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1998 Oxford University Press Nucleic Acids Research, 1998, Vol. 26, No. 12 2935–2940 Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation Anna V. Tinker and Carolyn J. Brown* Department of Medical Genetics, University of British Columbia, 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada Received March 12, 1998; Revised and Accepted May 4, 1998 ABSTRACT X chromosome inactivation occurs early in mammalian development to transcriptionally silence one of the pair of X chromosomes in females. The XIST RNA, a large untranslated RNA that is expressed solely from the inactive X chromosome, is implicated in the process of inactivation. As previous studies have shown that the XIST gene is methylated on the active X chromosome, we have treated a mouse/human somatic cell hybrid retaining an active human X chromosome with demethylating agents to determine whether expression of the human XIST gene could be induced. Stable expression of XIST was observed after several rounds of demethylation and stability of XIST expression correlated with the loss of methylation at the three sites analysed. We conclude that methylation is sufficient to inhibit expression of the XIST gene in somatic cell hybrids. No loss of expression was detected for eight other X-linked genes from the active X chromosome that was expressing XIST, suggesting that additional developmental or species-specific factors are required for the inactivation process. INTRODUCTION X chromosome inactivation in mammals involves a cis-limited transcriptional silencing of most of the genes on one X chromosome in females thus achieving dosage compensation for X-linked genes between females (who have two X chromosomes) and males (who have a single X chromosome and the sex-determining Y chromosome) (1). The inactivation occurs early in development, with the inactivated chromosome becoming late-replicating, hypermethylated, deficient in acetylated histones and heterochromatic (reviewed in 2). Analysis of methylation patterns, both by methylation-sensitive restriction enzyme digestion and genomic sequencing, has shown that the 5′ regions of many genes are methylated on the inactive X chromosome and unmethylated on the active X chromosome (reviewed in 3), while the 5′ regions associated with genes that escape X chromosome inactivation (i.e. genes that are expressed from both the active and inactive X chromosomes) remain unmethylated on both chromosomes (4–6). Treatment of cells with the cytidine analogs 5-azacytidine (5azaC) or 5-azadeoxycytidine (5azadC), which inhibit methyltransferase activity and thereby reduce levels of DNA methylation, causes reactivation of genes on the inactive X chromosome, particularly in somatic cell hybrids (reviewed in 7). The XIST gene is the only gene known to be expressed exclusively from the inactive X chromosome (8) and is localised to the smallest interval of the X chromosome required in cis for inactivation to occur (9,10). The XIST RNA, which is not translated, remains in the nucleus where it is associated with the inactive X chromosome (11,12). Expression of XIST (or its murine homologue Xist) is generally concordant with the presence of an inactive X chromosome (reviewed in 13), although expression appears to precede inactivation (14,15) and is observed at low levels in murine embryonic stem (ES) cells prior to inactivation (16,17). In ES cells, transgenes containing the murine Xist locus can induce inactivation (18,19) while chromosomes with targeted deletions of the Xist gene are unable to undergo inactivation (20,21) demonstrating that Xist is required for inactivation, and is also apparently sufficient to induce X chromosome inactivation during mouse development. The correlation between methylation and transcriptional inactivity extends to the XIST locus as well, as there is substantial evidence showing that the inactive allele on the active X chromosome is methylated. In human female somatic cells and mouse/human somatic cell hybrids, methylation was seen at several methylationsensitive restriction enzyme sites near the 5′ end of the gene on the active (XIST–) but not the inactive (XIST+) X chromosome (22). Differentially methylated sites have also been identified for the Xist gene in mice, and may serve as the ‘imprint’ directing the nonrandom Xist expression and paternal X chromosome inactivation seen in murine extraembryonic tissues (23–25). Additionally, ES cells deficient for the DNA methyltransferase enzyme responsible for maintaining DNA methylation show ectopic expression of Xist from the active X chromosome upon induction of differentiation (26), implicating DNA methylation in the silencing of the Xist locus in somatic tissues. We hypothesized that if XIST expression in somatic cells is prevented by DNA methylation then it should be induced by treatment with demethylating agents. Therefore we treated somatic cell hybrids retaining the human active