Available online at www.sciencedirect.
com
Origin and evolution of X chromosome inactivation
Joost Gribnau and J Anton Grootegoed
Evolution of the mammalian sex chromosomes heavily impacts
on the expression of X-encoded genes, both in marsupials and
placental mammals. The loss of genes from the Y chromosome
forced a two-fold upregulation of dose sensitive X-linked
homologues. As a corollary, female cells would experience a
lethal dose of X-linked genes, if this upregulation was not
counteracted by evolution of X chromosome inactivation (XCI)
that allows for only one active X chromosome per diploid
genome. Marsupials rely on imprinted XCI, which inactivates
always the paternally inherited X chromosome. In placental
mammals, random XCI (rXCI) is the predominant form,
inactivating either the maternal or paternal X. In this review, we
discuss recent new insights in the regulation of XCI. Based on
these findings, we propose an X inactivation center (Xic),
composed of a cis-Xic and trans-Xic that encompass all
elements and factors acting to control rXCI either in cis or in
trans. We also highlight that XCI may have evolved from a very
small nucleation site on the X chromosome in the vicinity of the
Sox3 gene. Finally, we discuss the possible evolutionary road
maps that resulted in imprinted XCI and rXCI as observed in
present day mammals.
Address
Department of Reproduction and Development, Erasmus MC, University
Medical Center, Rotterdam, The Netherlands
Corresponding author: Gribnau, Joost (j.gribnau@erasmusmc.nl)
Current Opinion in Cell Biology 2012, 24:397–404
This review comes from a themed issue on
Nucleus and gene expression
Edited by Asifa Akhtar and Karla Neugebauer
Available online 14th March 2012
0955-0674/$ – see front matter
# 2012 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2012.02.004
Introduction
Meiotic recombination keeps the autosomes organized
in homologous pairs, stabilizing the diploid genome of
mammalian species. For one pair of autosomes, this
advantage of the diploid state was partly lost, when this
pair of autosomes started to become the present mamma-
lian X and Y chromosomes around 160 million years ago,
shortly before separation of the metatherians (marsupials)
and eutherians (placental mammals) [1,2]. The initial
event probably has been a mutational change of one allele
of the Sox3 gene, resulting in the male sex-determining
gene Sry (sex-determining region Y) on the proto-Y of this
www.sciencedirect.com
chromosome pair (Figure 1a) [3]. It is thought that the
region around Sry gained male beneficial genes and other
modifications such as inversions, step by step limiting the
options for meiotic recombination between the evolving
heterologous sex chromosomes. As a result, the male
specific region of Y (MSY), clonally inherited from father
to son, never meets a meiotic pairing partner. The X
chromosome, however, spends two-third of its time in
XX females, where it pairs and recombines in meiotic
prophase of oogenesis. Further to the advantage of the
X chromosome, X-linked genes are hemizygous in males,
leading to rapid fixation of mutations associated with
positive natural and sexual selection. In anthropomorphic
terms, an arms race is going on between X and Y, resulting
in regression of the Y and enrichment of the gene content of
the X. Many of the events shaping the heterologous sex
chromosomes have occurred within a relatively short time
span after formation of the proto-X and proto-Y, before
radiation of the placental mammals [4–6]. Originating from
the spectacular evolutionary history of these particular
chromosomes and its consequences, the X and Y chromo-
somes of present-day placental mammals undergo marked
dynamic changes of their activities in development and
gametogenesis. These activity changes have a biological
function to support growth and fertility of both females and
males. In the present review, we aim to outline the overall
scheme of events and mechanisms, although we focus on X
chromosome inactivation (XCI) in female somatic cells.
Sex chromosomes and the need for gene
dosage compensation
The human X chromosome carries more than 1000 genes
[7]. By contrast, only around 100 single-copy and multi-
copy genes are found on the human Y chromosome,
where the MSY carries 78 genes encoding 27 different
proteins [8]. Hence, in diploid male somatic cells, there is
an X:autosome gene dosage imbalance for most genes on
the X. Problems resulting from this imbalance can be
prevented by a two-fold transcriptional upregulation of
the X chromosome. Indeed, this has been observed for
mouse and human, by comparing micro-array expression
data sets [9–11]. Another study, using an RNA-sequen-
cing approach, challenged these findings [12], but the
controversy is now explained [13,14]. When genes with
low transcriptional activity are excluded from the analysis,
it is observed that the moderately to highly expressed X-
chromosomal genes are two-fold upregulated [13,14].
In addition to excluding biological noise from leaky gene
expression, genes expressed at stochastically fluctuating
low levels may not require dosage compensation. How-
ever, genes expressed at higher levels will include dose
sensitive genes. From this, we suggest that evolutionary
Current Opinion in Cell Biology 2012, 24:397–404
398 Nucleus and gene expression

