Gene Map Locus: Xq13.2
Note: pressing the 
symbol will find the citations in MEDLINE whose text most closely
matches the text of the preceding OMIM paragraph, using the Entrez
MEDLINE neighboring function.
Therman et al. (1974) suggested that
condensation of the inactive X chromosome occurs around a center
(locus) on the long arm of the X chromosome near the centromere. They
based this on the observations that (1) the abnormal X chromosomes
with the assumed center in duplicate form have bipartite Barr bodies,
and (2) no X short arm isochromosomes (Xpi) had been confidently
identified (see later for an exception that proves the rule). They
suggested that Xpi is lethal because the cell has no method of dosage
compensation. The existence of such a locus in man is rendered
plausible by the demonstration in the mouse of a locus called Xce
(X-chromosome controlling element). Grahn
(1973) studied the position of the Xce locus on the mouse map.
Ohno et al. (1973) and Drews
et al. (1974) described an allele at the Xce locus. From studies
of 5 cases of structural anomalies involving the X chromosomes,
Mattei et al. (1981) concluded that the X
chromosome possesses only one inactivation center, which is probably
situated between Xq11.2 and Xq21.1.
Flejter et al. (1984) found that the
most frequent site of a bend in mitotic metaphase chromosomes is
Xq13.3-q21.1. It was observed in 1 member of the X-chromosome pair in
63% of 46,XX cells, and in only 2% of 46,XY cells. RBG-staining
showed that this specific bend is confined to the lyonized X
chromosome. The observations on cells from normal persons were
confirmed by studies of cells from 9 subjects with different
X-chromosome abnormalities. Noting that the 'center for Barr body
condensation' has been localized to the segment Xq11.2-q21.1 (Therman
et al., 1974, 1979;
Mattei et al., 1981), Flejter
et al. (1984) suggested that the highly specific bend is a
visible manifestation of the condensation process. It may represent
the first to be folded and the last to be unfolded portion of the
inactive X. Continuing this work, Flejter et
al. (1986) reasoned that the inactivation-associated fold might
be useful for identifying the inactive X and locating the
inactivation center in other mammalian species. They found that all 9
primate species examined expressed the fold. In most, the fold was at
the band homologous to human Xq13-q21.
By study of inactivation of the X chromosome in somatic cell
hybrids containing rearranged chromosomes, Brown
and Willard (1989) regionalized the human X-inactivation center,
symbolized XIC by them, to Xq13. Brown et al.
(1991) defined a minimal region of overlap of structurally
abnormal X chromosomes capable of being inactivated. The results were
consistent with models invoking a single XIC. One of the markers
localized to this region was the XIST gene (Brown
et al., 1991), which is expressed specifically from inactive, but
not active, X chromosomes (XIST = X-inactivation specific
transcript). By in situ hybridization, the XIST gene was located in
band Xq13, at the interface of bands Xq13 and q21.1. The order of
loci around XIST appeared to be:
cen--AR--CCG1--PHKA--XIST--PGK1--tel. Brown et
al. (1991) considered that the XIST gene is either involved in or
uniquely influenced by the process of X inactivation. The gene was
identified through a 750-bp cDNA clone prepared from a female
placental cDNA library. XIST cDNA probes hybridized to RNA prepared
from female samples or from somatic cell hybrids containing an
inactive human X chromosome, but not to RNA from males or from
hybrids containing only an active human X chromosome. The Xce locus
in the mouse is also closely situated to the Pgk-1 locus. The
expression of XIST in early development when X inactivation occurs
will be of interest.
Nomenclature: XIC (for X-inactivation center) in the human refers
to a region on the X chromosome; XIST refers to a specific gene in
that region which is necessary for X inactivation but alone is not
sufficient. The precise nature of Xce (for X-chromosome controlling
element) in the mouse and of its presumed homolog in human (see
300074) is unknown. By
linkage analysis, Simmler et al. (1993)
could show that the mouse Xce localizes outside the 170-kb region
immediately surrounding Xist and is, therefore, an entity distinct
from Xist.
Human XIST cDNAs containing at least 8 exons and totaling 17 kb
were isolated and sequenced within the region on the X chromosome
known to contain the X-inactivation center (Brown
et al., 1992). The XIST gene includes several tandem repeats, the
most 5-prime of which are evolutionarily conserved. The gene does not
contain any significant conserved open reading frames (ORFs) and thus
does not appear to encode a protein, suggesting that XIST may
function as a structural RNA within the nucleus. In support of this,
Brown et al. (1992) showed by fluorescence
in situ hydridization (FISH) experiments that XIST RNA is localized
within the nucleus in a position indistinguishable from the
X-inactivation-associated Barr body. Lafreniere
et al. (1993) demonstrated that the transcriptional orientation
of XIST is cen--3-prime--XIST--5-prime--qter.
