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The X chromosome in space

Teddy Jégu1,2, Eric Aeby1,2 and Jeannie T. Lee1,2
Abstract | Extensive 3D folding is required to package a genome into the tiny nuclear space, and
this packaging must be compatible with proper gene expression. Thus, in the well-hierarchized
nucleus, chromosomes occupy discrete territories and adopt specific 3D organizational structures
that facilitate interactions between regulatory elements for gene expression. The mammalian
X chromosome exemplifies this structure–function relationship. Recent studies have shown that,
upon X‑chromosome inactivation, active and inactive X chromosomes localize to different
subnuclear positions and adopt distinct chromosomal architectures that reflect their activity
states. Here, we review the roles of long non-coding RNAs, chromosomal organizational structures
and the subnuclear localization of chromosomes as they relate to X‑linked gene expression.

X‑chromosome inactivation
(XCI). A process to
epigenetically silence one
X chromosome in the somatic
cells of female mammals to
achieve similar expression
levels between the sexes.
Long non-coding RNAs
(lncRNAs). Transcripts of more
than 200 nucleotides that do
not encode proteins.
Xist
(X-inactive specific transcript).
A long non-coding RNA that is
transcribed from the
X-inactivation centre and is the
key regulator of mouse
X‑chromosome inactivation.
1
Howard Hughes Medical
Institute; Department of
Molecular Biology,
Massachusetts General
Hospital, Boston,
Massachusetts 02114, USA.
2
Department of Genetics,
Harvard Medical School,
Boston, Massachusetts
02115, USA.
Correspondence to J.T.L.
lee@molbio.mgh.harvard.edu
doi:10.1038/nrg.2017.17
Published online 8 May 2017
In mammals, sex is determined by a pair of sexually
dimorphic chromosomes (the X and Y chromosomes),
with males being the heterogametic sex (XY) and
females being the homogametic partner (XX). This
context induces an imbalance in the dosage of X‑linked
genes between the two sexes. In females, dosage com‑
pensation between the sexes is achieved by transcrip‑
tionally silencing most genes on one X chromosome, a
process called X‑chromosome inactivation (XCI)1–5. The
choice of which X chromosome to silence takes place
early in development in a random manner, generating
an active X chromo­some (Xa) and an inactive X chromo‑
some (Xi), which are maintained thus throughout adult
life. XCI induces exclusion of RNA polymerase II and
transcription factors from the Xi. This is accompanied
by the recruitment of histone and DNA modification
complexes to deposit repressive marks on the Xi such
as histone H3 lysine 27 trim­ethylation (H3K27me3),
H3K9me2, the H2A ­histone variant macroH2A and DNA
methylation6–10.
XCI is initiated by a single key locus, the X‑inactivation
centre (Xic), which in the mouse X chromosome is a
100–500 kb region that contains all required sequences for
this process11 (FIG. 1a). The core region of the Xic — the
region sufficient to direct silencing in cis when placed on
an autosome — is only 100 kb in size and, intriguingly,
contains no protein-coding genes. Instead, this region har‑
bours elements that produce long non-coding RNAs (lncR‑
NAs), including Xist, RepA, Tsix, Xite and Jpx (also known
as Enox (expressed neighbour of Xist))11,12. Outside the core
region lie genes encoding other lncRNAs (for example,
Ftx andTsx)13,14, as well as one protein-coding gene, Rnf12
(also known as Rlim)15. The minimal critical region of the
human XIC has yet to be defined, but the region is highly
conserved around the 2.3 Mb syntenic region, which
contains many of the same genes as in mice16.
Recent investigations in mice have focused inten‑
sively on the functional roles of lncRNAs. For example,
the Jpx RNA has been proposed as a numerator element
in controlling X‑chromosome counting 17. Tsix and Xite
together control allelic choice and also designate the
active X chromosome by preventing Xist expression on
this chromosome18–20. By contrast, Jpx and RepA RNAs
have been suggested to serve as positive regulators of
Xist 9,21. Once expressed, Xist RNA binds to a critical
nucleation site located in the Xist gene body 22 and spreads
from that site to the rest of the X chromosome to establish
chromosome-wide silencing 23–25. Xist RNA interacts with
a large proteome26–28 and collaborates with that proteome
to form heterochromatin on the X chromosome.
In the past few years, it has become clear that l­ nc­RNAs
of the Xic and beyond do more than target silencing pro‑
teins to the chromosome. In fact, the epigenetic regula‑
tion of XCI is about much more than getting chromatin
complexes and transcription factors to the right place at
the right time. Recent work suggests that the Xic also con‑
trols chromosome movement and the overall architecture
of the X chromosome in 3D nuclear space26,29–33. Here,
we discuss these recent advances as well as the essential
roles of chromatin architecture (BOX 1) and lncRNAs in
the XCI process. Most of the discussed studies focus on
mice, although we also provide an overview of human
studies where appropriate, in particular when reviewing
the implications of both the 3D structure and subnuclear
localization of X chromosomes during XCI.
Counting in time
XCI occurs very early in development. In mice, XCI is
observed in the late blastocyst stage, specifically in the
epiblast lineage at the time of uterine im­­plantation4.
At this time, all epiblast cells make a numerical
­determination of their X‑chromosome content.

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a 10 kb
Xic Pairing
Rnf12 Xpct Ftx Xist RepA centre

Jpx Tsix Tsx

Xpr Xite
Nucleation
centre

b
Pre-XCI X–X pairing XCI onset Xist
Xi
Xist
CTCF Xist Xite Jpx Tsix
Jpx Tsix Xite CTCF Xite
OCT4 CTCF CTCF CTCF
Jpx Tsix
OCT4 CTCF CTCF
Xist CTCF Xa Xist
CTCF Xist Xite CTCF
Jpx Tsix Xite Jpx
Tsix Xite

Xist activation Nucleation Xist spreading

YY1
Xist
Xist Xist
RNA YY1 RNA
Xi Pol II Xi Pol II Xi
Nucleation centre

Figure 1 | The potential roles of long non-coding RNAs in the initiation of X‑chromosome inactivation.
Nature Reviews | Genetics
a | The X‑inactivation centre (Xic) in mice. Non-coding genes with established functions in regulating X‑chromosome
inactivation (XCI) are shown in colour, whereas those in grey have been proposed as candidate regulators. The role of
the X‑pairing region (Xpr) in pairing is under debate. Rnf12 is a protein-coding gene located ~500 kb upstream of Xist.
Arrows indicate regions involved in the different steps of XCI. b | A proposed model that integrates counting,
X‑chromosome pairing, nucleation and Xist spreading mechanisms. Upon initiation of XCI, X–X pairing allows for
random expression of Xist from one allele only. X–X pairing involves the long non-coding RNA (lncRNA) Tsix, CTCF and
octamer-binding transcription factor 4 (OCT4). However, Xist expression also requires relief of CTCF negative
regulation at its promoter. This might be accomplished by the lncRNA Jpx (also known as Enox (expressed neighbour of
Xist)), which has been proposed to function as a X‑chromosome numerator in the counting process17. An alternative
model has also been reported39. Once activated, Xist is captured by the nucleation centre, where yin and yang 1 (YY1)
serves as receptor, and from here, Xist spreads in a cis-limited fashion to inactivate the X chromosome. CTCF- and
YY1-binding sites relevant to each XCI step are represented. RNA Pol II, RNA polymerase II; Xa, active X chromosome;
Xi, inactive X chromosome.