Figure 1 genes in female mammals is prevented by a second

compensatory mechanism, called XCI, which leads to
(a) degeneration of the Y inactivation of one of the two X chromosomes, resetting
the global X:autosome gene expression ratio at 1:1 in
recombination female cells (Figure 1c). Looking at the end result, XCI
block
can be viewed as a mechanism which equalizes X-chro-
Sox3 Sox3 Sox3 Sry male mosomal gene dosage between males and females.
beneficial
genes
X chromosome inactivation: cis and trans
proto mechanisms
autosomes X Y X Y X Y
In the female mouse embryo, an imprinted form of XCI
160 million years ago now
(iXCI) is initiated very early during pre-implantation
development, around the 4–8 cell stage, always targeting
(b) two-fold up-regulation in male
the X inherited from the father [16,17]. Following iXCI,
2x 2x this paternal X (Xp) remains inactive in the extra-embryo-
2x nic tissues, but is reactivated in the developing inner cell
mass that gives rise to the embryo proper [18]. This
proto reactivation asks for rapid intervention, which comes from
autosomes X Y X Y X Y
a strong wave of random XCI (rXCI), targeting either the
maternal X (Xm) or Xp, just after implantation. The
(c) X inactivation in female fascinating history of the discovery of rXCI, spearheaded
by Susumu Ohno and Mary Lyon around half a century
Xist Xist RNA
ago, is highlighted in several recent reviews [19–21].
2x 2x
Regarding the mechanism of rXCI, genetic studies per-
formed in the 1980s, involving X-to-autosomal transloca-
proto tions and X truncations, indicated a region of 3
autosomes X X X X Xa Xi megabases on the X, called the X inactivation center
Current Opinion in Cell Biology (Xic), instrumental in the initiation of rXCI [22]. Sub-
sequent studies focusing on this region revealed a non-
Evolution of mammalian sex chromosomes. (a) Degeneration of the Y coding gene, Xist in mouse and XIST in human, as the key
chromosome was triggered by changes on the Y, including the origin of
player in the XCI process [23–25]. Xist transcription is
Sry and the inclusion of male beneficial genes in a non-recombining
region. (b) The loss of Y-encoded genes was compensated by a two- upregulated on the future inactive X, and the spliced and
fold upregulation of expression of dose sensitive homologous X- poly-adenylated non-coding RNA molecules spread in cis,
encoded genes. (c) A two-fold upregulation of X-encoded genes would thereby recruiting chromatin remodelling complexes that
be lethal to female cells, and this has driven the evolution of XCI that render the X an inactive chromatin domain (reviewed in
silences one X chromosome in every female cell.
[26]). From what is known for mouse, Xist activation is
counteracted by the non-coding Tsix gene, which fully
overlaps with Xist but is transcribed anti-sense to Xist [27].
selection has employed mechanisms to keep the X:auto- To repress Xist transcription, transcription of Tsix needs to
some gene expression level at a ratio near 1:1, concomi- proceed through sequences overlapping with the Xist
tant with the evolution of the X and Y chromosomes, promoter [28]. This points to a transcriptional interfer-
leading to a global two-fold transcriptional upregulation ence mechanism, but the repression may also involve
of the X chromosome (Figure 1b). If the mechanism RNA-mediated recruitment of de novo methyltransferase
leading to this two-fold upregulation has become an DNMT3A [29,30]. With this basic machinery in place,
inherent and sex independent property of the X chromo- the hunt was on to find elements, genes and factors able to
some, female cells encounter a 2:1 X:autosome gene exert control over Xist and Tsix, in cis or in trans.
expression ratio. In some species, this is tolerated, as
for instance in the red flour beetle, Tribolium castaneum, Genuine promoter and enhancer sequences control Xist
where increased expression of both X chromosomes is and Tsix transcription, but flanking non-coding genes also
observed in XX female cells [15]. In mammals, the play an important role in activation of both genes.
developing oocyte contains two active X chromosomes, Located upstream of Tsix, the genes Xite and Tsx posi-
and a certain level of tolerance might also be found at tively regulate Tsix expression (Figure 2) [31,32].
some later steps of development and in specific tissues Chromosome conformation capture (3C) studies
and cell types. However, a general 2:1 X:autosome gene suggested that Xite and Tsx interact with Tsix within an
expression ratio is not tolerated by any of the mammalian active chromatin compartment or hub (ACH) [33]. Like-
species for which this was studied thus far. Overexpres- wise, the non-coding genes Jpx and Ftx, located upstream
sion of ‘male-dosage-compensated’ X-chromosomal of Xist, act as positive regulators of Xist (Figure 2) [34,35].

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 X chromosome inactivation Gribnau and Grootegoed 399

Figure 2

(a) cis-Xic (b) trans-Xic

RNF12
+ + +
boundary
mouse Xist Jpx Ftx Cnbp2 Xpr Rnf12 Xist Jpx FtxCnbp2 Xpr Rnf12
cen tel
Tsix Xite Tsx Tsix Xite Tsx
– – –
+ OCT4 OCT4
+ SOX2 SOX2
+ OCT4
SOX2 NANOG NANOG
Tsix ACH Xist ACH OCT4 CTCF
C REX1
SOX2 YY1
Y
KLF4 R
REX1
cis-Xic cMYC
KLF4
trans-Xic trans-Xic
Current Opinion in Cell Biology

The X inactivation center: cis and trans. (a) The cis-acting X inactivation center (cis-Xic) encompasses all cis-acting elements regulating Xist and Tsix.
The cis-Xic includes non-coding genes located upstream of Xist and Tsix, which are involved in setting up two active chromatin hub (ACH) structures.
(b) The trans-acting Xic includes all X-linked elements and genes that act in trans to activate Xist. Parts of the trans-Xic most probably are found at
more regions of the X, and the trans-Xic contains all XCI-activator genes including Rnf12, which seems to be a more potent XCI-activator (thick line)
than Jpx and Xpr (thin line) and the putative XCI activator Ftx (dashed line). Also shown are the binding sites, and the mode of action, of autosomally
encoded inhibitors of XCI.

