Genetic control of X chromosome inactivation in mice: definition of the Xce candidate interval

In early mammalian development, one of the two X chromosomes is silenced in each female cell as a result of X chromosome inactivation, the mammalian dosage compensation mechanism. In the mouse epiblast, the choice of which chromosome is inactivated is essentially random, but can be biased by alleles at the X-linked X controlling element (Xce). Although this locus was first described nearly four decades ago, the identity and precise genomic localization of Xce remains elusive. Within the X inactivation center region of the X chromosome, previous linkage disequilibrium studies comparing strains of known Xce genotypes have suggested that Xce is physically distinct from Xist, although this has not yet been established by genetic mapping or progeny testing. In this report, we used quantitative trait locus (QTL) mapping strategies to define the minimal Xce candidate interval. Subsequent analysis of recombinant chromosomes allowed for the establishment of a maximum 1.85-Mb candidate region for the Xce locus. Finally, we use QTL approaches in an effort to identify additional modifiers of the X chromosome choice, as we have previously demonstrated that choice in Xce heterozygous females is significantly influenced by genetic variation present on autosomes (Chadwick and Willard 2005). We did not identify any autosomal loci with significant associations and thus show conclusively that Xce is the only major locus to influence X inactivation patterns in the crosses analyzed. This study provides a foundation for future analyses into the genetic control of X chromosome inactivation and defines a 1.85-Mb interval encompassing all the major elements of the Xce locus.     

Further evidence for the importance of parental source of the Xce allele in X chromosome inactivation

Using mice that were mosaics for both Xce and phosphoglycerate kinase (Pgk-1) alleles, we present further evidence that the parental source of the X chromosome may affect the probability of that X chromosome remaining active. The reciprocal cross differences in PGK-1 activity described here are intermediate between those published previously for other alleles of Xce.     

Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus

The expression of maternally derived X-chromosomal Pgk-1 alleles was investigated in oocytes and early embryos of mice carrying different alleles (Xcea, Xcec) of the X-chromosome controlling element (Xce) locus. Pgk-1 allelic expression was determined by measuring their gene products using Cellogel electrophoresis and a sensitive fluorimetric enzyme assay. In addition to the already existing mouse strain of the genotypes Pgk-1a Xcec and Pgk-1b Xcea, a new line was bred carrying the combination Pgk-1b Xcec. The X chromosomes carrying the combinations Pgk-1a Xcec and Pgk-1b Xcec were of feral origin, whereas Pgk-1b Xcea was derived from a laboratory line. Our results using Xcec homozygous females confirm that maternal Pgk-1 is already expressed on day 4 of embryogenesis, thus substantiating data previously obtained using Xcea/Xcec heterozygous females. This finding also demonstrates that the timing of reactivation of maternal Pgk-1 is not influenced by the Xce locus. Furthermore, we found that oocytes from Xcec homozygous females have a balanced PGK-1 A/PGK-1 B allozyme ratio (50:50), whereas in oocytes obtained from Xcea/Xcec heterozygotes, the PGK-1 allozyme ratio is about 60:40. In tissues of adult Xce homozygous females, the PGK-1 allozymes are also balanced, whereas in Xcea/Xcec heterozygous females, the ratio is about 35:65. In addition to the relative activity of the PGK-1 allozymes, we also measured the absolute activity of PGK-1 in oocytes obtained from three types of Xce homozygous females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of evidence that the XqYq pairing tips at meiosis in the mouse show hypersensitivity to DNAse I

In situ nick translation procedures have been applied to meiotic metaphase I divisions of the normal and XY, Sxr mouse. Unlike in man, where the pairing tips of the XY bivalent show a special sensitivity to DNAse I nicking, no such sensitivity can be detected for either of these types of mouse. Hypersensitivity in the D-band equivalent region of the X chromosome does, however, exist, this site being early replicating in somatic cells and housing the X inactivation centre (Xce).     

Comparative analysis of the DXPas34 regulatory region in rodents

Mouse X chromosome inactivation center contains the DXPas34 minisatellite locus which plays an important role in expression regulation of the Tsix and Xist genes, involved into female dosage compensation. Comparative analysis of the DXPas34 locus from mouse, rat, and four common vole species revealed similar organization of this region in the form of tandem repeat blocks. A search for functionally important elements in this locus showed that all the species examined carried the conservative motif monomers, which could be involved in regulation of X inactivation.     

