Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks

Abstract

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.

Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells

“…4C). Analysis of recently published PBAT data (Kobayashi et al 2012) confirms that these H3K9me3-marked retroelements are relatively hypermethylated in blastocysts ( Fig. 2A).…”

Linkage disequilibrium analysis of allelic heterogeneity in DNA methylation

“…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.…”

“…Accordingly, we examined the deviation at the transitional zones found in the methylome of mouse germinal vesicle oocytes. 10,11 In almost all regions examined, the CpG pair in the transitional zone showed a clear deviation to concordant linkage (Fig. 4B).…”

“…In mouse oocytes, the transitional zone found at the boundary of a gene body often shows allelic heterogeneity. 10 There are 2 possible explanations for the methylation pattern underlying heterogeneity in the transitional zone: 1) methylation activity is stochastic in the transitional zone, resulting in each allele consisting of an interspersed mixture of methylated and unmethylated CpG sites, or 2) the transition between methylated and unmethylated regions occurs at a distinct boundary in each allele, whereas the boundary position differs among alleles (Fig. 4A).…”

“…3,10,11 The sequence reads were downloaded from the NCBI short read archives (accessions: DRA000484, DRA000495, DRA000570, and DRA000607).…”