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.

Figure 1. High-resolution DNA methylome map of mouse distal chromosome 7 imprinting cluster.

Illumina GenomeStudio viewer displays the locations of genes in distal chromosome 7 (149,700,000–151,000,000). Black vertical bars represent the location of 4 repetitive elements: LINE, SINE, LTR, and DNA transposons. Red, purple, blue, green, and khaki dots represent the methylation levels at individual CpGs in wild-type oocyte, Dnmt3L ^−/− oocyte, sperm, blastocyst, and ESC genomes, respectively. Black line plots depict the distribution of CpG densities (number of CpG per 200 nt) of individual CpGs. Open boxes represent the location of CpG islands (CGIs). Red, purple, blue, and green boxes represent the methylation levels at individual CGIs in wild-type oocyte, Dnmt3L ^−/− oocyte, sperm, and blastocyst genomes, respectively, determined by our results from shotgun bisulfite sequencing (SBS) method and Smallwood's results from reduced representation bisulfite sequencing (RRBS) method .

Figure 2. Genome-wide methylation profiling of mouse germ cells.

(A) Histograms of methylation levels of genomic CpGs in wild-type oocyte, Dnmt3L^−/− oocyte, sperm, blastocyst, and embryonic stem cell (ESC) genomes. (B) CpG methylation levels are plotted as a function of CpG density for the whole genome and 4 families of transposable elements (long interspersed nuclear element (LINE), short interspersed nuclear element (SINE), long terminal repeat (LTR), and DNA transposon).

Figure 3. High-resolution genome-wide mRNA expression and CpG methylation profiling.

GenomeStudio view of mRNA-seq data and CpG methylation map of the genomic region spanning the Nespas-Gnas maternally imprinted locus. (Top) Genomic stacked alignment plots of wild-type oocytes, Dnmt3L^−/− oocytes, and sperm. (Middle) Open boxes and black line plots represent the location of CGIs and the distribution of CpG densities of individual CpGs, respectively. (Bottom) Red, purple, blue, and green dots represent the methylation levels at individual CpGs in wild-type oocyte, Dnmt3L ^−/− oocyte, sperm, and blastocyst genomes, respectively. The red shaded areas show the extent of two maternal imprinting control regions (ICRs).

Figure 4. Relationship between gene expression and methylation in promoter and gene-body regions in mouse germ cells.

The expression level of genes in wild-type oocytes (A), sperm (B), and Dnmt3L^−/− oocytes (C) were divided into 5 percentile groups. The distribution of methylation is shown ±5 kb from the transcription termination site (TTS; left) and transcription start site (TSS; middle). The graphs on the right show the average methylation levels in the promoter and gene-body regions. Spearman's rank correlation coefficient (ρ) was used to test the statistical significance of the correlation between gene expression and DNA methylation levels (*: p<1×10⁻⁹).

Figure 5. Comparison of gene expression profiles between wild-type and Dnmt3L^−/− oocytes.

(A) Scatter plot and correlation coefficient (R²) of RPKM values of 20,854 genes in wild-type and Dnmt3L^−/− oocytes. Expression levels of oocyte-specific genes (B), DNA methyltransferase genes (C), maternally-imprinted genes that are potentially necessary to establish methylation imprints (D), and male germline-specific genes that contain oocyte-specific methylated CpG islands (CGIs) (E).

Figure 6. Identification of germline differentially methylated CGIs from DNA methylome profiles.

(A) Venn-like diagram of two groups of CGIs, namely, oocyte-methylated CGIs (light pink) and sperm-methylated CGIs (light blue) and two groups of gDMRs, namely, oocyte-methylated gDMRs (red) and sperm-methylated gDMRs (blue). (B) The genomic distribution of 1329 oocyte-methylated (left) and 349 sperm-methylated gDMRs (right). The gDMRs were classified into 5 genomic locations; promoter (within 500-bp upstream from the first exon) or first exon, last exon, other exon, intron, and intergenic region. (C) The locations of the intragenic 1045 oocyte-methylated (left) and 229 sperm-methylated gDMRs (right). The gDMRs were classified into 5 gene group locations; the genes were divided into 5 percentile groups according to their expression levels in wild-type oocytes and sperm, as shown in Figure 3. (D) Histograms of the methylation levels of the gDMRs in blastocysts. The number of newly identified oocyte-specific, sperm-specific methylated gDMRs, and known ICRs are shown in black, red, and blue, respectively. (E) Bisulfite sequencing at the Gpr1 gDMR in mouse blastocysts. (Top) Schematic representation of paternally-expressed Gpr1. The gene and gDMRs are shown in blue and green, respectively, and CpG sites are represented by vertical bars. (Bottom) Methylated and unmethylated CpGs are indicated by open and closed circles, respectively. The maternal and paternal alleles were distinguished by three polymorphisms between C57BL/6N and JF1 mice (G/A at 63,247,064; T/A at 63,247,072; and TA/AG at 63,247,089–63,247,090 on chromosome 1). (F) Histograms of the methylation levels of the demethylation-resistant oocyte-methylated gDMRs in Dnmt3L^−/− oocytes. The number of newly identified oocyte-specific, sperm-specific methylated gDMRs, and known ICRs are shown in black, red, and blue, respectively.