Expression and epigenomic landscape of the sex chromosomes in mouse post-meiotic male germ cells

Abstract

Background: During meiosis, the X and Y chromosomes are transcriptionally silenced. The persistence of repressive chromatin marks on the sex chromatin after meiosis initially led to the assumption that XY gene silencing persists to some extent in spermatids. Considering the many reports of XY-linked genes expressed and needed in the post-meiotic phase of mouse spermatogenesis, it is still unclear whether or not the mouse sex chromatin is a repressive or permissive environment, after meiosis.

Results: To determine the transcriptional and chromatin state of the sex chromosomes after meiosis, we re-analyzed ten ChIP-Seq datasets performed on mouse round spermatids and four RNA-seq datasets from male germ cells purified at different stages of spermatogenesis. For this, we used the last version of the genome (mm10/GRCm38) and included reads that map to several genomic locations in order to properly interpret the high proportion of sex chromosome-encoded multicopy genes. Our study shows that coverage of active epigenetic marks H3K4me3 and Kcr is similar on the sex chromosomes and on autosomes. The post-meiotic sex chromatin nevertheless differs from autosomal chromatin in its enrichment in H3K9me3 and its depletion in H3K27me3 and H4 acetylation. We also identified a posttranslational modification, H3K27ac, which specifically accumulates on the Y chromosome. In parallel, we found that the X and Y chromosomes are enriched in genes expressed post-meiotically and display a higher proportion of spermatid-specific genes compared to autosomes. Finally, we observed that portions of chromosome 14 and of the sex chromosomes share specific features, such as enrichment in H3K9me3 and the presence of multicopy genes that are specifically expressed in round spermatids, suggesting that parts of chromosome 14 are under the same evolutionary constraints than the sex chromosomes.

Conclusions: Based on our expression and epigenomic studies, we conclude that, after meiosis, the mouse sex chromosomes are no longer silenced but are nevertheless regulated differently than autosomes and accumulate different chromatin marks. We propose that post-meiotic selective constraints are at the basis of the enrichment of spermatid-specific genes and of the peculiar chromatin composition of the sex chromosomes and of parts of chromosome 14.

Keywords: Chromosome 14; Crotonylation; H3K27ac; H3K4me3; H3K9me3; MSCI; Mouse; Post-meiotic sex chromatin; Sex chromosomes; Spermiogenesis.

Fig. 1

Post-meiotic sex chromatin. a Immunofluorescence pictures of a round spermatid nucleus stained with DAPI (left in black and white; right in blue) and with an X chromosome paint (pink). The bright DAPI-dense region is the chromocenter; the arrow indicates the adjacent DAPI-dense region in which the X chromosome is located. A similar pattern is observed with a Y chromosome paint [–26, 34]. b Schematic diagram of XY gene expression during mouse spermatogenesis (adapted from [37]). In spermatogonia, X and Y gene expression is comparable to that of autosomes. At the pachytene stage of meiosis I, XY genes are transcriptionally shut down by MSCI (meiotic sex chromosome inactivation). After meiosis, XY gene expression is reactivated. The timing of appearance of the chromatin marks enriched on the sex chromosomes (as observed by immunofluorescence) during and after meiosis is represented under the schematic diagram. c Representation of the size of the mouse sex chromosomes in mm9/NCBIM37 (April 2007) and in mm10/GRCm38 (January 2012). In mm9/NCBIM37, the X and Y chromosomes are, respectively, 166.7 and 16 Mb, while in mm10/GRCm38 the X and Y chromosomes are, respectively, 171 and 91.8 Mb. Adapted from Ensembl http://www.ensembl.org/index.html

Fig. 2

Comparison of the chromatin composition of sex chromosomes and autosomes in mouse round spermatids. Scatter plot showing the chromosome coverage (% of each chromosome) of 9 histone PTM (i.e., H3K4me3, Kcr, H3K9ac, H4K8_hib, H4ac, K_acetylation, H3K9me3, H3K27ac, H3K27me3) and 5-hydroxymethylcytosine in round spermatids. Mean values for all chromosomes ± standard deviation are represented. See Additional file 1 for detailed statistical analyses and Additional files 2 and 3 for complementary graphic representations

Fig. 3

Post-meiotic Y chromosome is enriched in H3K27ac. Immunofluorescence detection of H3K27ac (red) in round spermatid nuclei. DAPI (blue) was used to stain nuclei. The most DAPI-dense round region is the chromocenter (i.e., the constitutive pericentromeric heterochromatin) the less DAPI-dense structure adjacent to the chromocenter is the post-meiotic sex chromatin (PMSC) and is indicated by an arrow. Anti-H3K9me3 (in green) marks the chromocenter and the PMSC in wild type (WT) round spermatids. Two types of staining were observed: either a brighter signal co-localizing with the PMSC (“WT PMSC+” panel), or a diffuse bright signal in the nucleus (“WT Diffuse” panel). As control, round spermatids with a large deletion of the Y chromosome (MSYq-) were used. See Additional file 4 for an extended panel