How does a cell know whether it has one, two
or three X chromosomes? It has long been established
that counting involves not only determining
the X‑chromosome number but also the ratio
of X chromo­s omes to autosomes (X:A). In diploid
cells, one X chromo­some is retained as the Xa and all
remaining X chromo­somes are inactivated. In tetraploid
cells, two X chromosomes are retained as Xas and all oth‑
ers are inactivated. Initially, the counting mechanism was
proposed to involve only a ‘blocking factor’ — defined
as a complex of autosomal factors that binds to one Xic
and prevents inactivation of the X chromosome in cis34–37.
Previously, the blocking factor was thought to bind and
transactivate Tsix 38,39, which in turn prevents Xist induc‑
tion and thereby keeps the X chromosome on in cis.
However, deleting Tsix in male cells does not result in Xist
upregulation and silencing of the only X chromosome
present18,40. This observation suggested that an additional
factor (or factors) is required to render female cells com‑
petent for XCI. The additional hypothetical factor was
named the ‘competence factor’ (REFS 18,19,39) — a com‑
plex of X‑linked and autosomal factors that would titrate
each other and effectuate the X:A ratio.
With its existence supported by genetic evidence
only, the competence factor remained elusive until
recently, when two candidates came to light. The E3
ubiquitin ligase RNF12 has been proposed to acti‑
vate Xist in trans by targeting the pluripotency factor
reduced expression protein 1 (REX1; also known as
ZFP42) for degradation; REX1 has been shown to acti‑
vate Tsix and negatively regulate Xist expression15,41,42.
Interestingly, studies recently reported that RNF12
is dispensable for random XCI in vivo, but demon‑
strated that RNF12 is required for imprinted XCI43,44,
a form of XCI that occurs in preimplantation embryos
and placental tissue of mice. Notably, imprinted XCI
does not involve a counting mechanism. Furthermore,
although Xist upregulation is affected, a significant frac‑
tion of Rnf12+/− female embryonic stem (ES) cells and
embryos can still undergo XCI15,44,45. Therefore, RNF12

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CTCF may not strictly be required for the counting process,
(CCCTC-binding factor). although it clearly affects X‑chromosome silencing in
A highly conserved other ways.
DNA-binding protein that
regulates chromatin
organization and is involved in
The role of Jpx in counting. The exact molecular nature of
transcriptional regulation and the X‑chromosome counting mechanism remains largely
insulation. unknown. One factor that has recently been implicated
as a numerator element of the X:A ratio in mice is the Jpx
Insulator
lncRNA (FIG. 1b). This lncRNA is encoded in the Xic locus
A DNA sequence element that
blocks heterochromatin
just upstream of Xist and is conserved in placental mam‑
spreading and interaction mals, including mice and humans16,46,47. Genetic analysis
between enhancers and showed that Jpx RNA serves as an activator of Xist in ES
promoters. cells and, furthermore, that Jpx is haploinsufficient 17,21.
Chromosome territories
When a single allele of Jpx was deleted, female mouse
The domains occupied by ES cells were no longer able to upregulate Xist RNA on
chromosomes in the differentiation21. By contrast, when extra copies of Jpx
interphase cell nucleus. were introduced into male ES cells, Xist turned on17, sug‑
gesting that XCI is sensitive to the dosage of Jpx RNA. In
fact, Jpx seems to function as a diffusible RNA, as the Jpx
gene could be placed on a different chromosome and still
function as an activator, and short hairpin RNAs directed
against Jpx abolished its activity 21. Additional genetic and
biochemical analyses showed that Jpx RNA functions by
evicting CTCF, an architectural protein and chromatin
insulator, from the Xist promoter 17 (FIG. 1b). The same study
showed that the transcriptional activation of Xist is regu‑
lated by a dynamic balance between levels of Jpx RNA and
CTCF protein, which is suggestive of a model in which Jpx
RNA serves as a numerator of the X:A ratio and CTCF
serves as a denominator 17 (FIG. 1b).
Box 1 | Chromatin: a key structure for looping
Although DNA in eukaryotic cells allows the storage of genetic information, it is its
packaging into chromatin that enables the fine organization of this genetic information
throughout the development of organisms. Chromatin is not organized randomly in the
nucleus, and each chromosome in the interphase occupies a spatially well-defined
territory called chromosome territory (see the figure, part a). Although occupying a
defined territory, chromosomes can be in contact with each other121. Indeed, chromatin
loops from one chromosome territory can intermingle with adjacent chromosome
territories. In addition to these interchromosomal interactions, chromosomes can form
multiple intrachromosomal interactions, which are regrouped into different topologically
associated domains (TADs) with an average size of 1 Mb (REF. 81) (see the figure, part b).
Chromosomes are partitioned into several TADs, which are spatially separated from each
other. TADs seem to regulate gene expression by facilitating or preventing interactions
between regulatory elements and, interestingly, gene expression within the same TAD is
more correlated than between two different TADs. Boundaries between each TAD
constitute key points of TAD organization. The TAD boundaries are enriched for binding
sites of the architectural proteins CTCF and cohesin122 (see the figure, part c). It has been
observed that cohesins can organize interactions within domains and that CTCF is
required to separate neighbouring folding domains123. Although CTCF and cohesin are
known to shape genome topology, the mechanisms by which they regulate 3D
chromosome organization remain to be elucidated in more detail.
a b c transcriptional fates, with one becoming the Xa chromo­
TADs
Cohesin
CTCF
Chromosome
territories
Nature Reviews | Genetics
It should be noted that there is an ongoing debate
regarding the relative contributions of Jpx and RNF12 to
the counting process. Barakat et al.45 created a Xic dele‑
tion that removed Jpx along with nearly all sequences at
the Xic and showed that mouse female ES cells with a
heterozygous deletion could still undergo XCI using the
wild-type Xist allele. The female cells showed no impair‑
ment of XCI on differentiation. However, in a separate
genetic experiment, the authors showed that a dele‑
tion containing both Jpx and Rnf12 worsened the XCI
defect when the deletion was restricted to Rnf12 (REF. 45),
supporting a role for Jpx as a positive regulator of Xist
expression. In vivo experiments in mice have also argued
against RNF12 as a counting factor during random XCI,
as the RNF12 protein is downregulated in epiblast cells
of wild-type embryos before random XCI occurs, and
females bearing a null mutation targeted to epiblast cells
exhibited no XCI defects44. Moreover, a re‑analysis of the
RNF12‑knockout made in ES cells15 suggests that it yields
a 45 kDa truncated protein that lacks the RING finger
domain (I. Bach, personal communication). Because a
RING-finger-mutated RNF12 has dominant-negative
activity 48, it is possible that the mutated RNF12 in ES
cells resulted in a gain‑of‑function mutation that could
indirectly influence XCI. Thus, although RNF12 influ‑
ences imprinted XCI43,49, its role in random XCI remains
unclear. In summary, further analyses will be necessary to
address the contributions of both Jpx and RNF12. Other
factors are likely to participate in the counting process.
For example, depletion of octamer-­binding transcription
factor 4 (OCT4; also known as POU5F1) was shown to
impair X‑chromosome pairing and resulted in increased
Xist upregulation from both X chromosomes in female
mouse ES cells50, which is indicative of a role for Oct4 as
a trans factor that regulates counting.
X–X pairing: a rendezvous in space
More than 10 years ago, it was discovered that two
female X chromosomes undergo an unusual homo­
logous chromo­some pairing event just before the ini‑
tiation of XCI51,52 (FIG. 1b). This pairing event has been
studied extensively in the ex vivo model, mouse ES cells.
Undifferentiated female ES cells harbour two active
X chromosomes, and the process of cell differentiation
induces the female cells to transition through the differ‑
ent steps of XCI. The two X chromosomes occupy dis‑
tinct chromosome territories in undifferentiated female
ES cells, but they become colocalized when the cells are
induced to differentiation (FIG. 1b). This phenomenon has
been called ‘X–X pairing’ (REFS 51,52) and occurs only
transiently, with a measured half-life of <30 minutes53,54.
When the X chromosomes separate, they adopt opposite
some and the other becoming the Xi chromosome.
This physical proximity is directed by the Xic and
occurs only at the Xic 51. Genetic analyses have shown
that Tsix and Xite, a known positive regulator of Tsix, are
both necessary and sufficient to direct the pairing inter‑
action51 (FIG. 1b). Indeed, deleting Tsix and Xite impairs
the X–X interactions and results in a chaotic choice of
Xa and Xi19,51,55. Under chaotic choice, Xa and Xi would

