Homozygous Tsix mutant mice reveal a sex-ratio distortion and revert to random X-inactivation

Tsix controls X-chromosome inactivation (XCI) by blocking the accumulation of Xist RNA on the future active X chromosome. Deleting Tsix on one X chromosome (X(Delta)X) skews XCI toward the mutated X chromosome in the female soma. Here I have generated homozygous Tsix-null mice (X(Delta)X(Delta)) to test how deleting the second allele affects the choice of XCI. Homozygosity leads to extremely low fertility and reveals two previously unknown non-mendelian patterns of inheritance. First, the sex ratio is skewed against female births so that one daughter is born for every two to three sons. Second, the pattern of XCI unexpectedly returns to random in surviving X(Delta)X(Delta) mice. Thus, with respect to choice, mutation of Tsix yields a phenotypic abnormality in heterozygotes but not homozygotes. To reconcile the paradox of female loss with apparent reversion to random choice, I propose that deleting both Tsix alleles results in chaotic choice and that randomness in X(Delta)X(Delta) survivors reflects a fortuitous selection of distinct X chromosomes as active and inactive.     

Xlr3b is a new imprinted candidate for X-linked parent-of-origin effects on cognitive function in mice

Imprinted genes show differential expression between maternal and paternal alleles as a consequence of epigenetic modification that can result in 'parent-of-origin' effects on phenotypic traits. There is increasing evidence from mouse and human studies that imprinted genes may influence behavior and cognitive functioning. Previous work in girls with Turner syndrome (45,XO) has suggested that there are X-linked parent-of-origin effects on brain development and cognitive functioning, although the interpretation of these data in terms of imprinted gene effects has been questioned. We used a 39,XO mouse model to examine the influence of the parental origin of the X chromosome on cognitive behaviors and expression of X-linked genes in brain. Our findings confirm the existence of X-linked imprinted effects on cognitive processes and identify a new maternally expressed imprinted gene candidate on the X chromosome, Xlr3b, which may be of importance in mediating the behavioral effects.     

Meiotic pairing and imprinted X chromatin assembly in Caenorhabditis elegans

The genetic imprinting of individual loci or whole chromosomes, as in imprinted X-chromosome inactivation in mammals, is established and reset during gametogenesis; defects in this process in the parent can result in disease in the offspring. We describe a sperm-specific chromatin-based imprinting of the X chromosome in the nematode Caenorhabditis elegans that is restricted to histone H3 modifications. The epigenetic imprint is established during spermatogenesis and its stability in the offspring is affected by the presence of a pairing partner during meiosis in the parental germ line. We observed that DNA lacking a pairing partner during meiosis, the normal situation for the X chromosome in males, is targeted for methylation of histone H3 at Lys9 (H3-Lys9) and can be silenced. Targeting unpaired DNA for silencing during meiosis, a potential hallmark of genome defense, could therefore have a conserved role in imprinted X-chromosome inactivation and, ultimately, in sex chromosome evolution.     

Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus

Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.     

The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression

According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)--the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells.     

Dosage compensation of the active X chromosome in mammals

Monosomy of the X chromosome owing to divergence between the sex chromosomes leads to dosage compensation mechanisms to restore balanced expression between the X and the autosomes. In Drosophila melanogaster, upregulation of the male X leads to dosage compensation. It has been hypothesized that mammals likewise upregulate their active X chromosome. Together with X inactivation, this mechanism would maintain balanced expression between the X chromosome and autosomes and between the sexes. Here, we show that doubling of the global expression level of the X chromosome leads to dosage compensation in somatic tissues from several mammalian species. X-linked genes are highly expressed in brain tissues, consistent with a role in cognitive functions. Furthermore, the X chromosome is expressed but not upregulated in spermatids and secondary oocytes, preserving balanced expression of the genome in these haploid cells. Upon fertilization, upregulation of the active X must occur to achieve the observed dosage compensation in early embryos.     

Methylation of CpG sites of two X-linked genes coincides with X-inactivation in the female mouse embryo but not in the germ line.

