FEBS Letters 588 (2014) 25142522

journal homepage: www.FEBSLetters.org

Review

X-chromosome inactivation in development and cancer

Ronan Chalign, Edith Heard
Mammalian Developmental Epigenetics Group, Genetics and Developmental Biology Unit, Institut Curie, CNRS UMR3215, INSERM U934, 75248 Paris, France

a r t i c l e i n f o a b s t r a c t

Article history: X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of
Received 5 June 2014 facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development.
Accepted 6 June 2014 Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combina-
Available online 14 June 2014
tion of chromatin associated proteins, DNA methylation and nuclear organization. However,
sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells
Edited by Wilhelm Just
and disappearance of the Barr body is frequently observed in cancer cells. In this review we summa-
rise current knowledge on the epigenetic changes that accompany X inactivation and discuss the
Keywords:
X-chromosome inactivation
extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast
Epigenetics cancer.
Cancer 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Development
Chromatin

1. Introduction some cases in cancer cells resulting in a double dose of X-linked

In mammals, dosage compensation for X-linked gene products
between the sexes is achieved by X-chromosome inactivation
(XCI) in females [1]. This process leads to the highly regulated tran-
scriptional silencing of one of the two X-chromosome during early
development, leading to the formation of the heterochromatic Barr
body. X inactivation is an outstanding example of chromosome-
wide epigenetic regulation involving the developmental silencing
of approximately one thousand genes. XCI shares many of the fea-
tures of others epigenetic mechanisms such as a mosaic cellular
phenotype; mitotic heritability but developmental reversibility of
X-chromosome inactivity; asynchronous DNA replication timing
compared to the rest of the genome; and nally a combination of
several epigenetic mechanisms including DNA promoter methyl-
ation, histones post-translational modications, and an unusual
nuclear organization. These various features are believed to act
synergistically to maintain the X inactive state. Thus, the inactive
X chromosome represents a remarkable illustration of the numer-
ous epigenetic mechanisms that can underlie the formation and
maintenance of facultative heterochromatin throughout the
lifetime of mammals. Pathologists have long noted that the hetero-
chromatic structure of the Barr body was frequently absent in
breast cancer cells, particularly in the most aggressive tumors
([2,3]). This observation was later found to be due to the frequent
genetic loss of the Xi, with reduplication of the Xa also occurring in
Corresponding author.
E-mail address: Edith.Heard@curie.fr (E. Heard).
genes. However, another mechanism for Barr body loss in cancer
that has been proposed involves the decompaction of its hetero-
chromatic structure, which may be accompanied by X-linked gene
reactivation.
Here we provide a brief overview of the current knowledge
pertaining to the establishment and maintenance of X inactivation,
as well as highlights of some of the known perturbations of the
inactive X chromosomes state in a cancer context. This review
does not provide an in depth analysis, for this, the reader is
referred to other recent reviews [46].
2. Setting up inactivation of the X chromosome in mammals
The X-inactivation center (Xic) is a key locus that is required for
the initiation of X inactivation. Studies involving X-chromosome
deletions and X-autosome translocations in mice and humans
mapped the precise region which contains the essential gene
for triggering the silencing process, the non-coding RNA Xist
(X-inactive-specic-transcript) (for review [7]). Xist is a 17 000
nucleotide (19 kb in Human), spliced, untranslated regulatory tran-
script that coats the X chromosome from which it is expressed in
cis [8,9]. Perhaps surprisingly, Xist is present only in eutherian
mammals, with no orthologous described in marsupials or mono-
tremes to date. However in marsupials, another non-coding RNA
Rsx (RNA on the silent X) was recently discovered, that appears
to have similar properties to Xist even though its sequence is unre-
lated to Xist [10]. In the mouse, deletions and transgenes have
demonstrated that Xist is required for both the imprinted and