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Heterokaryons
Cells containing genetically
different nuclei.
no longer be chosen in a mutually exclusive manner by
putative blocking and competence factors. By contrast,
inserting 1–3 kb sequences taken from within Tsix and/
or Xite into autosomes results in a remarkable ectopic
pairing between the X chromosome and autosome — an
action that precludes X–X pairing and results in a block
to the XCI process51. These genetic studies argue for a
biological role of X–X pairing in XCI.
Characterization of the genetic elements required for
X–X pairing point to an essential function of the Xic,
although the mechanisms remain mostly uncharac‑
terized. It is, however, known that active transcription
and CTCF are both required for X–X pairing 53. Acutely
inhibiting RNA polymerase II transcription blocks the
colocalization of the Xics during cell differen­tiation.
Interestingly, this requirement for transcription may
reflect the necessity of Tsix and Xite RNAs for the
recruitment of CTCF protein to the ~15 kb ‘pairing cen‑
tre’ (REF. 56) (FIG. 1b). Indeed, disrupting the RNA–CTCF
interaction prevented X–X pairing 56. The pluripotency
factor OCT4 also influences the timing of the pairing
event during cell differentiation by binding to the 5′ end
of Tsix 50 (FIG. 1b). Another locus, Xpr (X‑pairing region),
has been proposed to influence pairing 57 (FIG. 1a) .
However, a later study argued against a role for Xpr on
the basis of its inability to induce pairing on a transgene
array 58. A knockout of Xpr has, so far, not shed light on
its necessity for X–X interactions.
Although Xic–Xic pairing is unequivocally one of
the first observable events during female cell differen‑
tiation and precedes XCI, the molecular consequences
of pairing have been the subject of active debate. One
idea is that pairing is part of an allelic choice mecha‑
nism. That is, pairing could be a mechanism by which
the two X chromosomes adopt mutually exclusive and
opposite transcriptional states51. Indeed, inactivation
of one X chromosome must preclude silencing of the
second X chromosome, and there would need to be a
robust mechanism to render the decision mutually exclu‑
sive. Live-cell imaging that tracks the movement of two
X chromosomes supports the idea that Tsix expression
becomes monoallelic after X–X pairing 54. Interestingly,
allelic pairing in mammalian somatic cells has been
observed in other cases of allelism59–62 and could poten‑
tially be generalized to other allelic regulatory situations.
Alternatively, pairing may not be required in the case
of XCI. One study observed XCI occurring in the two
nuclei of heterokaryons in spite of the X chromosomes
being separated by a nuclear envelope45. However, it was
not ruled out that the heterokaryons continue to divide
and the nuclear envelope breaks downs during cell divi‑
sion, permitting the mixing of chromosomes between
two nuclei.
As with the counting mechanism, a number
of questions regarding the choice mechanism still
await answers. Although we favour a specific, robust
mechanism for the determination of allelic choice, a
stochastic model has been proposed in which the choice
of X chromo­somes for inactivation is made by chance,
with every X chromosome having an equal prob­ability
of becoming inactivated on titration of an X‑linked
activator 39. In this model, each X chromosome inde‑
pendently decides whether it will be inactivated. Thus,
some cells would inactivate no X chromosome, others
would inactivate both X chromosomes, and the remain‑
ing cells would inactivate only one of the two X chromo‑
somes. Only those cells that make the stochastic choice
of inactivating one X chromosome would survive the
developmental process and go on to contribute to the
embryo, supporting a mechanism that is less than robust
in a biological sense (that is, a mechanism that inher‑
ently includes errors). Hence, one would expect female
embryos undergoing XCI to demonstrate excessive cell
death in the epiblast lineage, a phenomenon that has not
been observed39.
With regard to allelic choice, recent evidence suggests
that the counting and choice mechanisms underlying
dosage compensation might differ in some respects
between mice and humans, despite a strong conserva‑
tion of the Xic and XIC elements between these species.
Indeed, a number of studies have suggested biallelic XIST
expression in human preimplantation embryos63,64, and
that biallelic expression is accompanied by a reduction of
gene expression from both chromosomes64,65. XCI does
not formally begin until one of the XIST loci is silenced.
The XACT lncRNA has been proposed to be a regulator
in this regard66,67. XACT expression is associated with the
Xa, and a recent study used transgenic and knockdown
analyses to propose that it may affect the choice of XCI
by opposing XIST silencing 67. Genetic deletion of XACT
will be required to determine whether it directly affects
XIST expression.
The Xi and its spatial transformation
Nucleation: a required step for Xist spreading and
Xi‑silencing launch. Once expressed from the Xi, Xist
coats the chromosome, and its spreading along the Xi
induces chromosome-wide silencing 23–25. Before this
cis spreading and during its transcription, Xist RNA
must be captured by a nucleation site located in the Xist
gene body in order to be retained on the Xi22 (FIG. 1b).
This capture is mediated by the transcription factor yin
and yang 1 (YY1), which binds to the nucleation site
in the Xist exon 1. The bivalent property of YY1 allows
the protein to bridge and tether Xist RNA to the nuclea‑
tion site DNA on the Xi22. Without the nucleation event,
Xist RNA is released from the site of transcription
and cannot spread in cis along the X chromosome22.
Although Xist RNA diffuses through the nucleus, it
does not bind to ectopic sites, nor does it bind to the
(future) Xa22. It is currently not clear what blocks the
Xa site and other YY1-binding sites from Xist binding,
although differential DNA methylation was proposed
to contribute to this68,69. Jeon and Lee22 observed that
introducing a Xist transgene into the genome of dif‑
ferentiated cells enables endogenous Xist RNA to dif‑
fuse throughout the nucleus and migrate (in trans) to
the transgenic Xist DNA locus, as the newly introduced
transgene lacks the blocking signal.
In contrast to these observations, one study sug‑
gested that the YY1 element could be involved in the
direct transcriptional upregulation of Xist in ES cells68.

