Transgenic mice carrying an Xist-containing YAC

The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.      

Familial skewed X-chromosome inactivation linked to a component of the cohesin complex, SA2

The gene dosage inequality between females with two X-chromosomes and males with one is compensated for by X-chromosome inactivation (XCI), which ensures the silencing of one X in every somatic cell of female mammals. XCI in humans results in a mosaic of two cell populations: those expressing the maternal X-chromosome and those expressing the paternal X-chromosome. We have previously shown that the degree of mosaicism (the X-inactivation pattern) in a Canadian family is directly related to disease severity in female carriers of the X-linked recessive bleeding disorder, haemophilia A. The distribution of X-inactivation patterns in this family was consistent with a genetic trait having a co-dominant mode of inheritance, suggesting that XCI choice may not be completely random. To identify genetic elements that could be responsible for biased XCI choice, a linkage analysis was undertaken using an approach tailored to accommodate the continuous nature of the X-inactivation pattern phenotype in the Canadian family. Several X-linked regions were identified, one of which overlaps with a region previously found to be linked to familial skewed XCI. SA2, a component of the cohesin complex is identified as a candidate gene that could participate in XCI through its association with CTCF.     

Lineage-specific function of the noncoding Tsix RNA for Xist repression and Xi reactivation in mice

The noncoding Tsix RNA is an antisense repressor of Xist and regulates X inactivation in mice. Tsix is essential for preventing the inactivation of the maternally inherited X chromosome in extraembryonic lineages where imprinted X-chromosome inactivation (XCI) occurs. Here we establish an inducible Tsix expression system for investigating Tsix function in development. We show that Tsix has a clear functional window in extraembryonic development. Within this window, Tsix can repress Xist, which is accompanied by DNA methylation of the Xist promoter. As a consequence of Xist repression, reactivation of the inactive X chromosome (Xi) is widely observed. In the parietal endoderm, Tsix represses Xist and causes reactivation of an Xi-linked GFP transgene throughout development, whereas Tsix progressively loses its Xist-repressing function from embryonic day 9.5 (E9.5) onward in trophoblast giant cells and spongiotrophoblast, suggesting that Tsix function depends on a lineage-specific environment. Our data also demonstrate that the maintenance of imprinted XCI requires Xist expression in specific extraembryonic tissues throughout development. This finding shows that reversible XCI is not exclusive to pluripotent cells, and that in some lineages cell differentiation is not accompanied by a stabilization of the Xi.     

A member of the caudal family of homeobox genes maps to the X-inactivation centre region of the mouse and human X chromosomes

X-inactivation is the process which allows equalization of thedosage of X chromosomal genes between males and females. A specificregion of the X chromosome, called the X-inactivation centre(XIC), is thought to have a key role in regulating this process.A gene, XIST, has been identified which maps to the XIC andso far has the unique property of being expressed exclusivelyfrom the inactive X chromosome. Although XIST is a good candidatefor a gene involved in regulating X-inactivation there is asyet no formal proof it has such a role. Here we describe anothergene, Cdx4, a member of the caudal-related family of homeoboxgenes, which is located within the minimal region assigned toXIC in humans. Furthermore, this gene is the closest known geneto XIST in both mouse and human. Unlike Xist, Cdx4 appears tobe normally X-inactivated in mice. Although it is not clearwhether the location of this gene within the XIC region is ofany significance in X-inactivation, the isolation of the genewill allow further definition of the region of inactive X-specificexpression surrounding XIST.     

Xist expression from an Xist YAC transgene carried on the mouse Y chromosome

We have constructed mouse transgenic lines carrying a YAC clone encompassing the Xist gene in order to investigate the factors influencing Xist expression and the initiation of X-inactivation. Two transgenic lines were derived, one carrying four copies integrated at an autosomal site and a second line carrying four copies integrated at a single site on the Y chromosome. Xist expression was not observed in mice carrying the autosomal insertion. However, Xist expression from the Y-inserted transgenes was observed and at levels commensurate with that found in normal female mice. Methylation sites in the autosomal transgene both 5' and 3' of the Xist gene are hypermethylated and appear to reflect methylation patterns observed on the active X chromosome. For the Y-linked transgene, methylation sites 5' and 3' of the Xist gene are hypomethylated reflecting patterns found on the inactive X chromosome. However, the 5' and 3' methylation levels have been decoupled at the active transgenic locus. The data suggest that sequences in the vicinity of Xist can initiate some of the features that are associated with the initiation process of X-inactivation.      

