The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells

Epigenetics & Chromatin

Nathalia Azevedo Portilho¹  Jacinthe Sirois²  Deepak Saini²  Ishtiaque Hossain²  William A. Pastor²  Christopher Moraes³ 

[1] Department of Biochemistry, McGill University, H3G 1Y6, Montreal, QC, Canada;Department of Chemical Engineering, McGill University, H3A 0C5, Montreal, QC, Canada;Department of Biochemistry, McGill University, H3G 1Y6, Montreal, QC, Canada;The Rosalind and Morris Goodman Cancer Institute, McGill University, H3A 1A3, Montreal, QC, Canada;Department of Chemical Engineering, McGill University, H3A 0C5, Montreal, QC, Canada;The Rosalind and Morris Goodman Cancer Institute, McGill University, H3A 1A3, Montreal, QC, Canada;

关键词: DNA methylation;    DNMT1;    GSK-3484862;    GSK-3685032;    5-Azacytidine;    Decitabine;    Demethylation;    Whole-genome bisulfite sequencing;   

DOI  :  10.1186/s13072-021-00429-0

来源: Springer

【 摘 要 】

BackgroundDNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss.ResultsWe determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs.ConclusionsGSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

【 授权许可】

CC BY