REVIEWS

Regulation of Xchromosome
inactivation by the Xinactivation centre
Sandrine Augui*, Elphge P. Nora* and Edith Heard

Abstract | X-chromosome inactivation (XCI) ensures dosage compensation in mammals

and is a paradigm for allele-specific gene expression on a chromosome-wide scale.
Important insights have been made into the developmental dynamics of this process.
Recent studies have identified several cis- and trans-acting factors that regulate
the initiation of XCI via the Xinactivation centre. Such studies have shed light on the
relationship between XCI and pluripotency. They have also revealed the existence of
dosage-dependent activators that trigger XCI when more than one Xchromosome
is present, as well as possible mechanisms underlying the monoallelic regulation of
this process. The recent discovery of the plasticity of the inactive state during early
development, or during cloning, and induced pluripotency have also contributed to
the Xchromosome becoming a gold standard in reprogramming studies.

Homogametic and
heterogametic sexes
In species with sexual
dimorphism, the sex that can
produce two different types of
gametes (X and Y or Z and W)
is called heterogametic,
whereas the sex that can
produce only one type of
gamete (X or Z) is called
homogametic.

Imprinted
Epigenetic marking of a locus
on the basis of its parental
origin, which can result in
differential expression of the
paternal and maternal alleles
in specific tissues or
developmental stages.

Mammalian Developmental
Epigenetics Group, Unit of
Genetics and Developmental
Biology, Institut Curie, CNRS
UMR3215, INSERM U934,
Paris F75248, France.
*These authors contributed
equally to this work.
Correspondence to E.H.
e-mail: Edith.Heard@curie.fr
doi:10.1038/nrg2987

Sex chromosome dimorphism leads to a genetic imbalance between the homogametic and heterogametic sexes,
which mammals compensate for by inactivating one of
the two Xchromosomes during female development.
Although this chromosome-wide silencing process was
originally described more than 50years ago (TIMELINE),
the underlying molecular mechanisms remain poorly
understood. One of the most intriguing aspects of
Xchromosome inactivation (XCI) is that two homologous Xchromosomes are differently treated within
the same nucleus. How the inactive state is set up and
faithfully transmitted through cell division remains a
central question for which answers are only now beginning to emerge. This Review will focus on the recent
progress that has been made in our understanding of
the initiation of XCI, as well as the reversibility of the
inactive state during specific stages of development
and in the context of reprogramming experiments.
In mice, which have been the favoured model
for XCI studies, there are two waves of XCI, the first
being imprinted (paternal XCI) and the second random.
Imprinted inactivation of the paternal X chromosome
(Xp) is initiated shortly after fertilization. This silent
state is maintained in extra-embryonic tissues but lost
in the inner cell mass (ICM), which gives rise to the
embryo proper. Shortly after this, random inactivation
of either the maternal X chromosome (Xm) or the Xp
is initiated in the cells of the ICM. Invitro differentiation of mouse embryonic stem cells (ESCs), which are

derived from the ICM and have two active X chromosomes, is accompanied by random XCI and has been
extensively used to dissect the early events underlying this process. As such, the regulation of random
XCI is more thoroughly understood and is the main
focus of the Review, although imprinted XCI is also
discussed.
A growing number of new molecular players have
been implicated in XCI over recent years. How they
function together to control XCI and how this fits in
with or challenges the original views of the pro
cess remains largely unclear. The aim of this Review is
to examine the role of these recently identified molecular players in the context of the initial historical notions
underlying the process of XCI; that is, the concepts
of counting, choice and sensing/competence (BOX1).
We will focus primarily on the Xinactivation centre
(Xic) and the key non-coding X-inactivation specific
transcript (Xist) it produces, which represents the trigger for chromosome-wide silencing. We first explain
briefly how the Xic was functionally and physically
identified. We then describe how Xist underlies some,
but not all, of the functions attributed to the Xic and
review our current knowledge on the increasingly
complex regulatory network controlling Xist expression. Finally, we discuss random and imprinted XCI
in the context of mouse development and the recent
insights that XCI has brought into reprogramming
processes.

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Timeline | Landmarks in our understanding of the initiation of random XCI
Discovery of a dense
structure in female
somatic nuclei called
the Barrbody140

Based on phenotypic variegation in

the coat colours of heterozygous
female mice, Lyon proposed that one
of the two Xchromosomes is stably
inactivated in femalecells142

Identification of an
Xcontrolling element
(Xce), which induces a
skew in choice of theXi40

Discovery of the
Xist/XIST gene as
a candidate for
the Xic68

Demonstration that large

single-copy Xist transgenes
are insufficient for full Xic
functions during random XCI34

(20002010) Discovery of
numerous Xist molecular
regulators (see main text)

1949
1960 1961 1963 1967 1983 1991 1996 1999 2000

The Barr body is proposed

to be an inactive
Xchromosome (Xi)141

(19631964) Lyon, Russell and

Grumbach propose that inactivation
spreads from a unique locus (the
Xinactivation centre, (Xic)) located
on the Xchromosome129131,143

(19831985) Definition of
the Xic and its functions1,2

(19961997) Demonstration that

Xist is essential for initiation of
XCI in mice10,11 and that multicopy
Xist transgenes can induce XCI
to some extent125127,144

The Xinactivation centre

Early studies of XCI patterns in mouse embryos or
embryonic cells that carried translocated or truncated X chromosomes revealed the existence of a
single Xlinked locus, the Xic, that needs to be physically linked to a chromosome to trigger its inactivation1
(FIG.1). Random XCI is only triggered in cells with at
least two Xic-bearing chromosomes2, suggesting that the
two copies of the Xic are able to potentiate each other in
trans, a phenomenon that has been referred to as competence, or sensing 35 (BOX1). In XX cells, either one of
the two Xchromosomes will be inactivated, a process
known as choice (BOX1). The autosomal ploidy of a cell
(the number of sets of autosomes that is contains) also
seems to affect the number of Xchromosomes that
will be inactivated, a phenomenon known as counting
(BOX1). The precise mechanisms underlying these pro
cesses are only now being unravelled and recent data suggest that they are highly interconnected, both genetically
and molecularly.

Polycomb group proteins

(PcG proteins). A class of
proteins originally described
in Drosophila melanogaster
that form large complexes
and maintain the stable and
heritable repression of several
genes throughout development.

Trithorax group proteins

(TrxG proteins). A class of
proteins originally described
in Drosophila melanogaster
that form large complexes
and maintain the stable and
heritable expression of several
genes throughout development.

