Downloaded from [Link]

org/ on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press

Dosage Compensation in Mammals

Neil Brockdorff1 and Bryan M. Turner2
1
Department of Biochemistry, University of Oxford Oxford OX1 3QU, United Kingdom; 2School of Cancer Sciences,
Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
Correspondence: turnerbm@[Link]

SUMMARY

Many organisms show major chromosomal differences between sexes. In mammals, females have two
copies of a large, gene-rich chromosome, the X, whereas males have one X and a small, gene-poor Y. The
imbalance in expression of several hundred genes is lethal if not dealt with by dosage compensation. The
male– female difference is addressed by silencing of genes on one female X early in development.
However, both males and females now have only one active X chromosome. This is compensated by
twofold up-regulation of genes on the active X. This complex system continues to provide important
insights into mechanisms of epigenetic regulation.

Outline

1 Introduction 5 X-chromosome reactivation and

reprogramming
2 Overview of X inactivation
6 Summary and future directions
3 Initiation of X inactivation
References
4 Propagation and maintenance of the inactive
state

Editors: C. David Allis, Marie-Laure Caparros, Thomas Jenuwein, and Danny Reinberg
Additional Perspectives on Epigenetics available at [Link]
Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a019406
Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

1
 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

OVERVIEW

In mammals, as in many other organisms, there is a major differences in metabolic control and other cellular pro-
chromosomal difference between the sexes. For example, hu- cesses. To avoid this, dosage compensation mechanisms
mans have 22 pairs of chromosomes known as autosomes that have evolved that balance the level of X-linked gene products
are present in both males and females; one member of each between the sexes. There are three general methods by which
pair is inherited from the mother and one from the father. But this can be performed: first, a twofold up-regulation in the
there are two other chromosomes, the sex chromosomes, des- expression of X-linked gene in males; second, a twofold
ignated X and Y, that differ between the sexes; females have down-regulation of genes on each of the two X chromosomes
two Xs, whereas males have one X and one Y. This matters in females; and finally, the complete inactivation of one of the
because although the X is a medium-sized chromosome with two X chromosomes in females. The first strategy has been
more than 1000 genes, the Y is small and gene poor. A similar adopted in the fruit fly, Drosophila (see Lucchesi and Kuroda
situation exists in other mammals, including rodents and mar- 2014), the second in the worm Caenorhabditis elegans (see
supials. Chromosomal differences are linked to the mecha- Strome et al. 2014), and, it now seems, both the first and the
nism by which sex is determined and seem to have evolved last in mammals.
over many millions of years. Over recent years, studies of dosage compensation in
Sex chromosome imbalance presents the organism with a mammals have provided crucial insights into fundamental
problem: The two sexes differ in the copy number of X-linked epigenetic mechanisms and how patterns of gene expression
genes. This can lead to an imbalance in the amount of gene are regulated through development. It can be confidently pre-
products (RNAs and proteins), which would, in turn, require dicted that they will continue to do so.

2 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

1 INTRODUCTION and in such diverse organisms, including mammals, birds,

and fruit flies? It may be that they have occurred as a by-
1.1 Sex Determination Creates a Need
product of the suppression of crossing-over required
for Dosage Compensation
to prevent intersex states (Fig. 1). Mathematical analysis
Sexual reproduction is common among eukaryotes. Even of the factors that influence the spread of alleles through
plants that can replicate perfectly well asexually by sending populations shows that suppression of crossing-over will
out shoots or runners often have an alternative sexual mode inevitably lead to the gradual accumulation of deleterious
of reproduction. A possible explanation is that sexual mutations around the region of crossover suppression. This
reproduction brings an enormous increase in genetic var- is largely because such mutations rarely become homozy-
iability on which natural selection can operate. The reshuf- gous, which is necessary if they are to be selected against.
fling of alleles that occurs with every sexual generation
produces a population better able to cope with environ-
mental shifts compared with a relatively homogeneous Female Proto-male
Pro
oto-male
e Male Male
population derived from asexual methods of reproduction.
But sex is complicated, requiring developmental pathways PAR
PAR
that lead to male and female sexual organs, as well as the
physiological and biochemical apparatus required for mei-
osis, germ cell maturation, the attraction of partners, and +
mating (see Marshall Graves and Shetty 2001, and refer-
ences therein, for further discussion of these issues).
Genetic mechanisms used in defining different sexes
vary widely from one organism to another. The simplest
system involves a single locus that is homozygous in one sex A A A A*
A proto-X
proto
p o-X
X proto-Y
pro to-Y X Y
(the “homogametic” sex) and heterozygous in the other
(the “heterogametic” sex; Fig. 1). This system has evolved Evolutionary
Evolu
utiona
ary tim
time
me
in different ways to reach varying levels of complexity in
different organisms. In some, mechanisms have been put in Male-determining Crossing-over Translocated
place that suppress meiotic recombination (crossing-over) allele suppressed regions
of the sex-determining alleles in the heterogametic sex
(Fig. 1), a step that helps prevent the generation of mixtures Figure 1. Evolution of the Y chromosome. Early in evolution, the two
of alleles leading to intersex states. The inability to recom- sexes may have differed at only a single, autosomal locus (marked by a
bine, in many cases, has spread to include part or all of one black box); one sex is homozygous at this locus (female) and the
chromosome, with an accompanying loss of genetic infor- other sex (male) is heterozygous (designated proto-male). The “male-
determining allele” is shown in yellow. If mating requires one mem-
mation. The evolutionary pressures that have driven this ber of each sex, then individuals homozygous for the male-determin-
chromosome degeneration are still not understood, but the ing allele cannot arise. At this early stage, physiological differences
end result in many species is that the two sexes show dif- between the sexes will be subtle, comparable to those that distinguish
ferences, not just in alleles at one or a few loci, but in the two mating types in yeast. To prevent the formation of intersex
complete chromosomes. In mammals, it is the males who states, crossing-over will be suppressed within and around the male-
determining locus (dark shading). Mutations, including deletions
carry the degenerate chromosome, whereas in birds, it is and inversions, will accumulate and cause the degenerate region in
the females (Marshall Graves and Shetty 2001). which crossing-over is suppressed to gradually expand (“Muller’s
Sexual differentiation is usually triggered by one or a ratchet”) until the chromosome has lost most of its active, functional
small number of crucial genes being switched on or off genes. (Mutations accumulate because suppression of crossing-over
during development. The products of these genes initiate reduces the probability that they will occur in homozygous form,
hence, reducing the selection pressure against them.) A small, active
a cascade of gene regulatory events that mediate progres-
region must remain that is homologous to the X chromosome to
sion down one or the other pathway of sex determination allow pairing and crossing-over at meiosis (indicated by a gray ×).
(see Strome et al. 2014 for details in C. elegans; see Lucchesi This is the pseudoautosomal region (PAR). The autosome, originally
and Kuroda 2014 for details in Drosophila). In humans, it is homologous to the future X (A in the diagram), will itself evolve
the protein product of the SRY gene on the Y chromosome sometimes through translocations from other chromosomes (shown
that sends the early embryo down the male pathway (re- as red shaded areas), eventually forming the distinctive X chromo-
some. The X, like other chromosomes, is a mosaic of DNA fragments
viewed by Quinn and Koopman 2012). A mechanism of this put in place at different periods through evolution; some of these are
sort does not need major chromosome differences to oper- ancient and some are relatively recent. On the human X, the more
ate successfully, so why have such differences arisen so often recent arrivals are enriched in genes that escape X inactivation.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 3

