Tsix, a gene antisense to Xist at the X-inactivation centre

In mammals, dosage compensation is achieved by X inactivation and is regulated in cis by the X-inactivation centre (Xic) and Xist. The Xic controls X-chromosome counting, choice of X to inactivate and initiation of silencing. Xic action culminates in a change in Xist RNA property from a scarce, unstable RNA to highly expressed Xist RNA that coats the future inactive X. Deleting a 65-kb region downstream of Xist results in constitutive Xist expression and X inactivation, implying the presence of a cis-regulatory element. In this region, we now report the discovery of a gene antisense to Xist. Tsix is a 40-kb RNA originating 15 kb downstream of Xist and transcribed across the Xist locus. Tsix sequence is conserved at the human XIC. Tsix RNA has no conserved ORFs, is seen exclusively in the nucleus and is localized at Xic. Before the onset of X inactivation, Tsix is expressed from both X chromosomes. At the onset of X inactivation, Tsix expression becomes monoallelic, is associated with the future active X and persists until Xist is turned off. Tsix is not found on the inactive X once cells enter the X-inactivation pathway. Tsix has features suggesting a role in regulating the early steps of X inactivation, but not the silencing step.     

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.     

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.     

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.     

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.     

The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation

Sex chromosomes are subject to sex-specific selective evolutionary forces. One model predicts that genes with sex-biased expression should be enriched on the X chromosome. In agreement with Rice's hypothesis, spermatogonial genes are over-represented on the X chromosome of mice and sex- and reproduction-related genes are over-represented on the human X chromosome. Male-biased genes are under-represented on the X chromosome in worms and flies, however. Here we show that mouse spermatogenesis genes are relatively under-represented on the X chromosome and female-biased genes are enriched on it. We used Spo11(-/-) mice blocked in spermatogenesis early in meiosis to evaluate the temporal pattern of gene expression in sperm development. Genes expressed before the Spo11 block are enriched on the X chromosome, whereas those expressed later in spermatogenesis are depleted. Inactivation of the X chromosome in male meiosis may be a universal driving force for X-chromosome demasculinization.     

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.     

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.     

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.     

Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells

Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing approach followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution and for a large set of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels in gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences on the X chromosome. The inactive X chromosome (Xi) was hypermethylated at only a subset of gene-rich regions and, unexpectedly, overall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed cells was similar to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell residing in gene-poor areas. Furthermore, analysis of 6,000 CpG islands showed that only a small set of promoters was methylated differentially, suggesting that aberrant methylation of CpG island promoters in malignancy might be less frequent than previously hypothesized.     

A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion

The identification of subtype-specific translocations has revolutionized the diagnostics of sarcoma and has provided new insight into oncogenesis. We used RNA-seq to investigate samples from individuals diagnosed with small round cell tumors of bone, possibly Ewing sarcoma, but which lacked the canonical EWSR1-ETS translocation. A new fusion was observed between BCOR (encoding the BCL6 co-repressor) and CCNB3 (encoding the testis-specific cyclin B3) on the X chromosome. RNA-seq results were confirmed by RT-PCR and through cloning of the tumor-specific genomic translocation breakpoints. In total, 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcoma cases. Gene profiling experiments indicated that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewing sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this subgroup of sarcoma and that overexpression of BCOR-CCNB3 or of truncated CCNB3 activates S phase in NIH3T3 cells. Thus, the intrachromosomal X-chromosome fusion described here represents a new subtype of bone sarcoma caused by a newly identified gene fusion mechanism.     

Comparative analysis of chimpanzee and human Y chromosomes unveils complex evolutionary pathway

The mammalian Y chromosome has unique characteristics compared with the autosomes or X chromosomes. Here we report the finished sequence of the chimpanzee Y chromosome (PTRY), including 271 kb of the Y-specific pseudoautosomal region 1 and 12.7 Mb of the male-specific region of the Y chromosome. Greater sequence divergence between the human Y chromosome (HSAY) and PTRY (1.78%) than between their respective whole genomes (1.23%) confirmed the accelerated evolutionary rate of the Y chromosome. Each of the 19 PTRY protein-coding genes analyzed had at least one nonsynonymous substitution, and 11 genes had higher nonsynonymous substitution rates than synonymous ones, suggesting relaxation of selective constraint, positive selection or both. We also identified lineage-specific changes, including deletion of a 200-kb fragment from the pericentromeric region of HSAY, expansion of young Alu families in HSAY and accumulation of young L1 elements and long terminal repeat retrotransposons in PTRY. Reconstruction of the common ancestral Y chromosome reflects the dynamic changes in our genomes in the 5-6 million years since speciation.     

Involvement of a novel Tnf receptor homologue in hair follicle induction.

Although inductive interactions are known to be essential for specification of cell fate in many vertebrate tissues, the signals and receptors responsible for transmitting this information remain largely unidentified. Mice with mutations in the downless (dl) gene have defects in hair follicle induction, lack sweat glands and have malformed teeth. These structures originate as ectodermal placodes, which invaginate into the underlying mesenchyme and differentiate to form specific organs. Positional cloning of the dl gene began with identification of the transgenic family OVE1. One branch of the family, dl(OVE1B), carries an approximately 600-kb deletion at the dl locus caused by transgene integration. The mutated locus has been physically mapped in this family, and a 200-kb mouse YAC clone, YAC D9, has been identified and shown to rescue the dl phenotype in the spontaneous dl(Jackson) (dl(J), recessive) and Dl(sleek) (Dl(slk), dominant negative) mutants. Here we report the positional cloning of the dl gene, which encodes a novel member of the tumour necrosis factor (Tnf) receptor (Tnfr) family. The mutant phenotype and dl expression pattern suggests that this gene encodes a receptor that specifies hair follicle fate. Its ligand is likely to be the product of the tabby (Ta) gene, as Ta mutants have a phenotype identical to that of dl mutants and Ta encodes a Tnf-like protein.