Download X inactivation Xplained

X inactivation Xplained
Anton Wutz1 and Joost Gribnau2
Random inactivation of one of the two female X chromosomes
establishes dosage compensation between XY males and XX
females in placental mammals. X inactivation is controlled by
the X inactivation center (Xic). Recent advances in genome
sequencing show that the Xic has evolved from an ancestral
vertebrate gene cluster in placental mammals and has
undergone separate rearrangements in marsupials. The Xic
ensures that all but one X chromosome per diploid genome are
inactivated. Which chromosome remains active is randomly
chosen. Pairing of Xic loci on the two X chromosomes and
alternate states of the X chromosomes before inactivation have
recently been implicated in the mechanism of random choice.
Chromosome-wide silencing is then initiated by the noncoding
Xist RNA, which evolved with the mammalian Xic and covers
the inactive X chromosome.
Addresses
1
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7,
1030 Vienna, Austria
2
Department of Reproduction and Development, Erasmus University
Medical Center, Rotterdam, The Netherlands
Corresponding author: Wutz, Anton ([email protected]) and
Gribnau, Joost ([email protected])
Current Opinion in Genetics & Development 2007, 17:387–393
This review comes from a themed issue on
Differentiation and gene regulation
Edited by Denis Duboule and Frank Grosveld
Available online 14th September 2007
0959-437X/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2007.08.001
Introduction
Mammals have evolved a complex set of functions for
gene regulation allowing elegant control of expression
during development. One of the most intriguing forms of
gene regulation leads to exclusive gene expression from
one single parental allele. Different mechanisms are used
to achieve monoallelic gene expression ranging from
parental imprinting [1] to odorant receptor choice in
the olfactory system [2]. These processes are tailored
to suit the requirements of different biological systems.
For instance, the immunoglobulin heavy chain rearrangements in B-lymphopoiesis generate a pool of cells each
expressing one specific antibody, which allows for clonal
selection in the immune system [3]. This process is
random with respect to the parental origin of the locus.
In contrast, imprinted genes show exclusive expression
from one parental allele [4]. In mammals dosage
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compensation of X-linked genes between the sexes is
established by inactivation of one of the two female Xs
(reviewed in [5]). In mice X chromosome inactivation
(XCI) is random in the embryo, and imprinted in extraembryonic tissues, where the paternally inherited X
chromosome is inactivated [6]. X inactivation, thus,
exemplifies the functions of the mammalian cell nucleus
in domain regulation and monoallelic expression. Here,
we review recent progress in understanding of the evolution of the mammalian dosage compensation system, the
mechanism of random choice and chromosome-wide
inactivation.
Evolution does dosage compensation
Dosage compensation systems adjust the gene doses
of sex chromosome linked genes between the sexes
and are observed throughout the animal kingdom.
Different strategies to accomplish dosage compensation
are used by fruit flies, worms, and mammals (reviewed
in [7]). Interestingly, no evidence for efficient dosage
compensation has been found by genome-wide expression analysis in chicken and finches [8]. Differences in
Z gene dosis between ZZ males and ZW females
result in elevated Z gene levels in males and might
contribute to sexual differentiation in birds. The observation of dosis compensation systems in other organisms must therefore be explained by an additional
advantage rather than an absolute necessity during
evolution. This hypothesis is highlighted by the observation that many genes escape XCI in human [9], and
supported by the intriguing finding that monoallelic
expression of the entire X chromosome has facilitated
the evolution of trichromatic color vision in monkeys
[10].
The mammalian dosage compensation system is controlled to a large extent by the X inactivation center
(Xic) located on the X chromosome. The Xic regulates
counting of the number of X chromosomes and contains
the noncoding Xist RNA gene, which localizes specifically
to the inactive X chromosome (Xi) and triggers chromosome-wide gene repression. Recent findings show that
this locus has apparently evolved exclusively in placental
mammals [11,12,13]. In marsupials and the more
distant vertebrates, such as frog or fish, the genomic
region around the Xic has an entirely different structure
with evidence for multiple rearrangements (Figure 1).
Interestingly, a different study indicates that the Xist
gene has newly emerged from an ancestral protein-coding
gene Lnx3 during mammalian evolution [14], indicating
that XCI in marsupials requires an alternative mechanism
for dosage compensation.