http://dx.doi.org/10.1016/j.febslet.2014.06.023
0014-5793/ 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522
random forms of X inactivation [1113]. Nevertheless, Xist alone
cannot recapitulate all the roles of the Xic which include sens-
ing/competence, whereby a cell initiates XCI only when more
than one Xic is present; counting, where only one X chromosome
stays active per diploid autosome set; and choice, whereby one
of the two X chromosomes is chosen for inactivation while the
other remains active. Some of these functions seem to be ensured
by other elements of the Xic, in the neighborhood of Xist. For exam-
ple, in mice the cis-acting Xist antisense transcription unit, Tsix
[14], plays a key role in the choice of which chromosome will be
inactivated. However, several additional cis-acting elements and
trans-acting factors are involved in the dosage-sensitive, monoall-
elic regulation of Xist expression during early differentiation. For
example, the Rnf12 gene lies upstream of Xist and its protein prod-
uct, an ubiquitin ligase that leads to the degradation of the Xist
repressor protein Rex1, has been shown to have a key role in the
XX-dosage dependent activation of Xist [15,16]. A recent study also
shed light on the precise chromosomal folding of the Xic region in
ESCs, revealing that the Xist and Tsix promoters lie in separate
Topologically Associated Domains (TADs) [17]. The Xist promoter
and all of Xists known positive regulators, including Rnf12, lie in
a single 500 kb TAD, while the Xist repressor Tsix and its regulators
lie in the neighboring 220 kb TAD, with the Xist/Tsix overlapping
gene bodies across the boundary between the two TADs. The spa-
tial separation between the Xist and Tsix promoters is likely to be
essential for the nely tuned reciprocal regulation and expression
of these two keys players in the XCI process [18,19].
So far, most studies of XCI during early development have been
carried out in mice, which are more amenable to a genetic
approach and where access to embryonic material is easier. In this
species, XCI occurs in two phases during embryogenesis, an initial
imprinted form and a later random form. In the imprinted form,
Xist itself is imprinted such that only the paternal allele is
expressed from two cell stage, whereas the maternal allele is
repressed by an as yet undened maternal imprint that is estab-
lished during oogenesis. The paternal X chromosome (Xp) becomes
silenced during pre-implantation development and remains inac-
tive in extra-embryonic tissues. However the Xp is reactivated in
the inner cell mass (ICM) of the mid-stage blastocyst, that will give
rise to the embryo proper [20]. XCI is then reinitiated and occurs
randomly by up-regulation of Xist from either the paternal or the
maternal X chromosome. This second wave of XCI, can be recapit-
ulated in vitro in differentiating mouse ESCs which provide a pow-
erful model system for the study of the molecular mechanisms
underlying random XCI. Remarkably, the differentiation process
is delayed or paused in female ES cells until one of the two
X chromosomes has become silenced, suggesting that a double
dose of X-linked genes interferes directly with differentiation, thus
creating a coupling between XCI and development [21]. Unexpect-
edly, in other mammals recent studies have revealed that the tim-
ing and pattern of XCI initiation observed in rodents is rather
different to that seen in other eutherian mammals such as humans
and rabbits [22,23], where XIST is not imprinted but is expressed
from all X chromosomes in males and females from the 4 or 8 cells
stages [24]. In rabbits, a signicant proportion of blastomeres dis-
play XIST RNA coating of both X chromosomes in early blastocysts.
This situation is rapidly resolved at slightly later stages, where
most cells display only one XIST RNA-coated chromosome. In
humans, the onset of XCI appears to be much slower than in mice
and rabbits, as the two X chromosome remain active both in the
ICM and the trophectoderm even though XIST coating is present.
By contrast, early XCI appears to be absolutely essential in mice,
in order to ensure proper dosage compensation given their fast
embryonic development [25]. The exact reasons for this evolution-
ary diversity are still not clear but the rapid evolution of rodent
genomes may mean that they have acquired mechanisms for nely
2515
regulating Xist, such as an early maternal imprint to prevent pre-
mature XCI, as well as reactivation of the Xp to allow for random
XCI (see [21,24] for discussion).
The mechanisms underlying the early events during XCI are still
not fully understood. Nevertheless, gene silencing is clearly trig-
gered by Xist RNA coating of the future inactive X chromosome.
It has been proposed that the Xist transcript might bind to high
afnity sites on the chromosome, thereby inducing local hetero-
chromatinisation, which can then facilitate the spread of XCI.
LINE1 (Long Interpersed Nuclear Elements 1) repeat elements,
which are highly enriched on the X chromosome compared to
autosomes, have been proposed to facilitate the spread of XCI, by
participating in the local propagation of inactivation and facilitat-
ing the recruitment of genes into the silent nuclear compartment
formed by Xist RNA [26]. However, direct genetic evidence proving
that L1s are required for efcient spreading or gene silencing is still
lacking. More recent ndings, based on mapping of Xist RNA along
the X chromosome and of the nuclear organization of the Xist RNA
coated inactive X, suggest that Xist propagation and coating are
highly related to the three-dimensional conformation of the inac-
tive X chromosome, and possibly also to the binding of Polycomb
Repressive Complex 2 (PRC2) protein complex [2729]. However,
although studies comparing PRC2/H3K27me3 density and Xist
RNA mapping suggest co-localisation [27,28], this is not in agree-
ment with super-resolution Xist RNA FISH/immunouorescence
studies, where PRC2 and Xist RNA are found to be spatially segre-
gated [30]. Furthermore, these studies do not demonstrate that
the regions of the X associated with Xist RNA are directly involved
in its spreading or silencing functions. Indeed, the mechanisms
underlying Xists cis-limited chromosome coating capacity, as well
as its ability to inactivate genes, remain mysterious.
Several studies have focused on the early changes in chromatin
structure that occur during the initiation of XCI with the hope of
providing insight into the role of Xist and the establishment of
the silent state of the inactive X. Loss of euchromatin-associated
histone modications (such as H3K9Ac, H3K4me2 and H3K4me3)
are amongst the earliest chromatin changes that occur following
Xist RNA coating [20,31,32] (Fig. 1). Global H4 hypoacetylation
occurs shortly afterwards [33]. Passive histone-loss during replica-
tion or else active removal (either by enzymatic activity, proteo-
lytic activity or histone exchange) may underlie the early
disappearance of those histones modications. In addition to these
early chromatin changes, the disappearance of factors associated
with transcription, such as RNA polymerase II and loss of nascent
transcripts are observed on the Xi immediately after Xist RNA coat-
ing. One or two cell cycles later, several new histone modications
appear on the Xist-coated chromosome. These include H3K27me3,
H3K9me2, H4k20me1 and H2Ak119ub1 (for review [22]) which all
become enriched with rather similar kinetics of Xi enrichment dur-
ing random XCI in differentiating ESCs. In pre-implantation
embryos, H3K27 tri-methylation precedes H3K9 di-methylation
on the X chromosome undergoing inactivation [20]. Intriguingly,
H3K27me3 and H3K9me3 seem to be enriched in different regions
of the human inactive X chromosome in somatic cells, suggesting
the existence of two different types of heterochromatin [34]. In
murine differentiating female ESCs and somatic cells, H3K27me3
and H3K9me2 are enriched rather uniformly along the Xi, though
slightly more in gene-rich regions [35]. The Xi prole of H2Aub1
has not yet been described.
The factors that lay down or bind to the different chromatin
marks present on the Xi are being unraveled in mice. Polycomb
repressive complex 1 (PRC1) mediates mono-ubiquitination of his-
tone H2A lysine 119 (H2Aub1) [36,37] and PRC2 catalyzes the tri-
methylation of H3K27 [38,39]. Xist RNA is believed to have a role in
the targeting of both PRC1 and PRC2 to the Xi, though whether this
is direct or indirect is still unclear. In the case of PRC2, the cofactor
2516 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522

