X-chromosome Inactivation Process Research Articles

The analysis of X-chromosome inactivation-related gene expression from single mouse embryo with sex-determination

Chromatin remodeling by histone and DNA modification is important for the initiation of X-chromosome inactivation (XCI). In this study, a thorough transcriptional analysis of five XCI-related genes was performed by single cell reverse-transcribed PCR. An analysis of the XCI-related gene ( Xist, Tsix, SUV39H1, SET7, and DNMT1) expression was performed to investigate the initiation process of XCI from early mouse single embryo (1-cell, 2-cell, 4-cell, 8-cell, and blastocyst). Detection of the expression of Xist and Tsix from single 2-cell embryo was feasible, although the expression of those genes was very low in single 1-cell embryo. Transcription of those genes may be activated from single 2-cell embryo. After determining the sex of single embryo by Y-chromosome-specific Zfy expression, we found that Tsix could be detected from both male and female single embryos, but it was only possible to detect Xist from female single embryo. XCI chromatin-remodeling genes, such as histone H3 methylation enzymes ( SUV39H1 and SET7) and DNA methylation enzyme ( DNMT1), were expressed during all early phases of embryogenesis. The expression of those genes in single embryo was not dependent on sex. Our study illustrated that the expression of these chromatin-remodeling genes, SUV39H1, DNMT1, and SET7, may be originated from germ cells, which were not dependent on zygotic activation of Xist from female single embryo.

Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome.

Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.

X-Chromosome inactivation (XCI) patterns in placental tissues of a paternally derived bal t(X;20) case

Objective: Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X;autosome translocations and is generally attributed to a selective growth of cells that inactivate the normal X chromosome. However, little is known concerning when in development the selection acts, and thus whether skewed XCI would also be seen in placental tissues. Furthermore, as males with X-autosome translocations are normally infertile, all translocations studied to date for XCI-skewing have been either maternal or de novo in origin. However, we have recently reported the transmission of a balanced X;autosome translocation involving chromosomes X and 20 from father to daughter with the use of intracytoplasmic sperm injection (ICSI). This case thus provides a unique opportunity to examine the process of X-chromosome inactivation during placental development, and we report the analysis of XCI patterns in placental tissues and cord blood from this female.

Association Between Nonrandom X-Chromosome Inactivation and BRCA1 Mutation in Germline DNA of Patients With Ovarian Cancer

Most human female cells contain two X chromosomes, only one of which is active. The process of X-chromosome inactivation, which occurs early in development, is usually random, producing tissues with equal mixtures of cells having active X chromosomes of either maternal or paternal origin. However, nonrandom inactivation may occur in a subset of females. If a tumor suppressor gene were located on the X chromosome and if females with a germline mutation in one copy of that suppressor gene experienced nonrandom X-chromosome inactivation, then some or all of the tissues of such women might lack the wild-type suppressor gene function. This scenario could represent a previously unrecognized mechanism for development of hereditary cancers. We investigated whether such a mechanism might contribute to the development of hereditary ovarian cancers. Patterns of X-chromosome inactivation were determined by means of polymerase chain reaction amplification of the CAG-nucleotide repeat of the androgen receptor (AR) gene after methylation-sensitive restriction endonuclease digestion of blood mononuclear cell DNA from patients with invasive (n = 213) or borderline (n = 44) ovarian cancer and control subjects without a personal or family history of cancer (n = 50). BRCA1 gene status was determined by means of single-strand conformational polymorphism analysis and DNA sequencing. All statistical tests were two-sided. Among individuals informative for the AR locus, nonrandom X-chromosome inactivation was found in the DNA of 53% of those with invasive cancer versus 28% of those with borderline cancer (P = .005) and 33% of healthy control subjects (P = .016). Nonrandom X-chromosome inactivation can be a heritable trait. Nine of 11 AR-informative carriers of germline BRCA1 mutations demonstrated nonrandom X-chromosome inactivation (.0002 < P < .008, for simultaneous occurrence of both). Nonrandom X-chromosome inactivation may be a predisposing factor for the development of invasive, but not borderline, ovarian cancer.

Role of the region 3' to Xist exon 6 in the counting process of X-chromosome inactivation.

During early embryogenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell, leading to the transcriptional silencing of thousands of genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region containing the Xist gene. Xist maps within Xic (refs 7-10) and is necessary in cis for inactivation. The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome. In order to characterize functional elements within Xic and the Xist gene, we created a 65-kb cre/loxP deletion extending 3' to Xist exon 6. In undifferentiated ES cells, Xist expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3' to Xist exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.

Steroid sulphatase deficiency: identification of heterozygotes using hydrolysis of dehydroepiandrosterone sulphate by peripheral leucocytes.

Diagnosis of X-linked recessive ichthyosis, which is expressed only in males, can readily be made by measurement of leucocyte steroid sulphatase activity. However, because the gene for steroid sulphatase activity partly escapes from the process of X-chromosome inactivation associated with gene dosage compensation, identification of heterozygotes (females) is more difficult. We have measured the steroid sulphatase (by hydrolysis of dehydroepiandrosterone sulphate) and beta-glucuronidase (by hydrolysis of methylumbelliferyl glucuronide) activities in leucocytes from 18 heterozygotes, 100 normal females, 100 normal males and 11 affected subjects. When the ratio of the activities of steroid sulphatase and beta-glucuronidase in mixed leucocytes was plotted as a function of the steroid sulphatase activity, 85% heterozygotes were distinguished from normal females. Measurement of steroid sulphatase activity alone with these cells enabled identification of 78% heterozygotes. Measurements on mononuclear leucocytes were much less effective. Thrombocytes showed 1% of the steroid sulphatase activity of leucocytes. In females, leucocyte steroid sulphatase activity was independent of the stage of the ovarian cycle at which the cells were collected.

Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxanthine guanine phosphoribosyl transferase expression in early mouse development.

Dosage compensation for X-linked genes occurs by the process of X-chromosome inactivation (XCI) in mammalian somatic cells (Lyon 1972). The inactivation of one X chromosome in females takes place early in development, although the exact time is unknown. One method of ascertaining the time of XCI is to determine the activity of the X chromosome at different stages of development as measured by the activity of an X-coded enzyme. Prior to XCI, and in the absence of other dosage compensating mechanisms, the activity for an embryonically expressed X-coded enzyme should be twice as high in female embryos with two X chromosomes, as in male embryos with one X chromosome. The distribution of enzyme activities for single embryos from a litter would have two equal-sized peaks that are separated by a factor of two. The convergence of the two peaks into one would indicate that XCI had occurred.

Expression of alpha-galactosidase in preimplantation mouse embryos.

THE most widely accepted model for X-chromosome dosage compensation in female mammals is the single-active X hypothesis (Lyon hypothesis)1. X-Chromosome dosage compensation probably occurs at about the time of implantation of the embryo into the uterus2,3 but it is not known whether this arises by a process of X-chromosome inactivation or by X-chromosome activation4. There is now good evidence for the expression of the embryonic genome during preimplantation development5–10 and Lyon2,3 has reviewed the evidence suggesting that X chromosomes are also expressed during this period. This includes both indirect observations11–13 and the more direct experiments with the X-linked enzyme hypoxanthine guanine phosphoribosyl-transferase (HPRT) (ref. 10). We have examined the expression of another X-linked enzyme, α-galactosidase14–15 and report here a 300-fold increase in activity during preimplantation development. This is probably due to the expression of embryonic X chromosomes.