Cytogenetic analyses show that the region Xq11.2-q21 is retained
in all structurally abnormal X chromosomes. From such observations
the conclusion is drawn that this 'critical region' contains the
locus controlling X inactivation. Structurally abnormal X chromosomes
without the X-inactivation center would allow nullisomy, disomy, or
trisomy for genes on the X chromosome--presumably nonviable states.
Pettigrew et al. (1991) studied a
28-year-old woman with primary amenorrhea and features of Turner
syndrome who had an isodicentric chromosome involving Xp. High
resolution chromosome analysis showed that the break in the long arm
was at Xq13.2. DNA analysis confirmed the breakpoint of the
isodicentric chromosome to be proximal to PGK1 (311800),
which is located at Xq13.
Borsani et al. (1991) isolated and
characterized the murine homolog, Xist, which is located in the mouse
X-inactivation center region and is expressed from the inactive X
chromosome. Brockdorff et al. (1991)
likewise found that the mouse counterpart of human XIST maps to the
XIC region. Using an interspecific Mus spretus/Mus musculus
domesticus F-1 hybrid mouse carrying an X;16 translocation, Brockdorff
et al. (1991) showed that Xist is exclusively expressed from the
inactive X chromosome. They suggested that XIST and its mouse homolog
are involved in X-chromosome inactivation.
Brockdorff et al. (1992) analyzed the
entire mouse Xist gene. The mature inactive X-specific transcript is
15 kb long and contains no conserved ORFs. A number of regions of the
Xist sequence comprised tandem repeats. Comparison with the human
XIST gene showed significant conservation of sequence and gene
structure. Brockdorff et al. (1992) found
that the Xist RNA in the mouse is not associated with the
translational machinery of the cell and is located almost exclusively
in the nucleus. Kay et al. (1993) showed
that the onset of Xist expression in development precedes X
chromosome inactivation and may therefore be a cause rather than
merely a consequence of X inactivation. The earliest Xist expression
in morulae and blastocysts is imprinted, resulting in specific
expression of the paternal Xist allele. Thus, imprinting may be the
cause of nonrandom inactivation of the paternal X in trophectoderm.
The imprint on Xist expression is lost shortly before gastrulation,
when random X inactivation occurs.
The severe phenotype of human females whose karyotype includes
tiny ring X chromosomes has been attributed to the inability of the
small ring X chromosome to inactivate. Migeon
et al. (1993) provided compelling evidence in support of this
suggestion. Using PCR, Southern blot analysis, and in situ
hybridization, they looked for the presence of the XIST locus in tiny
ring X chromosomes from 8 females who had multiple congenital
malformations and severe mental retardation. They found that some
rings lacked the XIST locus, while others had sequences homologous to
probes for XIST; however, in the latter group, the locus was either
not expressed or negligibly expressed, based on reverse
transcription-PCR analysis. As XIST transcription is an indicator of
X chromosome inactivity, the absence of XIST transcription strongly
suggested that tiny ring X chromosomes in females with severe
phenotypes are mutants in the X chromosome inactivation pathway and
that the inability of these rings to inactivate is responsible for
the severe phenotypes (Migeon et al.,
1994). They recorded that they found that 3 X-linked loci (AR,
313700; TIMP, 305370;
and PHKA1, 311870) were
expressed in the ring chromosomes from 2 subjects, indicating that
these chromosomes were indeed active. In addition, rings had active
chromatin (as indicated by labeling with acetylated histone H4). This
was taken as clear evidence that these rings represent chromosomal
mutations affecting cis inactivation and that the severe phenotype is
due to functional disomy resulting from lack of dosage compensation
for genes present within the ring chromosome. Two other subjects had
the XIST coding sequences present in the ring chromosome, suggesting
that XIST coding sequences alone may not be sufficient for X
inactivation. Breakpoints in formation of these ring chromosomes may
have disrupted neighboring regulatory or enhancer sequences, or there
may be a second gene in the XIC region essential for XIST
transcription.
