Figure 5. Constitutively silenced genes on Xp are silenced in spermatogenesis.
(A) Heatmap of X-linked and representative autosomal gene expression in single cells of the testis66. Chr1 and chr13, representative autosomes. Spermatogenesis stages shown in chronological order. A1: type A1 spermatogonia; In: intermediate spermatogonia; BS: S phase type B spermatogonia; BG2: G2/M phase type B spermatogonia; G1: G1 phase preleptotene; ePL: early S phase preleptotene; mPL: middle S phase preleptotene; lPL: late S phase preleptotene; L: leptotene; Z: zygotene; eP: early pachytene; mP: middle pachytene; lP: late pachytene; D: diplotene; MI: metaphase I; MII: metaphase II; RS2: steps 1–2 spermatids; RS4: steps 3–4 spermatids; RS6: steps 5–6 spermatids; RS8: steps 7–8 spermatids. MSCI, Meiotic sex chromosome inactivation; PMSC, post-meiotic sex chromatin.
(B) Averaged gene expression values for various X-linked categories and representative autosomal genes for stages shown in (a). *p= 0.0036 at eP, **p= 0.0046 at mP, and ***p= 6.48×10−6 at lP, between autosomal (chr1 and chr13 combined) and chrX genes, by Mann-Whitney U test.
(C) Left, The mean expression fold-change (FC) of X-linked gene categories at post-meiotic stages (from MII to RS8). P values were calculated by Mann-Whitney U test. Right, the Category 4 genes are exclusively and significantly enriched in the differentially downregulated genes from MII to RS8 among the X-linked gene categories. *p=0.042, by fisher’s exact test. Differentially expressed genes were defined as genes with FDR (False Discovery Rate) <0.05, in Wilcoxon ranked sum test (see Methods).
(D) Nascent RNA FISH of constitutive genes (Htatsf1 (top) and Zrsr2 (bottom)) across spermatogenesis. Cell types were determined based on morphologies of lateral synaptonemal complex by immunofluorescence using SYCP3 antibody. White arrowheads mark the nascent RNA FISH signal. Scale bar: 8μm.
(E) Quantification of nascent RNA FISH signal shown in (d) in all replicates. L: leptotene; Z: zygotene; P: pachytene; D: diplotene; S: spermatids.
(F) Top, metagene analysis of chromatin accessibility by ATAC-seq in mature spermatozoa67 for autosomal (chr1) and X-linked genes. Bottom, heatmap of ATAC-seq read coverages. Gene bodies ± 2kb shown. p=3.64×10−13 between chr1 and chrX, by Wilcoxon Signed-Rank test.
(G) Metagene analysis of ATAC-seq chromatin accessibility in spermatozoa67 for various X-linked gene categories. Gene bodies ± 2kb shown.
(H) p-values comparing chromatin accessibility of gene bodies shown in (g) between constitutive genes and other listed gene categories, by paired samples Wilcoxon test.
(I) Heatmap of ATAC-seq read coverages across gene bodies (± 2kb) in spermatozoa for different X-linked gene categories.