A
Initiation Propagation Maintenance
Transcriptionally active chromatin state Reversibly silenced chromatin state Stable silenced chromatin state

PRC2 CpG islands methylation

SUZ12
XIST RNA ?
PCL2
XIST RNA JARID2 Dnmt
EED HMTase ?
EZH
G9a Smchd1
?
CH3 +
? ?

H3K4me3 H3K9ac H3K27me3 CDYL H3K9me2 H3K27me3 CDYL H3K9me2

CH3
CH3
CH3

CH3
CH3
CH3 CH3
CH3
CH3
CBX7
BMI1
DNA RNAP II PHC
H4Ac H3K4me3 RING1 H2AUb H3K20me1 H2AUb H3K20me1
PRC1
RNA
canonical
RYBP RING1 PR Set7 macroH2A
BMI1
?

PRC1-like

B Formation of a Xi silent compartment

Xist RNA coats the Xi and Genes escaping XCI remain outside the Xi domain and
forms a nuclear silent compartment are engaged in long-range contacts with each other

Xa Xi Xi

Xa Xa Xa
Nucleus

Two active X-chromosomes

Active X-linked gene Inactive X-linked gene Xist RNA

Fig. 1. The kinetics of X-chromosome inactivation. Schematic view of the kinetics of events on the X chromosome based on ndings of many laboratories, performed mainly
in differentiating female embryonic stem cells. (A) During the initiation of XCI, Xist is expressed in cis from the future inactive X chromosome. Then Xist RNA exerts its
repressive effect through unknown factors. One of the earliest events following Xist RNA coating is loss of euchromatic histone marks, H3K4me2/3, H3K9Ac and H4Ac. During
this same time window, X-linked gene-silencing initiates. Several histone modications also become enriched on the Xist RNA-coated chromosome at this time. These include
H3K27me3, H3K9me2, H2Aub1 and H4K20me1. The PRC2 complex proteins Eed, Pcl2, Suz12, Ezh2 and Jarid2 are also detectable on the X at this stage. Ezh2 is responsible for
the appearance of H3K27me3 on the X. Jarid2 plays a role in the targeting of PRC2 to the chromatin. The histone methyl-transferases responsible for H4K20 mono-
methylation is PRSet7 but that for H3K9 di-methylation is not yet clear. By binding H3K27me3/H3K9me2 enriched Xi, Cdyl may also participate in propagating
heterochromatin marks such as H3K9me2 through G9a recruitment. PRC1 canonical complex may be recruited through binding of the chromodomain region of the Cbx7
protein to H3K27me3 enriched chromatin; while the non-canonical (Rybp containing) PRC1 complex appears to be Xist RNA dependent. The mono-ubiquitination of histone
H2A is induced thanks to the Ring1b protein, although the exact role of this mark in X inactivation is not clear. At later stages of differentiation, in the maintenance phase, the
PRC2 and PRC1 complexes no longer appear to be present on the Xi. However, macroH2A becomes associated with the inactive X at the nal differentiation stage. The latest
mark to appear is DNA methylation of promoters of X-linked genes. DNA methylation is deposit thanks to Dnmt proteins. Smchd1 may play a role in deposition or
maintenance of DNA methylation. (B) During the XCI process, Xist RNA coating on the inactive X chromosome induces the formation of a nuclear silent compartment. The
genes escaping silencing are excluded from this nuclear domain and then preferentially interact with each other in a long-range manner.

