Mouse Sex Chromosomes Research Articles

Kinase CDK2 in Mammalian Meiotic Prophase I: Screening for Hetero- and Homomorphic Sex Chromosomes.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution.

SummaryThe mouse sex chromosomes exhibit an extraordinary level of copy number amplification of postmeiotically expressed genes [1, 2], driven by an “arms race” (genomic conflict) between the X and Y chromosomes over the control of offspring sex ratio. The sex-linked ampliconic transcriptional regulators Slx and Sly [3, 4, 5, 6, 7] have opposing effects on global transcription levels of the sex chromosomes in haploid spermatids via regulation of postmeiotic sex chromatin (PMSC) [8, 9, 10, 11] and opposing effects on offspring sex ratio. Partial deletions of the Y chromosome (Yq) that reduce Sly copy number lead to global overexpression of sex-linked genes in spermatids and either a distorted sex ratio in favor of females (smaller deletions) or sterility (larger deletions) [12, 13, 14, 15, 16]. Despite a large body of work studying the role of the sex chromosomes in regulating spermatogenesis (recent reviews [17, 18, 19, 20]), most studies do not address differential fertility effects on X- and Y-bearing cells. Hence, in this study, we concentrate on identifying physiological differences between X- and Y-bearing sperm from Yq-deleted males that affect their relative fertilizing ability and consequently lead to sex ratio skewing. We show that X- and Y-bearing sperm in these males have differential motility and morphology but are equally able to penetrate the cumulus and fertilize the egg once at the site of fertilization. The altered motility is thus deduced to be the proximate cause of the skew. This represents the first demonstration of a specific difference in sperm function associated with sex ratio skewing.

Sequence and Structural Diversity of Mouse Y Chromosomes.

Over the 180 My since their origin, the sex chromosomes of mammals have evolved a gene repertoire highly specialized for function in the male germline. The mouse Y chromosome is unique among mammalian Y chromosomes characterized to date in that it is large, gene-rich and euchromatic. Yet, little is known about its diversity in natural populations. Here, we take advantage of published whole-genome sequencing data to survey the diversity of sequence and copy number of sex-linked genes in three subspecies of house mice. Copy number of genes on the repetitive long arm of both sex chromosomes is highly variable, but sequence diversity in nonrepetitive regions is decreased relative to expectations based on autosomes. We use simulations and theory to show that this reduction in sex-linked diversity is incompatible with neutral demographic processes alone, but is consistent with recent positive selection on genes active during spermatogenesis. Our results support the hypothesis that the mouse sex chromosomes are engaged in ongoing intragenomic conflict.

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

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.ConclusionsBased 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0099-8) contains supplementary material, which is available to authorized users.

Signs of Genomic Battles in Mouse Sex Chromosomes

Y chromosomes are challenged by a lack of recombination and are transmitted to the next generation only via males. Sequencing of the mouse Y reveals how these properties drive opposing evolutionary processes: massive decay of ancestral genes and convergent acquisition and amplification of spermatid-expressed gene families on the X and Y chromosome. The convergent acquisition and amplification of X-linked paralogs on the Y maintains a surprisingly gene-rich, euchromatic mammalian male chromosome.

Negative effects of GM-CSF signaling in a murine model of t(8;21)–induced leukemia

AbstractThe t(8;21)(q22;q22) is common in adult acute myeloid leukemia (AML). The RUNX1-ETO fusion protein that is expressed by this translocation is poorly leukemogenic and requires additional mutations for transformation. Loss of sex chromosome (LOS) is frequently observed in t(8;21) AML. In the present study, to evaluate whether LOS cooperates with t(8;21) in leukemogenesis, we first used a retroviral transduction/transplantation model to express RUNX1-ETO in hematopoietic cells from XO mice. The low frequency of leukemia in these mice suggests that the potentially critical gene for suppression of t(8;21) leukemia in humans is not conserved on mouse sex chromosomes. The gene encoding the GM-CSF receptor α subunit (CSF2RA) is located on X and Y chromosomes in humans but on chromosome 19 in mice. GM-CSF promotes myeloid cell survival, proliferation, and differentiation. To determine whether GM-CSF signaling affects RUNX1-ETO leukemogenesis, hematopoietic stem/progenitor cells that lack GM-CSF signaling were used to express RUNX1-ETO and transplanted into lethally irradiated mice, and a high penetrance of AML was observed in recipients. Furthermore, GM-CSF reduced the replating ability of RUNX1-ETO–expressing cells. These results suggest a possible tumor-suppressor role of GM-CSF in RUNX1-ETO leukemia. Loss of the CSF2RA gene may be a critical mutation explaining the high incidence of LOS associated with the t(8;21)(q22;q22) translocation.

