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The severe phenotype of human females whose karyotype includes tiny ring X chromosomes has been attributed to the inability of the small ring X chromosome to inactivate. The XIST locus is expressed only from the inactive X chromosome, resides at the putative X inactivation center, and is considered a prime player in the initiation of mammalian X dosage compensation. Using PCR, Southern blot analysis, and in situ hybridization, we have looked for the presence of the XIST locus in tiny ring X chromosomes from eight females who have multiple congenital malformations and severe mental retardation. Our studies reveal heterogeneity within this group; some rings lack the XIST locus, while others have sequences homologous to probes for XIST. However, in the latter, the locus is either not expressed or negligibly expressed, based on reverse transcription-PCR analysis. Therefore, what these tiny ring chromosomes have in common is a level of XIST transcription comparable to an active X. As XIST transcription is an indicator of X chromosome inactivity, the absence of XIST transcription strongly suggests that tiny ring X chromosomes in females with severe phenotypes are mutants in the X chromosome inactivation pathway and that the inability of these rings to inactivate is responsible for the severe phenotypes.

The authors report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X; 17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1--15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5[prime] end of F8C, but left the more proximal G6PD locus intact on the der(17)chromosome. As the deleted segment includes the 5[prime] half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5[prime] region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3[prime]F8C-5[prime]F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the pro-band, revealing a more complex rearrangement (three chromosomes involved) than that expected frommore » cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X. 27 refs., 5 figs., 1 tab.« less

As a result of selection following random X chromosome inactivation in human females, X chromosomes with visible deletions are usually inactive in every somatic cell. We have studied a female with mental retardation and dysmorphic features whose karyotype includes an X chromosome with a visible interstitial deletion in the proximal long arm. Based on cytogenetic analysis, the proximal breakpoint appeared to be in band Xq13.1, and the distal one in band q21.3. However, molecular analyses show that less of the q13 band is missing than cytogenetic studies indicated, as the deletion includes only loci from the region Xq13.3 to Xq21.31. Unexpectedly, studies of chromosome replication show that the pattern of X inactivation is random. Whereas the deleted X chromosome is late replicating in some cells from all tissues studied, it is early replicating in the majority of blood lymphocytes and skin fibroblasts, and is the active X chromosome in many of the hybrids derived from skin fibroblasts. As this chromosome is able to inactivate, it must include those DNA sequences from the X-inactivation center (XIC) that are essential for cis X inactivation. Molecular studies show that the XIC region, at Xq13.2, is present, so it is unlikely that the lack of consistent inactivation of this chromosome is attributable to close proximity of the breakpoint to the XIC. Supporting this conclusion is the similarity of the breakpoints to those of the other chromosomes we studied, whose deletions clearly do not interfere with the ability to inactivate. Our results show that deletions distal to DXS441 in Xq13.2 do not interfere with cis X inactivation. We attribute the random pattern of X inactivation reported here to the fact that in the tissues studied, cells with this interstitial deletion are not at a selective disadvantage.

X chromosome dosage compensation in Marsupials is like that in eutherian mammals except that the paternal X chromosome is always inactive, and silence of this chromosome is not well maintained. We previously showed that the unstable inactivation of the paternal G6PD allele is associated with the lack of DNA methylation in the 5′ CpG cluster. Even though this CpG island is unmethylated, the paternal allele (marked by an enzyme variant) is at least partially and often severely repressed in most tissues of the opossum, so that factors other than methylation must inactivate the locus. Here we report that when cell cultures are established from these tissues, the silent G6PD locus is derepressed. Although often complete, the extent of derepression differs among tissues and within different cell types in the same tissue, and is not accompanied by obvious changes in the pattern of chromosome replication. Studies of the HPRT locus in these cells show that the paternal HPRT allele also derepresses in cultured cells. These observations suggest that without DNA methylation to maintain the silence of the locus, tissue or cell-specific factors act to repress the silent locus, but are unable to maintain inactivity through cell division, or are lost as cells proliferate in culture.

In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells1. To test the hypothesis2 that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome3). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozy-gotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT+ allele on the inactive X was observed at a frequency of 6 X 10(-5) per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRT locus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.

We have established a cell line from mouse kidney cells expressing the tfm mutation and showed that these cells lack androgen binding activity. A subclone of these simian virus 40 (SV40)-transformed cells (6TGR-SV-tfm) selected in 6-thioguanine and lacking hypoxanthine phosphoribosyltransferase was used to produce a series of mouse--human hybrids containing the normal human X chromosome or various X autosome-translocation chromosomes (expressing only segments of the human X chromosome). When the androgen receptor locus (AR) was present in the hybrid, the number of receptor sites and kinetics of binding were similar to that in the human parental cells. Analysis of hybrids with partial human X chromosomes by using assays for X chromosome-linked enzymes and for the androgen receptor protein indicate that the AR locus on the human X chromosome is near the centromere between Xq13 and Xp11 and is proximal to the locus for phosphoglycerate kinase. Hybrids derived from 6TGR-SV-tfm mouse cells and human labial fibroblasts from an XY individual with the ar- form of androgen insensitivity have no binding activity. The lack of complementation indicates that the X chromosome-linked mutations in mouse and man affect homologous loci and supports the evolutionary conservation of X chromosomal loci in mammals; however, the position of the locus on the human X chromosome indicates that intrachromosomal rearrangement has occurred.