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It is entirely possible that the YY1 element has both tran‑ trans sites. Xist RNA would be expected to bind YY1
scriptional and nucleating functions. Another study did targets in cis with greater probability owing to the
not observe any diffusion capacity of Xist in mouse ES co‑transcriptional nature of the Xist–YY1 interaction22.
cells70, although the lack of diffusion might be explained The ability to observe trans diffusion would be increased
by stoichiometry and competition between cis and when the stoichiometry of the trans site is favoured over
the cis site. Indeed, the trans diffusion observed by Jeon
and Lee occurred in the context of multi-copy trans‑
A
genes22. Finally, how Xist RNA spreads from the nucle‑
ation centre to the rest of the X chromosome in cis is
currently not known. One idea is that its spreading is
guided by the 3D spatial organization of the Xi24,25, but
specific factors for spreading have yet to be identified.
Xi
Two distinct 3D X‑chromosome structures. Profound
Xa changes in overall chromosome structure during the
process of XCI perfectly exemplify the link between
the 3D chromosome organization and gene expression.
Differences in shape between Xa and Xi territories have
long been noted. The Xi territory is characterized by a
Ba Xa chromosome territory Bb Xi chromosome territory spherical shape and smooth surface, whereas the Xa ter‑
ritory displays a flatter shape and a larger and more irreg‑
IC TF CD or TAD ular surface71,72 (FIG. 2A). Although the Xi is significantly
Xist
more compacted than the Xa, the differences between
PR RNA Pol II these two homologues are surprisingly small; the overall
compaction of the Xi is 1.2‑fold higher than that of the
Speckles IC Collapsed Xa30,73,74. Except for local differences on the promoters
PR ANC
CDC of genes that are expressed from the Xa and silent on
INC the Xi, both chromosomes have a similar global 30 nm
Inactive chromatin structure. However, the two X chromosomes
gene display significant differences beyond this 30 nm fibre.
INC Indeed, large-scale chromatin structures on gene-rich
ANC PR regions are more condensed on the Xi than on the Xa74.
These observations were corroborated by 3D fluores‑
cence in situ hybridization (FISH) analysis showing that
Ca Xa TADs Cb Xi megadomains chromatin domains of approximately 1 Mb are similarly
compacted between the Xa and Xi, but a higher compac‑
tion in Xi territory is observed for specific chromosomal
segments of 20 Mb (REF. 73). Moreover, light and electron
microscopy have revealed that the condensed region of
the Xi has a distinctive ultrastructure and that the Xi
is not solid but rather has a sponge-like structure that is
organized in large, tightly packed chromatin domains
Dxz4 separated by interchromatin channels, which are contigu‑
ous with nuclear pores75. These observations are consist‑
ent with the concept of an active nuclear compartment
(ANC) and an inactive nuclear compartment (INC)76,
500 kb resolution 500 kb resolution a network model in which the chromosome territories
Figure 2 | The 3D organization of X chromosomes. A | The inactive X chromosome (Xi) are composed of two interacting networks with their
adopts a spherical shape with a smooth surface and is organizedNatureinto two megadomains,
Reviews | Genetics own dynamic organization. The INC is formed by the
whereas the active X chromosome (Xa) adopts a flatter shape with a more irregular transcriptionally silent and compacted core of chromatin
surface area. B | The active nuclear compartment (ANC) and inactive nuclear domain clusters (CDCs), whereas the ANC is formed by
compartment (INC)77 network model of chromosome territory structure proposes that the perichromatin region and the interchromatin channel
basic structural features of chromosome territory organization are present in both the Xa and is where transcription occurs77 (FIG. 2B).
(part Ba) and the Xi (part Bb). The INC is transcriptionally silent and contains compacted Recent super-resolution microscopy studies have
chromatin domain clusters (CDCs), whereas transcription occurs in the ANC, which is revealed that the Xi territory displays major differences
composed of the perichromatin region (PR) and the interchromatin channel (IC)77. In the Xi
from transcriptionally active chromosome territories,
chromosome territory, the ANC is partially collapsed and the CDCs are located closer to
each other than in the Xa chromosome territory. C | Allele-specific Hi‑C maps of the Xa although the basic structural features of chromosome
(part Ca) and Xi (part Cb) chromosomes, showing that the Xa is organized into territory organization seem to be maintained76. Indeed,
topologically associated domains (TADs) and the Xi is organized into two megadomains although the CDCs and interchromatin channels are
separated by the Dxz4 locus. CD, chromatin domain; RNA Pol II, RNA polymerase II. observable, the Xi chromosome territory displays par‑
Part C is reproduced with permission from REF. 30, Macmillan Publishers Ltd. tially collapsed ANCs with CDCs closer to each other