Jarid2 was recently found to be essential for the targeting of PRC2
to Xist RNA coated chromatin [40] (Fig. 1). In the case of PRC1, the
canonical complex may be recruited through binding of the chrom-
odomain region of the Cbx7 protein to H3K27me3 enriched chro-
matin; while the non-canonical (Rybp cofactor containing) PRC1
complex appears to be Xist RNA dependent but H3K27me3 inde-
pendent [41]. Intriguingly, both PRC2 and PRC1 complexes are
enriched on the Xi early on in differentiation but become depleted
at later stages of differentiation, suggesting that their association
with the Xi is linked more to early than to late maintenance events
[37,42]. The Cdyl protein, another chromodomain protein and tran-
scriptional corepressor, was recently identied as a new Xi partner
and potential reader of the H3K9me2 and H3K27me3 marks [43].
Cdyl relies on H3K9me2 for its general association to chromatin
in vivo, but requires both the presence of H3K9me2 and
H3K27me3 for its differentiation-dependent enrichment on the
Xi. Cdyl also associates with other factors such as MGA and the
H3K9 histone methyltransferase (HMTase) G9a. Although the pre-
cise mechanisms are still not clear, it has been proposed that the
combination of H3K9me2 and H3K27me3 on the Xi help to recruit
Cdyl, which in turn facilitates propagation of the H3K9me2 mark
by anchoring the G9a HMTase [43].
The chromatin changes so far described do not appear to be
sufcient for the initiation of gene silencing. For example, the dis-
ruption of PRC2 and PRC1 components does not impede the onset
of XCI and Xist RNA lacking the 50 conserved region of the transcript
(known as the A repeats) can still induce these chromatin
changes and yet does not induce gene silencing [4345]. Thus
repression of gene transcription that is triggered by Xist repeat A
must involve different pathways, for example through interaction
with proteins such as SATB1 and other factors [46]. The A-repeat
region of Xist has also been proposed to play a role in interacting
 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522
with PRC complexes, in the context of an independent transcript,
RepA [47,48]. However, the exact role of RepA remains unclear
and the A-repeat region of Xist is clearly dispensable for Polycomb
factor recruitment to the Xi, whereas another Xist region contain-
ing the conserved B and F repeats was recently identied as being
critical for inducing PRC2 recruitment, via Jarid2 [40]. The multiple
roles of Xist RNA in X inactivation are still poorly understood and
seem to be at several levels, involving chromatin, nuclear compart-
mentalization, as well as chromosomal organization. Although the
formation of a silent nuclear compartment by Xist RNA and the loss
of euchromatic marks correlate well with the onset of transcrip-
tional inactivity [44], whether these changes are cause or conse-
quence of XCI remains to be seen.
In addition to the recruitment of protein complexes such as
PRC2, PRC1, as well as Cdyl and its partners, some further changes
occur at later stages during the onset of XCI in differentiating ESC.
These include enrichment of histone variant macroH2A [49] which
has been shown to interfere with transcription factor afnity and
SWI-SNF nucleosome-remodeling in vitro [64,65]; the Trithorax
group protein Ash2L [45]; and the scaffold factor SAF-A [50], which
is thought to participate in restricting or recruiting Xist RNA to the
inactive X. Most X-linked genes on the Xi also acquire DNA meth-
ylation at their CpG islands, with different genes having rather dif-
ferent kinetics of hypermethylation [51]. The mechanism through
which DNA methylation is recruited to X-linked genes remains
unclear, although the Smchd1 and Dnmt1 DNA methyltransferase
proteins have been proposed to participate [52,53] (Fig. 1). Smchd1
was originally identied in a genetic screen for epigenetic modi-
ers, as a factor involved in the maintenance of X inactivation and
the hypermethylation of CpG islands associated with the inactive
X [51,52]. Smchd1 has also been shown to participate in the com-
paction of the Xi in somatic human cells, through an interaction
with HBiX1 [66]. These two proteins act in a PRC2-independent
manner.
The inactive X chromosome also displays a shift to asynchro-
nous replication timing during development. Asynchronous
replication is in fact one of the most conserved features of the inac-
tive X (see review [22]) and may provide a temporal segregation
that minimizes the exposure of the inactive X to transcription fac-
tors, thus improving the maintenance of transcriptional silencing.
This change in replication timing of the Xi occurs downstream of
chromatin changes such as loss of euchromatic marks and PRC
recruitment [31]. It is still not known whether asynchronous
replication has a role in XCI, or is simply a consequence of the
heterochromatinisation of the Xi. Thus several independent path-
ways seem to participate in the heterochromatinisation of the Xi.
Establishing the mechanisms of recruitment and action and the
possible links between them represents an important challenge
for the future.
3. Developmental plasticity of the inactive state and synergy of
epigenetic marks in the maintenance of X-chromosome
inactivity
In somatic cells, the inactive X chromosome is in a highly stable
state of transcriptional silence, with sporadic reactivation of
X-linked genes estimated to occur at frequencies of less than
108 [54]. However, during mouse development different lineages
display rather different degrees of X-linked gene silencing, and this
may be associated with slightly different epigenetic signatures.
During early development, the paternal X becomes associated with
Xist RNA, macroH2A, PRC2 and the histone modication it deposits,
H3K27me3. At the blastocyst stage, these marks are reversed in the
inner cell mass where the inactive paternal X (Xp) is reactivated
[20,55]. However these marks are maintained on the inactive Xp
in extra-embryonic cells of the trophoblast lineage. In trophoblast
2517
giant cells (TGC) that are derived from this extraembryonic lineage,
