Timing and sequence requirements defined for embryonic maintenance of imprinted DNA methylation at Rasgrf1

Epigenetic programming is critical for normal development of mammalian embryos. Errors cause misexpression of genes and aberrant development (E. Li, C. Beard, and R. Jaenisch, Nature 366:362-365, 1993). Imprinted genes are important targets of epigenetic regulation, but little is known about how the epigenetic patterns are established in the parental germ lines and maintained in the embryo. Paternal allele-specific expression at the imprinted Rasgrf1 locus in mice is controlled by paternal allele-specific methylation at a differentially methylated domain (DMD). DMD methylation is in turn controlled by a direct repeat sequence immediately downstream of the DMD which is required for establishing Rasgrf1 methylation in the male germ line (B. J. Yoon et al., Nat. Genet. 30:92-96, 2002). To determine if these repeats have a role in methylation maintenance, we developed a conditional deletion of the repeat sequence in mice and showed that the repeats are also required during a narrow interval to maintain paternal methylation of Rasgrf1 in developing embryos. Removing the repeats upon fertilization caused a total loss of methylation by the morula stage, but by the epiblast stage, the repeats were completely dispensable for methylation maintenance. This developmental interval coincides with genome-wide demethylation and remethylation in mice which most imprinted genes resist. Our data show that the Rasgrf1 repeats serve at least two functions: first, to establish Rasgrf1 DNA methylation in the male germ line, and second, to resist global demethylation in the preimplantation embryo.