Xist RNA triggers cis-inactivation

The Xic harbours the Xist gene68 (FIG.1B), which produces a non-coding RNA (ncRNA) that is retained in
the nucleus and that, in its spliced form, can coat the
chromosome from which it is expressed9. It is devoid
of any significant ORF and is only expressed from the
inactive Xchromosome (Xi) in somatic cells. During
both female mouse development and invitro differentiation of female mouse ESCs, Xist is monoallelically
upregulated. This upregulation is tightly correlated with
the onset of XCI and precedes the initiation of silencing (FIG.2). Deletions of Xist have demonstrated that it
is necessary in cis to induce chromosome-wide silencing 10,11. Furthermore, inducible expression of Xist cDNA
transgenes on autosomes demonstrated that Xist RNA is
sufficient to trigger cis-inactivation of the chromosome
from which it is expressed during an early developmental
time window 12.
How exactly Xist RNA induces gene silencing still
remains a mystery, but the highly conserved Arepeat
region of Xist is crucial for its silencing function, whereas

Identification
of the Xist
antisense unit,
Tsix56,145,146

Demonstration
that Xist RNA is
sufficient to initiate
cis-inactivation12

other parts of the RNA ensure its cis-coating capacity 13,14.

Expression of an Xist cDNA lacking the Arepeat region
in differentiating mouse ESCs has revealed that the transcript can induce several chromatin modifications on the
chromosome that it associates with, independently of
transcriptional repression. These modifications include
recruitment of Polycomb group proteins (PcG proteins),
the histone variant macroH2A, the Trithorax group protein
(TrxG protein) ASH2like (ASH2L) and heterogeneous
nuclear ribonucleoprotein U (hnRNPU; also known as
SAFA)12,1419. Wild-type Xist RNA has also been shown
to induce the spatial reorganization of the Xchromosome, creating a repressed nuclear compartment that is
depleted of the transcription machinery and into which
genes are recruited when they are silenced2022.
Based on the above evidence, Xist activation clearly
triggers the establishment of chromosome-wide silencing. Therefore, much of the research into the mechanisms of XCI initiation has focused on regulation of this
particular gene and the ncRNA it produces. However, an
important observation from studies of Xist knockouts is
that heterozygous Xist mutants are still able to initiate
XCI from the wild-type Xchromosome10,11. Thus, Xist
sequences alone cannot account for the competence
function of the Xic, which means other elements must
be responsible for female-specific (XX) Xist activation
and XCI initiation. As discussed below, it is now clear
that Xists unique expression pattern is controlled by a
complex interplay of long-range cis-acting elements and
trans-actingfactors.

Xist regulation during random XCI

How is female-specific, monoallelic Xist upregulation
achieved and why does it only occur within a precise
time window during development and differentiation?
In the following sections we describe what is known
about the different levels of control acting on Xist during
random XCI (see FIG.3A for a summary). Xist is expressed
at very low levels in undifferentiated male and female
ESCs, but becomes upregulated on one Xchromosome
upon differentiation of female cells. Although it is now
clear that Xist is controlled mainly at the transcriptional

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Box 1 | Key concepts in Xchromosome inactivation
Before the discovery of the many molecular actors in X-chromosome inactivation
(XCI), some key concepts relating to the steps necessary for inactivation to occur
were proposed. Although theoretical, these notions became, to an extent, dogmatic
over the years. However, these concepts are now being revised in the face of new
molecular insights.

Counting
This refers to the process by which a cell determines its X/autosome (X/A) ratio in
order to maintain only a single active Xchromosome per diploid autosome set. It was
first proposed by Lyon and Grumbach based on humans with abnormal numbers
of Xchromosomes118,119. A normal XY male, or an XO female, shows no inactivation of
the unique Xchromosome, whereas XXX and even XXXX individuals display one
active X chromosome and inactivation of all supernumerary Xchromosomes120124.
Choice
Refers to how one of the two X chromosomes is selected for inactivation. During
random XCI, the probability that the paternal or the maternal Xchromosome will be
chosen for XCI is equal, unless mutations or polymorphisms are present within the
X-inactivation centre (Xic)41. The selection of one Xchromosome for inactivation
must somehow preclude the initiation of XCI of the other Xchromosome and is thus
a part of the trans-function of the Xic.
Sensing/competence
This describes a permissive state for XCI that occurs only when there is more than one
X chromosome present in a cell. It must be noted that sensing/competence is implicit in
the original concept of counting (as defined above) and involves both XX-recognition,
as well as assessment of the X/A ratio. However, investigation of phenotypes of different
Xic mutants has led to a distinction being made between the two concepts4,5,47.

level23, post-transcriptional maturation events may also

participate. For example, recent studies have shown that
deletion of the Arepeat region of Xist prevents accumulation of the spliced form of Xist RNA during differentiation24 and somehow disrupts the genes correct
upregulation during development25.

Pluripotency factors
A class of proteins that
maintain pluripotency the
capacity to give rise to a wide
range of, but not all, cell
lineages of stem cells.

Repression of Xist in undifferentiated ESCs

What accounts for the low expression level of Xist in
undifferentiated ESCs? Several circumstantial lines of
evidence have pointed to pluripotency factors as negative
regulators of the XCI process (FIG.3Ba). In mouse ESCs,
inducible knockout of Nanog or Oct4 (also known as
Pou5f1) leads to ectopic Xist upregulation and chromosome coating in a fraction of differentiating male ESCs26.
Another study reported that knockdown of Oct4 leads
to Xist RNA accumulation on both Xchromosomes in
a fraction of differentiating female ESCs27. The binding
of OCT4, NANOG, SOX2, transcription factor 3 (TCF3;
also known as TCFE2A) and the PR domain containing protein PRDM14 within the first intron of Xist26,28,29
had led to the proposal that such factors might repress
Xist expression, via this region, in undifferentiated
ESCs. However, deletion of this intronic region of Xist
was recently shown to have no impact on Xist repression in undifferentiated ESCs30, although the chromosome with the deleted allele is mildly favoured for XCI
upon differentiation. Furthermore, it has recently been
shown, using reporter assays, that a construct containing just the Xist core promoter can be activated during
female mouse ESC differentiation23, and that OCT4,
NANOG and SOX2 do not bind the Xist promoter 26,28,31.
Therefore, these pluripotency factors probably control
Xist activity indirectly via intermediate regulators. As we

discuss later, both upregulation of RING finger protein

12 (RNF12; also known as RLIM) and XicXic homologous pairing events during differentiation may represent
such intermediates27,32.