Consistent with the fact that the mouse Xist gene is expressed
exclusively from the inactive X chromosome, Norris
et al. (1994) showed that in somatic tissues the 5-prime end of
the silent Xist allele on the active X chromosome is fully
methylated, while the expressed allele on the inactive X is
completely unmethylated. In tissues that undergo imprinted paternal
Xist expression and imprinted X inactivation, the paternal Xist
allele is unmethylated, and the silent maternal allele is fully
methylated. In the male germline, a developmentally regulated
demethylation of Xist occurs at the onset of meiosis and is retained
in mature spermatozoa. This may be the cause of imprinted expression
of the paternal Xist allele. A role for methylation in the control of
Xist expression is further supported by the finding that in
differentiating embryonic stem cells during the initiation of X
inactivation, differential methylation of Xist alleles precedes the
onset of Xist expression.
Torchia et al. (1994) used FISH to
examine the early versus late replication of loci on the X chromosome
and the relationship between activity of the gene and late
replication. Active autosomal genes tend to replicate early, whereas
inactive ones are more permissive and frequently replicate later. In
the assay used, an unreplicated locus was characterized by a single
hybridization signal, and a replicated locus by a doublet
hybridization signal. The percentage of doublets was used as a
measure of relative time of replication in S phase. Torchia
et al. (1994) concluded that silence of the XIST gene in males is
associated with late replication of the locus, whereas the locus
replicates asynchronously in the 2 X chromosomes in female cells. The
expansion of the FMR1 locus (309550)
in fragile X males led to late replication. The gene for factor VIII
(306700) was late
replicating in both normal and fragile X males and replicated at
nearly the same time on active and inactive X chromosomes in females,
consistent with inactivity of this gene in the tissue analyzed.
Hansen et al. (1995) presented data in
direct opposition to the conclusion of Torchia
et al. (1994). They demonstrated early replication of XIST on the
active X and late replication on the inactive X in the same cell
type, namely human fibroblasts. They believed that the discrepancy
could be explained by the indirect nature of the FISH-based method,
which is susceptible to errors because of the tendency of some loci
not to separate after replication, thus appearing to be unduplicated,
and for transcribed loci to yield false doublet (replicated) signals
when genomic probes are used that hybridized to nascent transcripts
(Hansen and Gartler, 1997).
The role of the Xist gene in X chromosome inactivation as the
master regulatory switch locus was supported solely by indirect
evidence until the experiments of Penny et al.
(1996), who provided direct evidence by gene targeting of Xist in
mouse embryonic stem (ES) cells. Their results provided evidence for
the absolute requirement of Xist in the process of X chromosome
inactivation. When ES cells that are chromosomally XX are maintained
in the undifferentiated state, both X chromosomes remain active and
Xist is expressed at very low levels; however, when they are allowed
to differentiate, X inactivation occurred and Xist expression
increased markedly. Penny et al. (1996)
knocked out 7 kb of DNA, including the first Xist exon, and showed
that this destroyed the activity of the gene. When ES cells
heterozygous for distinguishable alleles of Xist and other X-linked
genes were allowed to differentiate, X inactivation occurred, as
manifested by an asynchronously replicating chromosome, but only the
X not bearing the knockout underwent inactivation. Penny
et al. (1996) concluded that the counting mechanism still
recognized the XIC with the null Xist allele; that the normal or the
knockout X could be selected to remain active with a probability,
depending on the different Xce alleles they carried; that if the
knockout X was selected to remain active, the other X underwent
inactivation normally; but that if the normal X was selected, then
the knockout X failed to become inactive and the cell then had 2
active X chromosomes. The authors also studied the effect of the Xist
knockout in vivo in chimeric embryos made by aggregating ES cells
carrying the knockout with normal 8-cell embryos. Again, the counting
mechanism operated and X inactivation occurred, and once more only
the X with the normal Xist allele underwent inactivation. In contrast
to the ES cells, there was no evidence of cells with both X
chromosomes active, an observation consistent with earlier work
showing that cells with excess X-chromosome activity are rapidly
eliminated by cell selection. Thus, the work of Penny
et al. (1996) provided clear evidence that transcription of Xist
is required for the spreading of inactivation along the X chromosome
carrying it.
Lyon (1996) commented that the
experiments of Penny et al. (1996)
indicated that the counting and spreading functions of the XIC have
to some extent been separated. Migeon et al.