An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.     

X chromosome inactivation in nuclear transfer ES cells

Nuclear transfer ES (ntES) cells are established from cloned blastocysts generated by somatic cell nuclear transfer and are expected to be an important resource for regenerative medicine. However, cloned mammals, generated by similar methods, show various abnormalities, which suggest disordered gene regulation. Random X chromosome inactivation (XCI) has been observed to take place in cloned female mouse embryos, but XCI does not necessarily occur according to Xce strength, a genetic element that determines the likelihood of each X chromosome to be inactivated. This observation suggests incomplete reprogramming of epigenetic marks related to XCI. Here, we investigated XCI in ntES cell lines, which were established using differentiated embryoid bodies that originated from a female mouse ES cell line. We examined Xist RNA localization, histone modifications in the Xist locus, and XCI choice. We did not find substantial differences between the ntES lines and their parental ES line. This suggests that the Xist locus and the epigenetic marks involved in XCI are reprogrammed by nuclear transfer and subsequent ntES cell establishment. In contrast to skewed XCI in cloned mice, our observations indicate that normal XCI choice takes place in ntES cells, which supports the goal of safe therapeutic cloning for clinical use.     

X chromosome reactivation and regulation in cloned embryos

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Comparative analysis of the DXPas34 regulatory region in rodents

Mouse X chromosome inactivation center contains the DXPas34 minisatellite locus which plays an important role in expression regulation of the Tsix and Xist genes, involved into female dosage compensation. Comparative analysis of the DXPas34 locus from mouse, rat, and four common vole species revealed similar organization of this region in the form of tandem repeat blocks. A search for functionally important elements in this locus showed that all the species examined carried the conservative motif monomers, which could be involved in regulation of X inactivation.     

Exclusion of human chromosome 13q34 as the site of the cystic fibrosis mutation.

We have studied a family in which both cystic fibrosis (CF) and an unbalanced translocation between chromosomes 6 and 13 are found. As CF occurs in the child who is effectively monosomic for the translocated part of the long arm of chromosome 13, it was suggested that the locus of the gene mutation causing CF is on chromosome 13q34. The gene for human coagulation factor X is located at 13q34, and we have found a restriction fragment length polymorphism (RFLP) that is revealed by a cloned cDNA coding for this protein. Linkage analysis in eight CF families shows no evidence of cosegregation between CF and the gene for factor X, strongly suggesting that the locus for the defect causing cystic fibrosis is not at 13q34.     

A Cis-Acting Locus That Promotes Crossing over between X Chromosomes in Caenorhabditis Elegans

This study reports the characterization of a cis-acting locus on the Caenorhabditis elegans X chromosome that is crucial for promoting normal levels of crossing over specifically between the X homologs and for ensuring their proper disjunction at meiosis I. The function of this locus is disrupted by the mutation me8, which maps to the extreme left end of the X chromosome within the region previously implicated by studies of X;A translocations and X duplications to contain a meiotic pairing site. Hermaphrodites homozygous for a deletion of the locus (Df/Df) or heterozygous for a deletion and the me8 mutation (me8/Df) exhibit extremely high levels of X chromosome nondisjunction at the reductional division; this is correlated with a sharp decrease in crossing over between the X homologs as evidenced both by reductions in genetic map distances and by the presence of achiasmate chromosomes in cytological preparations of oocyte nuclei. Duplications of the wild-type region that are unlinked to the X chromosome cannot complement the recombination and disjunction defects in trans, indicating that this region must be present in cis to the X chromosome to ensure normal levels of crossing over and proper homolog disjunction. me8 homozygotes exhibit an altered distribution of crossovers along the X chromosome that suggests a defect in processivity along the X chromosome of an event that initiates at the chromosome end. Models are discussed in which the cis-acting locus deleted by the Dfs functions as a meiotic pairing center that recruits trans-acting factors onto the chromosomes to nucleate assembly of a crossover-competent complex between the X homologs. This pairing center might function in the process of homolog recognition, or in the initiation of homologous synapsis.     