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Sponge-like structure
(Also called a porous structure).
A model of chromosome
territory structure in which
chromolome territories are
composed of interconnected
chromatin domains permeated
by an interchromatin channel.
Interchromatin channels
Channels that pervade
chromatin domain clusters
both between and within
chromosome territories. The
channels are a chromatin-free
compartment containing RNA
polymerase II, transcription
factories, splicing speckles and
architectural proteins.
Chromatin domain clusters
(CDCs). Groups of chromatin
domains that comprise the
inactive nuclear compartment.
Perichromatin region
The decondensed and
transcriptionally competent
chromatin region residing at
the periphery of chromatin
domain clusters.
Chromosome conformation
capture
A technique to detect physical
interactions between distant
DNA sequences.
Topologically associated
domains
(TADs). Units of chromatin that
display a high frequency of
DNA long-range interactions
between loci located in the
same unit and a low frequency
of association between loci
located in adjacent units.
Macrosatellite
A specific class of large tandem
repeat DNA that is
characterized by the large size
of individual repeats, which can
each be several kilobases long.
Macrosatellites can span
hundreds of kilobases of DNA.
Superloops
Extremely long-range
intrachromosomal contacts
between two loci, up to 80 Mb
long.
Cohesins
Protein complexes composed
of the core subunits SMC3,
SMC1, RAD21 and STAG1
(also known as SA1) or STAG2
(also known as SA2). Cohesin
participates with CTCF to
mediate long-range chromatin
interactions.
(FIG. 2B) .Altogether, these observations argue for a
‘non-monotonous compaction’ model of the Xi in which
differences observed between the two X chromosomes
result from reorganization of the higher-order structures
rather than a modulation of ­chromatin compaction at
the nucleosome level.
The development of chromosome conformation
capture methods has enabled the fine-mapping of
X‑chromosome topology 78,79. At whole-chromosome
scale, recent studies have shed light on significant
differences in the folding of the Xa and Xi in human
and mouse cells 26,30,31,80–84. Hi‑C experiments have
revealed that the Xa is organized into >100 topologically
associated domains (TADs) on the whole chromosome,
whereas the Xi seems devoid of such megabase-scale
structures across its full length and is instead organized
into two megadomains of frequent intrachromosomal
contacts26,30,80,82,84 (FIG. 2C). The boundary region between
these two megadomains is conserved between human
and mouse and is located near the macrosatellite repeat
DXZ4 or Dxz4, respectively26,30,80,82,84.
Significant differences in loop structures between the
two X chromosomes can be observed. Indeed, in human
cells, there is a network of long-range superloops that is
present only on the Xi, and four of the main anchor
regions involved in the superloop network contain the
lncRNA genes XIST, DXZ4, FIRRE and LOC550643
(REFS 82,84,85). Interestingly, YY1 seems to bind these
regions only on the Xi, suggesting a potential role of YY1
in the regulation of Xi‑specific superloops86. Moreover,
the three genes DXZ4, FIRRE and LOC550643 are bound
by CTCF, and the superloops between these three loci
can occur simultaneously in the same cell, specifically on
the Xi84. Chromatin interaction analysis by paired-end
tag sequencing (ChIA-PET) experiments have supported
the direct role of CTCF in anchoring the super-
long-range interactions between these loci only on the
Xi87. This finding suggests a crucial role for the allelic
binding of CTCF in X‑chromosome topology regula‑
tion. CTCF therefore seems to be a recurrent player in
the regulation of XCI, from counting, to choice, to the
spread of silencing.
Xist RNA shapes the Xi conformation. Evidence indi‑
cates that asymmetric binding of CTCF and cohesins on
the Xa versus Xi could account for their distinct topol‑
ogies26,56,80,88–90. Interestingly, cohesins bind the X chro‑
mosomes in an allele-specific manner that favours the
Xa. There are 500–600 Xa‑specific cohesin sites and only
~20 Xi‑specific cohesin sites, and the rest of the ~200
sites are shared by the Xa and Xi26. In this same study,
it was shown that an Xi conditionally deleted for Xist
(XiΔXist) in mouse fibroblasts is sufficient to restore the
binding of a large number of cohesins on the Xi, sug‑
gesting that cohesin binding on the Xi is influenced
by Xist RNA. In fact, the anti-correlation between Xist
and cohesins implies that Xist has a repelling effect on
cohesins. Proteomic purification for Xist RNA showed
that Xist can directly interact with CTCF and co­­
hesins26. Thus, in this new model, Xist interaction with
co­­hesins is proposed to antagonize a set of cohesins on
the Xi by repelling their binding in cis, preventing the
­establishment of an Xa‑like binding pattern.
Previously, allele-specific chromosome conforma‑
tion capture-on-chip (4C) experiments showed that
silenced genes on the Xi lack 3D contacts that are seen
on the Xa, and deletion of Xist from the Xi causes con‑
tacts to re‑form in the same genomic locations as found
on the Xa31. Hi‑C experiments performed in an Xist
ablation mutant (XiΔXist) extended this observation and
demonstrated a crucial role for Xist in shaping the Xi 3D
conformation26. Indeed, deletion of Xist results in a mod‑
ulation of Xi folding, forming an Xa‑like chromosome
conformation through the restoration of TAD struc‑
tures on the Xi. This TAD restoration correlated with
the re‑establishment of Xi–cohesin binding, suggesting
that Xist-mediated repulsion of cohesins from the Xi
prevents an Xa conformation. More recently, it has been
observed that induction of Xist RNA expression on the
X chromosome in undifferentiated male mouse ES cells
generates the formation of the two megadomains sepa‑
rated by the boundary at the Dxz4 locus; the same study
found that the role of Xist in regulation of Xi topology
seems to be dependent on the Xist A‑repeat region30. Xist
shapes the topology of the Xi, while the 3D organiza‑
tion of the Xi in turn guides the binding of Xist lncRNA
during the establishment of XCI24,25. In the future, the
integration of a temporal dimension to the existing 3D
analysis using time course analysis during cell differen‑
tiation will enable understanding of the reciprocal regu‑
lation between Xist and Xi topology during the different
steps of XCI.
The idea that lncRNAs may generally regulate CTCF
and cohesin localization could be generalized to include
domains outside the X chromosome26,56,91–94. Thus, it is
tempting to speculate on the possibility that the inter‑
actions between the chromatin architectural proteins,
CTCF and cohesins, and specific lncRNAs constitute a
key regulatory aspect of 3D nuclear organization.
Different spaces and destinations for silent and escapee
genes on the Xi. Genes on both the mouse and human
X chromosomes have been observed to escape from XCI.
In humans, 15% of X‑linked genes are not fully subject to
XCI and are apparently less susceptible to the repressive
effect of XIST RNA95. In mice, a smaller number of genes
escape from XCI. Although escapee genes vary between
mouse tissues90,96–100, most are positioned close to each
other along the chromosome90, which suggests that
local rearrangements between tissues expose escapee
genes to the transcriptionally competent compartments.
This idea is corroborated by recent discoveries showing
that escapee genes that are close to each other can be
associated with the same individual TAD in mouse ES
cells100. Interestingly, although the Xi has two megad‑
omains in both Patski and F1 brain cells, the Xi mega­
domains in brain seem to be more condensed than
in Patski cells, which have more escapee genes80,90.
Although the Xi is devoid of TAD structures, escapee
genes have been proposed to form mini-TADs30 in
neural progenitor clones (NPCs). This study also sug‑
gested an unusually large number of escapee genes in

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a b Escapee of the Xi — the so‑called core, which is composed of

