生物
DNA甲基化
表观遗传学
甲基转移酶
DNMT1型
DNMT3B型
胚胎干细胞
DNA甲基转移酶
甲基化
遗传学
Cas9
细胞生物学
分子生物学
基因组
基因
基因表达
作者
Jing Liao,Rahul Karnik,Hongcang Gu,Michael J. Ziller,Kendell Clement,Alexander Tsankov,Veronika Akopian,Casey A. Gifford,Julie Donaghey,Christina Galonska,Ramona Pop,Deepak Reyon,Shengdar Q. Tsai,William Mallard,J. Keith Joung,John L. Rinn,Andreas Gnirke,Alexander Meissner
出处
期刊:Nature Genetics
[Springer Nature]
日期:2015-03-30
卷期号:47 (5): 469-478
被引量:366
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.
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