EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

Abstract

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.

Figure 1.

Global impact of EHMT2 on the DNA methylome of mouse ESCs. (A) Density histograms of RRBS methylation scores at CpGs in WT ESCs, Ehmt2^−/− ESCs, and Ehmt2^−/− ESCs rescued with a WT Ehmt2 transgene (Tg). (B) Average distribution of RRBS methylation over RefSeq genes and flanking sequences in WT, Ehmt2^−/−, and Ehmt2 rescued ESCs. (C) Heatmap representation of the RRBS methylation scores measured in the promoters (−1000 to +1000 bp) of selected germline and developmental genes. (D) Violin plots of RRBS methylation scores in retrotransposons.

Figure 2.

Impact of EHMT2 on the methylome in mouse embryos. (A) Expression of Ehmt2 mRNAs measured by RT-qPCR in early mouse embryos, depicted as a ratio relative to the expression of two housekeeping genes (Actb and Rpl13a; mean ± SEM, n = 2 technical replicates on five to 10 pooled embryos). (B) Promoter DNA methylation of candidate genes measured by COBRA in WT and Ehmt2^−/− E9.5 embryos. The restriction fragments marked with an asterisk are end products of the digestion (indicating DNA methylation). (C) Promoter DNA methylation of the Pou5f1 and Dppa3 genes measured by bisulfite sequencing in WT and Ehmt2^−/− E9.5 embryos. Circles represent methylated (black) or unmethylated (white) CpG dinucleotides; each horizontal line is one sequenced clone. (D) Pairwise comparison of methylated DNA immunoprecipitation (MeDIP) log₂ ratios at individual oligos in WT and Ehmt2^−/− E9.5 embryos. The density of data points increases from blue to dark red. The Pearson correlation coefficient (r) is indicated on the graph. (E) Density histograms of RRBS CpG methylation scores in WT and Ehmt2^−/− E8.5 embryos. (F) Average distribution of RRBS methylation over RefSeq genes and flanking sequences in WT and Ehmt2^−/− embryos. (G) Violin plots of RRBS methylation measured in CpG islands (CGIs) and retrotransposons. (H) Example of methylation profiles in the promoter of the Dazl gene in WT and Ehmt2^−/− embryos. The upper tracks depict smoothed MeDIP log₂ ratios of individual oligonucleotides, and the bottom tracks depict RRBS methylation scores at individual CpGs. The CpG density is shown in black.

Figure 3.

Identification of hypomethylated regions (HMRs) in Ehmt2^−/− embryos. (A) Histogram of the number of sequences that gain or lose >20% methylation in the RRBS from Ehmt2^−/− compared with WT embryos. (B) Pie charts representing the percentages of hypomethylated and hypermethylated sequences mapping to promoters, gene bodies, intergenic regions, and transposable elements (TEs). (C) Boxplot showing the dynamics of methylation at HMRs (filtered to lose >30% methylation in Ehmt2^−/− embryos) in early embryos, Ehmt2^−/− E8.5 embryos (green), and Dnmt3b^−/− E8.5 embryos (red). (D) Preferential site of expression of genes with a promoter-proximal HMR (−1000 to +1000 bp relative to the TSS). The dashed line represents the position for a P-value of 0.05. (E) Examples of promoter-proximal HMRs. The sites of hypomethylation identified by MeDIP (gray boxes) confirm the differences measured by RRBS. (F) Bisulfite cloning and sequencing of selected promoter HMRs in WT and Ehmt2^−/− E9.5 embryos. The values below the sequencing data indicate the percentage of methylated CpGs in the amplicon.

Figure 4.

Locus-specific control of DNA methylation imprints by EHMT2 at the Slc38a4 gDMR. (A) Methylation scores measured by RRBS in 16 known imprinted gDMRs in WT and Ehmt2^−/− embryos. (B) Bisulfite cloning and sequencing analysis of four gDMRs in WT and Ehmt2^−/− E9.5 embryos. (C) MeDIP profiles at the imprinted Slc38a4 locus (top) and RRBS scores in four WT and three Ehmt2^−/− embryos (bottom). The orange rectangle marks the position of the gDMR.

Figure 5.

HMRs are bound by EHMT2 and marked by H3K9me2. (A) RNA-seq quantification of the expression of genes encoding components of the DNA methylation machinery in Ehmt2^−/− and WT E9.5 embryos (mean FPKM ± SEM, n = 2 embryos). (B) ChIP-qPCR analysis of EHMT2 binding at HMRs in primary mouse embryonic fibroblasts (MEFs), represented as the percentage of input (mean ± SEM, n = 5). ChIP assays were performed with an antibody against EHMT2 and a control rabbit IgG. Actb served as a negative control, and Rhox11 was chosen as a positive control (Myant et al. 2011). (C) ChIP-qPCR analysis of EHMT2 binding at HMRs in E8.5 embryos (mean ± SEM, n = 4). (D) Browser views of EHMT2 ChIP-seq profiles in ESCs (Mozzetta et al. 2014) reveal that peaks of EHMT2 binding colocalize with promoter-proximal HMRs (red bars) at three germline genes. (E) Heatmap representation of the distribution of EHMT2 and H3K9 mono- and dimethylation at HMRs. The data represent the average density of ChIP-seq reads for EHMT2 in ESCs, H3K9me1/2 in ESCs, and H3K9me2 in E8.5 embryos normalized by the density of reads in the input control. (F) ChIP-qPCR analysis of H3K9me2 and H3K4me3 at HMRs in E8.5 embryos, represented as the percentage of input (mean ± SEM, n = 4). The promoters of the housekeeping genes Actb and Ube2f served as controls. (G) ChIP-qPCR analysis of H3K9me2 at four gene promoters in WT and Ehmt2^−/− E8.5 embryos (mean ± SEM, n = 6 embryos for WT, n = 4 embryos for Ehmt2^−/−). The heatmap on the bottom indicates CpG methylation measured by RRBS in the same promoters. (**) P < 0.01 (t-test).

Figure 6.

EHMT2 represses germline genes via DNA methylation in mouse embryos. (A) Comparison of RNA-seq expression levels for RefSeq genes in WT and Ehmt2^−/− embryos. Genes of the Magea family and differentially expressed genes are highlighted in colors. (B) Preferential tissue of expression of genes up-regulated at least threefold in Ehmt2^−/− embryos. (C) Examples of RNA-seq profiles at the Cyct and Asz1 genes in two biological replicates of WT and Ehmt2^−/− embryos. (D) Activation of germline genes in Ehmt2^−/− and Dnmt3b^−/− E8.5 embryos. The heatmap on the bottom indicates CpG methylation measured by RRBS in the corresponding promoters in Ehmt2^−/− and Dnmt3b^−/− E8.5 embryos. (E) ChIP-qPCR analysis of H3K9me2 in WT and Dnmt3b^−/− E8.5 embryos (mean ± SEM, n = 3 embryos for WT, n = 4 embryos for Dnmt3b^−/−), showing that the reduced DNA methylation does not impact the deposition of H3K9me2. (F) Model: EHMT2 deposits H3K9me2 and facilitates cytosine methylation at a subset of gene promoters in embryos. The inactivation of EHMT2 inhibits H3K9me2 and leads to reduced cytosine methylation, leading to aberrant gene activation. In Dnmt3b^−/− embryos, EHMT2 is able to bind to its target promoters but can no longer recruit cytosine methylation, which leads to incomplete gene silencing.