Female-specific activation of Xist

What is the mechanism underlying the specific upregulation of Xist in cells with more than one X chromosome? Several lines of evidence point to the existence
of long-range regulatory elements that are required
for Xists XXspecific upregulation. First, female cells
carrying a 58 kb deletion including Xist on
one Xchromosome can still initiate XCI on the wildtype Xchromosome33, implying that they can still sense
their XX status and are still competent for XCI. Second,
large 460 kb single-copy Xist transgenes in male ESCs
are unable to trigger Xist during differentiation, either
from the transgene or from the endogenous Xic34. This
implies that critical Xic sequences that are needed to
render cells competent for XCI must be missing from
these large DNA fragments (BOX2). Thus, the sequences
underlying XXspecific Xist activation must lie some
distance from the geneitself.
In a quest to identify these sequences, investigation of
the genomic neighbourhood of Xist led to the identification of at least three Xlinked loci that are possibly implicated in the activation of Xist during random XCI in
female mouse ESCs. One is the Xpairing region (Xpr),
which lies 200300kb 5 to Xist. Xpr is able to mediate homologous trans-interactions between the two Xic
loci (known as pairing) in female mouse ESCs before
Xist activation. This ability, which is also shown by Xpr
single-copy transgenes, was proposed to participate in
female-specific Xist expression, as XprXpr interactions
do not normally occur in male cells5. A recent report
describing the unusual genomic instability of the Xpr
region when present as a transgene in male cells35 could
be indicative of recombination pathways being involved
in Xpr pairing. However, the mechanisms underlying
Xpr pairing in female cells and the impact of this on Xist
transactivation remain to be elucidated.
A second locus that has clearly been shown to have
a role in XXspecific Xist activation is Rnf12, which
lies approximately 500kb 5 to Xist (FIG.3Bb). This gene
produces a trans-acting factor, RNF12, which has a
ubiquitin ligase activity. Overexpression of RNF12
can induce Xist RNA coating of the single X chromosome in differentiating male mouse ESCs and of both
Xchromosomes in differentiating female mouse ESCs32.
Based on such observations, it has been proposed that
RNF12 can activate Xist when present above a certain
threshold. In mouse ESCs, this threshold is proposed to
be reached only when two Xchromosomes are active.
How RNF12 activates Xist remains to be determined,
but one possibility is that its ubiquitin ligase activity
acts to degrade a repressor of Xist. Recently, RNF12
has been shown to be capable of activating the core
promoter of Xist30. Importantly, heterozygous deletion
of Rnf12 delays, but does not prevent, XCI in female
mouse ESCs32,36, implying that additional Xist activation
mechanisms, present in XX but not XY cells, must exist.

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Figure 1 | The Xinactivation centre. A|The X-inactivation centre (Xic) has been defined as the minimum region both
necessary and sufficient to trigger X-chromosome inactivation (XCI)2. The existence of a unique0CVWTG4GXKGYU^)GPGVKEU
locus controlling the
initiation of XCI was first proposed in 1964, based on studies of individuals or cell lines with balanced Xautosome
translocations. Aa|In normal female cells, there is random XCI such that there is an equal probability of either
Xchromosome undergoing inactivation. Ab | In studies of the reciprocal translocation T(X;16)16H (also known as T16H,
Searles translocation) only one of the two translocation products was found to be inactivated, suggesting the
existence of an Xlinked region (the Xic) that is required in cis for XCI to occur129131. Note that 16X is not found to be
inactivated, which is due to secondary counter selection. Ac | Surprisingly, when the same translocation is unbalanced
there is no inactivation process at all, suggesting that in the absence of two Xics, a cell does not detect the presence of
two X chromosomes132. Ad | Subsequent studies involving female embryonic cells where one of the two Xchromosomes
was truncated2,132 confirmed this hypothesis. It was revealed that neither the truncated X chromosome (HD2
truncation) nor the intact X chromosome showed any sign of XCI based on cytological staining. This indicates that at
least two Xics are required for a cell to initiate XCI. Ae | By contrast, for a truncation that does not remove Xic, random
XCI still takes place. B|In addition to providing a functional definition of the Xic, these chromosomal rearrangements
define the physical boundaries of the locus. In mice (shown), the minimum candidate region for the Xic has been defined,
based on studies in developing mouse embryos or differentiating embryo-derived (EK) cells133. The Xic lies between
the T16H breakpoint134,135 and the HD3 breakpoint1,2, a region spanning 8cM (1020Mb). Here, only the elements
around Xist are shown. Some of these elements, such as the Xist antisense gene (Tsix) or RING finger protein 12 (Rnf12;
also known as Rlim) gene are now known to be involved in Xist regulation. Xist and its antisense Tsix, as well
as regulators of Tsix Xite (X-inactivation intergenic transcription element) and DXPas34 are shown at higher
resolution under the Xic map. In humans (not shown), the XIC has been proposed to map between the T(X:14) and rea(X)
breakpoints, a region spanning 700kb136,137. However, the human XIC has been defined through the analysis of
X-inactivation status in somatic cells of patients with Xchromosomal deletions or translocations, rather than in
embryonic cells where XCI is actually initiated. Thus, it cannot be excluded that some of these rearrangements could
have arisen after initiation of XCI. Cdx4, caudal Xlinked gene 4; Chic1, cysteine rich hydrophobic 1; Cnbp2, cellular
nucleic acid binding protein 2; Ftx, five prime to Xist;Jpx, also known as Enox (expressed neighbour of Xist); Nap1l2,
nucleosome assembly protein 1like 2; Tsx, testis specific Xlinked; Xpct, Xlinked PEST-containing transporter.

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Figure 2 | The cycle of XCI in female mouse embryos and ESCs.

a | In mice, X-chromosome inactivation (XCI) begins at the fourcell stage
see the middle of the top part of this panel. Inactivation is initially
imprinted, with preferential inactivation of the paternal X chromosome (Xp).
Studies of parthenogenetic and gynogenetic embryos suggest that this
imprint is maternal. This is because, in the presence of two maternal
Xchromosomes (Xm), there is no XCI until blastocyst formation, implying
that the Xm cannot be inactivated prior to this, despite the presence of two
X chromosomes89,138. Furthermore, experiments performed with non-growing
oocytes showed that an Xchromosome from an immature oocyte (prior to
establishment of imprinting) behaves like an Xp during embryogenesis and
can undergo early X-inactivation104. Once established, the Xp remains
inactive in extra-embryonic tissues (trophectoderm and placenta) but is
reactivated in the inner cell mass (ICM) of the blastocyst in pre-epiblast cells,
which gives rise to the embryo. A second wave of inactivation then occurs in
the ICM and randomly affects either the Xp or the Xm. The inactive state is

then stably maintained and transmitted through cell divisions in the soma
but the inactive X chromosome (Xi) is reactivated
during the formation of
0CVWTG4GXKGYU^)GPGVKEU
the female germ line. Imprinted and random inactivation are both
Xist-dependent and both seem to involve RING finger protein 12 (RNF12;
also known as RLIM). A maternal pool of RNF12 may be required for initiation
of imprinted Xp inactivation and two copies of Rnf12 may be required to
activate Xist in female embryos during random XCI (see main text for
discussion). b | RNA fluorescent insitu hybridization (FISH) in mouse
embryonic stem cells (ESCs). In undifferentiated cells, the two
Xchromosomes are active, as shown here by biallelic expression of
-thalassaemia/mental retardation syndrome X-linked gene (AtrX). In these
cells, Xist is expressed at a low level and is hardly detectable. During
differentiation, one of the two Xist alleles is upregulated. Xist RNA coats the
X chromosome from which it is produced and triggers Xinactivation, which
leads to the monoallelic expression of Xlinked genes such as AtrX in
differentiated cells. Tsix, Xist antisense gene.

