Download Induction of XIST expression from the human active

 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
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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
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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. Therefore, while there is now substantial
evidence implicating XIST as a critical component of the
inactivation process, our results demonstrate that XIST is not
sufficient to cause inactivation of the human X chromosome in a
murine somatic cell, suggesting that additional factors are
required for the inactivation process.
ACKNOWLEDGEMENTS
We would like to thank Drs H. F. Willard, J. Lawrence, S. Gartler
and S. Hansen for informative discussions, and Sarah Baldry for
technical assistance. C.J.B. is the recipient of an MRC scholarship,
and this work was supported by Medical Research Council grant
MT-13690.
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