This would be in agreement with Ftx, Jpx and Xist setting
up an Xist ACH, neighbouring the Tsix ACH (Figure 2a).
From the above it follows that transcription of the key
genes Xist and Tsix additionally depends on an interplay
in cis with upstream non-coding genes, within two neigh-
bouring chromatin domains. The Xist ACH and Tsix ACH
are separated by a boundary element marked by a CTCF
binding site located just downstream of Xist. Indeed,
removal of this CTCF binding site blocks Xist induction
[36], possibly because both ACHs have accidently
merged, although this needs further investigation. Regu-
lation of Xist and Tsix transcription within the respective
ACHs may involve direct inter-genic promoter and
enhancer interactions, but the chromatin environment
may facilitate recruitment of factors implicated in tran-
scription initiation also by a mass action mechanism. It is
anticipated that the two ACHs are not engaged in com-
petition for proper spatial folding, but rather act as rela-
tively independent domains to promote transcription of
either Xist or Tsix. We suggest that the two chromatin
domains represent the cis acting machinery, determining
the probability that XCI is initiated. This probability has
been investigated in particular for rXCI, in relation to the
identification of trans-acting factors.
Trans-acting factors are probably taking part in control of
rXCI, to create regulatory mechanisms for counting and
initiation. Trans-acting activators would promote XCI
through activation of Xist or repression of Tsix, being
counteracted by trans-acting inhibitors [37]. In this model,
the activities of both the activators and the inhibitors are
expected to be highly dose-dependent. With activators
being encoded by X-linked genes, in contrast to inhibitors
of XCI that are autosomally encoded, the cell would obtain
a build-in stop mechanism to prevent inactivation of all X
chromosomes. Embryonic rXCI is forcefully initiated in
female cells only, which have a two-fold higher gene dose of
X-encoded activator(s), allowing the XCI-activator activity
to reach a threshold level required to overcome repression
by the XCI-inhibitors. Although allelic properties of the
two X chromosomes will play a role, the initiation of rXCI
on either one of the two X chromosomes in principle is a
stochastic event. Once rXCI is silencing one of the two X
chromosomes, the ensuing rapid downregulation of XCI-
activator gene(s) in cis should prevent initiation of rXCI on
the second X. Mutual exclusive initiation of rXCI may be
facilitated by spatial interaction between the two X
chromosomes, involving Tsix, Xite and Xpr (X pairing)
sequences [38–40], although a causal role for a transient
direct interaction still needs to be established.
In the developing embryo proper, rXCI does not start
before the onset of cell differentiation. Hence, it makes
biological sense that, in addition to the ubiquitously
expressed proteins CTCF and YY1, the most important
XCI inhibitors identified thus far happen to be the key
pluripotency and reprogramming factors OCT4, SOX2,
NANOG, REX1, KLF4 and MYC ([41–43,44];
reviewed by [45]). These factors are recruited to different
regions involved in repression of Xist, or activation of Tsix
or Xite (Figure 2b). By contrast, only few XCI-activators
have been identified. X-encoded RNF12 appears to be a
very potent activator of Xist, although the mechanism of