Current Opinion in Genetics & Development 2007, 17:387–393
388 Differentiation and gene regulation
Figure 1
Evolution of the X inactivation center. (a) A scheme of the Xic locus in placental mammals is shown. The Xist and regulatory Tsix transcript are
indicated. Genes that are derived from vertebrate protein-coding genes and have lost protein-coding potential in eutheria are indicated in blue.
Boxed genes serve as anchors for identifying the genomic region in vertebrates and marsupials. (b) The ancestral vertebrate gene cluster
corresponding to the mammalian Xic region has undergone an expansion during placental mammal evolution. The protein-coding LNX3 gene
shown in green shares sequence homology in a few exons with the noncoding Xist and is believed to be the ancestral Xist gene. (c) In the
marsupial lineage the ancestral vertebrate gene cluster has been rearranged and split in two clusters of genes, which map to distant regions on
the X. Xist has exclusively evolved in placental mammals and in marsupial genomes the LNX3 protein-coding gene is found.
Regulation of randomness
How random X inactivation is established is unclear at
present. Recent progress in the search for mechanisms
driving the XCI counting and choice process, which
mark the initiation of XCI, has produced evidence that
multiple regulatory systems may be involved. Different
observations made with aneuploid, and tetraploid cells
indicated that one X chromosome remains active per
diploid genome after completion of XCI. These and other
findings led to the hypothesis that counting and choice in
XCI can be explained by the action of an autosomal
blocking factor (BF), which protects one X chromosome
per diploid genome from inactivation (Figure 2a). Studies
aimed to identify the blocking factor binding site have
resulted in the identification of a 1.2 kb candidate
element, DXPas34, which is located 30 of Xist [15].
Deletion of this element resulted in ectopic XCI in male
cells, indicating a role for this element in the counting
process [16]. Surprisingly, a different study describing a
similar deletion of DXPas34 reported no ectopic XCI in
Current Opinion in Genetics & Development 2007, 17:387–393
male cells, indicating that more work has to be done to
clarify this topic [17].
The real nature of the blocking factor remains elusive so
far, and could be a nuclear episome, or a limited protein or
protein complex. Based on the blocking factor model
using theoretical considerations a symmetry-breaking
model has been proposed, in which many diffusible
molecules are quantitatively sequestered on the active
X chromosome by a mechanism that involves intermolecular BF interactions [18] (Figure 2b). Close proximity of
the Xic loci would benefit this model to allow binding
equilibration to occur fast enough to be able to discriminate between an active blocked and an inactive nonblocked X chromosome. Indeed, trans-interaction of X
chromosomes by Xic–Xic pairing has been observed
[19,20]. Based on this observation a mutual exclusive
choice model has been proposed that posits coordinate
trans-regulation of the interacting Xics [21] (Figure 2d).
Trans-vection is known from the fruit fly [22] and may aid
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X inactivation Xplained Wutz and Gribnau 389
Figure 2
Different models for the initiation of random XCI. (a) An autosomally encoded blocking factor (BF) protects one X chromosome from inactivation.
(b) Breaking of the symmetry in particle binding leads to accumulation of many diffusible molecules on one X chromosome protecting it from
inactivation. (c) Alternate epigenetic states mark the future Xa and Xi before the initiation of XCI. (d) Both X chromosomes meet in space and
determine the future Xa and Xi. (e) In a stochastic model each X chromosome has a certain probability to be inactivated.
blocking factor binding. The precise function of the Xic
interaction remains to be determined. Notably, XCI
counting is not affected in female cells with a 65 kb
deletion 30 of Xist which shows a loss of Xic pairing [19].
In several models, the XCI counting and choice process is
anticipated to be triggered by developmental or differentiation cues. Nevertheless, X chromosomes might be
distinguished before inactivation by assuming alternative
states (Figure 2c). Recently, it has been reported that
cohesion of sister chromatids is regulated differentially
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between the two female X chromosomes [23]. This
could highlight an inherent difference between the two
genetically identical chromosomes in at least part of the
cell population. The different states may represent blocking factor binding to one chromosome, or accessibility or
transcriptional differences between the two Xics,
although more work is required to establish the molecular
basis of this phenomenon.
