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
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.
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Nat Genet. 2015 May;47(5):469-78. doi: 10.1038/ng.3258. Epub 2015 Mar 30.
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.
DNA甲基化是一种关键的表观遗传修饰，参与调控基因表达并维持基因组完整性。在此，我们利用CRISPR/Cas9基因组编辑技术使人胚胎干细胞（ESC）中的所有三种具有催化活性的DNA甲基转移酶（DNMT）失活，以进一步研究这些酶的作用和基因组靶点。通过全基因组亚硫酸氢盐测序评估，单独破坏DNMT3A或DNMT3B以及同时破坏这两种酶，都会产生具有活力的多能细胞系，这些细胞系对DNA甲基化格局有不同影响。令人惊讶的是，与在小鼠中的研究结果相反，DNMT1的缺失导致人ESC迅速死亡。为克服这种直接致死性，我们构建了一个强力霉素响应型tTA-DNMT1拯救系，并轻易获得了纯合DNMT1突变系。然而，强力霉素介导的对外源DNMT1的抑制引发了DNA甲基化的快速、全面丧失，随后是广泛的细胞死亡。我们的数据提供了DNMT突变ESC的全面特征，包括这些酶靶点的全基因组单碱基图谱。
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