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.
Highlights
The unmethylated state of the imprinted control region (ICR) established in female germ cells is maintained during development, resisting the wave of genomewide de novo methylation
We previously demonstrated that a DNA fragment derived from this ICR-induced
We propose a link between the mechanisms for maintenance of the unmethylated state of the H19/insulin-like growth factor 2 (Igf2) ICR and the undifferentiated cell-specific induction of DNA demethylation
Summary
The Sox-Oct motif functions to induce DNA demethylation in P19 cells. Significance: The Sox-Oct motif might be a key element to induce region-specific DNA demethylation as well as activate transcription in undifferentiated cells. 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. To understand the mechanisms of maintaining the unmethylated state of the ICR, we previously screened for cis-acting sequences required to induce DNA demethylation in undifferentiated cells, and identified an. Two octamer motifs with 12 bp of intervening sequence (dyad Oct-binding sequence) in fragment b were required for the efficient induction of DNA demethylation and maintenance of the unmethylated state within the ICR on a transgene. We found that SO1 is a methylation-sensitive element The requirement for both a hypermethylation-sensitive Sox-Oct motif and the Oct3/4 protein for regional demethylation provides a novel insight into the maintenance mechanisms of the unmethylated state of the mouse H19/Igf ICR
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