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. 2021 Jan 4;12:73. doi: 10.1038/s41467-020-20345-3

Fig. 5. Activation of pachytene piRNA clusters in the male germline correlates with high 5hmC levels, whereas the extent of somatic suppression of these genes correlates with first-exon length and high levels of DNA methylation and H3K27me3.

Fig. 5

a piRNA and transcript abundance for pachytene piRNA clusters in pachytene spermatocytes. Promoter O/E CG is indicate by color: Blue, low (n = 49); green, intermediate (n = 33); yellow, high (n = 18). Two-sided Wilcoxon rank-sum test was used to determine p value; n.s. not significant. For all boxplots in this figure, whiskers show 95% confidence intervals, boxes represent the first and third quartiles, and the vertical midline is the median. b Level of histone modifications, ATAC, and RNA pol II for pachytene piRNA clusters in pachytene spermatocytes. Promoter O/E CG is as in a. Asterisks indicate two-sided Wilcoxon rank-sum test p values < 0.05 between 49 low-CG and 33 intermediate- or 18 high-CG promoters. Red (blue) indicates that the epigenetic level is significantly higher (lower) in low-CG pachytene piRNA clusters. c 5hmC levels in pachytene spermatocytes. Asterisks indicate two-sided Wilcoxon rank-sum test p values < 0.05 between 49 pachytene piRNA clusters and 397 testis-specific mRNAs, 95 testis-specific lncRNAs, all 6177 mRNAs, and all 2050 lncRNAs with low-CG promoters. d Top, DNA methylation levels for pachytene piRNA clusters and other groups of genes. Bottom, DNA methylation levels of pachytene piRNA clusters grouped as in a. Vertical line indicates the 50% DNA methylation level. e DNA methylation levels of pachytene piRNA clusters between forebrain and pachytene spermatocytes. Colors indicate promoter CG level; genes with forebrain expression level higher than 0.1 RPKM are denoted as triangles. f H3K27me3 levels, relative to control, are shown in adult testis and forebrain. Only the 23 pachytene piRNA clusters in the bottom-left quadrant defined in e are included and further classified by their expression levels in the forebrain: RPKM ≥ 0.1 (n = 12) or RPKM < 0.1 (n = 11). g First exon length of the same 23 pachytene piRNA clusters as in f, classified into the same groups as in f.