high rates of escape from XCI are observed for some, but not all,
X-linked genes [56]. The current hypothesis is that there is a need
for longer-term, more stable silencing in embryonic lineages as
these give rise to the embryo-proper, where cellular memory must
be maintained throughout the lifetime of the animal. On the other
hand, there is only a short-term requirement for XCI in extra-
embryonic tissues which are dispensable after birth. In particular,
DNA (cytosine) methylation of X-linked CpG islands appears to
be present in embryonic but not extra-embryonic lineages in
eutherians mammals and its absence correlates with reactivation
[57]. Indeed, epigenetic marks involved in maintaining the inactive
state appear to vary considerably in both embryonic and extraem-
bryonic lineages [58]. In TGCs, the Xi has an highly unusual chro-
matin content, not only lacking DNA methylation at CpG islands
but presenting both heterochromatic marks such as H3K27me3
and euchromatic marks such as histone H4 acetylation and H3K4
methylation. Moreover, Xist RNA does not form an overt silent
nuclear compartment and reactivation of Xi-linked has been
observed [56]. This may be partly due to the fact the trophecto-
derm cells undergo endoreplication resulting in an unusual
chromosome organization and possibly less stable propagation of
the silent chromatin state. Nevertheless, although the Xi shows a
substantial degree of gene reactivation in these cells it remains
globally silent. The presence of PRC2 and H3K27me3 on the Xi
may be sufcient to maintain silence in this context. Indeed mouse
embryos mutant for the PRC2 component, Eed, show much higher
reactivation rates of an X-linked GFP transgene specically in the
trophectoderm [59]. In the same mutants, no impact was found
in the epiblast, presumably due to the presence of other epigenetic
factors, including PRC1 complexes and DNA methylation. Although
a direct role of PRC1 complex proteins in the maintenance of X
inactivity has not so far been demonstrated, two different com-
plexes have been found associated with the Xi [41]. The timing
of recruitment of PRC1 complexes to the Xi during the same time
window as PRC2 in developing embryos [36,37,60] suggests that
this complex is likely involved in the maintenance of inactivity.
This is potentially by SWI-SNF complexes inhibition, blocking tran-
scriptional activity or by recruiting silencing components, as
described in other systems [6163].
The extent that different epigenetic marks participate in the
maintenance of the inactive state has been addressed in experi-
ments aiming to prevent or reverse them. Studies have disrupted
several marks on the Xi simultaneously such as DNA methylation
(by 5-azacytidine treatment or in DNA methyltransferase mutant
cells, Dnmt1/), histone hypoacetylation (by pan-histone deace-
tylase inhibitor treatment, TSA) and Xist RNA (by a conditional
KO) can lead to a signicant increase of Xi-linked genes expres-
sion [54]. However disruption of each of these marks alone has lit-
tle effect. This clearly demonstrates that several epigenetic
modications lock in the inactive state of the Xi. Nevertheless,
given the relatively low reactivation frequencies even after the
combined removal of these three marks, other epigenetic players
are likely to be involved in the maintenance of silencing. Indeed,
so far, full reactivation of the Xi has never been achieved experi-
mentally other than via fusion with ES cells, which have the repro-
gramming potential of the inner cell mass [20,37], or by nuclear
transfer where the reprogramming potential of the oocyte is
involved [67,68], or through the expression of the Yamanaka cock-
tail pluripotency factors (Oct4, Klf4, c-Myc and Sox2) that can
drive somatic cells into an induced pluripotent state resembling
ES cells [67,6971]. The reactivation of the Xi is thought to be
one of the later events during reprogramming, presumably due
to the need to reverse several layers of epigenetic marking, some
of which may require active mechanisms (specic transcription
factors or chromatin enzymatic activities) and others may require
2518 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522
passive DNA replication to enable gradual removal. Further studies
on the key players involved in the X-chromosome reactivation pro-
cess during reprogramming, both during development (in the ICM
and in the germ line) and also experimentally (via nuclear transfer
or IPS formation) should provide insights into the nature and
extent of the maintenance mechanisms that are at play in different
cellular states and for different parts of the X chromosome (for
review [5]).
4. Variability in X-inactivation status and escape from XCI
In eutherian mammals, such as humans and mice, the majority
of X-linked genes are subject to transcriptional repression during
XCI, although a variable subset of X-linked genes can escape silenc-
ing (escapees) and are bi-allelically expressed showing higher
expression in females when compared to males [7275]. Only Xist
appears to be expressed exclusively from inactive X in female
somatic cells. Two main groups of escapees are present on the X
chromosome. One group comprises escapees that lie within the
pseudo autosomal region (PAR) and have an exact homolog on
the Y chromosome, and show equal expression levels between
the sexes [73]. A second group consists of genes that are located
outside the PARs. These include genes such as Kdm5c (Smcx or Jar-
id1c) and Utx, which have Y-chromosomal homologs lying outside
of the PAR and presumably represent evolutionary remnants from
the proto-sex chromosomes. Some other escapees have no obvious
Y-linked homologs. The actual degree of escape from XCI for such
genes can vary considerably and depend on the tissue or the spe-
cies [74,76,77]. Such escapees usually display a lower level of
expression from the inactive X compared to the active X, thus lead-
ing to female expression that is less than twice the dose compared
to males [78]. The lower expression may be due to the gene only