Association of Sly with sex-linked gene amplification during mouse evolution: a side effect of genomic conflict in spermatids?

In common with other mammalian sex chromosomes, the mouse sex chromosomes are enriched for genes with male-specific function such as testis genes. However, in mouse there has been an unprecedented expansion of ampliconic sequence containing spermatid-expressed genes. We show via a phylogenetic analysis of gene amplification on the mouse sex chromosomes that multiple families of sex-linked spermatid-expressed genes are highly amplified in Mus musculus subspecies and in two further species from the Palaearctic clade of mouse species. Ampliconic X-linked genes expressed in other cell types showed a different evolutionary trajectory, without the distinctive simultaneous amplification seen in spermatid-expressed genes. The Palaearctic gene amplification occurred concurrently with the appearance of Sly, a Yq-linked regulator of post-meiotic sex chromatin (PMSC) which acts to repress sex chromosome transcription in spermatids. Despite the gene amplification, there was comparatively little effect on transcript abundance, suggesting that the genes in question became amplified in order to overcome Sly-mediated transcriptional repression and maintain steady expression levels in spermatids. Together with the known sex-ratio effects of Yq/Sly deficiency, our results suggest that Sly is involved in a genomic conflict with one or more X-linked sex-ratio distorter genes. The recent evolution of the novel PMSC regulator Sly in mouse lineages has significant implications for the use of mouse-model systems in investigating sex chromosome dynamics in spermatids.

Deficiency in the multicopy Sycp3-like X-linked genes Slx and Slxl1 causes major defects in spermatid differentiation.

The human and mouse sex chromosomes are enriched in multicopy genes required for postmeiotic differentiation of round spermatids into sperm. The gene Sly is present in multiple copies on the mouse Y chromosome and encodes a protein that is required for the epigenetic regulation of postmeiotic sex chromosome expression. The X chromosome carries two multicopy genes related to Sly: Slx and Slxl1. Here we investigate the role of Slx/Slxl1 using transgenically-delivered small interfering RNAs to disrupt their function. We show that Slx and Slxl1 are important for normal sperm differentiation and male fertility. Slx/Slxl1 deficiency leads to delay in spermatid elongation and sperm release. A high proportion of delayed spermatids are eliminated via apoptosis, with a consequent reduced sperm count. The remaining spermatozoa are abnormal with impaired motility and fertilizing abilities. Microarray analyses reveal that Slx/Slxl1 deficiency affects the metabolic processes occurring in the spermatid cytoplasm but does not lead to a global perturbation of sex chromosome expression; this is in contrast with the effect of Sly deficiency which leads to an up-regulation of X and Y chromosome genes. This difference may be due to the fact that SLX/SLXL1 are cytoplasmic while SLY is found in the nucleus and cytoplasm of spermatids.

Evolutionary genetics: Stickleback's view of sex chromosome evolution

Evolutionary genetics: Stickleback's view of sex chromosome evolution

Characterization of the OFD1/Ofd1 genes on the human and mouse sex chromosomes and exclusion of Ofd1 for the Xpl mouse mutant☆

Oral–facial–digital type 1 (OFD1) syndrome is an X-linked dominant condition characterized by malformations of the face, oral cavity, and digits. The responsible gene, OFD1, maps to human Xp22 and has an unknown function. We isolated and characterized the mouse Ofd1 gene and showed that it is subject to X-inactivation, in contrast to the human gene. Furthermore, we excluded a role for Ofd1 in the pathogenesis of the spontaneous mouse mutant Xpl, which had been proposed as a mouse model for this condition. Comparative sequence analysis demonstrated that OFD1 is conserved among vertebrates and absent in invertebrates. This analysis allowed the identification of evolutionarily conserved domains in the protein. Finally, we report the identification of 18 apparently nonfunctional OFD1 copies, organized in repeat units on the human Y chromosome. These degenerate OFD1-Y genes probably derived from the ancestral Y homologue of the X-linked gene. The high level of sequence identity among the different units suggests that duplication events have recently occurred during evolution.