The potential risk of female lethality dictates a need for a
precise counting process. This assumption has spurred a
Current Opinion in Genetics & Development 2007, 17:387–393
390 Differentiation and gene regulation
hunt for regulatory mechanisms. Nevertheless, any given
mechanism of XCI counting and choice requires a stochastic element. In principle, this could be achieved
simply by inactivating each X chromosome with a certain
fixed and fine-tuned probability. It has been shown that
chaotic choice results if Tsix is disrupted on both Xic
alleles [24]. Surprisingly, this can result in viable female
mice, albeit with a low frequency [24] (Figure 2e). Therefore, chaotic choice may represent the basic mechanism
underlying the XCI counting and choice process. Currently, the molecular identity for factors involved in the
counting process remains unknown [5]. Suggestions that
beside an autosomally encoded blocking factor an
X-linked competence factor might stimulate X inactivation further complicate the scenarios [21]. The molecular underpinning is clearly complex and it is hard to
see how to discriminate between different models if not
by analysis of molecular components.
XCI starts with the accumulation of Xist along the future
inactive X chromosome. Recent work indicates that Xist
up-regulation is the consequence of sex-specific induction of Xist transcription on the future Xi and not stabilization of the Xist RNA [25]. In differentiated cells Xist
repression on the Xa is mediated by DNA methylation
[26]. A recent study shows that the methyl-DNA binding
protein Mbd2 is required for Xist repression and, thus,
provides a link to repressor complexes [27]. During the
early stages of XCI, Xist expression is regulated by the
Tsix gene. Tsix is a long untranslated RNA, which acts
predominantly in the nucleus and is transcribed in antisense direction over the Xist gene. A recent report shows
that Tsix does not require splicing for its function [28],
leaving open the question whether the act of transcription
or the Tsix RNA itself are required for Xist repression. Tsix
function requires the intronic DXPas34 element [16,17].
Deletion of this element results in nonrandom XCI of the
mutated X chromosome and a defect in imprinted XCI
[17]. DXPas34 harbors many putative CTCF binding
sites, frequently flanked by putative YY1 sites [29]. Several of these CTCF sites show differential methylation in
gametes and somatic cells, implying a direct role for this
element in imprinted and random XCI [30]. Moreover,
analysis of YY1 mutant embryos has indicated a role for
this protein in the regulation of Tsix and Xist [29], but it
remains to be established if YY1 and CTCF act as bona
fide blocking factors, or are required to perform structural
roles for Xic regulation.
A conundrum is the lack of conservation of Tsix and
DXPas34 between the mouse and human XIC. It has
been observed in human and bovine cells that the antisense TSIX RNA is coexpressed with XIST and, thus,
apparently does not act to repress XIST in all mammals
[31,32]. This raises the possibility that evolution has
selected different molecular components for regulating
randomness in different mammals.
Current Opinion in Genetics & Development 2007, 17:387–393
Building a cis-limited repressive nuclear
compartment
The human Xi is comprised of a heterogeneous chromatin
landscape [33]. Not all genes are repressed on the Xi and a
relatively large set of genes escape from inactivation in
human. Recently, a gene activity profile has been derived
for the human Xi [9]. The availability of the human
X chromosome sequence [34] allowed the identification
of X enriched sequence elements [35,36]. Importantly, a
prediction for gene inactivation and escape of inactivation
was reported [35]. The identified sequences originate in
part from genomic repeat elements of the L1 long interspersed elements (LINE) class, which were already
implicated in spreading of X inactivation [37]. Experimental support for this hypothesis comes from analyses
of mice carrying the T(X;4)37H X;autosome translocation
chromosome. This study shows that spreading of Xist
RNA is attenuated at the autosomal boundary of the
translocation, which has a low abundance of LINE
elements [38].
Albeit L1 elements are not exclusive to the X chromosome,
they might be part of the features on the X chromosome
that facilitate spreading of the dosage compensation complex. Recent studies indicate that in the interphase nucleus
the human Xi is organized into an outer rim of genes and a
repeat-rich core, to which Xist localizes [39]. In differentiated mouse ES cells, Xist forms a repressive compartment
from which the transcription machinery is excluded as a
first step in X inactivation [40]. This compartment
initially does not contain genes and is also not sufficient
for gene silencing [40]. Genes become repressed and
associate with the repressive compartment upon silencing,
which requires the repeat-A sequences on the 50 -end of Xist
RNA [41] (Figure 3a). How silencing is established and
how it is restricted to one specific chromosome requires
further studies. The function of Xist in gene silencing has
been reported to be restricted to early development and
specific somatic cell types such as precursors in the blood
system [42] (Figure 3b). This suggests that the pathways
for gene silencing are regulated in a cell type-specific
manner.
Once X inactivation has been established the silent
X chromosome is stably maintained in somatic cells.