X chromosome with 5azadC. Numerous clones expressing XIST were recovered, thereby demonstrating that silencing of XIST is maintained by *To whom correspondence should be addressed. Tel: +1 604 822 0908; Fax: +1 604 822 5348; Email: [email protected] 2936 Nucleic Acids Research, 1998, Vol. 26, No. 12 methylation. Furthermore, these clones allowed us to analyse the effect of XIST expression upon other X-linked genes. Eight other genes were examined and none showed altered expression after XIST induction, suggesting that expression of XIST is not sufficient to cause inactivation of the human X chromosome in mouse/human somatic cell hybrids. A MATERIALS AND METHODS Cell culture and treatment with 5azadC The hybrid AHA-11aB1, a subclone of the monochromosomal hybrid line AHA 11a (GM10324), was obtained from Dr H. F. Willard (Case Western Reserve University). Cells were maintained without selection in α-MEM with 7.5% fetal calf serum and penicillin/streptomycin (Gibco-BRL) supplement at 37C. Cells were plated at 104 cells/60 mm culture dish and allowed to attach for 4–6 h. The media was then supplemented with 0.2 µg/ml 5-azadC for 24 h. Fresh media was added, and the cells were grown for an additional 1–4 days before RNA isolation, or individual colonies were isolated by trypsinization in cloning cylinders and RNA isolated after these clones had been cultured an additional 2 weeks. To identify clones which had lost hypoxanthine phosphoribosyl transferase (HPRT) activity, cells were plated in media supplemented with azaguanine–thioguanine (AG/TG; 2 × 10–4 M 8-azaguanine, 6 × 10–5 M 6-thioguanine) and grown for 2 weeks before isolating individual colonies or staining with methylene blue for counting of colonies. B RNA isolation and analysis of expression Total RNA was isolated from cultured cells using an acid–guanidinium/phenol extraction protocol as previously described (27). RNA was also isolated from nuclear or cytoplasmic fractions using the same technique. The nuclear/cytoplasmic fractionations were as previously described (11), and were relatively crude so that some cross-contamination of the fractions may have occurred. cDNA was made from 5 µg of the RNA by reversetranscription with MMLV reverse transcriptase (GIBCO-BRL) using random hexamers as primers (28). PCR was performed with primers for XIST and MIC2 as previously described (29). Both sets of primers span splice junctions and therefore only amplify cDNA not genomic DNA. MIC2 is a pseudoautosomal gene which was included as a control for amplifiable cDNA as it is expressed from both the active and the inactive X chromosome (30). To examine expression of XIST, PCR was performed with primers along the length of the gene as diagrammed in Figure 3A. The primer pairs used were as follows: 1, AT1:29r; 2, A5:29r; 3, 26:C39-2; 4, C16-7:C16-9; 5, 8:11r; 6, 16:17r; 7, 3:5r. Most of these primers (29r, 26, 8, 11r, 16, 17r, 3 and 5r) have previously been described (11). Additional primers include: AT1: GAA CCA ACC AAA TCA CAA AGA; A5: TTT CTT ACT CTC TCG GGG CT; C39-2: TTA GCG TGC ATA ATC TTA GG; C16-9: TGC CCA CTC CTT ATG ATC TT; and C16-7: GAC ACC CTG TTG TTG TAC TAC AA. Primers for the examination of expression of the other X-linked genes have been described (31) with the exception of the T-plastin gene for which the primers used were: 1: TGA CCT TGT GAA GAG TGG C and 2: ACC TGC GAA TCA TGC ACC T (32). Seven of the genes examined are normally subject to X chromosome inactivation including one gene (TIMP1) from the short arm of the X chromosome (28), two genes (AR and CCG1) proximal to XIST on the long arm of the Figure 1. Expression of XIST after treatment of an active X-containing somatic cell hybrid with 5azadC. (A) Schematic outline of the clones isolated after demethylation of AHA-11aB1 by treatment with 5azadC. Shorter clone designations used in the text and a summary of XIST expression for these clones are shown in the boxes to the right. (B) XIST expression in the clones. RT–PCR of RNA from the clones diagrammed in (A) with primers for the human MIC2 and XIST genes. MIC2 is expressed from both the active and inactive human X chromosome and is included as a control for amplifiable cDNA. PCR product from both genes is cDNA-specific as the primers flank introns. X chromosome, and four genes (PGK1, IDSX, G6PD and T-plastin) distal to XIST on the long arm of the X chromosome (31; A.T. and C.B., unpublished observation). The RPS4X gene is located proximally to XIST on the long arm of the X chromosome and is expressed from both the active and the inactive X chromosome (33). As described above, MIC2 expression was also examined as a control for amplifiable cDNA. Northern analysis was performed with 10 µg of total RNA as previously described (28) using a radiolabelled PGK1 cDNA clone (34) as the probe. RNA for the northern analysis was isolated from AHA-11aB1; from AHA-A5-2b, an XIST-expressing derivative as shown in Figure 1; from a female lymphoblast (GM07059); and from a mouse/human somatic cell hybrid retaining an inactive X chromosome (t11-4Aaz5; ref. 28). DNA isolation and PCR-based analysis of methylation DNA was isolated from cultured somatic cells using salt precipitation as previously described (35). DNA was digested with an excess of EcoRI restriction enzyme (GIBCO-BRL) to reduce viscosity, then treated with RNase followed by phenol/ chloroform extraction and precipitation in ethanol. The digested 2937 Nucleic Acids Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.112 Nucleic 2937 5azadC. The derivation of the clones is diagrammed in Figure 1. Clonal cell lines derived from two demethylation treatments showed some instability of XIST reactivation, as the line AHA-B2 eventually lost its XIST expression. One line, however, (AHA-A5-2b) from the third round of demethylation has been maintained in culture for over a year and continues to express XIST. Methylation of XIST Figure 2. Methylation of the XIST gene after treatment with 5azadC. (A) Outline of the location of the three methylation-sensitive restriction enzyme sites assayed by PCR with primers AT-2 and 29r following digestion. Methylated DNA will be resistant to restriction enzyme digestion and subsequent PCR will generate product. Unmethylated DNA will be cut by the restriction enzymes so that there will not be intact template and no product will be observed after PCR. The black rectangle represents the first exon of the XIST gene. (B) Products observed after PCR amplification with primers AT-2 and 29r following digestion of DNA from the clones listed beside each panel with the enzymes listed across the top of the panels. All DNAs, including the ‘uncut’ lane were predigested with EcoRI (which does not have a recognition site within the amplified region) to reduce viscosity. AHA-11a is the parental active X containing somatic cell hybrid from which the other clones were derived after one to three treatments with 5azadC (derivation of the clones is shown in Fig. 1). AHA-B2 expressed XIST early in culture (XIST+), but lost expression over time (XIST–) (see also Fig. 1B). DNA was resuspended in water and additional digests with HhaI (New England Biolabs), SstII (GIBCO-BRL) or AvaI (New England Biolabs) were performed with excess enzyme according to manufacturer’s instructions. Approximately 50 ng of singly- or doubly-digested DNA was amplified by PCR using primers AT-2: ATG CTC TCT CCG CCC TCA (from sequence in 36) and 29r: GGT ATC GGA TAC CTG CTG AT (11), as shown in Figure 2A. RESULTS Induction of XIST expression by demethylation We treated the mouse/human hybrid cell line AHA-11aB1 (AHA-11a) with a concentration of 5azadC previously shown to induce expression from the HPRT gene on the inactive X chromosome. AHA-11a retains the human active X chromosome as its only human chromosome and therefore does not express XIST. Gene expression in the treated cell lines was monitored by RT–PCR using primers for the XIST gene and for the MIC2 gene as a control for amplifiable cDNA (Fig. 1). XIST expression was detected after one round of 5azadC treatment. The treated cells were cloned from single cell isolates and approximately one in five clones was found to show XIST expression; however, continued culturing of these clones resulted in loss of expression. We sought to obtain more stably reactivated clones by retreating one of the reactivated clones, AHA-1f (see Fig. 1 for full subclone names), with 5azadC, resulting in the isolation of several more clones expressing XIST. Two of these, AHA-A5 and AHA-B2, were subjected to a third round of demethylation treatment with The transcriptionally silent allele of XIST on the active X chromosome has been shown to be methylated at a series of restriction enzyme sites near the 5′ end of the gene (22). We used a PCR-based assay to examine methylation at a subset of these sites (Fig. 2). Primers AT-2 and 29r amplify DNA from the 5′ flanking region into the first exon of XIST generating a 555 bp product that spans methylation-sensitive restriction enzyme sites for HhaI, SstII and AvaI. To determine the methylation status of these sites, genomic DNA isolated from the clones diagrammed in Figure 1 was digested with each restriction enzyme, and then amplified by PCR. Methylated DNA is resistant to digestion, and therefore the template remains uncut and can be amplified to yield a PCR product. As unmethylated DNA is cut by the enzymes, the template needed for amplification is eliminated and no product is generated. Due to the sensitivity of the PCR, loss of amplification is detected only when the majority of cells in the population are unmethylated at the sites analysed. Amplification was observed with DNA from AHA-11A after digestion with all three enzymes, suggesting that the original clone was methylated at all three restriction sites. This is consistent with previous reports that the silent copy of XIST on the active X chromosome is methylated at these sites (22). AHA-1f DNA did not amplify after digestion with AvaI while it did after digestion with HhaI or SstII, suggesting that the AvaI site had become demethylated. AHA-B2 (XIST+; the ‘early’ clone of Fig. 1B) did not amplify after digestion with AvaI and SstII; however upon silencing of XIST expression (XIST–; the ‘late’ clone of Fig. 1B) amplification was restored after predigestion with SstII, suggesting partial remethylation of this region. AHA-A5 and AHA-A5-2b did not amplify after digestion with all three methylation-sensitive enzymes. The XIST transcript induced by demethylation shows normal expression To determine if the XIST RNA induced by demethylation is processed similarly to that observed in female somatic cells, RT–PCR was performed with primers along the length of the transcript (Fig. 3). Expression patterns for the demethylated cell line AHA-A5-2b were compared to that observed for a female lymphoblast line. Amplification from the upstream primer AT1 was only observed with genomic DNA, not with either cDNA, consistent with the previously located major transcriptional start site (22). Six other primer pairs, which span the length of the gene, were capable of amplifying cDNA from both AHA-A5-2b and the female cell line. Alternative splicing was observed in both cDNAs with primer pair 7 (and faintly with primer pair 5) as has been previously described for both females and inactive X-containing mouse/human somatic cell hybrids (8,11). To examine whether the induced XIST RNA was localized to the nucleus rather than transported into the cytoplasm, RNA was isolated from nuclear and cytoplasmic fractions of the AHA-A5 cell line. Amplification of XIST was detected primarily in the nuclear fraction, while similar analysis of the PGK1 gene showed 2938 Nucleic Acids Research, 1998, Vol. 26, No. 12 Figure 3. Expression of XIST from hybrid AHA-A5-2b compared to expression from female cells. (A) Diagram of the XIST gene showing the location of the primers used. The previously determined splicing pattern is shown, with less frequently observed splicing events indicated by dashed lines. (B) Amplification of cDNA from AHA-A5-2b (A5-2b) and from the human female lymphoblast cell line GM07350 (female) using the seven pairs of primers outlined in (A). (C) cDNA derived from the cytoplasmic and nuclear fraction of AHA-A5 was amplified with primers for the PGK1 gene (PGK) and for the XIST gene (XIST). a significantly different pattern with product observed in both the cytoplasmic and nuclear fractions (Fig. 3), as would be expected of a mRNA which is transported into the cytoplasm for translation. Induced XIST expression does not alter expression of other X-linked genes The induction of XIST expression from the active X chromosome might be anticipated to silence the expression of other genes on the chromosome. To address this, the transcriptional status of seven genes subject to X chromosome inactivation (PGK1, CCG1, TIMP1, T-plastin, AR, IDSX and G6PD) and one gene which escapes X chromosome inactivation (RPS4X) was analysed by RT–PCR in RNA isolated from the various clones (Fig. 4). The initial hybrid (AHA-11a) and derivative XIST-expressing clones from each of the three rounds of demethylation treatments showed expression of all eight genes. This result was confirmed by northern blot analysis for two of the genes, PGK1 (Fig. 4B) and TIMP1 (data not shown). In both cases expression was similar in the active X parental line (AHA-11a) and the XIST-expressing clone AHA-A5-2b, and hybridisation was specific to the human transcript as no signal was detected for RNA from a hybrid cell line containing an inactive X chromosome. If X chromosome inactivation was induced by XIST expression at a relatively low frequency, then the changes in gene expression could well be undetectable by RT–PCR, northern analysis or random sampling of clones. To detect such a low frequency inactivation, the clones were tested for their ability to grow in azaguanine–thioguanine (AG/TG) supplemented media. Only cells which have lost HPRT expression (through transcriptional silencing, null mutation or chromosome loss) can survive to form colonies in this media. Therefore if XIST expression from the active X chromosome can cause a low frequency of X chromosome inactivation (thereby silencing the HPRT locus) then the XIST reactivated clones would be expected to show an increased frequency of AG/TG resistant Figure 4. Expression of X-linked genes from clones before and after induction of XIST expression. (A) Expression of XIST and an additional eight genes as listed across the top was examined by RT–PCR with human-specific primers. As the active X-containing hybrid AHA-11a does not express XIST, a control lane of cDNA from the female cell line GM07350 amplified with the XIST primers was included as a positive control in the first panel. (B) Expression of PGK1 was examined by northern blot analysis in an active X-containing somatic cell hybrid (AHA-11a) and a derivative line which stably expressed XIST (AHA-A5-2b) as well as a human female lymphoblast (Human) and an inactive X-containing mouse/human somatic cell hybrid (Xi Hybrid). The panel on the right shows the ethidium-bromide stained gel and the panel on the left shows an autoradiograph after hybridisation with a PGK1 cDNA probe. colonies. Control cell lines included the parental AHA-11a line as well as two lines which had been subjected to 5azadC treatment but for which XIST expression had either never been detected (AHA-1a) or had been lost (AHA-B2-1b). The results are summarized in Table 1. None of the lines expressing XIST showed a significant increase 2939 Nucleic Acids Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.112 Nucleic in colony formation in AG/TG supplemented media. As the variability in frequency of AG/TG resistant colonies might have obscured a small increase in the frequency, six AG/TG resistant single cell clones were picked from both the AHA-11a parental line and the AHA-A5-2b XIST-expressing cell line. All clones were found to retain both the short arm and proximal long arm of the X chromosome, but none were observed to have inactivated genes (PGK1, TIMP1) in these regions (data not shown). Therefore XIST expression from the active human X chromosome in a somatic cell hybrid does not cause inactivation of other genes on that chromosome. Table 1. Frequency of loss of HPRT activity correlated with XIST expression Cell line XIST status AHA-11a AHA-1a AHA-B2-1b AHA-1b AHA-A5 AHA-A5-2b off off offa on on on No. of 5azadC treatments 0 1 3 1 2 3 No. of colonies (×106) 11.0 ± 1.4 7.1 ± 1.3 11.0 ± 6.5 5.6 ± 2.4 11.7 ± 11.3 5.5 ± 1.1 Frequency of colonies observed after azaguanine/thioguanine culture of derivatives of a somatic cell hybrid which contained an active X chromosome and had been demethylated by treatment with 5azadC. XIST expression was determined by RT–PCR as shown in Figure 1. aAHA-B2-1b is a derivative of AHA-B2 which had initially reactivated XIST, but had lost XIST expression when tested for colony formation in azaguanine/ thioguanine. DISCUSSION Methylation and XIST expression on the active X chromosome We have shown that expression of the normally silent XIST locus can be induced from the human active X chromosome in somatic cell hybrids by treatment with demethylating agents. The expression of XIST is correlated with demethylation of sites at the 5′ end of the gene while subsequent transcriptional silencing is associated with remethylation of at least some of these sites. No single site examined showed complete concordance between methylation and silencing of expression, however, the extent of demethylation at the 5′ end of the gene correlated well with the stability of expression, such that those clones which were the least methylated have also expressed XIST for the longest period of time. These results are consistent with methylation keeping the XIST locus on the active X chromosome transcriptionally silent. Treatment of the inactive X chromosome with demethylating agents has been shown to reactivate many genes; there is, however, substantial variation in the frequencies at which reactivation occurs. It is difficult to compare reactivation frequencies between experiments as the various detection methods used (such as assays of enzyme activity, colony formation in selective media, northern analysis or RT–PCR) have different sensitivities. Additionally, the time at which expression is assayed following treatment also varies significantly, and it has been suggested that the frequency may be specific to the cell line or cell type (37). These variabilities notwithstanding, the observed frequency of XIST reactivation (>10%) suggests that XIST may be relatively sensitive to reactivation. This may be due to the limited number of methylated CpGs associated with the 5′ end of the gene in contrast to other X-linked 2939 genes which have been examined (such as HPRT and PGK1 which have strong CpG dense regions or islands at their 5′ end; reviewed in 2,7), or could also reflect a lack of other factors involved in maintaining the silenced state. Late replication is a common feature of transcriptionally silent genes (38), however the silent Xist/XIST allele on the active X chromosome replicates earlier than the expressed allele on the inactive X chromosome (39,40), perhaps predisposing this gene to loss of silencing. In addition to the activation of silent genes, treatment with 5azadC and 5azaC causes significant cell lethality, decondensation of chromatin (41) and alteration of replication timing (42). The expression of XIST could therefore have arisen as a secondary effect of the demethylating agent. However, as the expression of XIST induced by 5azadC correlated with the loss of methylation at the 5′ end of the gene it seems more likely that the induction of expression was directly due to the loss of methylation. Furthermore, the transcriptional start site, the