www.sciencedirect.com Current Opinion in Cell Biology 2012, 24:397–404

400 Nucleus and gene expression
action of this E3 ubiquitin ligase needs to be resolved in
more detail [46,47]. Rnf12 is located 500 kb upstream of
Xist, and this close proximity to the source of Xist RNA
production, in combination with a short half-life of
RNF12, is thought to guarantee a fast and stringent stop
mechanism of XCI-activator production once rXCI has
started. Analysis of Rnf12+/ and Rnf12 / female ES
cells showed reduced initiation of rXCI upon differen-
tiation, although this effect seems to be influenced by the
genetic background [47,48]. The Rnf12 knockout
mice indicated that RNF12 is essential also for iXCI
[48]. It is concluded that RNF12 is essential for both
iXCI and rXCI, but that more activators of XCI are
present, such as the candidate XCI-activators Jpx and
Ftx. Knockout and rescue studies have implicated a role
for Jpx RNA in trans, but also uncovered important cis
effects [35]. So far, regulation of XCI by Ftx was studied
only in male cells, which did not allow discrimination
between cis and trans effects. Rnf12 transgenic male ES
cell lines showed a robust XCI phenotype of the single X
present in these cells, reinforcing the action of RNF12 in
trans. However, male cells carrying Jpx, Ftx, Xpr or Tsx
transgenes did not show ectopic induction of XCI [46]. It
is quite possible, therefore, that Jpx, Ftx, Xpr and Tsx
exert their effect on XCI mainly in cis [46].
Taken together, the present model points out that XCI, as
investigated mainly for rXCI, is regulated by trans-acting
activators that modulate Xist and Tsix directly or through
the two neighbouring cis-acting chromatin domains, the
ACHs, separated by a boundary element. Composition
and structure of these domains is a key determinant in
expression of Xist and Tsix. The Xic can be viewed as a
functional entity, but quite a large and complex entity,
composed of a cis-acting Xic (cis-Xic) and a trans-acting
Xic (trans-Xic). Next to Xist and Tsix, the cis-Xic includes
all cis regulatory regions involved in regulation of Xist and
Tsix directly or by modulating the activity of the ACHs.
Polymorphisms of the DNA incorporated into the ACHs
might affect their distinctive activities, leading to skewed
XCI on a hybrid genetic background. Skewed XCI in
mouse is genetically linked to the X choosing element
(Xce) locus. The Xce has been mapped to a 1.8 megabase
region including Xist [49], and may represent any nucleo-
tide change in the cis-Xic that extends into the Xist-ACH.
The requirement for a feedback stop mechanism may
have promoted the evolutionary selection of genes encod-
ing XCI-activators in close vicinity to the cis-Xic, as found
for Rnf12, but this is probably not a rule of thumb.
Temporal spreading of global silencing from the Xic
region is not strictly linear along the X [11], which would
allow the trans-Xic to be located at various sites on the X.
The abovementioned early genetic studies defining the
Xic indicated that the most important sequences control-
ling XCI are retained within a 3 megabase region, but
this does not exclude that some genes contributing to a
robust threshold of XCI-activator total activity are located
Current Opinion in Cell Biology 2012, 24:397–404
at a larger distance, so that the trans-XIC may include
various regions of the X chromosome.
Evolution of XCI
Upon formation of the mammalian proto-X and proto-Y by
mutational change of Sox3 giving rise to the Sry gene with
male-specific expression, the early non-recombining
region must have included Sox3 and probably some neigh-
bouring genes on the brand-new X chromosome. At a high
dose, SOX3 is still capable of mimicking the action of SRY
in triggering transcription of Sox9, the immediate down-
stream gene that is required for testis formation [3].
Hence, Sox3 is a dose sensitive gene, which may have
experienced transcriptional upregulation from the proto-
X, leading to an ensuing need for dosage compensation
specifically in female cells. Perhaps, this represents the
evolutionary origin of XCI, where the genes that are
mechanistically involved in XCI have evolved in a small
region around Sox3 (Figure 1c). The non-coding Xist gene
originates, at least partly, from the Lnx3 gene [50], encod-
ing a member of an old family of E3 ubiquitin ligases that
dates far back to a metazoan ancestor [51]. The transcribed
and processed Xist RNA gained a completely new func-
tion, not related to the function of Lnx3. Spreading of Xist
RNA on the early X may have been very localized,
reminiscent of the action of the non-coding RNAs
Kcnq1ot1 and Air, which are transcribed from one parental
allele of imprinted autosomal loci and mediate silencing of
a region 300 kb around the Kcnq1ot1 and Air genes,
respectively [52,53]. XCI may have evolved from such a
local silencing, step by step extending into larger regions
of the X chromosome during eutherian evolution, by
recruiting more factors to participate in the process.
From the model discussed above, it is anticipated that
several genes involved in the control of XCI might have
been present in close proximity to Sox3 right from the start.
Are Xist and Rnf12 located in close proximity to Sox3, in
placental mammals? When examined for human, mouse
and cow, this is not the case (Figure 3a), but when we
searched for the chromosomal location of Lnx3 relative to
the locations of Sox3 and Rnf12 in birds, we found these
three genes close together, on chromosomes 4A and 4 of
zebra finch and chicken, respectively (Figure 3a). The
syntenic region including these genes was on the autosome
pair that became the proto-X and proto-Y of mammals [2],
so that this finding would be in agreement with the present
hypothesis of the evolutionary origin of XCI in the vicinity
of Sox3. Possibly, orthologs of the oldest activators of XCI
in placental mammals, other than Rnf12 and still unknown,
remain to be found close to Sox3 on chicken chromosome 4
(or on zebra finch chromosome 4A).
In marsupials, who have maintained Lnx3 and hence lack
Xist, imprinted inactivation of the paternally inherited X
chromosome (iXp) is the only form of XCI, at all stages of
development and in all tissues. However, the imprinted
www.sciencedirect.com
 X chromosome inactivation Gribnau and Grootegoed 401

Figure 3

(a) Xist Rnf12 Sox3

human chr. X
Tsix
0,7mb 68mb

Sox3 Xist Rnf12

mouse chr. X
48mb Tsix 0.5mb

0,7mb Tsix

cow chr. X
Sox3 55mb Rnf12 Xist

Lnx3 Rnf12

zebra finch chr. 4A

0,1mb 1.2mb Sox3

Lnx3 Rnf12

chicken chr. 4
Sox3 1.7mb 0,1mb

(b) 200 mya 180 160 140 now

monotremes

iXp
marsupials

iXp?

Sox3 -> Sry Lnx3 -> Xist

placentals
genomic imprinting
(Lnx3 imprinted?) rXCI

?
iXp-Xist? iXCl
?
Current Opinion in Cell Biology

Evolution of X chromosome inactivation. (a) Sox3 is located at a quite large distance from Rnf12 and Xist on the X chromosome of mammals. By
contrast, Lnx3, the ancestral gene giving rise to Xist, is located in close proximity of Rnf12 and Sox3 on the X syntenic region of chromosomes 4A and 4
of zebra finch and chicken, respectively. (b) Timing of the evolution of Sry, Xist, imprinted inactivation of Xp and rXCI. The evolution of imprinted
inactivation of Xp may have occurred at least two times: (1) iXp in the marsupial lineage; and (2) iXp-Xist in the eutherian lineage, based on imprinted
Lnx3 or Xist. The evolution of rXCI might have been driven by relaxation of iXp-Xist, during placental radiation. The iXCI in mouse species might
represent a re-invention of iXp-Xist.

inactivation of Xp in mouse (and possibly in few or many
other placental mammals), indicated herein with iXCI,
is dependent on Xist, to obtain stable silencing [54,55].
It has been hypothesized that the Xp is inherited in a
pre-inactive state, in marsupials and eutherians, as a
consequence of meiotic sex chromosome inactivation
(MSCI) in spermatogenesis [16,56]. MSCI silences the
incompletely synapsed X and Y chromosomes in male
meiotic prophase, forming the so-called XY body, inde-
pendent of Xist [57]. This silencing mechanism
represents a specialized form of an evolutionary ancient
mechanism leading to meiotic silencing of unsynapsed
chromatin regions (MSUC) [58,59]. However, MSUC
may not target small unsynapsed regions effectively. In