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Figure 3 | Summary of Xist regulation at the onset of XCI. Numerous factors are implicated0CVWTG4GXKGYU^)GPGVKEU
in Xist regulation.
A | Network of genetic interactions. Note that here arrows do not necessarily imply direct regulation. Repression of Xist
by SOX2, although not formally assessed, is to be expected, given that it shares the vast majority of its targets with
OCT4 and NANOG. B | Possible molecular mechanisms involved in regulating Xist. Ba | Binding sites of pluripotencyassociated transcription factors within elements of the network. It is still unclear whether binding to these sites actually
mediates control of Xist and Tsix (Xist antisense gene) expression see main text for details. Bb | The activation of Xist
also requires Xlinked activators such as RING finger protein 12 (RNF12; also known as RLIM) in a dose-dependent
manner. The upregulation of RNF12 during differentiation is thought to activate Xist, thereby triggering cis-inactivation
and ultimately lowering RNF12 levels. This feedback loop ensures that one X chromosome remains active.
Bc | Different modes of cis-regulation operate on each chromosome. On the future active X chromosome, Tsix
expression is stimulated by the X-inactivation intergenic transcription element (Xite) and DXPas34. On the future
inactive X chromosome, the A-repeat region is required for the accumulation of the spliced form of Xist, which
mediates silencing. However, an effect of this region on the Xist promoter in XX-differentiating cells still cannot be
excluded (dashed arrow). Bd | The regulatory activity of the A-repeat region has been proposed to involve the
expression of a short RNA, RepA. RepA has been proposed to bind Polycomb repressive complex 2 (PRC2) and recruit it
to the Xist promoter, somehow resulting in the activation of Xist. This binding of RepA to PRC2 would be antagonized
by Tsix on the future active Xchromosome. CTCF, CCCTC-binding factor; Jpx, also known as Enox (expressed neighbour
of Xist); OCT4, also known as POU5F1; PcG, Polycomb group; PRDM14, PR domain zinc finger protein 14; REX1,
reduced expression protein 1 (also known as ZFP42); YY1, transcriptional repressor protein Yin and Yang 1.

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Box 2 | Transgenesis studies of the X-inactivation centre
Numerous experiments involving transgenesis have attempted to identify the
minimum region necessary to recapitulate the functions of the X-inactivation centre
(Xic). Single-copy Xist transgenes of up to 460kb are unable to trigger Xist
upregulation during differentiation of male embryonic stem cells (ESCs), either from
the transgene or from the endogenous Xic34. This shows that crucial Xic sequences
required to render cells competent for X-chromosome inactivation (XCI) are missing
from these large DNA fragments. Importantly, such single-copy transgenes can
trigger imprinted XCI when paternally inherited (see the main text), implying that Xic
sequence requirements are different between the two forms of XCI34,96,125.
Surprisingly, the lack of random XCI functions for single-copy transgenes can be
bypassed at least partially using multicopy arrays, which can initiate inactivation in
cis in male and female cells126,127. However, their capacity to trigger Xist expression
from the endogenous X chromosome is limited, and inactivation of the endogenous X
chromosome or the transgene is neither random nor exclusive in such lines32,34,128.
Importantly, female mouse ESCs were also reported to be unable to trigger Xist
expression from these single-copy transgenes, even though they are competent to
trigger random XCI of their endogenous Xchromosomes34,125. Thus, not only do such
ectopic single-copy Xic fragments lack sequences to efficiently trigger the
endogenous Xist allele(s) in trans, they also cannot respond to trans-activating
competence signals, such as RING finger protein 12 (RNF12; also known as RLIM)32,
originating from the endogenous Xchromosomes in XX cells. Most of these missing
elements still remain to be identified.

Intriguingly, contrasting outcomes have been reported

in two different studies concerning the effects of
homozygous deletion of Rnf12 in ESCs. In one case,
complete abrogation of XCI is reported30, whereas in
the other only a slightly reduced efficiency of XCI is
observed36. Whatever the cause of these differences,
RNF12 is clearly a key activator of Xist. It also has an
essential role during imprinted XCI, as will be discussed
later. However, additional XCI activation mechanisms
that can act in a partially redundant fashion during
random XCI must exist and remain to be identified.
Another element that has recently been implicated in
Xist activation in XX cells is the Jpx locus (also known
as Enox (expressed neighbour of Xist)) which is situated
immediately 5 to Xist37. This locus lies within a 120kb
region that is hyperacetylated in undifferentiated female
(but not male) mouse ESCs 38. Like many other loci
within the Xic, Jpx produces an ncRNA and has been
proposed to enable female-specific Xist activation, as its
heterozygous deletion impedes XCI initiation. However,
unlike Rnf12 transgenes, Jpx transgenes that are unlinked
to Xist do not activate endogenous Xist expression in
male ESCs32. How exactly Jpx, or its surrounding chromatin environment, influences Xist activation in female
cells remains to be determined. A recent study reported
that another long ncRNA Ftx (five prime to Xist) can
control the expression levels of Xist, Tsix and Jpx in
male cells. However, the role of Ftx in XCI remains to be
investigated in femalecells39.
In conclusion, it is becoming increasingly evident that
the competence of XX cells for Xist upregulation is not
mediated by one, but by several loci that act at the DNA
level and/or at the level of the proteins or RNAs that they
produce, with partially redundant activity. It should be
emphasized that, to date, no Xic sequence introduced as
a single extra copy in a male ESC has yet been reported
to induce XCI in a fashion that is reminiscent of normal

XX ESCs (BOX 2). Identifying the remaining unknown

Xic elements and factors, and understanding how
their interplay controls Xist expression, is an exciting
challenge for thefuture.

