Abstract
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.
Highlights
DNA methylation is a key regulator of gene expression in mammals [1]
Our results indicate that GSK-3484862 mediates striking demethylation in Murine embryonic stem cells (mESCs), comparable to what is observed in a complete DNMT1 knockout, with minimal non-specific toxicity
Results mESCs tolerate high concentrations of GSK‐3484862 As of this writing, GSK-3484862 and related inhibitors are not available for sale from GlaxoSmithKline, so we conducted experiments using GSK-3484862 purchased from ChemieTek and MedChemExpress
Summary
DNA methylation is a key regulator of gene expression in mammals [1]. DNA methyltransferases (DNMTs) transfer a methyl group to the fifth carbon of the DNAAzevedo Portilho et al Epigenetics & Chromatin (2021) 14:56 methylation patterns through many cycles of cell division [4, 5].DNA methylation is critical for maintaining silencing of certain genes, genes associated with germline development, and transposons [1, 6, 7]. There is high interest in demethylating drugs for both research and therapeutic purposes [9]. Two such drugs are widely used in research and as therapeutics: 5-azacytidine and decitabine (5-aza-2’-deoxycytidine). They were synthesized in the 1960s as potential chemotherapeutics that might interfere with nucleic acid metabolism in rapidly dividing cells [10, 11]. 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. 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
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