Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

Abstract

SUMMARYMammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.

The evidence for functional non-CpG methylation in mammalian cells

“…Several studies have revealed that in mouse GVOs, non-CpG methylation is prevalent at maternally methylated imprint control regions. 43,44 In mouse adult brain tissue, parentof-origin-dependent non-CpG methylation was associated with transcriptionally repressed alleles at a further eight imprinted loci. 17 There has also been a report implicating non-CpG methylation in regulating interchromosomal interactions between an enhancer element and odorant receptor (OR) genes within olfactory sensory neurons.…”

Methylation Status of H19/IGF2 Differentially Methylated Region in in vitro Human Blastocysts Donated by Healthy Couples

“…Whether the observed methylation status of the blastocyst is considered as a normal phenotype for these embryos remains to be answered, but if we consider this phenomenon as a divergence from the norm, numerous explanations can be provided that include: 1) paternally-derived altered methylation [35][36][37] , 2) maternally-derived altered methylation [38] , 3) reduced maintenance of DNA methylation [29,39] , 4) Non-rigid and dynamic methylation statuses of DMR [40][41][42] , 5) reduced or altered methylation due to ART procedure [30,43] , and 6) altered methylation due to technical issues. Each of these propositions requires to be evaluated in the context of new experiments.…”

“…Despite a general consensus that the methylation status of DMRs is persistent to genome-wide demethylation, recent evidence suggests that DMR can be affected at least in part by the genome-wide demethylation during mouse pre-implantation development [42] . In this regard, Tomizawa et al [40] have demonstrated that murine DMRs are not fully protected from the major epigenetic reprogramming events occur during preimplantation development. Instead, the DMRs appear to be demethylated and show dynamic changes in CpG methylation in blastocysts.…”

DNA methylation establishment during oocyte growth: mechanisms and significance

“…However, the functional role of non-CpG methylation is largely unclear, in particular because non-CpG methylation cannot be copied directly onto the daughter DNA strands after replication by Dnmt1. Non-CpG methylation is especially abundant in oocyte DNA (Tomizawa et al, 2011;Kobayashi et al, 2012), where it can be considered to be the result of accumulation of the non-CpG methylation activity of the de novo methyltransferases acting in a non-replicating cell. Whether it has any functional significance is not clear: there is no evidence that prominent sites of non-CpG methylation in oocytes retain high levels of methylation after fertilisation in preimplantation embryos (Tomizawa et al, 2011;Kobayashi et al, 2012).…”