Figures and data
Global characteristics of DNA methylation during spermatogenesis.
(A) Schematic of spermatogenesis and the four representative stages analyzed in this study (THY1+, KIT+, PS, and RS). The time window of the transient reduction of DNA methylation is indicated.
(B) ELISA-based quantification of 5-mC during spermatogenesis. Percentage of 5mC in total DNA is shown.
(C, D) Immunostaining of paraffin sections of adult testes with anti-5mC antibody and antibodies against stage-specific markers (ZBTB16 and H1T). Slides were counterstained with DAPI. Images were acquired with a confocal microscope. Regions bordered by dashed yellow squares are magnified in the right panels. Scale bar in the left panel: 20 µm; scale bar in the right panel: 5 µm.
MethylCap-seq analysis during spermatogenesis.
(A) Dynamic changes in DNA methylation during spermatogenesis. MAnorm analysis of MethylCap-seq at each transition of spermatogenesis. The genomic distribution of each peak is shown with colored bars.
(B) Track view of MethylCap-seq data of a representative genomic region for each stage of spermatogenesis. Two biological replicates are shown. Sites with loss of DNA methylation are underlaid in green and indicated by green arrows; sites with gain of DNA methylation are underlaid in red and indicated by red arrows.
(C) Average tag density plots of MethylCap-seq reads in each representative group of genes defined in a previous study (Sin et al., 2015).
Changes in DNA methylation during spermatogenesis.
(A) Enrichment analysis of MethylCap-seq around TSSs (±2 kb) at each transition during spermatogenesis. Pearson correlation values are shown.
(B) Volcano plots for the enrichment and P-values of distal MethylCap-seq outside ±1 kb of TSSs during the transition from KIT+ spermatogonia to PS. Peaks were detected by MAnorm.
(C) GO enrichment analyses for 909 genes losing 5mC and 753 genes gaining 5mC from KIT+ spermatogonia to PS.
(D) RNA-seq heatmap for 909 genes losing 5mC from KIT+ spermatogonia to PS.
(E) Pie charts showing gene expression changes in three groups of genes (909 genes losing 5mC, 753 genes gaining 5mC, and all genes).
Sites of DNA demethylation preset sites of nucleosome retention during spermatogenesis.
(A) Box-and-whisker plots showing mononucleosome enrichment at each genomic locus for each class of genes. Central bars represent medians, the boxes encompass 50% of the data points, and the whiskers indicate 90% of the data points. * P-value < 0.0001, Mann–Whitney U test.
(B) Genomic distribution of mononucleosome peaks in sperm (total 36,911 peaks): MNase-seq data (Erkek et al., 2013). Peaks are detected by MACS2 (Padj-value<0.01, Fold enrichment>5). (C, D) Changes in the enrichment of H3K4me3 (X-axis) and mononucleosomes in spermatozoa (Y-axes) at demethylated promoters (C) and exons (D) at the mitosis-to-meiosis transition.
Sites of DNA demethylation preset sites of bivalent genomic domains in spermatozoa.
(A) Mononucleosome enrichment in spermatozoa at genomic sites of MethylCap-seq peaks of KIT+ spermatogonia. Class I (green): genomic sites that are demethylated at the mitosis-to-meiosis transition, and Class II (red): all other sites.
(B) H3.3 enrichment in the two classes of genomic sites in sperm.
(C) Enrichment of H3.3 and nucleosomes
(D) Enrichment of SCML2 in cultured germline stem (GS) cells, and H3.3 enrichment in sperm (left) and mononucleosome enrichment in sperm (right)
(E) Enrichment of H3K27me3 and H3K4me3 during spermatogenesis and in spermatozoa.
(F) RNA-seq analysis in embryos for three groups of genes. Top 1,000 nucleosome-enriched genes among class I and II peak-containing genes at promoters (7,577 and 7,261 genes) and bottom 3,000 nucleosome-enriched genes (Nucleosome-depleted genes).
Model: Sites of DNA demethylation preset sites of nucleosome retention during spermatogenesis.
Genomic regions that are demethylated during the transition from mitotic spermatogonia to meiotic spermatocytes acquire H3K4me3 in meiotic spermatocytes, leading to SCML2-mediated deposition of H3K27me3, thereby establishing persisting bivalent marks at these hypomethylated nucleosome retention sites in spermatozoa.