(1996) provided evidence of the separate counting mechanism in
humans. Counting may be a later evolutionary development found only
in eutherians. What is the significance of the expression of XIST in
the adult? In cells that have already undergone X inactivation, loss
of the XIST gene does not cause reactivation. Spreading is a long
range process apparently operative over megabases, but with some form
of local response in that some genes escape inactivation. The
XIST/Xist gene does not code for a protein, and its 15- to 17-kb RNA
transcript is retained in the nucleus and is associated with the
inactive X chromosome itself.
Willard (1996) gave a review of the X-inactivation center and the role of XIST in X chromosome inactivation.
To investigate the function of the Xist gene product, Marahrens
et al. (1997) generated male and female mice that carried a
deletion in the structural gene but maintained a functional Xist
promoter. They found that males with the mutated allele developed
normally and were fertile. Females who inherited the mutant gene from
their mothers also developed normally, with the wildtype paternal X
being exclusively inactivated in every cell. However, female mice
inheriting the mutant Xist allele on the paternal X chromosome were
severely growth-retarded and died early in embryogenesis. The
wildtype maternal X chromosome was inactive in every cell of the
growth-retarded embryo proper, whereas both chromosomes were
expressed in the mutant female trophoblast where X inactivation is
imprinted. However, an XO mouse with a paternally inherited Xist
mutation was healthy and appeared normal. Marahrens
et al. (1997) concluded that the imprinted lethal phenotype of
the mutant females was due to the inability of extraembryonic tissue
with 2 active X chromosomes to sustain the embryo. The results
indicated that Xist RNA is required for female dosage compensation
but plays no role in spermatogenesis. In a review of Xist, Solter
and Wei (1997) commented: 'After reading the paper by Maharens
and colleagues, it becomes even more obvious that despite the
tremendous progress made in the field over the last few years, the
problem of X inactivation in mammals remains as complex and
tantalizing as ever.'
During mouse preimplantation development, the exclusive expression
of the Xist gene from the paternal inherited allele is thought to
play a role in the inactivation of the paternally inherited X
chromosome in the extraembryonic cell lineages of the developing
female embryo. Preferential paternal X inactivation occurs in
first-trimester human trophoblastic cells also (Goto
et al., 1997), a situation that persists until birth when
preferential paternal X inactivation is demonstrable in full-term
placentas (Harrison, 1989). Daniels
et al. (1997) determined whether the pattern of XIST expression
in human preimplantation embryos is similarly correlated with
paternal X inactivation. They developed procedures sensitive to a
single cell, for the simultaneous analysis of XIST and HPRT
expression and of sexing, initially using human fibroblast cells.
Application of these procedures to human cleavage-stage embryos
derived by in vitro fertilization revealed a pattern of XIST
expression different from that in the mouse. Transcripts of the XIST
gene were detected as early as the 1-cell zygote and, with increasing
efficiency, through to the 8-cell stage of preimplantation
development. In addition, transcripts of XIST were detected in both
male (hence from the maternally inherited allele) and female
preimplantation embryos. This pattern of expression is not consistent
with a role for the early expression of XIST in the choice of
paternal X inactivation in the extraembryonic cell lineages of the
developing human embryo. Ray et al. (1997)
likewise found expression of the XIST gene in human preimplantation
embryos from the 5- to 10-cell stage onwards consistent with its role
in the initiation of inactivation. They found also that, in contrast
to the mouse, transcripts were detected in both male and female
embryos demonstrating XIST expression from the maternally derived X
chromosome in male embryos, X(M)Y. Brown and
Robinson (1997) discussed this mouse/human paradox.
To understand transcriptional regulation of the XIST gene,
Hendrich et al. (1997) identified and
characterized the human XIST promoter and 2 repeated DNA elements
that modulate promoter activity. As determined by reporter gene
constructs, the XIST minimal promoter is constitutively active at
high levels in human male and female cell lines and in transgenic
mice. Promoter activity is dependent in vitro on the binding of the
common transcription factors SP1 (189906),
YY1 (600013), and TBP
(600075). The authors
further identified 2 cis-acting repeated DNA sequences that influence
reporter gene activity. DNA fragments containing a set of highly
conserved repeats located within the 5-prime end of XIST stimulated
reporter activity 3-fold in transiently transfected cell lines.
Additionally, a 450-bp alternating purine-pyrimidine repeat located
25 kb upstream of the XIST promoter partially suppressed promoter
activity by approximately 70% in transient transfection assays.
Hendrich et al. (1997) concluded that the
XIST promoter is constitutively active and that critical steps in the
X-inactivation process must involve silencing of XIST on the active X
chromosome by factors that interact with and/or recognize sequences
located outside the minimal promoter.
