Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.     

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.     

Evidence for a relationship between DNA methylation and DNA replication from studies of the 5-azacytidine-reactivated allocyclic X chromosome

We examined the sequence of DNA synthesis of the human active, inactive and reactivated X chromosomes in mouse-human hybrid cells. The two independent reactivants, induced by 5-azacytidine (5-azaC), expressed human hypoxanthinephosphoribosyl transferase (HPRT), and one also expressed human glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK). Restriction enzyme analysis of DNA methylation at the re-expressed loci revealed hypomethylation of CpG clusters, that characterizes the relevant genes on the active X. The transfer of active and inactive X chromosomes from the native environment of the human fibroblast to the foreign environment of the hybrid cell did not affect the specific replication sequence of either human X chromosome. The silent X chromosome when reactivated, remained allocyclic, and the first bands to replicate were the same as prior to reactivation. In one reactivant, however, further progression of replication was significantly altered with respect to the order in which bands were synthesized. This alteration in the replication of the silent X following 5-azaC-induced reactivation suggests that DNA methylation may modulate the replication kinetics of chromosomal DNA.     

Frequent derepression of G6PD and HPRT on the marsupial inactive X chromosome associated with cell proliferation in vitro

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 depressed. 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.     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987     

Spontaneous reactivation of the inactive X chromosome in mouse embryonal carcinoma cells

The mouse embryonal carcinoma cell line MC12 carries two X chromosomes, one of which replicates late in S phase and shares properties with the normal inactive X chromosome and, therefore, is considered to be inactivated. Since the hypoxanthine phosphoribosyl transferase (HPRT) gene on the active X chromosome is mutated (HPRT(NDASH;)), MC12 cells lack HPRT activity. After subjecting MC12 cells to selection in HAT medium, however, a number of HAT-resistant clones (HAT(R)) appeared. The high frequency of HAT resistance (3.18 x 10(-4)) suggested reactivation of HPRT(PLUS;) on the inactive X chromosome rather than reversion of HPRT(NDASH;). Consistent with this view, cytological analyses showed that the reactivation occurred over the length of the inactive X chromosome in 11 of 20 HAT(R) clones isolated. The remaining nine clones retained a normal heterochromatic inactive X chromosome. The spontaneous reactivation rate of the HPRT(PLUS;) on the inactive X chromosome was relatively high (1.34 x 10(-6)) and comparable to that observed for XIST-deleted somatic cells (Csankovszki et al., 2001), suggesting that the inactivated state is poorly maintained in MC12 cells.     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.     

Karyotypic abnormality of the X chromosome is rare in mutant HPRT-lymphocyte clones

Lymphocyte clones mutated at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus on the X chromosome were studied by synchronization and G banding to determine the proportion of mutant clones having visible karyotypic change. 47 spontaneously mutant clones, 17 mutant clones induced by X-irradiation and 33 wild-type clones were studied. All clones were karyotypically normal except for 1 clone induced by X-irradiation in which an interstitial deletion of the short arm of the X chromosome had been inserted into the long arm of the same chromosome between q23 and q24; this change may have been coincidental or may have resulted in a position effect mutation. It was concluded that the great majority of mutations were not associated with a visible chromosome abnormality. This conclusion complements molecular studies which suggest that gene changes at the HPRT locus in HPRT- mutants generally extend over segments of DNA too small to be resolved by karyotypic analysis.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.     

Abnormal methylation does not prevent X inactivation in ICF patients.

DNA undermethylation is a characteristic feature of ICF syndrome and has been implicated in the formation of the juxtacentromeric chromosomal abnormalities of this rare syndrome. We have previously shown that in female ICF patients the inactive X chromosome (Xi) is also undermethylated. This result was unexpected since female ICF patients are not more severely affected than male patients. Here we show that CpG island methylation is abnormal in some ICF patients but in other ICF patients, the difference in methylation pattern between Xi and Xa (active X) is maintained. The consequences of Xi undermethylation on gene expression were investigated by enzyme assays. They showed that significant gene expression did not correlate with CpG island methylation status. The widespread Xi undermethylation does not affect overall Xi replication timing and does not prevent Barr body formation suggesting that a normal methylation pattern is not required for normal chromatin organization of Xi. Molecular investigation of some X-chromosome intron regions showed that the methylation changes in ICF female patients extend to non CpG islands sequences. Our results suggest that the genetic alteration of DNA methylation in ICF syndrome has little consequence on X chromosome gene expression and chromatin organization.     

Methylation profile of genes on the human X chromosome

X chromosome inactivation occurs in female mammals for the purpose of equalisation of dosage of X linked genes between the two sexes. In eutherian mammals, one of the two copies of the X chromosome present in female individuals is silenced. Epigenetic modifications of both DNA and histones have been implicated to play a crucial role in this inactivation phenomenon. In this work, we have employed a novel method published earlier by us, to assess the DNA methylation levels of genes on the inactive X chromosome in the human system. We have used genomic DNA from cells with the following karyotype namely, 47,XXX and 45,X to compare methylation levels from the active and inactive X. We report differential methylation of genes from the active and the inactive X chromosome with higher number of methylated genes being present on the inactive X chromosome. Our work has also led to identification of motifs that show a significant similarity to microRNA sequences which are enriched in methylated regions specific to the inactive X.