Escapee
repeat-dense regions and inactivated genes bound by
Xist lncRNA. This relocalization seems to be dependent
on the Xist A‑repeat region and may be associated with
long interspersed nuclear elements (LINEs)103,104. Taken
together, these studies suggest that the inside of the Xi
Xist territory forms a transcriptionally silent compartment,
Xist marked by Xist, whereas the periphery of the Xi creates
an environ­ment that is more permissive for transcrip‑
Inactive tion, with specific interactions involved in gene escape
gene (FIG. 3). A potential concern for this model, which argues
Inactive
gene for a compacted central core and a sharp geographical
division between repressed (interior) and expressed
(exterior) genes, is that repressed and expressed genes
(such as escapees) are often separated only by a few
c d kilobases, and it may be difficult to envision how two
Xist neighbouring genes can be partitioned so distantly and
ANC sharply in space.
Escapee Indeed, additional studies have reached somewhat
Escapee different conclusions with regard to how the Xi terri‑
tory is organized at the cytological level. Clemson and
collaborators agree that the internal core of the Xi terri‑
Xist tory is composed of non-genic sequences, but found that
the outer rim is formed by gene-rich regions regardless
Inactive
Inactive gene of their transcriptional activity 105 (FIG. 3). Another study
gene has shown that Xi‑silencing genes can be located and
Collapsed ANC maintained outside the Xi territory and that transcrip‑
tion can occur inside the Xi99 (FIG. 3). In separate studies,
Figure 3 | Models for the position of silent and escapee genes on the Xi. The ‘core’ the Cremer and Belmont groups also argue against the
model103,104 (part a) posits that inactive genes are in a transcriptionally silent compartment
model whereby inactive genes are inside the Xi territory
marked by Xist, and escapees are exterior to this core. The ‘outerNature
rim’ model
Reviews(part
105
b)
| Genetics
proposes that non-genic sequences form a core marked by Xist, whereas inactive and and escapee genes are localized at the periphery, instead
escapee genes are localized to the outer rim of the inactive X chromosome (Xi). A model proposing that genes escaping XCI are distributed
proposed by Calabrese et al.99 (part c) suggests that X‑silencing can be located and throughout the Xi territory 73,75,76 (FIG. 3). In this model,
maintained outside the Xist core and that transcription can occur inside this core. In the silenced chromatin is closer to transcriptionally compe‑
‘ANC-INC’ model (part d) proposed by Smeets et al.76, the Xi chromosome territory is tent compartments than in the central core model, and
pervaded by a collapsed ANC, which is enriched in Xist and inactive genes. Escapees are therefore allows for a close proximity between active
located both inside and outside the Xi chromosome territory. escapees and adjacent silenced genes. This model, in
which active and silent genes can ‘intermix’ in space,
would circumvent the physical problem associated with
the central core model.
one NPC clone and a high variability between inde‑ How escapees, which often reside next to silent
pendent NPC clones. For this reason, further investiga‑ genes, can be immune to the silencing effect of Xist is
tions are required to better understand the link between currently not known. However, studies have shown that
escapee gene expression and 3D chromosome structure. escapees often have CTCF-binding sites located near
A 3D model of the Xi with 1 Mb resolution showed their promoters30, and CTCF seems to separate inactive
that CTCF-enriched regions and active escapee genes and escape domains by binding to the boundary regions
Xist A‑repeat region are located preferentially at the periphery of the Xi ter‑ between escapees and closely juxtaposed inactive
A domain of Xist RNA that has ritory in mouse F1 brain80. Another study also proposed genes90,106,107. Interestingly, CTCF may be enriched in the
been identified as necessary that whereas silenced genes on the Xi are engaged in perichromatin regions77, suggesting that CTCF could
for Xist-mediated silencing but
not for localization of Xist to
very few interchromosomal contacts and reside in the bind to these boundary regions in the peri­chromatin
the X chromosome. chromosomal territory’s interior 31, escapee genes are regions to sequester inactive genes in the INC and
located at the periphery of the Xi. Escapee genes dis‑ expose adjacent escapees in the ANC. Notably, studies
Escapee genes play more specific contacts compared with genes that using allele-specific chromatin immunoprecipitation
X‑Linked genes that are
are subject to XCI and are engaged in long-range con‑ followed by sequencing in mouse cells have shown
biallelically expressed in
female somatic cells. tacts with each other and in many interchromosomal that CTCF binds to the Xa and Xi in a distinct man‑
interactions31,80. ner, helping to organize the Xa and Xi into functional
Long interspersed nuclear These studies confirm previous observations show‑ compartments for gene expression and repression26,56,99.
elements ing that silenced genes have a more internal position Recently, it has been hypothesized that YY1 may have
(LINEs). A type of repetitive
DNA in the mammalian
in the Xi101–103. After cell differentiation and the initi‑ a role in facilitating escape from XCI in human cells,
genome that is derived from ation of XCI, X‑linked genes are re‑localized from the potentially by binding near the transcription start site
transposons. periphery of the Xi territory to a more internal location of escapee genes86.

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Human Mouse Dxz4 macrosatellite, the DsTR minisatellite, their asso‑

ciated lncRNAs and CTCF-binding sites. In mice, the
DXZ4 Dxz4 DsTR Firre locus and its corresponding lncRNA also seem to
Xi Xi be involved in the regulation of Xi topology 111.
CTCF CTCF CTCF CTCF CTCF