Chromosome-wide chromatin analysis of X-linked epigenetic modifications confirmed many cytological observations, including hypoacetylation of histone H4,
accumulation of histone H3, K9, and K27 tri-methylation
and accumulation of histone variant macroH2A on the Xi
[43,44] (Figure 3b). These results suggest that a wide
variety of chromatin modifiers including Polycomb group
complexes, histone deacetylases, and DNA methylases act
in concert to maintain repression of the Xi [45]. Beside
profiles of histone modification marks along the X chromosome, a DNA methylation profile of the X chromosome
has also been established [46]. This study shows that in
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X inactivation Xplained Wutz and Gribnau 391
Figure 3
Silencing of one X chromosome by Xist. (a) The two X chromosome territories are depicted. Xist RNA produced from the Xic locus accumulates
on the future Xi. This leads to the formation of a nuclear compartment that is devoid of the transcription machinery, such as RNA polymerase II
(Pol II). Polycomb group complexes are recruited to the Xist-covered chromosome and establish chromosome-wide histone modifications. The
genes localize to the outside this compartment and are silenced in a manner that requires the Xist repeat-A sequence. Silencing of genes
coincides with their association with the Xist compartment. (b) A summary of chromatin modifications during initiation of X inactivation. Xist RNA
accumulation is followed by recruitment of Polycomb repressor complex 1 (PRC1), which mediates histone H2A lysine 119 ubiquitinylation
(H2AK119ub1), and PRC2, which mediates histone H3 lysine 27 tri-methylation (H3K27me3). Accumulation of histone H4 lysine 20 monomethylation can also be observed on the X. In this phase Xist triggers gene silencing. Initially X inactivation is reversible and genes become
reactivated if Xist is lost. If cell differentiation progresses histone macroH2A becomes enriched on the Xi and histone H4 is hypoacetylated. Later
during the XCI process DNA cytosine methylation marks the promoters of genes on the Xi. In this phase X inactivation is irreversible and Xist
is not required for maintenance of the silent state. In somatic cells Xist does not have the capacity to initiate chromosome-wide silencing. Thus,
the silencing function of Xist depends on the Xist repeat-A sequence and a defined cellular context.
contrast to CpG island methylation found on the Xi, DNA
methylation is surprisingly more abundant on the Xa and
appears to be confined to gene body sequences on the Xa.
This finding may be related to the doubling of gene
expression of the single Xa relative to autosomes [47].
Intriguingly, in female XX ES cells global DNA hypomethylation is observed [48]. Could this possibly indicate a
potential function of demethylation in the counting process? The same epigenetic marks appear to be used at
different places for different regulatory functions in X
inactivation. This highlights a crucial role for spatial organization in complex gene regulatory mechanisms.
The Barr body has been originally observed to locate
sometimes close to the nucleolus [27]. A recent investigation has quantified the location of the Xi in differentiating ES cells and demonstrated that the association of the Xi
with the nucleolus occurs in S phase [49]. This has led to
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speculation about a role for the subnuclear localization in
providing an environment for stable replication of heterochromatin. It has been shown before that the Xi replicates
out of synchrony with other chromosomes either early in
S-phase in preimplantation embryos and late in S-phase in
embryonic cells after implantation [6]. This highlights a
spatial and temporal separation of the facultative heterochromatin of the Xi. Deletion of the Xist gene has been
shown to result in loss of perinucleolar position of the Xi,
but reactivation of the chromosome is limited to single
genes in occasional cells [49]. Moreover late replication and
chromosome-wide histone H4 hypoacetylation are maintained independent of Xist, yet, histone methylation marks
and macroH2A are lost without Xist [50–52]. These
results indicate that many superimposed epigenetic mechanisms maintain silencing of the Xi, and the exact role of
the temporal association of the Xi with the nucleolus in
maintaining the silent state remains to be determined.
Current Opinion in Genetics & Development 2007, 17:387–393
392 Differentiation and gene regulation
Conclusion and outlook
There is increasing evidence that indexing of the information stored in the genome is aided by chromatin
modifications such as histone methylation and acetylation. A combination of these signals has been proposed
to constitute a histone code [53]. Recently, it has become
clear that this is only one side of the full story and that
nuclear positioning and compartmentalization carry an
equal if not greater weight [54]. The chromosome-wide
phenomenon of X inactivation might hold promise to
further unravel the interplay between chromatin modifications and compartmentalization and will shed light on
the gene regulatory repertoire of mammals.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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