each protein. These data highlight the differences between murine (m) ESCs and hESCs, including the presence of a MACROH2A-enriched inactive X chromosome in undifferentiated XX hESC lines. We also provide the first evidence for MACROH2A accumulation on the Y-chromosome in XY hESCs.
authors.affiliation: Developmental Epigenetics, Department of Paediatrics, Murdoch Childrens Research Institute, Royal Children's Hospital, University of Melbourne, Parkville, Melbourne, Victoria, Australia.
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Shaw
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Williams
authors.forename: Susan
authors.initials: S
authors.lastname: Hawes
authors.forename: Richard
authors.initials: R
authors.lastname: Saffery
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D049951:X Chromosome Inactivation
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; C000619701:Cenpa protein, mouse; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C120756:macroH2A histone