Reduction of H3K9 methylation by G9a inhibitor improves development of mouse SCNT embryos

Inefficient mammalian cloning can be improved by removal of somatic H3K9me3 from the embryonic genome. However, this strategy involves injection of histone demethylase mRNA into embryos, which itself is a limiting step due to the invasive and labor-consuming nature. Here we report that treatment with an inhibitor against G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved development of cloned embryos in mice. Intriguingly, G9ai treatment resulted in immediate reduction of H3K9me1/2 and secondary loss of H3K9me3 in SCNT embryos. By G9ai treatment, the birth rate of cloned pups increased about fivefold (up to 3.9%) and, when combined with trichostatin A, a histone deacetylase inhibitor, it was further improved to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic activation of Obox family genes. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications. Overall design: Comparative analysis of gene expression in embryos derived from IVF, non-treated SCNT, TSA-treated SCNT, G9a inhibitor-treated SCNT, and TSA&G9a inhibitor-treated SCNT.