Rapid evolution of human pseudoautosomal genes and their mouse homologs

Comparative studies of genes in the pseudoautosomal region (PAR) of human and mouse sex chromosomes have thus far been very limited. The only comparisons that can presently be made indicate that the PARs of humans and mice are not identical in terms of gene content. Here we describe additional comparative studies of human pseudoautosomal genes and their mouse homologs. Using a somatic cell hybrid mapping panel, we have assigned the mouse homolog of the human pseudoautosomal interleukin 3 receptor alpha subunit (IL3RA) gene to mouse Chromosome (Chr) 14. Attempts to clone the mouse homolog of the human pseudoautosomal adenine nucleotide translocase-3 (ANT3) gene resulted in the isolation of the murine homologs of the human ANT1 and ANT2 genes. The mouse Ant1 and Ant2 genes are very similar in sequence to their human homologs, and we have mapped them to mouse Chromosomes (Chrs) (8 and X respectively) that exhibit conserved synteny with the chromosomes on which the human genes are located. In contrast, the homolog of ANT3 appears to be either very divergent or absent from the mouse genome. Southern blot analysis of DNA from a variety of mammalian species shows restricted conservation of human pseudoautosomal genes, a trend that also applies to the two cloned mouse homologs of these genes and to neighboring human genes in distal Xp22.3. Our observations combined with those of other workers lead us to propose a model for the evolution of the PAR that includes both rapid sequence evolution and the incremental reduction in size of the region during mammalian evolution.

Different chromosomal localization of the Clcn4 gene in Mus spretus and C57BL/6J mice

We report the unprecedented finding of a gene with a different map position in two mouse strains. The Clcn4 gene was found to map to the X chromosome in the wild Mediterrean mouse, Mus spretus but to chromosome 7 in the inbred strain of laboratory mouse C57BL/6J. These data indicate that a recent evolutionary rearrangement occurred on the mouse sex chromosomes, very close to the pseudoautosomal region. Our data provide molecular evidence for a major divergence near the pseudoautosomal region, consistent with the hypothesis that hybrid sterility in these species results from abnormal pairing of sex chromosomes during male meiosis.

Structure and function of ribosomal protein S4 genes on the human and mouse sex chromosomes.

The human sex-linked genes RPS4X and RPS4Y encode distinct isoforms of ribosomal protein S4. Insufficient expression of S4 may play a role in the development of Turner syndrome, the complex human phenotype associated with monosomy X. In mice, the S4 protein is encoded by an X-linked gene, Rps4, and is identical to human S4X; there is no mouse Y homolog. We report here the organization of the human RPS4X and RPS4Y and mouse Rps4 genes. Each gene comprises seven exons; the positions of introns are conserved. The 5' flanking sequences of human RPS4X and mouse Rps4 are very similar, while RPS4Y diverges shortly upstream of the transcription start site. In chickens, S4 is encoded by a single gene that is not sex linked. The chicken protein differs from human S4X by four amino acid substitutions, all within a region encoded by a single exon. Three of the four substitutions are also present in human S4Y, suggesting that the chicken S4 gene may have arisen by recombination between S4X- and S4Y-like sequences. Using isoform-specific antisera, we determined that human S4X and S4Y are both present in translationally active ribosomes. S4Y is about 10 to 15% as abundant as S4X in ribosomes from normal male placental tissue and 46,XY cultured cells. In 49,XYYYY cells, S4Y is about half as abundant as S4X. In 49,XXXXY cells, S4Y is barely detectable. These results bear on the hypothesized role of S4 deficiency in Turner syndrome.

Structure and Function of Ribosomal Protein S4 Genes on the Human and Mouse Sex Chromosomes

The human sex-linked genes RPS4X and RPS4Y encode distinct isoforms of ribosomal protein S4. Insufficient expression of S4 may play a role in the development of Turner syndrome, the complex human phenotype associated with monosomy X. In mice, the S4 protein is encoded by an X-linked gene, Rps4, and is identical to human S4X; there is no mouse Y homolog. We report here the organization of the human RPS4X and RPS4Y and mouse Rps4 genes. Each gene comprises seven exons; the positions of introns are conserved. The 5' flanking sequences of human RPS4X and mouse Rps4 are very similar, while RPS4Y diverges shortly upstream of the transcription start site. In chickens, S4 is encoded by a single gene that is not sex linked. The chicken protein differs from human S4X by four amino acid substitutions, all within a region encoded by a single exon. Three of the four substitutions are also present in human S4Y, suggesting that the chicken S4 gene may have arisen by recombination between S4X- and S4Y-like sequences. Using isoform-specific antisera, we determined that human S4X and S4Y are both present in translationally active ribosomes. S4Y is about 10 to 15% as abundant as S4X in ribosomes from normal male placental tissue and 46,XY cultured cells. In 49,XYYYY cells, S4Y is about half as abundant as S4X. In 49,XXXXY cells, S4Y is barely detectable. These results bear on the hypothesized role of S4 deficiency in Turner syndrome.