being expressed from the Xi in a proportion of cells and/or to lower
transcription levels from the Xi. Allele-specic analyses of genes
that escape X inactivation reveal that they occupy an unique chro-
matin conguration, with no/low enrichment of heterochromatin-
associated marks and no coating by Xist RNA [27,29,35,74,76]. RNA
FISH studies have shown that escapees tend to be located outside
the Xist RNA coated territory [28,44]. More recently, chromosome
conformation capture analyses have revealed that Xi escapees tend
to interact with each other preferentially in the nucleus [29]. The
ability to escape may require a specic genomic context, as it has
been shown that Kdm5c gene has a capacity to escape from
XCI that is independent of its location on the inactive X chromo-
some [79]. This raises the question of how genes that are subject
to XCI, can resist the inuence of nearby escapees. Recent studies
suggested that regions of the X chromosome that are subject to
XCI appear to be protected from the spreading of transcriptionally
active escapee domains thanks to CTCF insulator elements
[80,81] and the presence of young active LINE-1 elements has also
been speculated to be involved in facilitating XCI of genes in
regions that are otherwise prone to escape [28].
Surprisingly, many more escapees have been found in humans
when compared to mice, with an estimation of up to 15% of
X-linked genes escaping XCI to some extent in humans. An addi-
tional 10% of human X-linked genes show variable degree of
escape, based on investigation in cell lines [82,83]. Many of these
human escapee genes lie on the more recently added short arm
of the X chromosome. Indeed, it has been hypothesized that their
incomplete silencing could be due to a barrier effect caused by
the centromeric heterochromatin that separates the XIC on the
long arm (Xq13) from the short arm where most escapees are
located (for review [78]). It should be noted however, that to date,
only a few of these human Xi escapees have been conrmed in vivo
given the difculties in obtaining human tissue samples and in
accurately quantifying allelic expression of X-linked genes. Never-
theless, comparative analysis of CpG island DNA methylation has
been successfully used to determine indirectly the XCI status of
X-linked genes between different tissues, supporting the idea of
substantial tissue-specic escape from XCI [84]. There is also
evidence for human individual-specic genes that escape from X
inactivation. TIMP1 is good example of this phenomenon where it
has been shown that H3 acetylation status correlates well with
females in which escape of this gene is found [85].
Apart from a small number of genes for which escape from XCI
appears to be evolutionarily conserved (such as KDM5C/JARID1C),
for most escapees on the human X, escape from XCI is not neces-
sarily consistent between individuals or between tissues or even
between cells within the same individual. Nevertheless, the exis-
tence of escape from X inactivation, particularly in human remains
largely unexplained [78]. Escape genes may play essential roles for
normal development. Indeed, Turner syndrome is very deleterious
to patient with a single X chromosome, perhaps due to haploinsuf-
ciency of escape genes, even though XO mice are largely not
affected [8688].
A comprehensive study of X-linked allelic expression in hun-
dreds of human lymphoblastoid cell lines has revealed that 5% of
X-linked genes show increased expression level in females when
compared to male. Based on this in vitro system, it seems that only
a few escape genes have a role in compensating sex biases even
though this 5% could have a strong impact when they are lost
[83]. A recent in vivo study using RNA sequencing done on post-
mortem human brain tissues revealed that X-linked genes show
gene expression dimorphisms. Sex-biased gene expression was
widespread in terms of gene number, chromosomes and range of
brain regions involved. However, the percent of X- and Y-linked
genes involved in the sex-bias seem highly related to particular
regions of the brain [89]. For a more comprehensive view of the
biological roles of genes that escape X inactivation in mammals,
and particularly in the context of human health and disease, more
primary tissue-specic and developmental analyses of XCI status
will be required. Further investigation of the DNA sequence con-
text, chromatin environment, nuclear organization and chromo-
some conformation, as well as developmental regulation will be
needed to assess the mechanisms underlying escape from XCI.
More detailed investigation of some of the known factors that
ensure the maintenance of Xi gene silencing, for example Smchd1
which results in partial escape from XCI when deleted in the
mouse [93], will also be needed to understand the molecular basis
for developmental and tissue-specic escape from XCI. Finally,
there are some examples of X-linked gene reactivation from the
Xi that have been associated with the aging, for example as shown
for the Otc gene in mice [94]. The mechanism for this age-related
escape is unclear although it may be due to epigenetic "erosion"
due to inefcient maintenance of chromatin states over extended
cell division or in adult stem cells. Although loss of the Barr body
in ageing females has been reported, the degree to which
age-related epigenetic relaxation of the Xi occurs in humans and
mice has not so far been investigated systematically.
5. Unstable X-chromosome inactivation in cancer?
As described above, Xi reactivation is observed in a develop-
mental context in mouse extra-embryonic lineages and in the germ
line, and escape from XCI is observed for a subset of X-linked genes
in somatic cells particularly in humans. However, the Xi is globally
stably silent in somatic tissues suggesting that dosage compensa-
tion is important, although the impact that failure to maintain Xi
inactivity in adult tissues would have, has not so far been assessed
in any systematic way. The synergy and partial redundancy of
 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522 2519

