Abstract
A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.
Highlights
Genomic imprinting is an epigenetic phenomenon that employs DNA methylation to direct unequal expression of the two parental alleles of a gene
Our results suggest that the stem cell/germline factors Sox2 and Oct4 participate in imprinting control regions (ICR) hypomethylation
Using methylation sensitive Southern analysis, we found that integrated transgenes containing the wild type ICR and H19 promoter did not acquire methylation in F9 cells, while mutation of the octamers resulted in a small amount of de novo ICR methylation, which is shown by the incomplete digestion of the BglII fragment with HpaII (Fig. 1A and B)
Summary
Genomic imprinting is an epigenetic phenomenon that employs DNA methylation to direct unequal expression of the two parental alleles of a gene. A subset of DMRs are the key elements directing monoallelic transcription of one or more imprinted genes and are often termed ‘imprinting control regions’ (ICRs). Their parental-specific DNA methylation imprints generally are established during oogenesis or spermatogenesis and are maintained after fertilization in somatic cell lineages. ICRs regulate transcription of imprinted genes by several mechanisms, including methylation-dependent repression, expression of non-coding RNAs, and long-range chromatin interactions [1]. In addition to DNA methylation, certain histone modifications are frequently associated with ICRs, and there is evidence that they can act as an imprint [2]
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