title: 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.
authors.affiliation: Division of Molecular Biology, Faculty of Medicine, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University, Tottori 683-8503, Japan. horinao@med.tottori-u.ac.jp
authors.forename: Naohiro
authors.initials: N
authors.lastname: Hori
authors.forename: Mariko
authors.initials: M
authors.lastname: Yamane
authors.forename: Kaori
authors.initials: K
authors.lastname: Kouno
authors.forename: Kenzo
authors.initials: K
authors.lastname: Sato
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000502:physiology*; D005260:Female; D018392:Genomic Imprinting / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D062085:RNA, Long Noncoding / Q000096:biosynthesis* / Q000235:genetics; D020218:Response Elements / Q000502:physiology*
chemical_list: C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D062085:RNA, Long Noncoding; D007335:Insulin-Like Growth Factor II