heterochromatin-associated marks that cooperatively maintain the
integrity of heterochromatic inactive X were discussed above. The
loss of Xist from the Xi in terminally differentiated cells does not
lead to global reactivation of the inactive X, as genes remain
repressed presumably via the above-mentioned synergistic epige-
netic modications [54]. Thus although Xist is required for initia-
tion of X inactivation, it was thought not to be necessary for the
maintenance of transcriptional silencing on the Xi up until recently
[90,91]. However, a recent study showed that the conditional
knock-out of Xist in the hematopoietic compartment of the mouse,
led to an aggressive form of hematologic cancer in females [92].
Although allele-specic analyses were not performed in this study
and thus Xi reactivation could not be demonstrated directly, the
authors hypothesized that X-linked gene over-expression could
be the result of X inactivation relaxation in tumor cells and some-
how trigger cancer.
In humans, there is still only limited evidence available for the
status of the Xi in cancer to date, even though it was described
more than 50 years ago that the Barr body is frequently lost in
breast cancers [2,95] (Fig. 2), leading to the proposal that Xi
reactivation may be a common event in some cancers. More recent
studies suggest that the disappearance of the Barr body is
sometimes associated with over-expression of X-linked genes, sug-
gesting a potential role of the X chromosome in cancer progression
[96]. In some cases, the tumors lacking an inactive X chromosome,
also present a duplication of the active X chromosome [9698].
Thus, at least two mechanisms could explain the loss of Barr body
in cancer cells (Fig. 2). On one hand, epigenetic instability of Xi may
occur, with decondensation of heterochromatic Barr body, leading
to Xi-linked gene reactivation. Although this mechanism has been
frequently hypothesized, so far no evidence supports a model of
global Xi reactivation and heterochromatin loss in cancer
[99101]. In some cases, XIST RNA mislocalisation and sporadic
Xi reactivation has been observed [102,103]. For example, one
study on an ovarian cancer cell line, showed a disruption of XIST
expression and potential reactivation of the MPP1 (p55) gene
[97]. In the context of breast cancer, bi-allelic expression of a single
X-linked gene, VBP1, associated with promoter DNA hypomethyla-
tion was reported in one primary breast tumor sample [96]. How-
ever, without global analyses of expression status and chromatin
structure of the inactive X chromosome, it is difcult to assess
the precise extent of Xi instability in a cancer context. On the other
hand, Barr body disruption can be due to physical loss of the Xi in
female cancers [103,104]. Indeed, recent evidences show that the
inactive X chromosome is genetically unstable in cancer as this
study reporting an higher mutations rate on the inactive X com-
pare to rest of the genome [105]. The consequence of both models
would result with double dose of some or all X-linked genes. In
early embryos, the presence of two active X chromosomes is
ultimately lethal [12]. However in somatic cells the increased dose
of a subset of genes on the X chromosome could potentially
provide a selective advantage and promote cancer development,
considering that even a rare cell expressing a gene with a prolifer-
ative advantage could contribute to cancer progression. Future
studies on the causes or consequences of Xi reactivation in differ-
ent situations, during normal development and in cancer, will be
needed. Indeed, the emerging role of aberrant gene dosage in dis-
eases, whether of the X chromosome or for autosomes, brings with
it the possible application of drugs that impact on epigenetic reg-
ulators in potential therapeutic strategies [106108].
A direct role for BRCA1, a tumor suppressor protein that is fre-
quently mutated in familial cases of breast and ovarian cancer, was
proposed some years ago to explain loss of the Xi [109]. This study
proposed a direct action of BRCA1 on XIST RNA coating of the Xi

