Complete reactivation of X chromosomes from human chorionic villi with a switch to early DNA replication.

Abstract

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 vilii and derivative hybrids provide in vitro models for exploring early events that program the single active X chromosome.In mammals, the sex difference in dosage of X chromosomes is compensated by silencing all but one X chromosome in female somatic cells (1). Dosage compensation is not established simultaneously in all tissues (2) but is programmed along with other tissue-specific functions. At the time of tissue differentiation, only one X chromosome achieves potential transcriptional activity; the others become inactive, condense at interphase, and replicate asynchronously [later than the autosomes and the active X chromosome (3)]. The inactivity of the allocyclic X chromosome is rigorously maintained (4), and spontaneous reversal of the processi.e., reactivating the entire chromosome and reversing allocyclic replication-has not been observed in somatic cells. The spontaneous reactivation events that have been noted (5, 6) affect some loci but not others, and the chromosome remains late-replicating (5). On the other hand, reactivation of the entire X chromosome is believed to occur regularly during the ontogeny of oocytes. Primordial germ cells in the yolk sac of female rodents have a condensed X chromosome (7), and there is evidence from mouse embryos that glucose-6-phosphate dehydrogenase (G6PD) genes on both X chromosomes are expressed shortly after germ cells am've at the gonadal ridges, whereas only one locus was active previously (8).Although recent observations provide insights into maintenance of X chromosome dosage compensation (9-11), little is known about initiation of the process. Determining the initial steps in X chromosome differentiation-thos...

DNA methylation stabilizes X chromosome inactivation in eutherians but not in marsupials: evidence for multistep maintenance of mammalian X dosage compensation.

“…The only sex differences in methylation of opossum G6PD occur in the body of the gene, where the paternal allele is less methylated than the maternal one. Several lines of evidence suggest that such differential methylation is not functional: (i) differences occur in nuclease-insensitive regions; (ii) in eutherians, methylation outside of CpG clusters fails to show a consistent correlation with gene activity or inactivity (21,24,25,27,34); and (iii) methylation patterns in this region persist even when the silent locus is reactivated (15,21) or the late-replicating chromosome becomes early-replicating (16). Most likely, sex differences in methylation of the body of the gene result from differences in secondary structure of these regions on the active and inactive X at the time of de novo methylation and are heritable vestiges of early embryonic events rather than regulatory in nature.…”

“…Preferential inactivation of the paternal X has been observed in trophectoderm and primitive endoderm of rodents (12,13), whereas either X may be inactive in the corresponding human tissues (14,15). Further, the inactive X is frequently expressed in human trophectoderm derivatives (chorionic villi) (15,16).…”

“…Further, the methylation status of clustered CpGs 3' to G6PD is correlated with expression of that locus (27,28). When alleles on the silent X chromosome are reactivated either spontaneously, as in chorionic villi (15,16), or by 5-azacytidine, not only does the relevant CpG cluster become less methylated, but the chromatin in the region of the cluster acquires nuclease hypersensitivity (29). Hence, in the absence of DNA methylation within the cluster, the silence of alleles on the inactive X chromosome is not well maintained.…”

Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression.

“…Once established, X inactivation in somatic cells is clonally inherited and extremely stable. However, in germ cells reactivation occurs just prior to meiosis (13,22), and it has been reported that reactivation of the inactive X chromosome frequently occurs in human trophectoderm derivatives (30). The ontogeny of X inactivation is very likely a multistep process whose initiation is little understood at the molecular level.…”

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

“…A growing body of evidence indicates that methylation is involved in the stable maintenance of X-chromosome inactivation, although whether it is a cause or an effect of inactivation remains unclear (11,16). DNA-mediated gene transfer has shown that the hypoxanthine phosphoribosyl transferase (Hprt) gene on the active X chromosome of embryonic lineages and adult organs, but not on the inactive X, is competent in transferring the Hprt+ phenotype to Hprt-recipient cells (5).…”