TADs in Xic and the role of RS14. In the X chromo‑

DXZ4 Dxz4 DsTR
Xa Xa
CTCF CTCF CTCF
Figure 4 | Organization of the DXZ4 and Dxz4 loci in human and mouse models,
respectively. The human DXZ4 locus consists of 50–100 copies ofNature
a CpG-rich 3 kb repeat,
Reviews | Genetics
which is hypomethylated, organized in euchromatin (green circles) and bound by CTCF on
the inactive X chromosome (Xi). On the active X chromosome (Xa), the locus is
hypermethylated (red dashed line) and packaged into heterochromatin (red circles) without
CTCF binding109. In mice, Dxz4 consists of approximately six repeats and is organized in
euchromatin only on the Xa, but is biallelically bound by CTCF26,108. The minisatellite DsTR is
found 50 kb downstream of mouse Dxz4, but does not occur in humans.
The DXZ4 locus: a platform for Xi folding. DXZ4 is an
X‑linked locus that is localized at the boundary between
the two megadomains of the Xi and has a fundamental
role in Xi topology in mouse and human cells. In both
species, deletion of the DXZ4 locus disrupts mega­domain
formation but does not seem to affect the establishment
of XCI30,84. The structure and organization of this macro­
satellite are somewhat different between the two species108
(FIG. 4). The human DXZ4 consists of 50–100 copies of a
CpG-rich 3 kb repeat, which is hypo­methylated, organ‑
ized in euchromatin and bound by CTCF on the Xi, but
is hypermethylated, packaged into hetero­chromatin and
not bound by CTCF on the Xa109. In mice, Dxz4 con‑
sists of approximately six repeats that are not particu‑
larly GC‑rich, and it is organized in euchromatin only
on Xa and biallelically bound by CTCF26,108. The mini‑
satellite DsTR lies 50 kb downstream of the mouse Dxz4
locus, and could be expressed biallelically but seems to
be mainly bound by CTCF on the Xa80,108. There is no
evidence for the presence of this minisatellite in humans.
In human cells, the Xi is organized into two,
non-overlapping types of heterochromatin, which are
sequestered into distinct territories, with macroH2A,
H3K27me3 and Xist representing one type, and
H3K9me3 and HP1 marking the second type110. DXZ4,
FIRRE and LOC550643 loci form a euchromatin domain
on the Xi that is bound by CTCF and localized at the
boundary of the two distinct heterochromatin territo‑
ries85. Deletion of the DXZ4 locus in somatic human
cells leads to a partial loss of heterochromatin, as the
H3K27me3 mark seems to recede84. By contrast, deletion
of the Dxz4 locus on the mouse Xi is proposed to cause
more silencing on the Xi, especially for escapee genes30.
However, a caveat is that this effect was observed pre‑
dominantly in one cell line, and there are variable effects
in other cell lines. Therefore, apart from partitioning the
Xi into two megadomains, the true function of DXZ4
and Dxz4 is not currently known, nor is it known which
specific element (or elements) in the Dxz4/DXZ4 locus
is responsible for the cis effects observed in the two spe‑
cies. Indeed, published deletions are large (~200 kb) and
encompass multiple potential elements, including the
some, the Xic can be partitioned into two distinct TADs,
which have opposite effects on Xist expression (FIG. 5).
The Xist TAD contains the Xist promoter, its 5′ sequence
and its positive regulators, including Jpx, Ftx, Xpr and
Rnf12 loci in a domain of ~550 kb. The Tsix TAD con‑
tains the Tsix promoter, the 5′ sequence of Tsix and its
associated regulators, including Xite, Tsx, Dxpas34,
Chic1 and Linx (also known as Islr2), spanning a total
of 260 kb. The 3D organization of the Xic into the Xist
TAD and the Tsix TAD could facilitate the interactions
of regulatory elements in each TAD in order to activate
or repress Xist, respectively 81,112. Expression profiles of
genes located in the same TAD might be correlated dur‑
ing differentiation. During XCI, genes in the Xist TAD
become upregulated, whereas genes located in the Tsix
TAD decrease in expression81. It has been proposed that
before XCI, Tsix and its enhancer Xite interact to pro‑
mote Tsix expression and prevent the activation of Xist,
which seems to make a looping contact with Jpx in a
‘poised’ interaction112 (FIG. 5).
On the Xa, the Tsix–Xite interaction is maintained,
favouring Tsix expression and Xist repression. At the
same time, the interaction between Xist and Jpx is lost
on the Xa. By contrast, on the future Xi, the interaction
between Tsix and Xite is lost 112. This leads to repres‑
sion of Tsix, which in turn enables the activation of
Xist expression, and the Xist–Jpx interaction becomes
activated112 (FIG. 5). Similar interactions are observed
in human cells, in which the interaction between XIST
and its putative regulator JPX is anchored by RNA poly­
merase II87 and is only detected on the Xi, supporting
the role of this chromatin loop in the regulation of XIST
expression from one of the two X chromosomes only.
The Xist and Tsix TADs are separated by a region
called RS14, which is located at the 3′ end of Xist. RS14
contains a strong DNase I hypersensitive site (DHS),
can bind CTCF and forms a border between the Xist
and Tsix TADs81,112–114 (FIG. 5). RS14 plays a part in XCI
initiation, as deleting it results in compromised Xist
induction114. From a molecular point of view, the dis‑
ruption of this boundary leads to an altered organization
of the Xist and Tsix TADs, and induces ectopic contacts
between sequences from previously separated TADs81,113.
Disorganization of the Xist and Tsix TADs in turn causes
transcriptional misregulation of the genes contained in
them81. Interestingly, although the RS14 boundary can
control the organization of the Xist and Tsix TADs, a
reciprocal regulation also seems possible because inter‑
actions within the TADs could contribute to defining
the boundary between them. Indeed, using a physical
polymer model-based deconvolution of chromosome
conformation capture carbon copy (5C) data, a virtual
disruption of Tsix and its regulators Linx and Chic1 was
shown to be able to affect the sharpness of the boundary
between the Xist and Tsix TADs113.

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Tsix TAD Xist TAD more specifically during mid‑to‑late S phase when the Xi
is undergoing DNA replication along with other blocks
of heterochromatin across the genome (euchromatin is
Linx
replicated in early S phase). It has been proposed that
Xpr the separation of DNA replication in time and in space
Pre-XCI
Chic1 + Tsix enables the faithful replication and maintenance of the
Xite Xist Xi‑heterochromatin state29. Notably, the perinucleolar
Rnf12
Tsx Jpx space is enriched for sucrose non-fermenting protein 2
homologue (SNF2H; also known as SMARCA5), an
Ftx ISWI chromatin remodelling subunit that has a key role
in heterochromatin replication29.
RS14
Regulation and role of the Xi perinuclear localization.
CTCF CTCF
Although the perinuclear localization of the Xi has been
observed for many decades, the role of this subnuclear
localization during XCI and the molecular mechanisms
DHS
involved are poorly understood. During cell differen‑
tiation, some TADs on the X chromosomes become
Linx Xite lamina-associated domains81. Recent studies showed that
Xpr Xist can interact with membrane attachment nuclear
After XCI
(on Xi) envelope proteins (lamin B receptor (LBR), lamina-
Chic1 associated proteins (LAPs) and SUN domain-containing
Xist
Tsix + Rnf12 protein 2 (SUN2)) (FIG. 5). Among these proteins, LBR
Jpx
Tsx may have a role in Xist-mediated transcriptional silenc‑
ing26,27, which suggests a role of the Xist protein part‑
Xi ners in Xi perinuclear targeting. Although the nuclear
Ftx
periphery is often associated with transcriptional silenc‑
ing 118, the Xa can also be observed at the nuclear enve‑
Figure 5 | The X-inactivation centre is partitioned into two topologically associated lope73, which suggests that the perinuclear localization
domains. Before X-chromosome inactivation (XCI), the X‑inactivationNaturecentre (Xic)
Reviews is
| Genetics of the Xi at the nuclear periphery is not sufficient to
divided into two topologically associated domains (TADs), the Tsix TAD and the Xist TAD,
which are separated by the RS14 boundary. RS14 contains a strong DNase I hypersensitive explain, by itself, the opposite epigenetic states between
site (DHS) and is bound by CTCF. Each TAD contains promoter and 5′ sequences of Tsix or the Xa and Xi. A possible mechanism is that the nuclear
Xist with their respective positive regulators. Before XCI, Tsix and its enhancer Xite interact periphery has two chromatin compartments. One com‑
to promote Tsix expression, whereas the interaction between Xist and Jpx is poised. After partment harbours active chromatin and is associated
differentiation, the interaction between Tsix and Xite is lost, whereas the Xist–Jpx with the nuclear pores that facilitate splicing and the
interaction becomes active. Xi, inactive X chromosome; Xpr, X-pairing region. export of mRNAs for translation in the cytoplasm, and
another compartment harbours heterochromatin and is
associated with lamin B119.
The final journey
XCI is a multistage journey through space. The X chromo­ Regulation and role of the Xi perinucleolar localization.
some not only undergoes regional and local changes Several studies in the past decade have suggested that
in chromosome structure but also travels through the Xic plays a major part in targeting the Xi to the peri‑
nucleus to find its final destination. nucleolar space29,32,33. Zhang and colleagues29 showed
that inserting Xic into an autosome will cause locali‑
Perinuclear and perinucleolar Xi localizations. After zation of the autosome to the perinucleolar space in a
XCI takes place, the Xa and Xi move to separate sub­ cell-cycle-dependent manner. Thus, the Xic is sufficient
nuclear positions as they adopt opposite epigenetic to target chromosomes to the perinucleolar ring during
states (FIG. 6). The Xi (historically known as the hetero‑ mid‑to‑late S phase. It is specifically Xist that is respon‑
chromatic Barr body) was observed to be a ‘nucleolar sible for this localization29 (FIG. 6), as an Xi conditionally
satellite’ present in female, but not male, cells of the deleted for Xist (XiΔXist) in mouse fibroblasts loses the
cat 115. Like other non-sex chromosomes, the Xa does ability to localize to the perinucleolar space. This role of
not have a specified position in the nucleus, but the Xi is Xist is conserved between mammalian species because
distinct in having two favoured subnuclear localization ectopic expression of XIST on an autosome in human
Nuclear periphery
domains — one at the nuclear periphery (perinuclear) and cells is also sufficient to promote the relocalization of
A subcompartment of the
nucleus that is composed of the other next to the nucleolus (perinucleolar)29,116,117 the XIST-bearing chromosome near to the nucleolus32,33.
the nuclear-membrane bilayer, (FIG. 6). Analysis by both electron microscopy and in situ An interesting open question is whether it is the Xist
its associated proteins and the hybridization showed that the Xi is located at the nuclear lncRNA or the Xist locus that is responsible for the
nuclear-pore complexes. envelope in 75–80% of interphase cells116,117. At a spe‑ ­targeting mechanism.
Lamina-associated domains
cific time during the cell cycle, however, the Xi moves More recently, it was observed that Firre, Dxz4 and
Genomic regions that interact to a perinucleolar location29. Indeed, 80–90% of the Xi DsTR are also associated with the nucleolus and repre‑
with the nuclear lamina. migrates to the perinucleolar region during S phase, sent nucleolus-associated domains that can be bound

