Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells

被引：362

作者：

Liao, Jing ^{[1
,2
,3
]}

Karnik, Rahul ^{[1
,2
,3
]}

Gu, Hongcang ^{[1
]}

Ziller, Michael J. ^{[1
,2
,3
]}

Clement, Kendell ^{[1
,2
,3
]}

Tsankov, Alexander M. ^{[1
,2
,3
]}

Akopian, Veronika ^{[1
,2
,3
]}

Gifford, Casey A. ^{[1
,2
,3
]}

Donaghey, Julie ^{[1
,2
,3
]}

Galonska, Christina ^{[1
,2
,3
]}

Pop, Ramona ^{[1
,2
,3
]}

Reyon, Deepak ^{[4
]}

Tsai, Shengdar Q. ^{[4
]}

Mallard, William ^{[1
,2
,3
]}

Joung, J. Keith ^{[4
]}

Rinn, John L. ^{[1
,2
,3
]}

Gnirke, Andreas ^{[1
]}

Meissner, Alexander ^{[1
,2
,3
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge, MA 02142 USA

[2] Harvard Stem Cell Inst, Cambridge, MA USA

[3] Harvard Univ, Dept Stem Cell & Regenerat Biol, Cambridge, MA 02138 USA

[4] Massachusetts Gen Hosp, Dept Pathol, Mol Pathol Unit, Charlestown, MA USA

来源：

NATURE GENETICS | 2015年 / 47卷 / 05期

关键词：

DE-NOVO METHYLATION; DNA METHYLATION; SELF-RENEWAL; HUMAN ES; PLURIPOTENT; EXPRESSION; MAINTENANCE; MUTATIONS; INDUCTION; CPG;

D O I：

10.1038/ng.3258

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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.

引用

页码：469 / U64

页数：12

共 66 条

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