title: Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jing
authors.initials: J
authors.lastname: Liao
authors.affiliation: 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.
authors.forename: Rahul
authors.identifier: 000000020400635X
authors.initials: R
authors.lastname: Karnik
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Hongcang
authors.initials: H
authors.lastname: Gu
authors.affiliation: 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.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ziller
authors.affiliation: 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.
authors.forename: Kendell
authors.initials: K
authors.lastname: Clement
authors.affiliation: 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.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Tsankov
authors.affiliation: 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.
authors.forename: Veronika
authors.initials: V
authors.lastname: Akopian
authors.affiliation: 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.
authors.forename: Casey A
authors.initials: CA
authors.lastname: Gifford
authors.affiliation: 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.
authors.forename: Julie
authors.initials: J
authors.lastname: Donaghey
authors.affiliation: 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.
authors.forename: Christina
authors.initials: C
authors.lastname: Galonska
authors.affiliation: 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.
authors.forename: Ramona
authors.initials: R
authors.lastname: Pop
authors.affiliation: Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.affiliation: Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.affiliation: 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.
authors.forename: William
authors.initials: W
authors.lastname: Mallard
authors.affiliation: Department of Pathology, Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.affiliation: 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.
authors.forename: John L
authors.initials: JL
authors.lastname: Rinn
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gnirke
authors.affiliation: 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.
authors.forename: Alexander
authors.initials: A
authors.lastname: Meissner
mesh_terms: D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D020134:Catalytic Domain; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D053595:Embryonic Stem Cells / Q000201:enzymology* / Q000502:physiology; D044127:Epigenesis, Genetic; D015870:Gene Expression; D055786:Gene Knockout Techniques; D006801:Humans; D051379:Mice; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse
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