splicing pattern of the RNA and the nuclear localisation of the RNA was similar to those seen for the inactive X chromosome in both females (Fig. 3) and somatic cell hybrids (11). Since the loss of methylation can induce apparently normal XIST expression, we conclude that methylation is preventing XIST expression in somatic cells. It has been proposed that methylation may serve as the imprint by which paternal and maternal genomes are differentiated. In mice, non-random X chromosome inactivation is observed in the extraembryonic tissues, and methylation of the Xist promoter is implicated in the marking of the maternal X (23–25). Whether humans have a similar nonrandom inactivation in extraembryonic tissues is less clear (reviewed in 43); our results, however, are consistent with methylation being able to inhibit expression and therefore having the capacity to serve in the differential expression of the gene. Lack of inactivation of other X-linked genes after XIST induction There is now extensive evidence implicating XIST in the inactivation process, and recent experiments in mice have shown that transgenic insertions of the Xist gene are capable of inducing many of the features of X chromosome inactivation (reviewed in 44). Thus it was possible that the induction of XIST expression from the active X chromosome might be sufficient to cause inactivation of the chromosome. As the expression of genes from the human X chromosome should not be required for survival of a somatic cell hybrid such inactivation should not be detrimental to the cell. To determine whether XIST expression was inducing silencing the expression of other X-linked genes was examined by several techniques. RT–PCR, which is a very sensitive procedure, was used to examine the expression of eight genes which are located along the length of the human X chromosome, and no loss of expression was observed for these genes in XIST-expressing clones. Northern analysis, which would detect moderate reductions in the level of expression, was used to analyse two genes. Again no reduction in expression was observed with expression of XIST. Finally, growth in selective media was examined to detect very infrequent loss of HPRT gene expression. As the untreated hybrid forms colonies at a frequency of ∼10–5, any loss of expression at this frequency or higher should have been detected by this assay; however, no such silencing was detected. Therefore, we conclude that expression of XIST from the human active X chromosome in mouse/human somatic cell hybrids is insufficient to cause a detectable reduction in expression of other X-linked genes. 2940 Nucleic Acids Research, 1998, Vol. 26, No. 12 The lack of inactivation of X-linked genes in the presence of XIST expression may be due to a variety of causes, and we consider four possibilities. (i) XIST expression may not induce X chromosome inactivation. This seems unlikely as it has recently been demonstrated that Xist with only ∼15 kb of flanking sequences is able to induce inactivation in murine ES cells (19). (ii) The active X chromosome may have become resistant to inactivation. Inactivation results in the inactive X chromosome becoming methylated, late-replicating and reduced in acetylated histones (reviewed in 2), and while it is generally believed that the active X chromosome is unaffected by the inactivation process, it is possible that the inactivation process also alters the active X chromosome, thereby conferring resistance to inactivation. (iii) XIST expression may only induce inactivation in conjunction with factors expressed exclusively during early development. The lack of X chromosome inactivation in our mouse–human hybrid system may therefore be due to the absence of such factors in somatic cells. A difference in the role of XIST between embryonic and somatic cells was demonstrated by the lack of reactivation when the XIST locus is deleted from the inactive X chromosome in either mouse/human somatic cell hybrids or human somatic cells (45,46) despite the demonstrated requirement for Xist in X chromosome inactivation during development (20,21). (iv) The factors necessary for inactivation may be present, but the murine homologues may be sufficiently different from the human such that they are unable to interact with the human XIST RNA [which shows ∼30% sequence divergence from the murine Xist RNA (11,47)]. Such an incompatibility is supported by the observation that reversal of inactivation of the human X chromosome upon microcell fusion with murine embryonal carcinoma cells occurs in the presence of XIST expression (48). In summary, we conclude that methylation is sufficient to maintain transcriptional silencing of the XIST gene in mouse/ human somatic cell hybrids containing an active human X chromosome. Disruption of methylation by treatment with 5azadC induced expression of XIST, but did not result in silencing of other X-linked genes. 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