Fig. 2. Genetic and epigenetic instability of the Xi in cancer. Schematic view of genetic and epigenetic alterations that can affect the X chromosome in breast cancer cells. The
left panel lists the different genetic situations that have been be observed concerning the X chromosomes in cancer cells i.e. Xi loss and Xa reduplication (for example [2]); Xi
hypermutation [105]; and no Xi loss (for example [104]). On the right, we represent potential epigenetic instability that has been hypothesized to affect the Xi in some cancer
cell. So far, few studies have evaluated X-linked gene reactivation in breast cancer (for example [96]). Further studies are needed to explore to what extent epigenetic instabily
is present on the inactive X chromosome in cancer cells and what could be their impact in carcinogenesis.
2520 R. Chalign, E. Heard / FEBS Letters 588 (2014) 25142522
and suggested that in BRCA1 mutated tumors, the Xi might be epi-
genetically relaxed. However subsequent studies [102,104]
revealed that cells of BRCA1-mutated primary breast tumors can
contain one or several XIST RNA coated chromosomes. Neverthe-
less, this does not invalidate a role for BRCA1 in XIST expression
[102], as, among its many roles it may act as a transcription
regulator [110]. Another chromatin regulator frequently over-
expressed in cancer, Aurora B Kinase (AURKB), has also been
proposed to regulate the association of XIST to the Xi [111,112].
However, what the exact consequences are on Xi status are not
clear. Thus, the disappearance of the Barr body in aggressive breast
tumors noted more than 50 years ago, still remains a fascinating
but poorly understood phenomenon. Further investigation will be
required to elucidate the nature and extent of the inactive X
chromosomes genetic and epigenetic instability in cancer, with
obvious clinical interest.
6. Conclusion
The inactive X chromosome provides a remarkable example of
chromosome-wide gene repression that reveals the diversity of
processes that can contribute to the formation of facultative
heterochromatin. XCI also highlights the complex issue of gene
dosage. The repeated inactivation and reactivation of the X chro-
mosome during development illustrates the importance of a ne
regulation of all the mechanisms involved. Recent studies have
revealed that a combination of chromatin factors, chromosome
conformation and nuclear compartmentalization together ensure
maintenance of the inactive state. The mechanism that triggers
gene silencing in the rst place, via Xist RNA remains tantalizingly
obscure. Once XCI is established, silencing appears to be rather sta-
ble in terminally differentiated cells, although XCI is less complete
in humans than in mice and some genes can escape this silencing
process, either for a purpose or accidentally. Increasing evidence
suggests that silencing efciency of X-linked genes is dependent
on tissue types and individuals and points to a complex interplay
between genetic variation and epigenetic status. The growing real-
ization that both genetic and epigenetic defects may affect the
inactive X chromosome in tumors opens up new avenues of
research to understand how Xi epigenetic instability in cancer
arises and whether this instability can actually contribute to cancer
progression and would thus be of therapeutic interest.
Acknowledgments
We would like to thank Simao Teixeira da Rocha for critical
reading of the manuscript. Support is gratefully acknowledged
from lAssociation pour la Recherche sur le Cancer (ARC)
(post-doc fellowship to R.C.); FRM (Equipe FRM to E.H); Equipe
labellise La Ligue Contre Le Cancer (Equipe Labllis to E.H) [27] Engreitz, J.M., Pandya-Jones, A., McDonel, P., Shishkin, A., Sirokman, K., Surka,
and EU FP7 MODHEP EU grant no. 259743 (to E.H.) and Labex DEEP
(ANR-11-LBX-0044) part of the IDEX Idex PSL (ANR-10-IDEX-0001-
02 PSL).
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