DNA microarray analysis of G9a knockout embryonic stem cells

In embryonic stem cells (ESCs), the expression of development-related genes, including germ cell–related genes, is globally repressed. The transcription factor MAX represses germ cell–related gene expression in ESCs via PCGF6-polycomb repressive complex (PRC)1, which consists of several epigenetic factors. However, we predicted that MAX represses germ cell–related gene expression through several additional mechanisms because PCGF6-PRC1 regulates the expression of only a subset of genes repressed by MAX. Here, we report that MAX associated with DNA methyltransferases (DNMTs) and the histone methyltransferase SETDB1 cooperatively control germ cell–related gene expression in ESCs. Both DNA methylation and histone H3 lysine 9 tri-methylation of the promoter regions of several germ cell–related genes were not affected by knockout of the PRC1 components, indicating that the MAX-DNMT and MAX-SETDB1 pathways are independent of the PCGF6-PRC1 pathway. Our findings provide insights into our understanding of MAX-based repressive mechanisms of germ cell–related genes in ESCs. Strategy for conditional G9a-KO was described in a previous report (Tachibana et al., 2007, EMBO J. 26:3346-59). In brief, we constructed a conditional G9a KO vector, in which exons 26, fused 27/28 (cDNA-derived), and PGK-Neo cassette were flanked by two loxP sequences. The targeting vector was introduced into TT2 ES cells carrying a G9a mutant alle (Tachibana et al., 2002, Genes Dev, 16, 1779-1791) and a wild-type allele (G9a-/+). Transfected cells were selected with medium containing G418 and ganciclovir. Successfully targeted recombinant cells, that carryed a G9a mutant allele and G9a-flox allele (G9a-/2lox), were screened by Southern hybridization. In this strategy, Cre recombinase treatment leads the deletion of exons 26 and 27 (encoding the SET domain catalytic core) form the G9a-flox allele, thereby procucing G9a-deficient cells (G9a-/1lox).