Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2) Imprinted Control Region

Abstract

DNA demethylation is used to establish and maintain an unmethylated state. The molecular mechanisms to induce DNA demethylation at a particular genomic locus remain unclear. The mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region (ICR) is a methylation state-sensitive insulator that regulates transcriptional activation of both genes. The unmethylated state of the ICR established in female germ cells is maintained during development, resisting the wave of genome-wide de novo methylation. We previously demonstrated that a DNA fragment (fragment b) derived from this ICR-induced DNA demethylation when it was transfected into undifferentiated mouse embryonal carcinoma cell lines. Moreover, two octamer motifs within fragment b were necessary to induce this DNA demethylation. Here, we demonstrated that both octamer motifs and their flanking sequences constitute Sox-Oct motifs (SO1 and SO2) and that the SO1 region, which requires at least four additional elements, including the SO2 region, contributes significantly to the induction of high-frequency DNA demethylation as a Sox-Oct motif. Moreover, RNAi-mediated inhibition of Oct3/4 expression in P19 cells resulted in a reduced DNA demethylation frequency of fragment b but not of the adenine phosphoribosyltransferase gene CpG island. The Sox motif of SO1 could function as a sensor for a hypermethylated state of the ICR to repress demethylation activity. These results indicate that Sox-Oct motifs in the ICR determine the cell type, DNA region, and allele specificity of DNA demethylation. We propose a link between the mechanisms for maintenance of the unmethylated state of the H19/Igf2 ICR and the undifferentiated cell-specific induction of DNA demethylation.