Monoallelic regulation of Xist during random XCI

During random XCI in mice, expression of Xist is
restricted to a single allele. This is the result of both
counting and choice (BOX1) and is influenced by regulatory elements within the Xic. Some of these elements are intimately linked to the regulation of XCI by
pluripotency factors and the XX competence-regulation
mechanisms describedabove.
The Xce locus. In female inbred mice, the two Xchromosomes have an equal chance of being chosen for
XCI. However, XCI choice can be biased by alleles at
the Xlinked Xcontrolling element (Xce), which maps
within Xic and causes non-random XCI in heterozygotes4042. At least three natural alleles of Xce have been
identified, based on skewed XCI patterns in Xce heterozygotes. Although Xce has been genetically mapped
to the region 3 to Xist43, its exact nature, location and
mechanism of action are still not known. Furthermore,
given the complexity of the Xist regulatory landscape,
Xce alleles may correspond not just to one, but to several
polymorphic controlling elements within this subregion
of the Xic4446.
Tsix-mediated repression of Xist. Analysis of targeted
deletions and studies with transgenes have revealed that
the region lying immediately 3 to Xist is essential for
correct monoallelic regulation of this gene. Heterozygous
deletion of a 65kb region 3 to Xist in female mouse ESCs
results in nonrandom Xist upregulation and inactivation
of the mutated Xchromosome47. Subsequent sequence
replacement48,49 and reinsertion50,51 strategies have shown
that the major Xist repressor in this region is its antisense
transcription unit, Tsix (FIG.1). Indeed, disruption of
Tsix transcription by a 3.7kb deletion encompassing its
promoter recapitulates the skewing observed with the
65kb deletion, although additional elements regulating
Xist may lie within this larger region48,50,51. Although
deletion of the Tsix promoter and enhancers leads to
skewed XCI of the mutated copy of the Xchromosome,
this does not enhance the overall expression level of
Xist50. Thus, antisense transcription appears to control
the binary decision of whether to upregulate Xist during differentiation. In fact, the ratio of sense/antisense
transcription across Xist seems to be crucial in determining which allele will be upregulated. When antisense
transcription is artificially driven across Xist, this prevents its upregulation in cis52,53. Conversely, enforced Xist
transcription is sufficient to induce preferential inactivation of the mutated chromosome, without altering Tsix
transcript levels54,55 (FIG.3Bc). However, Xist repression in
undifferentiated cells does not rely on Tsix alone. This is
because Tsix deletion does not lead to high-frequency
ectopic Xist activation before differentiation; rather,
it does so only after differentiation is induced, when
pluripotency-factor downregulation begins47,48,52,5658.

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REVIEWS

Dicer
An RNase III family
endonuclease that processes
dsRNA and precursor
microRNAs into small
interfering RNAs and
microRNAs, respectively.

CCCTC-binding factor
(CTCF). A highly conserved
DNA-binding protein with 11
zinc fingers that, in mammalian
genomes, binds to regulatory
elements such as insulators.

How does Tsix prevent Xist upregulation? Several

reports have now clearly established that antisense
transcription across the Xist promoter is accompanied
by modifications of its chromatin structure23,57,5963,
although the precise function of these chromatin
changes is unclear. Whether it is the act of transcription
or the Tsix RNA itself that participates in these chromatin changes is also still an open question. The possible
existence of XistTsix RNA duplexes and small RNAs
(~2542 nucleotides long) that match the Xist promoter
and the Arepeat region has been reported in differentiating mouse ESCs64, suggesting the potential involvement of an RNAi-like mechanism. However, although
a Dicer mutation was reported to increase Xist levels in
this study, more recent analyses have shown that this is
likely to be due to demethylation of the Xist promoter 65.
This demethylation is due to the downregulation of
microRNAs that regulate the DNA methyltransferase
3A (DNMT3A) when the microRNA machinery is
impaired65,66.
The repressive action of Tsix has also been proposed
to rely on competition for Xist-activating factors. In particular, the Xist Arepeat region, which is essential for
Xist RNA accumulation as well as for Xist-mediated
gene silencing in cis at the level of the Xist RNA has
also been reported to produce a 1.6kb non-coding transcript, RepA, independently of the main Xist promoter 67.
It has been proposed that this RepA transcript enhances
Xist expression via recruitment of the Polycomb repressive complex 2 (PRC2) and that Tsix RNA prevents Xist
activation by somehow competing with RepA RNA for
PRC2 recruitment 23,67 (FIG.3Bd). However, the exact role
that PRC2 serves in the activation of Xist is still unclear.
Indeed, it had been suggested that PRC2 also participates
in Xist repression in male cells, in synergy with Tsix62.
Additionally, PRC2 does not seem to be required for Xist
upregulation, as a lack of EED protein a key component of PRC2 does not prevent XCI initiation in
female embryos, nor in male Tsix mutants62,68. Thus, the
functional relevance of the connections among RepA,
PRC2 and the initiation of XCI remainsunclear.
Although the exact molecular mechanisms underlying the role of Tsix in regulating Xist are still not fully
understood, the genetic evidence suggests that antisense
transcription has a key role in the cis-regulation of Xist.
Importantly, however, Tsix heterozygous mutants do not
simply induce Xist expression more frequently from the
mutated chromosome. Somehow, inactivation of the Xist
wild-type Xchromosome also seems to be prevented, or
bypassed, owing to accelerated XCI on the deleted X
chromosome, which results in rapidly reduced RNF12
levels33. This finding highlights the fact that there must
be some mechanism to coordinate XCI between the
two chromosomes. Indeed, this coordination seems to
be lost in Tsix homozygous mutants, as an increased
number of cells activate Xist from both Xchromosomes4. The general picture that is emerging from such
studies is that Tsix plays a central part in the accurate
monoallelic expression of Xist during random XCI initiation. This, of course, begs the question of how Tsix
itself is regulated.