Derepression with decreased expression of the G6PD locus on the inactive X chromosome in normal human cells

Studies of a unique clone of skin fibroblasts from a normal 46 XX female reveal that the G6PD locus on the inactive X chromosome has been derepressed. The reactivation event occurs spontaneously, and is associated with normal karyotype, including the presence of a late-replicating X chromosome. Analysis of mouse-human hybrids with the relevant chromosome provides evidence that the derepressed locus is on the inactive X, and that reactivation is not extensive (the PGK locus is not derepressed). Nor is any general change in DNA methylation of this chromosome detectable with Hpa II and an X-specific DNA probe. Studies of the glucose-6-phosphate dehydrogenase phenotype in these heterozygous cells indicate that the reactivated X produces only half the enzyme subunits as are produced by the active X. Although this dosage difference may be related to the mutational event responsible for derepression of the locus, these observations along with other evidence suggest that loci on the inactive X, when expressed, have less activity than corresponding loci on the active X.     

Chromosomal localization of GABAA receptor subunit genes: relationship to human genetic disease

Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a beta subunit and three isoforms of the alpha subunit. The alpha 2 and beta genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The alpha 1 gene is on chromosome 5 (bands q34-q35) and the alpha 3 gene is on the X chromosome. The alpha 3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human alpha 3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression.     

Mapping of locus for X-linked congenital stationary night blindness (CSNB1) proximal to DXS7.

A recombinant chromosome in a male affected with X-linked congenital stationary night blindness (CSNB1) provides new information on the location of the CSNB1 locus. A four-generation family with five males affected with X-linked CSNB was analyzed with five polymorphic markers for four X-chromosome loci spanning the region OTC (Xp21.1) to DXS255 (Xp11.22). Four of the males inherited the same X chromosome; one male inherited a chromosome that from OTC to DXS7, inclusive, was derived from the normal X chromosome of his unaffected grandfather and that from a location between DXS7 and DXS426 proximally was derived from the chromosome carrying the CSNB1 locus. This recombinant maps the CSNB1 locus in this family to a region on the short arm of the X chromosome proximal to the DXS7 locus.     

X-linked dominant Charcot-Marie-Tooth disease: Suggestion of linkage with a cloned DNA sequence from the proximal Xq

Summary A large kindred with the X-linked dominant form of peroneal muscular atrophy (Charcot-Marie-Tooth disease) was analyzed for individual variation in the length of DNA fragments after restriction endonuclease digestion. A systematic search was performed for linkage with a series of cloned single-copy DNA sequences of known regional assignment to the human X chromosome. Close linkage was found with the pDP34 probe (DXYS1 locus, Xq13-q21), suggesting that the gene responsible for the disease is located on the proximal long arm of the X chromosome.     

Genetic and molecular evidence of an X-chromosome deletion spanning the tabby (Ta) and testicular feminization (Tfm) loci in the mouse

A new radiation-induced mutation in the mouse, tabby-25H (Ta25H), has proved to be a deletion which spans both the tabby and testicular feminization (Tfm) loci on the X chromosome. The Ta phenotype closely resembles that of the original TaFa mutation in both the heterozygous and hemizygous conditions but Ta25H/Y animals additionally show the Tfm/Y phenotype, being externally female but possessing abdominally located testes. There is a shortage of both Ta25H/+ and Ta25H/Y classes relative to their normal sibs among the progeny of Ta25H/+ females at weaning age and this was indicated to be due to prenatal or neonatal losses. Exencephaly was observed in some members of both classes prior to birth. Both Ta25H classes tend to be runted at weaning but, remarkably, Ta25H/+ females often show a range of abnormalities not evident in Ta25H/Y animals. When probes for the Zfx, Ccg-1, Phk, and DXPas19 loci, which lie close to Ta, were hybridised to DNAs from Ta25H hemizygotes, the profiles of the X-linked bands were similar to those of control DNAs, suggesting these loci lie outside the deletion. However, a clear absence of an X-linked band was found with human androgen receptor probes, indicating that the Tfm locus is indeed missing. The deletion, therefore, extends a minimum of 1.5 cM and, with its proximal and distal boundaries partially defined, it could be as large as 4 cM. As Ta25H/+ females show the striped X-inactivation coat pattern, the putative X-inactivation centre, Xce, which lies close to Ta, cannot be located within the region deleted. The greasy (Gs) locus similarly appears to lie outside the deletion.