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

Nat Genet. 2015 May;47(5):469-78. doi: 10.1038/ng.3258. Epub 2015 Mar 30.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Base Sequence
  • Catalytic Domain
  • Cell Differentiation
  • Cell Proliferation
  • Cell Survival
  • Cells, Cultured
  • Coculture Techniques
  • CpG Islands
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases / genetics*
  • DNA Methylation*
  • DNA Methyltransferase 3A
  • Embryonic Stem Cells / enzymology*
  • Embryonic Stem Cells / physiology
  • Epigenesis, Genetic
  • Gene Expression
  • Gene Knockout Techniques
  • Humans
  • Mice

Substances

  • DNMT3A protein, human
  • Dnmt3a protein, mouse
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA Methyltransferase 3A
  • DNA methyltransferase 3B
  • DNMT1 protein, human
  • Dnmt1 protein, mouse

Associated data

  • GEO/GSE63281