Regulation of Tsix expression. Perhaps surprisingly, the

Tsix core promoter is dispensable for basal transcription of this RNA and its deletion does not skew choice69.
Conversely, several critical regulatory elements of Tsix
have been defined. For example, the DXPas34 minisatellite, which was initially identified based on differential
methylation patterns of the active X chromosome versus
the Xi44,70, acts as an enhancer of the Tsix promoter in
reporter assays71. Furthermore, DXPas34 removal results
in loss of Tsix transcription and nonrandom Xist upregulation in mouse ESCs and mice56,69,72. This suggests that
loss of this regulatory element is responsible for the phenotypes observed with the larger deletions of the Tsix
promoter region that encompass the DXPas34 minisatellite47,48. Interestingly, transcription can be initiated at
DXPas34 (REF.69) and the 5 ends of some Tsix isoforms
map to the minisatellite73. In addition, several of the
transcription factors that have been shown to regulate
Tsix expression, such as CCCTC-binding factor (CTCF)
and its PcG co-factor YY1, as well as the stem-cell
factor reduced expression protein 1 (REX1; also known
as ZPF42), bind to DXPas34 (REFS31,74).
Another enhancer of Tsix, Xite (X-inactivation intergenic transcription element), lies 1015kb upstream
of the Tsix start site. Deletion of Xite results in mildly
skewed XCI and accelerated downregulation of Tsix
upon differentiation75. Several pluripotency factors have
been found to target Xite and their binding sites are necessary for Xite reporter constructs to be transactivated27.
These pluripotency factors include SOX2 (REFS27,28),
OCT4 (REFS27,28) andNANOG28.
In summary, Tsix is a regulator of Xist and is itself
regulated by several long-range cis-acting elements, as
well as by pluripotency factors (FIG.3A). Although mutations in Tsix or Xite on one Xchromosome can clearly
skew the choice of Xchromosome to be inactivated,
the question remains as to how asymmetry in Xist
expression patterns is established when two genetically
identical Tsix alleles arepresent.
Ensuring monoallelic expression of Xist. After Xist has
been upregulated and XCI triggered on one Xchromosome in female cells, repression of the second allele of
Xist (on the active X chromosome) is maintained by
DNA methylation of its promoter. This is supported by
the fact that impairment of both Dnmt3a and Dnmt3b
leads to ectopic Xist activation at late developmental
stages in both males in females76. However, the mechanisms ensuring that only one Xist allele is expressed at
the outset of XCI are still not fully understood. One
pathway that has been proposed to explain the asymmetric expression of Xist is a negative feedback loop
that involves Rnf12 (and other possible Xlinked Xist
activators)32 and is triggered by Xist expression itself 30
(for review, see REF. 77). Upon initiation of XCI on one
Xchromosome, rapid Xist RNA-mediated silencing of
Rnf12 would result in downregulation of the protein,
thereby diminishing the activating effect of RNF12 on
Xist transcription (FIGS3Bb,4a). This feedback model is
based on the hypothesis that Rnf12 (and other potential Xlinked XCI-promoting factors) must be rapidly

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REVIEWS
C 2QUV:%+HGGFDCEMNQQR

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5KUVGTEJTQOCVKFEQJGUKQPRCVVGTPU

Figure 4 | Models for monoallelic regulation of Xist.

Monoallelic Xist expression may be achieved through
several (not mutually exclusive) mechanisms. a | The
feedback model proposes that each Xchromosome
produces an X-chromosome inactivation (XCI) promoting
factor (or factors), which will activate Xist in a
dose-dependent fashion. Initiation of XCI will lead to the
downregulation of such a factor on one allele, bringing its
concentration below the threshold required to activate the
second Xist allele33,77. It has been proposed that RING finger
protein 12 (RNF12; also known as RLIM) is one such factor32.
To serve as a robust feedback mechanism, the
downregulation of the XCI-promoting factors would need
to occur very rapidly, before Xist activation on the second
allele. b | The X-inactivation centre (Xic) pairing model
proposes that physical interactions between the two
X-pairing regions (Xpr) may render the two Xchromosomes
competent for Xist expression. Subsequent
trans-interactions between homologous Tsix (Xist antisense
gene) and Xite (X-inactivation intergenic transcription
element) regions could enable monoallelic Xist
upregulation5,78,79 by promoting a symmetry-breaking event
between the two alleles139. This model was recently
supported by live-cell imaging of Tsix-pairing events
followed by Xist/Tsix RNA fluorescent in situ hybridization
(FISH)81. c | The stochastic and secondary selection-based
model proposes that each X chromosome has a low and
intrinsic probability of activating Xist. Because only cells
with one active X chromosome per diploid set of
autosomes can survive, this leads to counter-selection
against cells with two active or two inactive
Xchromosomes. Counter-selection may either involve cell
death or the ability to shift to an XCI pattern that is
compatible with cell proliferation33. Such a model requires
that cells remain competent for Xist activation for
numerous cell cycles85, a property that has not yet been
examined invivo (in peri-implantation mouse embryos).
d | Pre-emptive states corresponding to different
propensities for Xist activation have been proposed to exist
prior to XCI. In this model each chromosome can alternate
between these states, which have been proposed to
involve alternative structural configurations at the level of
sister chromatid cohesion between the Xchromosomes82.

0CVWTG4GXKGYU^)GPGVKEU

downregulated by Xist RNA to avoid activation of the

second Xist allele (FIG.4a).
Transient homologous Xic pairing has also been proposed to play a part in the coordination and asymmetric
treatment of the two Xics during initiation of XCI (FIG.4b).
Indeed, initial XicXic pairing events mediated by Xpr
are followed by pairing at Tsix/Xite region in differentiating XX ESCs at the moment of Xist upregulation5,78,79.
Although pairing at Tsix/Xite is clearly not necessary for
Xist activation78,79, several observations indicate these
events may be linked to the monoallelic regulation of
Xist. First, Tsix/Xite trans-interactions occur at the time
of Xist activation78,79. Second, Tsix deletions, which have
been shown to skew choice, also abrogate pairing at the
Tsix/Xite region78,79. Third, single-copy transgenes of
the region containing Xist, Tsix and Xite that are unable
to activate Xist either in cis or in trans are also unable to
associate with the endogenous Xic78. Last, ectopically
provided Tsix and Xite multicopy arrays can mediate

efficient pairing with the endogenous Xic regions and

inhibit XCI in females4,79,80. Insights into the events
immediately downstream of pairing have recently been
obtained using live cell imaging of tagged Tsix loci81. Tsix
expression was found to become transiently monoallelic
after separation of the loci, thus providing a window of
opportunity for Xist upregulation in cis to the silent Tsix
allele. Depletion of CTCF and OCT4 by knockdown,
as well as transcriptional inhibition, has been shown
to disrupt Tsix/Xite pairing and perturb Xist expression. However, it is unclear whether the Xist deregulation observed is directly due to the disruption of these
chromosomal interactions or due to other effects27,80.
The mechanisms that drive pairing, and its exact role
(or roles) in XCI, remain to be precisely elucidated.
Another mechanism that has been proposed to
account for monoallelic Xist upregulation is that Xist
activation would occur stochastically at a low frequency
and that this would be followed by a counter-selection of

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REVIEWS
cells that have not established XCI correctly during differentiation. This means that they have either inactivated
both Xchromosomes or neither Xchromosome, both of
which would be deleterious situations with aberrant X
chromosome dosage (FIG.4c). However, although some
degree of selection following inaccurate XCI may occur
during invitro differentiation of mouse ESCs4,33, it rarely
seems to occur invivo in mice (C. Corbel, I. Okamoto
and E.H., unpublished observations).
A final proposed model is that, before random XCI,
the two Xic loci are differently poised for Xist activation (FIG.4d). Indeed, in undifferentiated mouse ESCs,
the two Xchromosomes exist in alternative and
alternating structural states at the level of their sister
chromatid cohesion82. These states appear to be anticorrelated between the two Xchromosomes in the same
cell. Furthermore, chromosome-wide patterns of asynchronous sister chromatid cohesion states are altered
by mutations within Xist or Tsix. However, the basis for
these coordinated, alternating states and their possible
role in random monoallelic choice during XCI remain
to be determined.
In conclusion, multiple models have been proposed
for the asymmetric expression and monoallelic regulation of Xist. These are not mutually exclusive; in fact,
they may be exploited at different levels and to varying
extents, in order to accomplish appropriate Xist and
XCI patterns during development. Indeed, in different species, some of these routes to achieve monoallelic XCI may be exploited more than others, as will be
discussed later.

