Dynamic CpG and Non-CpG Methylation of the Peg1/Mest Gene in the Mouse Oocyte and Preimplantation Embryo

In somatic tissues, the CpG island of the imprinted Peg1/Mest gene is methylated on the maternal allele. We have examined the methylation of CpG and non-CpG sites of this differentially methylated CpG island in freshly ovulated oocytes, in vitro aged oocytes, and preimplantation embryos. The CpG methylation pattern was heterogeneous in freshly ovulated oocytes, despite the fact that they all were arrested in metaphase II. After short in vitro culture, Peg1/Mest became hypermethylated, whereas prolonged in vitro culture resulted in demethylation in a fraction of oocytes. Non-CpG methylation also occurred in a stage-specific manner. On alleles that were fully methylated at CpG sites, this modification was found, and it became reduced in two-cell stage embryos and blastocysts. These observations suggest that the process of establishment of the methylation imprint at this locus is more dynamic than previously thought. In somatic tissues, the CpG island of the imprinted Peg1/Mest gene is methylated on the maternal allele. We have examined the methylation of CpG and non-CpG sites of this differentially methylated CpG island in freshly ovulated oocytes, in vitro aged oocytes, and preimplantation embryos. The CpG methylation pattern was heterogeneous in freshly ovulated oocytes, despite the fact that they all were arrested in metaphase II. After short in vitro culture, Peg1/Mest became hypermethylated, whereas prolonged in vitro culture resulted in demethylation in a fraction of oocytes. Non-CpG methylation also occurred in a stage-specific manner. On alleles that were fully methylated at CpG sites, this modification was found, and it became reduced in two-cell stage embryos and blastocysts. These observations suggest that the process of establishment of the methylation imprint at this locus is more dynamic than previously thought. Establishment of the mature epigenetic configuration of the genome is part of the maturation process of the gametes and is essential for normal development after fertilization. DNA methylation of CpG sites is one of the epigenetic modifications that regulates gene expression (for review, see (1Shiota K. Yanagimachi R. Differentiation. 2002; 69: 162-166Crossref PubMed Scopus (61) Google Scholar)). The genome undergoes widespread changes in CpG methylation during germ cell maturation. Imprinted genes are of particular interest, because they are frequently associated with CpG-rich regions that are methylated differentially on the paternal and maternal chromosomes (for a review on imprinting, see Ref. 2Paldi A. Curr. Top. Dev. Biol. 2003; 53: 115-138Crossref PubMed Google Scholar). These differentially methylated regions (DMRs) 1The abbreviations used are: DMR, differentially methylated region; MII, meiotic division II. are believed to play an important role in the parental origin-dependent regulation of individual imprinted genes and of entire genomic regions during embryonic development. The methylation profile typical for the paternal or maternal chromosome is believed to be established during maturation of the male and female germ cells, but the exact kinetics of this process is still unclear. Some observations have even suggested that some imprinted genes reach their mature methylation profile only after fertilization (3El-Maarri O. Buiting K. Peery E.G. Kroisel P.M. Balaban B. Wagner K. Urman B. Heyd J. Lich C. Brannan C.I. Walter J. Horsthemke B. Nat. Genet. 2001; 27: 341-344Crossref PubMed Scopus (174) Google Scholar). Peg1/Mest is a typical imprinted gene that is predominantly expressed from the paternal allele in the mesoderm and its derivatives (4Kaneko-Ishino T. Kuroiwa Y. Miyoshi N. Kohda T. Suzuki R. Yokoyama M. Viville S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1995; 11: 52-59Crossref PubMed Scopus (254) Google Scholar). The methylation analysis of Peg1/Mest revealed that the CpG island in the promoter region was completely methylated on the maternal and unmethylated on the paternal chromosomes (5Lefebvre L. Viville S. Barton S.C. Ishino F. Surani M.A. Hum. Mol. Genet. 1997; 6: 1907-1915Crossref PubMed Scopus (69) Google Scholar). It has been shown that the human PEG1/MEST is already unmethylated in spermatogonia (6Kerjean A. Dupont J.M. Vasseur C. Le Tessier D. Cuisset L. Paldi A. Jouannet P. Jeanpierre M. Hum. Mol. Genet. 2000; 9: 2183-2187Crossref PubMed Scopus (184) Google Scholar). In the maternal germ line, Peg1/Mest is fully methylated in ovulated oocytes that are arrested in metaphase of the second meiotic division (MII) (7Lucifero D. Mertineit C. Clarke H.J. Bestor T.H. Trasler J.M. Genomics. 2002; 79: 530-538Crossref PubMed Scopus (355) Google Scholar). In a previous study (8Kerjean A. Couvert P. Heams T. Chalas C. Poirier K. Chelly J. Jouannet P. Paldi A. Poirot C. Eur. J. Hum. Genet. 2003; 11: 493-496Crossref PubMed Scopus (80) Google Scholar), we have detected methylation heterogeneity in growing oocytes at several imprinted loci, including Peg1/Mest. The heterogeneity was substantially increased in the oocytes matured in vitro, suggesting that the methylation imprint, at this stage, is unstable and can be influenced by the cellular environment. In the present study, we investigated whether changes in the methylation imprint occurred at the Peg1/Mest locus during the last stages of oocyte maturation. Although DNA methylation has been found predominantly on cytosines that are part of CpG dyads, it has been shown that cytosines in non-CpG sites can also be modified (9Ramsahoye B.H. Biniszkiewicz D. Lyko F. Clark V. Bird A.P. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5237-5242Crossref PubMed Scopus (757) Google Scholar, 10Haines T.R. Rodenhiser D.I. Ainsworth P.J. Dev. Biol. 2001; 240: 585-598Crossref PubMed Scopus (92) Google Scholar, 11Malone C.S. Miner M.D. Doerr J.R. Jackson J.P. Jacobsen S.E. Wall R. Teitell M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10404-10409Crossref PubMed Scopus (86) Google Scholar, 12Imamura T. Yamam