To further our understanding of initiation and imprinting of X-chromosome inactivation, we have examined methylation of specific CpG sites of X-linked Pgk-1 and G6pd genes throughout female mouse development. Methylation occurs around the time of inactivation and earlier for Pgk-1, which is closer to the X-inactivation centre. In female primordial germ cells, the inactive X chromosome escapes methylation; this may underly the reversibility of inactivation at meiosis. Similarly, the genes are unmethylated on the inactive X chromosome in sperm; hence, the imprint specifying preferential X-inactivation in extra-embryonic tissues must reside elsewhere.     

Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1

Genomic imprinting is an epigenetic modification that results in expression from only one of the two parental copies of a gene. Differences in methylation between the two parental chromosomes are often observed at or near imprinted genes. Beckwith-Wiedemann syndrome (BWS), which predisposes to cancer and excessive growth, results from a disruption of imprinted gene expression in chromosome band 11p15.5. One third of individuals with BWS lose maternal-specific methylation at KvDMR1, a putative imprinting control region within intron 10 of the KCNQ1 gene, and it has been proposed that this epimutation results in aberrant imprinting and, consequently, BWS1, 2. Here we show that paternal inheritance of a deletion of KvDMR1 results in the de-repression in cis of six genes, including Cdkn1c, which encodes cyclin-dependent kinase inhibitor 1C. Furthermore, fetuses and adult mice that inherited the deletion from their fathers were 20-25% smaller than their wildtype littermates. By contrast, maternal inheritance of this deletion had no effect on imprinted gene expression or growth. Thus, the unmethylated paternal KvDMR1 allele regulates imprinted expression by silencing genes on the paternal chromosome. These findings support the hypothesis that loss of methylation in BWS patients activates the repressive function of KvDMR1 on the maternal chromosome, resulting in abnormal silencing of CDKN1C and the development of BWS.     

Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.     

RNA sequencing shows no dosage compensation of the active X-chromosome

Mammalian cells from both sexes typically contain one active X chromosome but two sets of autosomes. It has previously been hypothesized that X-linked genes are expressed at twice the level of autosomal genes per active allele to balance the gene dose between the X chromosome and autosomes (termed 'Ohno's hypothesis'). This hypothesis was supported by the observation that microarray-based gene expression levels were indistinguishable between one X chromosome and two autosomes (the X to two autosomes ratio (X:AA) ~1). Here we show that RNA sequencing (RNA-Seq) is more sensitive than microarray and that RNA-Seq data reveal an X:AA ratio of ~0.5 in human and mouse. In Caenorhabditis elegans hermaphrodites, the X:AA ratio reduces progressively from ~1 in larvae to ~0.5 in adults. Proteomic data are consistent with the RNA-Seq results and further suggest the lack of X upregulation at the protein level. Together, our findings reject Ohno’s hypothesis, necessitating a major revision of the current model of dosage compensation in the evolution of sex chromosomes.     

Global hypomethylation of the genome in XX embryonic stem cells

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.     

Expression of Xist in mouse germ cells correlates with X-chromosome inactivation.

Mammals compensate for different doses of X-chromosome-linked genes in male (XY) and female (XX) somatic cells by terminally inactivating all but one X chromosome in each cell. A transiently inactive X chromosome is also found in germ cells, specifically in premeiotic oogenic cells and in meiotic and postmeiotic spermatogenic cells. Here we show that the Xist gene, which is a expressed predominantly from the inactive X-chromosome in female somatic cells, is also expressed in germ cells of both sexes, but only at those stages when an inactive X chromosome is present. This suggests support for the putative role of Xist as a regulator of X-chromosome inactivation and suggest a common mechanism for the initiation and/or maintenance of X-chromosome inactivation in all cell types.     

Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.

Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.     

Kallmann syndrome gene on the X and Y chromosomes: implications for evolutionary divergence of human sex chromosomes.

The recently identified gene for X-linked Kallmann syndrome (hypogonadotropic hypogonadism and anosmia) has a closely related homologue on the Y chromosome. The X and Y copies of this gene are located in a large region of X/Y homology, on Xp22.3 and Yq11.2, respectively. Comparison of the structure of the X-linked Kallmann syndrome gene and its Y homologue shed light on the evolutionary history of this region of the human sex chromosomes. Our data show that the Y homologue is not functional. Comparative analysis of X/Y sequence identity at several loci on Xp22.3 and Yq11.2 suggests that the homology between these two regions is the result of a complex series of events which occurred in the recent evolution of sex chromosomes.