Abstract
DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.
Highlights
Mouse H19 and insulin-like growth factor 2 (Igf2) genes are ϳ70 kb distant from each other and are a set of imprinted genes that are reciprocally expressed only from the maternal and paternal allele, respectively, in identical tissues with the same timing
These results indicate that the unmethylated state of the maternal imprinted control region (ICR) is actively maintained during development by a cis-acting sequence existing within an 0.75-kb portion that is in the dDMD region but not in the dSilencer region (Fig. 1a)
Cis-acting Element Directing Demethylation within the ICR—To determine the ability to maintain the unmethylated state within the ICR, we introduced a 3.8-kb construct in various states of methylation into the undifferentiated embryonal carcinoma cell line P19. pEE consists of a proximal promoter region of H19 and a portion of the ICR that contains the entire dDMD region and three CTCF-binding sites (Fig. 1a)
Summary
Mouse H19 and Igf (insulin-like growth factor 2) genes are ϳ70 kb distant from each other and are a set of imprinted genes that are reciprocally expressed only from the maternal and paternal allele, respectively, in identical tissues with the same timing Monoallelic expression of both genes depends on differential methylation of an imprinted control region (ICR), located 2– 4-kb upstream of the H19 gene, that is hypermethylated in the paternal chromosome but unmethylated in the maternal chromosome. When a 1.2-kb region (dSilencer) that comprises the hypermethylationdependent silencer within the ICR is deleted, the unmethylated status of the ICR is maintained on the maternal allele (4) These results indicate that the unmethylated state of the maternal ICR is actively maintained during development by a cis-acting sequence existing within an 0.75-kb portion (putative differential methylation control region) that is in the dDMD region but not in the dSilencer region (Fig. 1a). Oct-binding sequence (DOS) in the putative differential methylation control region is involved in demethylation of low-density methylated DNA and that DOS is required to maintain the unmethylated state in both the CTCF-binding site and CpG sequences widely distributed within the ICR
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