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402 Nucleus and gene expression
studies on mice carrying X-to-autosome translocations
resulting in meiotically unsynapsed regions of 40 mega-
bases or even larger regions, MSUC of those regions was
found only in a proportion of meiotic prophase cells
[60,61]. As proposed above, XCI may have originated
in a small non-recombining region of the early X chromo-
some, and that region would have been too small to be
recognized by the MSUC machinery. In transgene stu-
dies, it was found that all sequences required for
imprinted inactivation of autosomes with an Xist/Tsix
transgene integration site are located within the trans-
gene, which is also in agreement with the notion that
MSCI is not required for iXCI [17]. Furthermore, detailed
studies on mouse have shown that many Xp-linked genes
undergo zygotic activation [62,63]. Taken together, the
weight of the evidence suggests that MSCI has no
mechanistic impact on iXCI in mouse, and that imprinted
inactivation of Xp has not evolved from MSCI.
Parent-of-origin expression, by genomic imprinting,
originates from shortly before the separation of marsupials
and placental mammals [64]. Possibly, Lnx3 was an
imprinted gene before its transformation to Xist
(Figure 3b), so that Xist may have maintained this geno-
mic imprint during the beginning of the eutherian radi-
ation. The first placental mammals may have shown
imprinted X inactivation, probably targeting Xp rather
than Xm to oppose accidental inactivation of the single X
from maternal origin in male embryos. It is anticipated
that several events have occurred within a relatively short
period, around the marsupial-eutherian split (Figure 3b).
These events include mutation of Sox3 to proto-Sry,
mutation of Lnx3 to proto-Xist, and the onset of X inac-
tivation. It is not at all unlikely that two mechanisms for X
inactivation came into existence: Xist independent
imprinted inactivation of Xp in the marsupial lineage,
and Xist dependent imprinted inactivation of Xp in the
eutherian lineage. In mouse, many autosomal genes
maintain regulatory genomic imprints in extra-embryonic
tissues, in regulation of placental growth. However, for a
proportion of these genes, the expression becomes more
relaxed, independent from the original imprint, in the
developing embryo proper [65]. The early eutherian
imprinted XCI mechanism may have undergone a similar
relaxation during evolution of the placental mammals,
leading to reactivation of Xp in the embryo proper, forcing
selection towards the invention of rXCI.
Recent studies indicate that XCI in pre-implantation
human and rabbit embryos shows quite some differences,
with respect to timing and mechanism, from what is
known for the mouse [66]. No strong evidence for iXCI
in human and rabbit has been obtained. Mice experience
zygotic genome activation at an earlier time point in pre-
implantation development than human and rabbit, and
such an early genome activation might have been an
important factor driving the evolution of iXCI [66].
Current Opinion in Cell Biology 2012, 24:397–404
Can it be excluded that iXCI as observed in mouse in
fact is a recent mechanism, turning up next to rXCI? It has
been described that rXCI in the mouse, where only few
X-linked genes escape from silencing, is much more
complete than rXCI in women [67]. Possibly, driven by
a short generation time and strong natural selection,
mouse species may have reached an advanced level of
rXCI not seen in many other placental mammals. Inter-
estingly, a Tsix gene that fully overlaps with Xist seems to
be a unique feature of the mouse, as all other eutherian
species examined have a Tsix gene that overlaps only with
the last two exons of Xist [68]. Hence, Tsix might be less
functional, or act through a different mechanism, in
eutherian species other than mice. In addition to devel-
oping an optimized rXCI mechanism, natural selection
may have pushed mouse species to call on a mechanism
for iXCI, either new or re-invented. Exploiting an
advanced rXCI mechanism in combination with iXCI
covering dosage compensation in the very early embryo,
mouse species might be exceptionally well equipped for
balanced growth of both male and female embryos shar-
ing the same uterine horn during a short gestation period.
Conclusion and more questions
The mammalian solution to the evolution of heterologous
sex chromosomes appears to involve the co-evolution of
two layers of gene dosage compensation: a two-fold upre-
gulation of dose sensitive X-linked genes that were lost
from the Y chromosome, and X chromosome inactivation in
female cells. Although important progress has been made
deciphering the mechanisms driving XCI in mouse, which
is the best studied placental mammal regarding these
aspects of development, important factors and regulatory
pathways still need to be identified. Which factors have
contributed to evolution of XCI after the separation of
marsupials and eutherians? How many XCI activators are
involved and how do they act on Xist or Tsix? What is the
precise nature and role of the proposed active chromatin
hubs? How special is the mouse, employing both iXCI and
rXCI? What will we learn from studying XCI in other
mammalian species, including human? The forthcoming
years of research will be challenging and exciting.
Acknowledgements
We would like to thank all lab. members for stimulating discussions. J.G.
was supported by NWO VICI and ERC starting grants.’
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Luo ZX, Yuan CX, Meng QJ, Ji Q: A Jurassic eutherian mammal
and divergence of marsupials and placentals. Nature 2011,
476:442-445.
2. Marshall Graves JA: Weird animal genomes and the evolution of
vertebrate sex and sex chromosomes. Annu Rev Genet 2008,
42:565-586.
www.sciencedirect.com