Fig. 4

Dynamics of XY gene expression and autosomal gene expression during spermatogenesis. a Percentage of expressed genes in spermatogonia B (SB), pachytene spermatocytes (PS), round spermatids (RS) and elongating spermatids for the sex chromosomes (X and Y), five representative chromosomes (chromosomes 3, 6, 14, 16 and 18) and all autosomes. In spermatogonia B, the proportion of expressed genes is similar between the X chromosome and autosomes. At the pachytene stage of meiosis I, the proportion of X-linked expressed genes is decreased due to MSCI. After meiosis, the proportion of expressed genes is similar between the X chromosome and autosomes. The Y chromosome is depleted in genes expressed before meiosis but enriched in round spermatids expressed genes. The percentage values are indicated in the table. The level of statistical significance is marked with three asterisks (*) if p < 0.001. b RPKM mean values of expressed genes in spermatogonia B, pachytene spermatocytes, round spermatids and elongating spermatids for the sex chromosomes (X and Y), five representative chromosomes (chromosomes 3, 6, 14, 16 and 18) and all autosomes. The expression level of X- and Y-linked genes is similar to that of autosomal genes in spermatogonia B. The value is indicated in the table. c Distribution of RPKM values of expressed genes in spermatogonia B, pachytene spermatocytes and round spermatids. The X-axis represents categories of RPKM range of values (0–2, 2–4, 4–6, etc.), the Y-axis, the percentage of genes in each category. X-linked genes expression values are indicated in pink, Y-linked genes expression values are in green, and autosomal values are in gray. After meiosis, the sex chromosomes are enriched in genes with low expression values. See Additional files 5 and 6 for detailed statistical analyses

Fig. 5

XY and autosomal gene expression in pachytene spermatocytes and in round spermatids. Proportion of pachytene-repressed genes and of non-reactivated genes in round spermatids (RS) for the sex chromosomes (X and Y), five representative chromosomes (chromosomes 3, 6, 14, 16 and 18) and all autosomes. The X chromosome is enriched in pachytene-repressed genes. Similar proportions of non-reactivated genes are found for all chromosomes. Values are indicated in the table. The level of statistical significance is marked with three asterisks (***) if p < 0.001 or with (ns) if not significant. See Additional file 7 for detailed statistical analyses

Fig. 6

Sex chromosomes and chromosome 14 are enriched in genes expressed after meiosis. a Proportion of round spermatid (RS) enriched genes and round spermatid (RS)-specific genes for the sex chromosomes (X and Y), five representative chromosomes (chromosomes 3, 6, 14, 16 and 18) and all autosomes. The proportion of round spermatid-enriched genes and round spermatid-specific genes is significantly higher for the sex chromosomes and chromosome 14 than for the other autosomes. The level of statistical significance is marked with one asterisk (*) if p < 0.05, two (**) if p<0.01.(*) and three (***) if p<0.001. b RPKM mean values of round spermatid-enriched genes and specific genes for sex chromosomes (X and Y), five representative chromosomes (chromosomes 3, 6, 14, 16 and 18) and all autosomes. Round spermatid-specific genes encoded by the sex chromosomes display significantly lower values than autosomes. The values are indicated in the table. c Distribution of RPKM values of round spermatid-enriched genes and round spermatid-specific genes. The X-axis represents categories of RPKM range of values (0–2, 2–4, 4–6, etc.), and the Y-axis, the percentage of genes in each category. X-linked genes expression values are indicated in pink, Y-linked genes expression values are in green, and autosomal values are in gray. See Additional files 8 and 9 for detailed statistical analyses

Fig. 7

Sex chromosomes and chromosome 14 have common chromatin features in round spermatids. Graphic representation of the ChIP-Seq profiles of H3K9me3 and RNA-Pol II enzyme over chromosomes 3, 5, 6, 10, 14, 16, 18, X and Y in round spermatids. RNA-Pol II (in green) marks the regions where round spermatid-expressed genes are located. The genomic location of round spermatid-specific genes is represented as narrow black bars on the chromosomes. The genomic location of Speer genes is indicated by blue bars underneath the chromosomes. Ampliconic multicopy spermatid-specific genes on portions of chromosomes 5, 14 and X are circled. Note that they are enriched in H3K9me3 (in red). The figure was made using NCBI Genome Decoration Page Web site (using mm10/GRC38 Ensembl gene 81)

Fig. 8

Mouse chromosome 14 encodes many spermatid-specific genes belonging to α-takusan family and located in two main regions. Graphic view of the chromosomal location of the 277 genes related to α-takusan family found using Ensembl BLAST/BLAT. Those genes are located in clusters at the tip of the chromosome [chr14: 3,000,000–7,600,000 (indicated by two orange arrowheads) and chr14: 19,000,000–19,000,000 (indicated by one yellow arrowhead)] or more distal [chr14: 41,200,000–51,800,000 (indicated by two red arrowheads)]. The figure was made using Ensembl BLAST/BLAT Web site (using mm10/GRC38 Ensembl gene 81)