Eutherians
Mammals in which the
development of progeny
takes place in the mothers
body thanks to the placenta,
a fetal membrane that
facilitates nutrient and waste
exchange between the fetus
and the mother.

Meiotic sex chromosome

inactivation
(MSCI). Silencing and heterochromatinization of sex
chromosomes in the male
germ line during meiosis.

Androgenetic
Androgenetic embryos are
produced by the fusion of two
haploid paternal genomes.

Parthenogenote
A uniparental embryo
produced by the development
of an unfertilized egg.

Gynogenote
An embryo produced by
the fusion of two haploid
maternal genomes.

Impact of the X/autosome ratio on XCI

Experiments involving triploid and tetraploid embryos
demonstrated that the number of inactive Xchromosomes seems to depend on autosomal ploidy, with the
majority of cells retaining one active X chromosome per
diploid set of autosomes83,84 (see Counting in BOX1).
More recently, kinetic measurements revealed that, for
the same number of Xchromosomes, Xist upregulation
happens more rapidly in differentiating mouse ESCs with
a high X/autosome (X/A) ratio than in cells with a low
X/A ratio85. This suggests that autosomal factors directly
regulate the probability of Xist activation; secondary
selection is, however, clearly measurable at later stages
in these cell populations. What could the nature of these
autosomal counting factors be? Given that most pluripotency factors are autosomal, it is tempting to speculate
that they might be part of this X/A counting mechanism.
The exact nature of counting still remains mysterious
and it should be noted that all of the Xic mutations so far
proposed to affect counting have only been investigated
in diploid cells. Therefore, they have not been tested
for their sensitivity to autosomal dosage, which is how
defects in X/A counting should be assessed.
The regulation of imprinted XCI
Although random XCI is believed to be the norm in
eutherians, in mice XCI is initially subject to imprinting
during pre-implantation development, with exclusive
inactivation of the Xp (FIG.2). At the time of zygotic

genome activation (ZGA), both X chromosomes

are active but the Xp rapidly initiates XCI following
imprinted Xist expression from the 24 cell stage
onwards8688. XCI seems to be complete by the blastocyst
stage. Imprinted inactivation of the Xp is maintained in
the extra-embryonic tissues but is reversed in the ICM,
where random XCI subsequently takes place8687.
How is this imprinted form of XCI controlled? A
robust maternal imprint that prevents inactivation of the
Xm exists in mice, given that XCI does not occur in Xm
disomies, leading to early lethality owing to defects in
extra-embryonic development 8991. Xist is clearly essential for imprinted XCI, as a deletion of the paternal Xist
allele also leads to early lethality 8. However, it has been
proposed that the Xp may also be predisposed to silencing due to its heterochromatinization in the XY body
during meiotic sex chromosome inactivation (MSCI) in
the male germ line92,93. In support of this, a recent study
suggested that some genes on the Xp may be silenced
independently of Xist during cleavage stages94. However,
a subsequent study came to the conclusion that Xist is in
fact required for initiation of XCI of Xlinked genes95,
although silencing of repetitive elements on the Xp may
persist independently of Xist from the male germ line into
the zygote on the Xp. The demonstration that Xist is sufficient for the initiation of imprinted XCI in mice came
from a study96 showing that autosomal Xist transgenes
can initiate imprinted cis-inactivation independently
of MSCI when they are paternally transmitted.
How then is Xist regulated during early mouse
embryogenesis? Contrary to the situation for random
XCI, Xist expression is strictly dependent on parental
origin immediately after fertilization. Whereas Xist
is exclusively transcribed from the paternal allele, the
maternal allele of Xist is never expressed during early
pre-implantation development 97100. Importantly, paternal Xist expression occurs regardless of Xchromosome
number, unlike the situation during random XCI. For
example, in androgenetic XX embryos, Xist RNA coating
of both X chromosomes is seen, although this is resolved
to monoallelic Xist expression by the blastocyst stage101.
Furthermore, XO androgenotes also initiate Xist RNA
coating, but this later disappears. Conversely, parthenogenotes (XmXm) show a complete absence of Xist
expression up to the morula stage, after which some
Xist upregulation is observed102,103. Taken together, these
data suggest that only the paternal but not the maternal Xist allele can respond to the transcription factor
environment present in cleavage-stage mouse embryos.
Recently, the maternal pool of RNF12 was shown to be
essential for paternal Xist expression, as imprinted XCI
is not initiated in Rnf12+/female embryos derived from
Rnf12deficient oocytes36.
What prevents Xist expression from the Xm in cleavagestage mouse embryos? Evidence that a repressive imprint
is deposited during egg maturation came from the observation that an Xchromosome derived from non-growing, rather than fully grown, oocytes can be inactivated
in gynogenotes104. However, the nature of this imprint is
still unknown. Unlike many autosomal imprinted loci,
Xist imprinting does not seem to rely on differential

