Abstract
Histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation is thought to be a major influential factor in cellular reprogramming, such as somatic cell nuclear transfer (SCNT) and induction of pluripotent stem cells (iPSCs). The diazepin-quinazolin-amine derivative (BIX-01294) specifically inhibits the activity of histone methyltransferase EHMT2 (euchromatic histone-lysine N-methyltransferase 2) and reduces H3K9me2 levels in cells. The imprinted gene small nuclear ribonucleoprotein N (Snrpn) is of particular interest because of its important biological functions. The objective of the present study was to investigate the effect of BIX-01294 on H3K9me2 levels and changes in Snrpn DNA methylation and histone H3K9me2 in mouse embryonic fibroblasts (MEFs). Results showed that 1.3 μM BIX-01294 markedly reduced global levels of H3K9me2 with almost no cellular toxicity. There was a significant decrease in H3K9me2 in promoter regions of the Snrpn gene after BIX-01294 treatment. A significant increase in methylation of the Snrpn differentially methylated region 1 (DMR1) and slightly decreased transcript levels of Snrpn were found in BIX-01294-treated MEFs. These results suggest that BIX-01294 may reduce global levels of H3K9me2 and affect epigenetic modifications of Snrpn in MEFs.
Highlights
Studies have shown that somatic cell nuclear transfer (SCNT) embryos consistently display aberrant histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation [1]
Estimation of BIX-01294 cytotoxicity on cultured mouse embryonic fibroblast (MEF) The cultured MEFs became sparse and exhibited anomalous morphology after incubation with BIX-01294 at 2 μM or higher, indicating that BIX-01294 is cytotoxic to MEFs at these concentrations
Based on the relative growth rate (RGR) (Table 2) [18], MTT assays indicated that 2 μM BIX-01294 (RGR = 78.14 %) or higher produced mild cytotoxicity in cultured MEFs (Figure 1)
Summary
Studies have shown that somatic cell nuclear transfer (SCNT) embryos consistently display aberrant histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation [1]. Chen et al [2] showed that H3K9 methylation is a barrier to somatic cell reprogramming into iPSCs, indicating that H3K9me hypermethylation might be a major influential factor in embryonic reprogramming. This hypothesis is supported by Sridharan et al.’s [3] demonstration that inhibition of H3K9 methylation enhances reprogramming. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2, known as G9a) is believed to be dominant in catalysing H3K9me and regulates gene repression [4].
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