High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice

Abstract

Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genomewide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra-and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germlinespecific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.

Linkage disequilibrium analysis of allelic heterogeneity in DNA methylation

“…The embryonic germ cells undergo epigenetic reprogramming during which methylation levels change dramatically. 3 Thus, it is possible that positive D values observed in germline cells may be the result of global changes in methylation status. In contrast, methylation patterns are relatively stable in adult somatic cells.…”

“…3,10,11 To characterize the methylation pattern within each allele underlying heterogeneity, we focused on the methylation status of the 2 adjacent CpG sites (CpG pair) residing in a single sequencing read, which is derived from a single allele. We examined the linkage of methylation states in a CpG pair using a modified version of the coefficient of linkage disequilibrium, hereafter designated as D. The coefficient D corresponds to the deviation of the observed frequency of alleles from their equilibrium frequency.…”

“…We examined the deviation in male primordial germ cells (embryonic day 13.5 and 16.5) and in sperm cells, each corresponding to the progressive stages of reprogramming. 3 On day 13.5, no deviation in male primordial germ cells is observed when methylation is fully erased and reprogramming has not yet started (Fig. 5B).…”

“…[18][19][20] The allelic heterogeneity becomes most obvious at the period between these 2 phases. 3 Knowledge of the methylation pattern underlying allelic heterogeneity may assist in understanding the mechanism of de novo methylation acting in the epigenetic reprogramming. We considered 2 hypotheses: 1) de novo methylation progresses stochastically at individual CpG sites, resulting in the spatially interspersed methylation patterns in all alleles, or 2) de novo methylation occurs stochastically among alleles; however, once present, it propagates rapidly, resulting in the mixture of fully methylated and unmethylated alleles (Fig.…”

“…While the methylation status of individual CpG sites is generally consistent among alleles, resulting in methylated or unmethylated CpG sites in all alleles, there are certain regions that show heterogeneity in methylation status among alleles. The methylation heterogeneity (intermediate methylation 1 or partially methylated domain 2 ) is reported in primordial germ cells under epigenetic reprogramming, 3 sperms, 4 embryonic stem cells, 5 induced pluripotent stem cells, 6 placental cells, 2 and other somatic cells under normal and disease conditions. [7][8][9] These regions are highly gene enriched and often interact with other epigenetic markers such as histone modifications.…”