3. Sutton E, Hughes J, White S, Sekido R, Tan J, Arboleda V,
Rogers N, Knower K, Rowley L, Eyre H et al.: Identification of
SOX3 as an XX male sex reversal gene in mice and humans. J
Clin Invest 2011, 121:328-341.
4. Brawand D, Soumillon M, Necsulea A, Julien P, Csardi G,
Harrigan P, Weier M, Liechti A, Aximu-Petri A, Kircher M et al.: The
evolution of gene expression levels in mammalian organs.
Nature 2011, 478:343-348.
5. Potrzebowski L, Vinckenbosch N, Kaessmann H: The emergence
of new genes on the young therian X. Trends Genet 2009, 26:1-4.
6. Zhang YE, Vibranovski MD, Landback P, Marais GA, Long M:
Chromosomal redistribution of male-biased genes in
mammalian evolution with two bursts of gene gain on the X
chromosome. PLoS Biol 2010, 8.
7. Ross MT, Grafham DV, Coffey AJ, Scherer S, McLay K, Muzny D,
Platzer M, Howell GR, Burrows C, Bird CP et al.: The DNA
sequence of the human X chromosome. Nature 2005,
434:325-337.
8. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L,
Brown LG, Repping S, Pyntikova T, Ali J, Bieri T et al.: The male-
specific region of the human Y chromosome is a mosaic of
discrete sequence classes. Nature 2003, 423:825-837.
9. Johnston CM, Lovell FL, Leongamornlert DA, Stranger BE,
Dermitzakis ET, Ross MT: Large-scale population study of
human cell lines indicates that dosage compensation is
virtually complete. PLoS Genet 2008, 4:e9.
10. Nguyen DK, Disteche CM: Dosage compensation of the active X
chromosome in mammals. Nat Genet 2006, 38:47-53.
11. Lin H, Gupta V, Vermilyea MD, Falciani F, Lee JT, O’Neill LP,
Turner BM: Dosage compensation in the mouse balances up-
regulation and silencing of X-linked genes. PLoS Biol 2007,
5:e326.
12. Xiong Y, Chen X, Chen Z, Wang X, Shi S, Wang X, Zhang J, He X:
RNA sequencing shows no dosage compensation of the active
X-chromosome. Nat Genet 2010, 42:1043-1047.
13. Deng X, Hiatt JB, Nguyen DK, Ercan S, Sturgill D, Hillier LW,
 Schlesinger F, Davis CA, Reinke VJ, Gingeras TR et al.: Evidence
for compensatory upregulation of expressed X-linked genes in
mammals, Caenorhabditis elegans and Drosophila
melanogaster. Nat Genet 2011, 43:1179-1185.
Using a new strategy to analyse Chip and RNA-seq data, this manuscript
settles the debate about X to autosomal gene expression levels in
mammalian cells, by showing that X-linked genes are upregulated com-
pared to autosomal genes.
14. Lin H, Halsall JA, Antczak P, O’Neill LP, Falciani F, Turner BM:
 Relative overexpression of X-linked genes in mouse
embryonic stem cells is consistent with Ohno’s hypothesis.
Nat Genet 2011, 43:1169-1170.
This correspondence letter is in agreement with the findings reported in