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Xa TAD
Xa

Nucleolus

XCI initiation

Firre
Nucleolus Xite Xite
Tsix Tsix Dxz4
CTCF CTCF
CTCF Xist
CTCF
X–X pairing
Xic
SNF2H

XCI establishment

Dxz4/DXZ4
LBR
Xi

Xi
Xa Nucleolus Xa Nucleolus
Xist
Two megadomains

Mid-to-late S phase

Figure 6 | The X chromosome in space. Before X-chromosome inactivation (XCI), the two active X chromosomes (Xas)
assume random positions relative to each other. At the onset of XCI, the homologous X chromosomes come into contact
Nature Reviews | Genetics
transiently to choose the future inactive X chromosome (Xi). After X–X pairing, the Xi is located at the nuclear periphery;
this localization might be regulated by the interaction between Xist and the membrane attachment nuclear envelope
protein lamin B receptor (LBR). The Xa can also be located at the nuclear periphery. During mid‑to‑late S phase, the Xi is
located at the nucleolar periphery, where Xist, Dxz4 and Firre loci represent nucleolus-associated domains. This Xi
perinucleolar localization is regulated by the long non-coding RNAs (lncRNAs) Xist and Firre and by CTCF, and seems to be
involved in heterochromatin maintenance. SNF2H, sucrose non-fermenting protein 2 homologue; TAD, topologically
associated domain. Xic, X-inactivation centre.

by the nucleolar protein nucleophosmin80,88. However,
the mechanism by which these loci are targeted to the
nucleoli is poorly understood, and it is also unknown
whether these sequences are involved in the establish‑
ment or maintenance phase of silencing. Yang and col‑
leagues have proposed that Firre and Dxz4 loci can serve
as additional attachment points to target the Xi to the
nucleolus88 (FIG. 6). A reduction of Firre lncRNA expres‑
sion using short interfering RNAs decreased the asso‑
ciation frequency between Firre and Dxz4 loci with the
nucleolus88. Interestingly, Firre and Dxz4 loci are bound
by CTCF26,56,80,88,108, and knockdown of CTCF signifi‑
cantly reduces the association of Firre and Dxz4 with
the perinucleolar region88 Given the previous observa‑
tion that CTCF can interact with nucleophosmin and
tether an insulator region to the nucleolus120, CTCF
could have a similar role in Xi perinucleolar localiza‑
tion, that is, tethering Xist, Firre, Dxz4 and DsTR loci
to the nucleolus.
The precise mechanistic relationships remain to be
investigated. It is known that disrupting the Xi peri­
nucleolar localization observed in Firre lncRNA and
Ctcf knockdowns leads to a decrease of the repressive
mark H3K27me3 on the Xi, but this reduction does not
result in the reactivation of X‑linked genes88. Conversely,
the loss of Xi nucleolar localization in Xist-deleted cell

10 | ADVANCE ONLINE PUBLICATION www.nature.com/nrg

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lines (XiΔXist) is accompanied by a decrease in H3K27me3
enrichment and an increase in Xi gene expression29.
Furthermore, ectopic localization of an Xist-bearing
autosome to the perinucleolar region is correlated with
deposition of the repressive H3K27me3 mark and gene
repression33. Taken together, these results suggest that
the targeting of the Xi to the nucleolus could play a part
in heterochromatin maintenance and gene silencing.
Conclusions
Technological and methodological developments over
the past decade have improved our ability to study 3D
genome organization and have enabled a better under‑
standing of the relationship between structure and
function. This understanding is helping to shape our
knowledge of XCI as well as other epigenetic processes.
However, many questions remain: for example, how can
escapees be immune to the Xi silencing during XCI?
And what is the relationship between their 3D organi‑
zation and their transcription? What specific factors and
molecular mechanisms are involved in the regulatory
processes? It is predicted that lncRNAs play a key part
in the regulation of architectural protein factors such
as CTCF and cohesins. We therefore anticipate that, in
the next few years, the development of new approaches
to study protein–RNA interactions will pave the
way to a better understanding of the molecular linkage
between gene expression, conformational changes in
higher-order chromosome structures and nuclear ­spatial
compartments.

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Acknowledgements
The authors thank M.-E. Gilles, Y. Jeon and F. Ariel for many
inspirational discussions. T.J. is a European Molecular Biology
Organization (EMBO) postdoctoral fellow (EMBO ALTF 1313–
2015); E.A. is funded by the Swiss National Science Foundation.
J.T.L. is funded by the US National Institutes of Health
(R37‑GM58839) and is an investigator of the Howard Hughes
Medical Institute.
Competing interests statement
The authors declare no competing interests.

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