Panning et al. (1997) demonstrated that
low-level XIST expression is detected from both active X chromosomes
prior to X inactivation in female embryonic stem (ES) cells. A
similar low-level expression is detected from the single active X
chromosome in male ES cells. After differentiation, high-level XIST
expression occurs only in the inactive X chromosome. Differentiating
female cells increase XIST expression from the inactive X chromosome,
prior to silencing low-level XIST expression on the active X. The
transition from low-level to high-level XIST expression is achieved
by stabilization of XIST transcripts at the inactive X. Panning
et al. (1997) suggested that these developmentally modulated
changes in XIST expression are regulated by several different
mechanisms: factors that stabilize XIST transcripts at the inactive
X, an activity that blocks the stabilization at the active X, and a
mechanism that silences low-level XIST expression from the active X.
Although it is commonly believed that the initiation of X
inactivation is random, with an equal probability (50:50) that either
X chromosome will be the inactive X in a given cell, significant
variation in the proportion of cells with either X inactive is
observed both in mice heterozygous for alleles at the Xce locus and
among normal human females in the population; see familial skewed X
inactivation (300087).
Families in which multiple females demonstrate extremely skewed
inactivation patterns that are otherwise quite rare in the general
population are thought to reflect possible genetic influences on the
X-inactivation process. Plenge et al.
(1997) reported a rare C-to-G transversion mutation in the XIST
minimal promoter that underlay both epigenetic and functional
differences between the two X chromosomes in 9 females from 2
unrelated families. All females demonstrated preferential
inactivation of the X chromosome carrying the mutation, suggesting
that there is an association between alterations in the regulation of
XIST expression and X-chromosome inactivation.
Marahrens et al. (1998) created
laboratory mice by targeted disruption of the mouse Xist gene.
Females heterozygous for an internal deletion in the Xist gene, which
included part of exon 1 and extended to exon 5, underwent primary
nonrandom inactivation of the wildtype X chromosome. The authors
concluded that the Xist gene not only has a role in chromatin
remodeling, but also includes an element required for the choice of
which X chromosome to inactivate. In conflict with the prevailing
view of how choosing occurs, the element identified by the deletion
plays a positive role in the choice mechanism.
In order to characterize functional elements in the Xist gene
important to X-chromosome inactivation, Clerc
and Avner (1998) created a deletion extending 3-prime to the Xist
exon 6. In undifferentiated ES cells, Xist expression from the
deleted X chromosome was markedly reduced. In differentiated XX ES
cells containing 1 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 X inactivation center (Xic) was capable of initiating X
inactivation, even in the absence of another Xic. These results
demonstrated a role for the region 3-prime to the Xist exon 6 in the
counting process and suggested that counting is mediated by a
repressive mechanism that prevents inactivation of a single X
chromosome in diploid cells. Carrel and
Willard (1998) discussed the implications of the experiments of
Clerc and Avner (1998) and presented with
diagrams the 3 general models proposed for the initiation of X
inactivation and the establishment of active and inactive X
chromosomes.
Johnston et al. (1998) showed that
alternate promoter usage of the murine Xist gene resulted in distinct
stable and unstable RNA isoforms. Unstable Xist transcripts initiated
at a novel upstream promoter (P0), whereas stable Xist RNA was
transcribed from the previously identified promoter (P1) and from a
novel downstream promoter (P2). Analysis of cells undergoing X
inactivation indicated that a developmentally regulated promoter
switch mediated stabilization and accumulation of Xist RNA on the
inactive X chromosome.
Plenge et al. (1997) identified a
mutation in the XIST minimal promoter in multiple females in a
family reported by Rupert et al. (1995)
as showing nonrandom X-chromosome inactivation. The mutation was a
C-to-G transversion at position -43 in the minimal promoter on the
preferentially inactive X chromosome. The -43 mutation created a
novel HhaI restriction site that was used to test for the presence
of the mutation in a large series of unrelated females,
representing 1,166 independent X chromosomes. The mutation was
found on only 1 additional chromosome in this data set, ruling out
that the mutation represented a common polymorphism. The
heterozygous female identified by this screen was from a large
family with Snyder-Robinson mental retardation syndrome (309583),
which maps to Xp22.12-p21.3. Haplotype analysis indicated that the
2 families were not related. Subsequent analysis in the second
family revealed 6 additional females and 4 males who had inherited
the XIST mutation.