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Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.
Liao, Jing; Karnik, Rahul; Gu, Hongcang; Ziller, Michael J; Clement, Kendell; Tsankov, Alexander M; Akopian, Veronika; Gifford, Casey A; Donaghey, Julie; Galonska, Christina; Pop, Ramona; Reyon, Deepak; Tsai, Shengdar Q; Mallard, William; Joung, J Keith; Rinn, John L; Gnirke, Andreas; Meissner, Alexander.
Afiliação
  • Liao J; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Karnik R; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Gu H; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
  • Ziller MJ; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Clement K; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Tsankov AM; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Akopian V; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Gifford CA; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Donaghey J; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Galonska C; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Pop R; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Reyon D; Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
  • Tsai SQ; Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
  • Mallard W; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Joung JK; Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
  • Rinn JL; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
  • Gnirke A; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
  • Meissner A; 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
Nat Genet ; 47(5): 469-78, 2015 May.
Article em En | MEDLINE | ID: mdl-25822089
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Metilação de DNA / DNA (Citosina-5-)-Metiltransferases / Células-Tronco Embrionárias Limite: Animals / Humans Idioma: En Ano de publicação: 2015 Tipo de documento: Article
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Metilação de DNA / DNA (Citosina-5-)-Metiltransferases / Células-Tronco Embrionárias Limite: Animals / Humans Idioma: En Ano de publicação: 2015 Tipo de documento: Article