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Differentiation of embryonic stem (ES) cells is accompanied by silencing of the  Oct-4  gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of  Oct-4  in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the  Oct-4  promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the  Oct-4  regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.

Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells