Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells
Published Date:Mar 30 2015
Source:Nat Genet. 47(5):469-478.
Embryonic Stem Cells
Gene Knockout Techniques
Pubmed Central ID:PMC4414868
Funding:DP1 GM105378/DP/NCCDPHP CDC HHS/United States
P01 GM099117/GM/NIGMS NIH HHS/United States
P01GM099117/GM/NIGMS NIH HHS/United States
R01 DA036898/DA/NIDA NIH HHS/United States
Description: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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