[13 ].
15. Prince EG, Kirkland D, Demuth JP: Hyperexpression of the X
chromosome in both sexes results in extensive female bias of
X-linked genes in the flour beetle. Genome Biol Evol 2010,
2:336-346.
16. Huynh KD, Lee JT: Inheritance of a pre-inactivated paternal X
chromosome in early mouse embryos. Nature 2003, 426:857-862.
17. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E: Epigenetic
dynamics of imprinted X inactivation during early mouse
development. Science 2004, 303:644-649.
18. Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S,
Otte AP, Brockdorff N: Reactivation of the paternal X
chromosome in early mouse embryos. Science 2004,
303:666-669.
19. Augui S, Nora EP, Heard E: Regulation of X-chromosome
inactivation by the X-inactivation centre. Nat Rev Genet 2011,
12:429-442.
20. Morey C, Avner P: The demoiselle of X-inactivation: 50 years
old and as trendy and mesmerising as ever. PLoS Genet 2011,
7:e1002212.
www.sciencedirect.com
X chromosome inactivation Gribnau and Grootegoed 403
21. Lee JT: Gracefully ageing at 50, X-chromosome inactivation
becomes a paradigm for RNA and chromatin control. Nat Rev
Mol Cell Biol 2011, 12:815-826.
22. Brown CJ, Lafreniere RG, Powers VE, Sebastio G, Ballabio A,
Pettigrew AL, Ledbetter DH, Levy E, Craig IW, Willard HF:
Localization of the X inactivation centre on the human X
chromosome in Xq13. Nature 1991, 349:82-84.
23. Borsani G, Tonlorenzi R, Simmler MC, Dandolo L, Arnaud D,
Capra V, Grompe M, Pizzuti A, Muzny D, Lawrence C et al.:
Characterization of a murine gene expressed from the inactive
X chromosome. Nature 1991, 351:325-329.
24. Brockdorff N, Ashworth A, Kay GF, McCabe VM, Norris DP,
Cooper PJ, Swift S, Rastan S: The product of the mouse Xist
gene is a 15 kb inactive X-specific transcript containing no
conserved ORF and located in the nucleus. Cell 1992,
71:515-526.
25. Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M,
Tonlorenzi R, Willard HF: A gene from the region of the human X
inactivation centre is expressed exclusively from the inactive
X chromosome. Nature 1991, 349:38-44.
26. Wutz A: Gene silencing in X-chromosome inactivation:
advances in understanding facultative heterochromatin
formation. Nat Rev Genet 2011, 12:542-553.
27. Lee JT, Davidow LS, Warshawsky D: Tsix, a gene antisense to
Xist at the X-inactivation centre. Nat Genet 1999, 21:400-404.
28. Ohhata T, Hoki Y, Sasaki H, Sado T: Crucial role of antisense
transcription across the Xist promoter in Tsix-mediated Xist
chromatin modification. Development 2008, 135:227-235.
29. Shibata S, Yokota T, Wutz A: Synergy of Eed and Tsix in the
repression of Xist gene and X-chromosome inactivation.
EMBO J 2008, 27:1816-1826.
30. Sun BK, Deaton AM, Lee JT: A transient heterochromatic state
in Xist preempts X inactivation choice without RNA
stabilization. Mol Cell 2006, 21:617-628.
31. Anguera MC, Ma W, Clift D, Namekawa S, Kelleher RJ 3rd, Lee JT:
Tsx produces a long noncoding RNA and has general
functions in the germline, stem cells, and brain. PLoS Genet
2011, 7:e1002248.
32. Ogawa Y, Lee JT: Xite, X-inactivation intergenic transcription
elements that regulate the probability of choice. Mol Cell 2003,
11:731-743.
33. Tsai CL, Rowntree RK, Cohen DE, Lee JT: Higher order
chromatin structure at the X-inactivation center via looping
DNA. Dev Biol 2008, 319:416-425.
34. Chureau C, Chantalat S, Romito A, Galvani A, Duret L, Avner P,
Rougeulle C: Ftx is a non-coding RNA which affects Xist
expression and chromatin structure within the X-inactivation
center region. Hum Mol Genet 2011, 20:705-718.
35. Tian D, Sun S, Lee JT: The long noncoding RNA, Jpx, is a
molecular switch for X chromosome inactivation. Cell 2011,
143:390-403.
36. Spencer RJ, del Rosario BC, Pinter SF, Lessing D, Sadreyev RI,
 Lee JT: A boundary element between Tsix and Xist binds the
chromatin insulator Ctcf and contributes to initiation of X-
chromosome inactivation. Genetics 2011, 189:441-454.
By demonstrating the presence of a boundary element, the data in this
paper are instrumental to support the presence of Xist and Tsix active
chromatin hubs, described in [33]. Removal of the boundary element
disturbs Xist activation.
37. Monkhorst K, Jonkers I, Rentmeester E, Grosveld F, Gribnau J: X
inactivation counting and choice is a stochastic process:
evidence for involvement of an X-linked activator. Cell 2008,
132:410-421.
38. Augui S, Filion GJ, Huart S, Nora E, Guggiari M, Maresca M,
Stewart AF, Heard E: Sensing X, chromosome pairs before X
inactivation via a novel X-pairing region of the Xic. Science
2007, 318:1632-1636.
39. Bacher CP, Guggiari M, Brors B, Augui S, Clerc P, Avner P, Eils R,
Heard E: Transient colocalization of X-inactivation centres
Current Opinion in Cell Biology 2012, 24:397–404
404 Nucleus and gene expression
accompanies the initiation of X inactivation. Nat Cell Biol 2006,
8:293-299.
40. Xu N, Tsai CL, Lee JT: Transient homologous chromosome
pairing marks the onset of X inactivation. Science 2006,
311:1149-1152.
41. Donohoe ME, Silva SS, Pinter SF, Xu N, Lee JT: The pluripotency
factor Oct4 interacts with Ctcf and also controls X-
chromosome pairing and counting. Nature 2009,
460:128-132.
42. Donohoe ME, Zhang LF, Xu N, Shi Y, Lee JT: Identification of a
Ctcf cofactor, Yy1, for the X chromosome binary switch. Mol
Cell 2007, 25:43-56.
43. Navarro P, Chambers I, Karwacki-Neisius V, Chureau C, Morey C,
Rougeulle C, Avner P: Molecular coupling of Xist regulation and
pluripotency. Science 2008, 321:1693-1695.
44. Navarro P, Oldfield A, Legoupi J, Festuccia N, Dubois A, Attia M,
 Schoorlemmer J, Rougeulle C, Chambers I, Avner P: Molecular
coupling of Tsix regulation and pluripotency. Nature 2010,
468:457-460.