Mapping and expression of the ubiquitin-activating enzyme E1 (Ube1) gene in the mouse.

The nucleotide sequence of the human cDNA encoding ubiquitin-activating enzyme E1 is more than 99% identical with the human A1S9T cDNA, a gene that has been shown to complement the temperature-sensitive mutant mouse cell line, tsA1S9. The amino acid sequences of the proteins encoded by these two cDNA sequences are identical, and both cDNAs were previously shown to be located in the same region of the human X chromosome; thus, ubiquitin-activating enzyme E1 and A1S9T appear to be the same gene, designated UBE1. By in situ hybridization to metaphase chromosomes from male mice and by Southern blot analysis of male and female mouse DNA, we show that, in the mouse, a human UBE1 cDNA probe identified both X- and Y-linked loci. Ube1 is located at band A2 of the mouse X Chromosome (Chr) and Ube2 on the short arm of the Y Chr. This is in contrast to the situation in the human, where there is no evidence for Y-linked sequences related to UBE1. Mapping of the Ube1 gene in interspecific backcrosses between Mus spretus and C57BL/6 shows that the Ube1 locus maps close to Timp, in a conserved region of the mouse and human X Chrs that include Otc, Cybb, Syn1, Timp, and Araf. Expression of Ube1 on the inactive X Chr was examined to determine whether this gene is subject to X-Chr inactivation in the mouse, as there is previous evidence that the human UBE1 gene escapes, at least partially, X inactivation. Sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products from M. spretus, C57BL/6J, and T(X;16)16H x M. spretus F1 female mice indicates that the mouse Ube1 gene is subject to X-Chr inactivation in vivo. This represents a new example of differences between the sex chromosomes of mouse and human.

Structure and chromosomal mapping of a highly polymorphic repetitive DNA sequence from the pseudoautosomal region of the mouse sex chromosomes

The pseudoautosomal region of the Mov15 mouse strain is marked by a Moloney murine leukemia provirus. The sequences flanking the Mov15 provirus were molecularly cloned and shown to consist of a tandemly repeated sequence of 31 nucleotides. Copy number variation of this repeat most likely accounts for the polymorphism in the mouse pseudoautosomal region detected with a probe from the flanking sequences. In situ hybridization to metaphase chromosomes showed heavy labeling of the pairing region of the X and Y chromosomes. The repetitive sequence was also found at the subtelomeric region of three autosomes. A similar level of amplification as the one seen on the sex chromosomes seems to be present on chromosomes 9 and 13. Lower copy number appear to be present on chromosome 4.

Regions of active chromatin conformation in 'inactive' male meiotic sex chromosomes of the mouse.

The sensitivity to DNase I of the meiotic sex chromosomes of the male mouse was determined by in situ nick translation. At pachytene and diakinesis-metaphase I, six segments, four at the ends of the X and Y chromosomes and two at internal sites on the X chromosome, were found to be more sensitive than the other parts of these chromosomes. The sensitive segments presumably reflect an active or potentially active chromatin conformation which is maintained in the sex chromosomes despite the earlier reported, almost complete cessation of uridine incorporation. The distribution of regions which are sensitive to DNase I corresponds to that of early DNA replication bands. Active conformation patterns like those figured here, probably exist in the sex chromosomes of other mammals as well.

Replication of the mouse sex chromosomes early in the S period.

The late-replicating X of the mouse can be clearly identified in a large proportion of metaphase spreads as an unlabelled chromosome when labelling of cells is done early in the S period. The Y chromosome follows this same pattern. Studies on Cattanach’s translocation are also reported, and it has been shown that the X<sup>t</sup> can be distinguished from the normal X in early labelled material.

Asynchronous replication of the mouse sex chromosomes

The Y-chromosome in male cells and one of the X-chromosomes in female cells were identified in C57BL/10Jax strain mouse embryo tissue culture on the basis of their late replication, utilizing a technique involving the uptake of tritiated thymidine, and autoradiography.