Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos

Abstract

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Dynamics of DNA Methylomes for Gametes and Early Embryos

(A) The dynamics of the average DNA methylation level. The average methylation level is calculated as the percentage of methylated CpG in all CpG content. “Z” means the mean of methylation level between oocyte and sperm. (B) Differentially methylated CpG sites between consecutive stages. (C) Sites with increasing or decreasing methylation levels co-occur during each consecutive transition. The last two bars represent the comparisons between sperm or oocyte with the 1,000-cell stage, respectively. See also Figure S1.

Figure 2. Early Embryos Reset the Methylation Landscape to a Pattern that Is Almost Identical to Sperm

(A and B) Box plots of methylation values of CpG sites highlight the dynamic patterns of oocyte-specific unmethylated sites (methylation level ≥ 0.75 in sperm and ≤ 0.25 in oocyte) and oocyte-specific methylated sites (methylation level ≥ 0.75 in oocyte and ≤ 0.25 in sperm) across early embryogenesis. Black line indicates median, edges stand for the 25th/75th percentile, and whiskers stand for the 2.5th/97.5th percentile. (C and D) Box plots of DNA methylation level for sites with similar methylation level in both gametes across early embryogenesis. (E) Dot plots show pairwise comparisons of average methylation level in CGIs. (F) Graphical representation of methylation pattern at two CGIs within a locus in gametes, 32-cell, and 1,000-cell embryos. Green bars highlight the positions of CGI. Vertical line height indicates the methylation level. (G) Pearson correlations coefficient heatmap among methylomes. Correlation coefficients are colored yellow to red to indicate low to high, respectively. See also Figure S2 and Table S1.

Figure 3. The Methylome of Paternal DNA Is Maintained, and the Methylome of Maternal DNA Is Gradually Reset to Sperm Pattern

(A) Distribution of the average methylation values of paired reads, which include at least four consecutive oocyte-specific unmethylated CpG sites (mCpG percentage ≥ 75% in sperm and ≤ 25% in oocyte) in gametes and early embryos. n is the number of paired reads in each stage. (B) Distribution of average methylation values of paired reads covering at least four consecutive oocyte-specific methylated sites (mCpG percentage ≥ 75% in oocyte and ≤ 25% in sperm). x axis represents methylation level. Bins are at 0.25 intervals (bins at 0.05 are shown in Figures S1C and S1D). (C) Dynamic changes of DNA methylation for a representative locus located in chr7:21,315,648-21,315,662. Paired reads from whole-genome data covering all five CpGs are shown. Open circles represent unmethylated CpGs, and filled circles represent methylated CpGs. (D) Dynamic changes of DNA methylation for a representative locus in chr6: 27,393,375-27,393,560 tracked with SNPs to distinguish maternal and paternal DNA. The data are validated using bisulfite PCR. Ten reads were randomly picked for sperm and oocyte. Five reads from paternal DNA and five reads from maternal DNA are randomly picked in 16-cell, 64-cell, and 1,000-cell stages of embryos. See also Table S4. (E) Number of differentially methylated loci covered by oocyte-specific methylated and unmethylated paired reads. (F) Genome-wide analysis of the proportion of differentially methylated loci covered by paired reads progressively resetting to sperm methylation state. Gray bars represent oocyte-specific hypermethylated loci, and black bars represent oocyte-specific hypomethylated loci. (G) Number of DMRs genome wide, methylation difference > 0.2. (H) Genome-wide analysis of the proportion of DMRs progressively resetting to sperm methylation state. Methylation level difference of the DMRs was set higher than 0.2, 0.3, and 0.5, respectively. See also Figures S3 and S4 and Tables S2 and S3.

Figure 4. DNA Demethylation Rate of Oocyte-Specific HypoRs and De Novo Methylation Rate of HyperRs

(A and B) Fitting curves of the DNA demethylation rate of oocyte-specific HypoRs and de novo methylation rate of HyperRs. The methylation level difference of HypoRs and HyperRs is greater than 0.2. HypoRs and HyperRs represent hypomethylated and hypermethylated regions versus sperm, respectively. OSMS represents oocyte-specific methylation state, which is compared with sperm. Box plot of proportion DNA with OSMS was drawn for each stage. Black line indicates median, edges stand for the 25th/75th percentile, and whiskers represent the 2.5th/97.5th percentile. (C) Graphical representation of methylation dynamics of one region containing one oocyte-specific HyperR (demethylation locus, black bar) and one oocyte-specific HypoR (de novo methylation locus, gray bar). The left panel shows the dynamics of the methylation level. The right panel shows the relative proportion of DNA copies with oocyte methylation state for each CG site within the DMRs. (D) Dynamics of a representative locus in chr16: 46,387,770-46,387,944 covered by paired reads containing common methylated sites and differentially methylated sites (sperm versus oocyte). Paired reads with oocyte methylation landscape decrease and reprogram to sperm methylation state. See also Table S5.

Figure 5. Functional Significance of Sperm Methylome Pattern

(A) Dot plots show pairwise comparisons of average methylation level in promoters (2 kb up TSS) based on RefSeq gene annotations. Differentially methylated promoters are highlighted in green and red. See also Table S6. (B) The heatmap represents the combination of GO term enrichment in genes with oocyte-specific, sperm-specific, MBT-specific, and gastrula-specific hypomethylated promoters. The statistical significance of the heatmap is colored red to orange to indicate high to low, respectively. Yellow is background. See also Tables S6 and S7. (C) Graph represents methylation at one HOXa gene cluster region in gametes and early embryos. Vertical line height indicates the methylation level. (D) RT-qPCR validates the expression changes for genes with methylation level significantly changed in promoters among developmental stages transition. Data show mean ± SD from one representative experiment in triplicate; at least two independent experiments were performed with similar results. See also Figure S5.