Providing clear evidence for involvement of pluripotency factors in
repression of XCI, as a follow up of a previous study [43], these authors
opened new avenues in our understanding of the link between cell
differentiation and XCI.
45. Navarro P, Avner P: An embryonic story: analysis of the gene
regulative network controlling Xist expression in mouse
embryonic stem cells. Bioessays 2010, 32:581-588.
46. Jonkers I, Barakat TS, Achame EM, Monkhorst K, Kenter A,
Rentmeester E, Grosveld F, Grootegoed JA, Gribnau J: RNF12 is
an X-Encoded dose-dependent activator of X chromosome
inactivation. Cell 2009, 139:999-1011.
47. Barakat TS, Gunhanlar N, Pardo CG, Achame EM, Ghazvini M,
 Boers R, Kenter A, Rentmeester E, Grootegoed JA, Gribnau J:
RNF12 activates Xist and is essential for X chromosome
inactivation. PLoS Genet 2011, 7:e1002001.
Following identification of RNF12 as a trans-acting XCI-activator [46], this
study describes the molecular mechanism by which RNF12 exerts its role
as an activator.
48. Shin J, Bossenz M, Chung Y, Ma H, Byron M, Taniguchi-Ishigaki N,
 Zhu X, Jiao B, Hall LL, Green MR et al.: Maternal Rnf12/RLIM is
required for imprinted X-chromosome inactivation in mice.
Nature 2010, 467:977-981.
Similarities and differences between iXCI and rXCI are a complex field of
study in which this manuscript provides clear evidence for a role of RNF12
in iXCI.
49. Chadwick LH, Pertz LM, Broman KW, Bartolomei MS, Willard HF:
Genetic control of X chromosome inactivation in mice:
definition of the Xce candidate interval. Genetics 2006,
173:2103-2110.
50. Duret L, Chureau C, Samain S, Weissenbach J, Avner P: The Xist
RNA gene evolved in eutherians by pseudogenization of a
protein-coding gene. Science 2006, 312:1653-1655.
51. Flynn M, Saha O, Young P: Molecular evolution of the LNX gene
family. BMC Evol Biol 2011, 11:235.
52. Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L,
Komorowski J, Nagano T, Mancini-Dinardo D, Kanduri C:
Kcnq1ot1 antisense noncoding RNA mediates lineage-
specific transcriptional silencing through chromatin-level
regulation. Mol Cell 2008, 32:232-246.
53. Nagano T, Mitchell JA, Sanz LA, Pauler FM, Ferguson-Smith AC,
Feil R, Fraser P: The Air noncoding RNA epigenetically silences
transcription by targeting G9a to chromatin. Science 2008,
322:1717-1720.
Current Opinion in Cell Biology 2012, 24:397–404
54. Kalantry S, Purushothaman S, Bowen RB, Starmer J, Magnuson T:
Evidence of Xist RNA-independent initiation of mouse imprinted
X-chromosome inactivation. Nature 2009, 460:647-651.
55. Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R: Xist-
deficient mice are defective in dosage compensation but not
spermatogenesis. Genes Dev 1997, 11:156-166.
56. Namekawa SH, VandeBerg JL, McCarrey JR, Lee JT: Sex
chromosome silencing in the marsupial male germ line. Proc
Natl Acad Sci USA 2007, 104:9730-9735.
57. McCarrey JR, Watson C, Atencio J, Ostermeier GC, Marahrens Y,
Jaenisch R, Krawetz SA: X-chromosome inactivation during
spermatogenesis is regulated by an Xist/Tsix-independent
mechanism in the mouse. Genesis 2002, 34:257-266.
58. Baarends WM, Wassenaar E, van der Laan R, Hoogerbrugge J,
Sleddens-Linkels E, Hoeijmakers JH, de Boer P, Grootegoed JA:
Silencing of unpaired chromatin and histone H2A
ubiquitination in mammalian meiosis. Mol Cell Biol 2005,
25:1041-1053.
59. Turner JM, Mahadevaiah SK, Fernandez-Capetillo O,
Nussenzweig A, Xu X, Deng CX, Burgoyne PS: Silencing of
unsynapsed meiotic chromosomes in the mouse. Nat Genet
2005, 37:41-47.
60. Schoenmakers S, Wassenaar E, van Cappellen WA, Derijck AA, de
Boer P, Laven JS, Grootegoed JA, Baarends WM: Increased
frequency of asynapsis and associated meiotic silencing of
heterologous chromatin in the presence of irradiation-
induced extra DNA double strand breaks. Dev Biol 2008,
317:270-281.
61. Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS:
Pachytene asynapsis drives meiotic sex chromosome
inactivation and leads to substantial postmeiotic repression in
spermatids. Dev Cell 2006, 10:521-529.
62. Patrat C, Okamoto I, Diabangouaya P, Vialon V, Le Baccon P,
Chow J, Heard E: Dynamic changes in paternal X-chromosome
activity during imprinted X-chromosome inactivation in mice.
Proc Natl Acad Sci USA 2009, 106:5198-5203.
63. Okamoto I, Arnaud D, Le Baccon P, Otte AP, Disteche CM,
Avner P, Heard E: Evidence for de novo imprinted X-
chromosome inactivation independent of meiotic inactivation
in mice. Nature 2005, 438:369-373.
64. Renfree MB, Hore TA, Shaw G, Graves JA, Pask AJ: Evolution of
genomic imprinting: insights from marsupials and
monotremes. Annu Rev Genomics Hum Genet 2009, 10:241-262.
65. Ferron SR, Charalambous M, Radford E, McEwen K, Wildner H,
Hind E, Morante-Redolat JM, Laborda J, Guillemot F, Bauer SR
et al.: Postnatal loss of Dlk1 imprinting in stem cells and niche
astrocytes regulates neurogenesis. Nature 2011, 475:381-385.
66. Okamoto I, Patrat C, Thepot D, Peynot N, Fauque P, Daniel N,
 Diabangouaya P, Wolf JP, Renard JP, Duranthon V et al.:
Eutherian mammals use diverse strategies to initiate X-
chromosome inactivation during development. Nature 2011,
472:370-374.
Studies with pre-implantation human and mouse embryos indicate
important differences in the XCI mechanism between mouse and other
mammalian species, and therefore this paper emphasizes the importance
of studying iXCI and rXCI in an evolutionary context.
67. Berletch JB, Yang F, Disteche CM: Escape from X inactivation in
mice and humans. Genome Biol 2010, 11:213.
68. Horvath JE, Sheedy CB, Merrett SL, Diallo AB, Swofford DL,
Program NCS, Green ED, Willard HF: Comparative analysis of the
primate X-inactivation center region and reconstruction of the
ancestral primate XIST locus. Genome Res 2011, 21:850-862.
www.sciencedirect.com