XY chromosome behaviour in the germ-line of the human male: A FISH analysis of spatial orientation,chromatin condensation and pairing

We have used multicolour fluorescencein situ hybridization to study the behaviour of the X and Y chromosomes in relation to a representative autosome, chromosome 1, on air-dried testicular preparations from normal fertile human males. In a proportion of Sertoli cells at interphase as well as spermatogonial metaphases there is an apparent selective undercondensation of the heterochromatic block of the long arm of the Y, which may be of functional significance with respect to Y-specific gene activity, initiating and maintaining spermatogenesis; we suggest that this may involve a mechanism similar to heterochromatin position-effect variegation inDrosophila. In the supporting Sertoli as well as pre-meiotic and leptotene cells the X and Y occupy relatively restricted domains at opposite poles of the nuclear membrane, while the chromosome 1 centromere regions are located interstitially and appear prealigned. The XY pairing and sex vesicle formation comprises a complex series of spatial movement and differential condensation patterns. On the basis of these observations we propose that: the XIST/Xist gene, known to be involved in somatic X inactivation, imposes a chromatin reorganization leading to bending at the X-inactivation centre both at first meiotic prophase in males and in the soma in females; and the differential X and Y segments are protected from potentially deleterious meiotic exchanges by their separate spatial orientation. In addition, there is an indication that the timing of pairing and first meiotic segregation of the sex chromosomes is different, and precocious in comparison to the pairing and segregation of the autosomes, which may explain the high incidence of sex chromosome aneuploidy in sperm.     

Cloning and characterization of a murine brain specific gene Bpx and its human homologue lying within the Xic candidate region

The X inactivation centre (Xic) is a cis-acting locus thought to play a key role in the initiation of X-inactivation. We have cloned and characterized a new gene, Bpx, lying distal to the murine Xist. Bpx, which is specifically expressed in the brain, shows strong homology to genes encoding nucleosome assembly proteins and is normally X- inactivated in mice. Isolation and localization of BPX, its human homologue, has shown the gene to be located centromeric to XIST in man. The Xq13 region, whose orientation is apparently globally conserved between man and mouse, must therefore contain an inversion of at least 600 kb spanning the XIST sequence and including the CDX4 and BPX genes.      

Molecular and cytogenetic analysis of familial Xp deletions

Five families in which an Xp deletion is segregating and two families in which an X chromosome rearrangement including a deletion of the short arm is segregating were ascertained for study. Normal fertility was seen in all families. Members from 5 of the 7 families manifested short stature (height <5th centile), while normal height was present in two families. Studies of both the FMR-1 and the androgen receptor loci using PCR based X-inactivation analysis demonstrated that in all families analyzed, there is preferential inactivation of one X chromosome. Molecular cytogenetic analysis showed that members of 3 of the 7 families share a common breakpoint in an approximate 2-3 Mb region at Xp22.12, suggesting a possible hotspot for chromatin breakage. Previous genotype-phenotype correlations and deletion mapping have indicated that a gene for stature resides within the pseudoautosomal region in Xp22.33. Our findings indicate that the loss of this region is not always associated with short stature, suggesting that other factors may be involved.     

Familial cases of point mutations in the XIST promoter reveal a correlation between CTCF binding and pre-emptive choices of X chromosome inactivation

The choice mechanisms that determine the future inactive X chromosome in somatic cells of female mammals involve the regulated expression of the XIST gene. A familial C(-43)G mutation in the XIST promoter results in skewing of X chromosome inactivation (XCI) towards the inactive X chromosome of heterozygous females, whereas a C(-43)A mutation found primarily in the active X chromosome results in the opposite skewing pattern. Both mutations point to the existence of a factor that might be responsible for the skewed patterns. Here we identify this factor as CTCF, a conserved protein with a 11 Zn-finger (ZF) domain that can mediate multiple sequence-specificity and interactions between DNA-bound CTCF molecules. We show that mouse and human Xist/XIST promoters contain one homologous CTCF-binding sequence with the matching dG-contacts, which in the human XIST include the -43 position within the DNase I footprint of CTCF. While the C(-43)A mutation abrogates CTCF binding, the C(-43)G mutation results in a dramatic increase in CTCF-binding efficiency by altering ZF-usage mode required for recognition of the altered dG-contacts of the mutant site. Thus, the skewing effect of the two -43C mutations correlates with their effects on CTCF binding. Finally, CTCF interacts with the XIST/Xist promoter only in female human and mouse cells. The interpretation that this reflected a preferential interaction with the promoter of the active Xist allele was confirmed in mouse fetal placenta. These observations are in keeping with the possibility that the choice of X chromosome inactivation reflects stabilization of a higher order chromatin conformation impinging on the CTCF-XIST promoter complex.