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REVIEWS
gametic DNA methylation105. Given the transient nature
of this Xist imprint (it is reversed by the morula or early
blastocyst stage), it may rely on the different chromatin states of the two parental genomes that have been
reported to exist during early embryogenesis106, or else
on a maternally bound repressor that becomes gradually diluted. Whatever its nature, the imprint must lie
either within Xist or close to it, because paternally or
maternally derived 210 kb, single-copy, Xist-containing
autosomal transgenes show correctly imprinted Xist
expression patterns. Tsix does not appear to represent
the maternal repressive Xist imprint that is present only
in cleavage-stage embryos, as it is not expressed until
the morula stage, well after paternal Xist expression
and XCI have initiated49,56 (FIG.2). However, Tsix does
seem to be required later to maintain the silent state of
the maternal Xist allele in extra-embryonic male and
female tissues107.
In conclusion, although the imprinted form of XCI
shares certain conserved features with the random form
in the epiblast (such as the implication of Xist and its
activation by RNF12), the two processes appear to differ
substantially in the mechanism by which Xist is monoallelically controlled. The initiation of imprinted XCI in
pre-implantation embryos bypasses the requirement
for sensing, counting and choice by ensuring parent-oforigin-specific Xist expression through imprinting. By
the morula or early blastocyst stage, however, this imprint
appears to be lost or ignored and the sensing, counting
and choice mechanisms that characterize random XCI
seem to be enabled (FIG.2a). Importantly, a recent study
has shown that in rabbit and human embryos, contrary to the situation in mice, Xist is not imprinted108.
Furthermore, this study showed that, unlike mice, both
Xist alleles can be activated, often simultaneously, during
early embryogenesis in these species. This suggests that
very different strategies of Xist regulation are deployed
in other mammals, with a lack of imprinting and with a
choice of X chromosome to inactivate occurring downstream of Xist upregulation108. This highlights the evolutionary diversity of X-inactivation initiation mechanisms
in mammals and suggests that imprinted XCI via an Xist
imprint may have evolved specifically in mice.

Reprogramming of the inactive Xchromosome

During mouse pre-implantation embryogenesis, the
inactive Xp is specifically reactivated in the ICM at
the blastocyst stage86,87. Another round of Xi reactivation also occurs following random XCI in the female
germ line just before meiosis109,110 (FIG.2a). Reactivation
of the Xi can also be induced when somatic cells are
fused to ESCs, as well as during cloning, somatic cell
nuclear transfer (SCNT) and induced pluripotency. An
important question is whether reprogramming is simply
a mirror image of the steps involved in the initiation of
XCI, or do other pathways come into play?
One key difference in these two reprogramming
events is that the epigenetic status of the Xi is probably less firmly locked-in, in the ICM of the blastocyst, compared to primordial germ cells of the embryo.
However, in both cases, loss of Xist expression seems to

be a very early event. How is Xist repression achieved?

In the blastocyst, Tsix is expressed from the Xp in ICM
cells. However, it is not required to reprogramme the
Xist locus as deletion of Tsix does not interfere with Xist
downregulation in the ICM58. Conversely, Xist repression from the Xp coincides with the increase in NANOG
expression86 and Nanog mutants do not show loss of
PRC2 recruitment, a marker of the inactive state, on the
Xp111. This argues for a role for NANOG in Xp reactivation in the ICM. Similarly, a role for pluripotency factors during X chromosome reactivation in the germ line
has also been proposed, based on the early expression
of OCT4 in primordial germ cells109,110,112, although this
remains to be demonstrated formally. Thus, loss of Xist
expression seems to be an early event in both waves of
reprogramming that occur invivo. However, the manner in which the reversal of repressive chromatin marks,
nuclear reorganization of the Xi and gene reactivation are
achieved remains to be defined. Whether similar mechanisms are exploited in both of these reprogramming
waves also remains to beseen.
The Xi can also be reactivated when somatic cells are
treated with a cocktail of factors, including some pluripotency factors, to generate induced pluripotent stem
cells113. However, the degree of reprogramming is often
incomplete, as many genes still remain silent despite
partial loss of Xist RNA coating and PcG-associated
histone H3 lysine 27 trimethylation (H3K27me3)114. In
cloning experiments, it has been shown that the inactive state of the previously inactive X chromosome is
preserved to some extent in pre-implantation stages and
extra-embryonic tissues, suggesting that reprogramming of Xist and reactivation of Xlinked genes is not
fully accomplished during SCNT115,116. The fact that at
subsequent stages, in post-implantation SCNT embryos,
XCI is found to be random presumably indicates that,
in the ICM, the inactive state of the previously inactive
somatic X chromosome is completely reset 115. This is as
expected, given the capacity of the ICM to reactivate the
Xp during normal mouse development. Interestingly, in
pre-implantation SCNT embryos, the silent Xist allele
on the previously active somatic Xchromosome is often
aberrantly upregulated116. This aberrant Xist upregulation on the previously active X chromosome, as well
as Xist expression on the previously inactive X chromosome, presumably results in aberrant XCI of both
Xchromosomes and has a deleterious effect on early
development. A demonstration of this came from a
study of Xist deletion, which resulted in dramatically
increased cloning efficiencies117. Finally, it should be
noted that substantial species differences may exist in
the status of the X chromosome in the ICM and possibly
in ESCs and in induced pluripotent stem cells. A recent
study revealed that, in rabbits, XCI initiates in the ICM
rather than X-reactivation occurring in the ICM as happens in mice108. The situation is even more surprising
in humans, where XIST RNA is clearly expressed in the
ICM but the X chromosome appears to remain globally
active108. Thus, the regulation of XIST by pluripotency
factors and reprogramming events in blastocysts may
be strikingly different between mammals.

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REVIEWS
Perspectives
Our understanding of the molecular events underlying
the initiation of XCI has increased substantially over the
past decade. It is now apparent that Xist and its regulators
are embedded in a network of stem-cell-specific factors,
the interplay of which will require genetic and biochemical studies in order to be elucidated. The ESC model
system for random XCI should allow this goal to be
achieved in the near future. However, a full understanding of the regulation of imprinted XCI remains a challenge, as no invitro model exists for the initiation of this
process. Biochemical approaches in ESCs will hopefully
shed some light on the molecular partners and targets of
RNF12 and its role as an XX dosage-sensitive Xist activator. However, it is clear that other dosage-dependent
activators exist and will hopefully be identified using
genetic screens and transgenesis techniques.
Despite the recent exciting advances in our understanding of Xist regulation, there are still many gaps
in our knowledge of the repertoire of cis- and transacting elements involved in regulating the onset of
XCI. Much is now known about Xist and its antisense
transcript Tsix; however, the full extent of the Xic still
remains to be defined. Indeed, the regulatory landscape
of Xist that is emerging is highly complex and careful
genetic dissection of long-range regulatory elements
will be required. Furthermore, the discovery that transacting factors such as RNF12 are encoded at locations
so closely linked to Xist begs the question of whether

1.

2.

3.
4.
5.
6.
7.

8.
9.

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all Xlinked loci that are involved in XCI process will

be close to, or part of, theXic.
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evolutionary tinkering that can be used to achieve the
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Acknowledgements

We would like to thank all of our colleagues in the Mammalian

Developmental Epigenetics team for their discussions and
input and extend our apologies to any authors whose work
could not be discussed owing to space constraints. Our work is
supported by the FRM, ANR, ARC, the EU Integrated projects
HEROIC and SYBOSS, EU Networks of excellence Epigenome
and Epigenesys, as well as an ERC Advanced Investigator
award.

Competing interests statement

The authors declare no competing financial interests.

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