Mammalian sex-dosage compensation is mediated by maintaining activity of only one X chromosome. The asynchronous DNA synthesis characterizing the silent human X chromosome is thought to be reversible only during ontogeny of oocytes. We have previously shown that the glucose-6-phosphate dehydrogenase (G6PD) locus (G6PD) on the allocyclic X chromosome in chorionic villi is partially expressed. We now show that in hybrids derived from a clone of chorionic villi cells (heterozygous for G6PD A) and mouse A9 cells, the loci for G6PD, hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase are expressed on both human X chromosomes; the human X chromosomes carrying either G6PD A or B replicate synchronously with each other and with murine chromosomes. The X chromosome with G6PD A was identified as the original late-replicating X, because methylation in the body of the HPRT gene on this chromosome remained characteristic of the inactive X chromosome. These results indicate that X-chromosome inactivation is completely reversible in cells of trophoblast origin; induction of full transcriptional activity is accompanied by acquisition of isocyclic replication, showing an intimate relationship between these processes. The molecular events responsible for this reversal may be similar to those occurring during maturation of oocytes. Chorionic villi and derivative hybrids provide in vitro models for exploring early events that program the single active X chromosome.

The X chromosome in mammalian somatic cells is subject to unique regulation—usually genes on a single X chromosome are expressed while those on other X chromosomes are inactivated1. The X-locus for steroid sulphatase (STS; EC 3.1.6.2), the microsomal enzyme that catalyses the hydrolysis of various 3β-hydroxysteroid sulphates, is exceptional because it seems to escape inactivation. Evidence for this comes from fibroblast clones in females heterozygous for mutations that result in a severe deficiency of this enzyme in affected males; all clones from these heterozygotes have STS activity, and enzyme-deficient clones that are expected if the locus were subject to inactivation2, have not been found3. Further evidence that the STS locus escapes inactivation is that the human inactive X chromosome contributes STS activity to mouse–human hybrid cells4. On the basis of these hybrid studies5,6 the STS locus has been mapped to the distal half of the short arm (p22–pter) of the human X chromosome. Although the STS locus on both X chromosomes in human female cells is expressed, quantitative measurements of STS activity in males and females do not accurately reflect the sex differences in number of X chromosomes7–13 (Table 1). The ratio of mean values for normal females to that of normal males is greater than 1:1 but less than the ratio of 2:1 expected if STS loci on all X chromosomes were equally expressed. The incomplete dosage effect suggests that the STS locus on the inactive X chromosome might not be fully expressed. To test this hypothesis, we examined two heterozygotes for X-linked STS deficiency who were also heterozygous for the common electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD A and B). Studies of fibroblast clones from these females provide evidence, presented here, for differential expression of STS loci on the active and inactive X chromosome.

Studies of glucose-6-phosphate dehydrogenase (G6PD) in heterozygous cells from chorionic villi of five fetal and one newborn placenta show that the locus on the allocyclic X is expressed in many cells of this trophectoderm derivative. Heterodimers were present in clonal populations of cells with normal diploid karyotype and a late replicating X chromosome. The expression of the two X chromosomes was unequal, based on ratios of homodimers and heterodimers in clones. Studies of DNA, digested with Hpa II and probed with cloned genomic G6PD sequences, indicate that expression of the locus in chorionic villi is associated with hypomethylation of 3' CpG clusters. These findings suggest that dosage compensation, at least at the G6PD locus, has not been well established or maintained (or both) in placental tissue. Furthermore, the active X chromosome in these human cells of trophoblastic origin can be either the paternal or maternal one; therefore, paternal X inactivation in extraembryonic lineages is not an essential feature of mammalian X dosage compensation.

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.

Skin fibroblasts of human males affected with adrenoleukodystrophy (ALD) have previously been shown to be abnormal with respect to C26 fatty acid content. Skin fibroblast clones from heterozygotes in three families segregating this mutation have been analyzed and are of two types: clones with normal ratios of C26 to C22 fatty acids and clones with an excess of C26 fatty acids similar to that found in cells of affected males. This indicates not only that the locus is X linked but also that it is subject to inactivation. In most of the heterozygotes there were significantly more clones of abnormal type than those expressing the normal allele, indicating a proliferative advantage in vitro for skin fibroblasts of mutant type. The increased levels of fatty acids in plasma in most heterozygotes and the phenotype of blood cells of women heterozygous for both ALD and glucose-6-phosphate dehydrogenase (G6PD) in one family are evidence that selection favoring the mutant allele may occur in vivo as well as in vitro and may explain why many heterozygotes manifest clinical symptoms of the disease. These studies have also revealed the close linkage between ALD and G6PD loci, because there are no recombinants among 18 informative offspring of doubly heterozygous mothers. Therefore, the ALD locus can be mapped on the human X chromosome near the G6PD locus at Xq28.

To explore the molecular basis of X chromosome inactivation, we have examined the human locus for glucose-6-phosphate dehydro-genase (G6PD) in various human tissues. Studies of DNA from males and females and from somatic cell hybrids with active or inactive X chromosomes, show that two remarkably dense clusters of CpG dinucleotides in the 3' coding sequences are hypomethylated in active G6PD genes but extensively methylated in inactive ones. Reacquisition of G6PD activity, either spontaneous or induced by 5-azacytidine, is accompanied by demethylation of both clusters; however, the clusters remain methylated in reactivants that express HPRT but not G6PD. Our observations implicate these 3' CpG clusters in the transcription of G6PD and in maintenance of dosage compensation for X linked housekeeping genes.