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

Mutations, including deletions and translocations, gradu- genes encoding components of large multiprotein com-
ally spread beyond the original suppressed region, leading plexes are particularly likely to exert a phenotypic effect
to the progressive degeneration of one of the two originally (Pessia et al. 2012). Also, having half the normal level of
homologous chromosomes (Fig. 1). This irreversible, de- “multiple” gene products can have a cumulative effect. For
generative process has been termed “Müller’s ratchet,” in example, if several components of a metabolic or signaling
recognition of the geneticist who first proposed it and mod- pathway are all reduced twofold, the end product of that
eled it mathematically. There is no selection for this process, pathway may be reduced severalfold, with likely phenotypic
it just happens as a consequence of local crossover suppres- effects (Fig. 2) (Oliver 2007). Thus, problems will inevita-
sion, which, in turn, was made necessary by the adoption of bly accumulate for the heterogametic sex as the proto-Y
a two-sex strategy for reproduction (discussed in Charles- chromosome degenerates over evolutionary time, both
worth 1996; Charlesworth and Charlesworth 2005). But through loss of X-linked genes that are individually dosage
whatever the evolutionary drive behind chromosome de- sensitive and the progressive loss of genes that are individ-
generation, the fact that it has occurred (and is presumably ually dosage tolerant, but collectively less so (Fig. 2).
continuing) has required the coevolution of mechanisms to The problem faced by males was recognized more than
cope with two contingent problems: First, there is a major 40 years ago by the geneticist Susumu Ohno, who specu-
chromosomal difference between members of the same spe- lated that the problem could be solved by up-regulating
cies, and second, the heterogametic sex is monosomic for a twofold, expression of genes on the single male X chromo-
large chromosome and thus monoallelic for a large number some (Ohno 1967). He also noted that this could occur on
of genes. Both these issues must be addressed by mecha- a gene-by-gene basis over evolutionary time. Certainly, loss
nisms of dosage compensation. of a dosage-sensitive gene (or a gene in a pathway in which
some members had already been lost) would result in a
strong selection pressure in favor of up-regulating (i.e.,
1.2 Solving the Male Problem: Up-Regulation
compensating) expression of the remaining gene copy.
of X-Linked Genes
However, if the male (heterogametic, XY) sex were to adopt
In both mammals and Drosophila, males have one copy of this strategy, it would cause problems for the female
each sex chromosome, an X and a Y, whereas females have (homogametic, XX) members of the species, who would
two copies of the X. In both groups of organisms, the Y is then have to cope with increasing numbers of genes whose
gene poor and largely heterochromatic. It contains just a expression had been “increased” twofold. It may be, there-
few genes needed for male development or fertility. In con- fore, that silencing of one of the two X chromosomes in
trast, the X is a large, gene-rich chromosome. In all organ- female mammals, the subject of most of this article, is a
isms gene products (RNAs and proteins) are made in direct response to this overexpression.
proportion to the number of copies of the gene per cell. The general feasibility of Ohno’s suggestion was estab-
Thus, the presence of one or two X chromosomes would lished by work on dosage compensation in the fruit fly
cause a twofold difference in the intracellular concentra- Drosophila melanogaster in which genetic, biochemical,
tions of many gene products between the sexes. Further- and recent high-throughput gene expression studies have
more, XY males are monoallelic for the great majority of confirmed that dosage compensation occurs through up-
X-linked genes (only a few genes have homologs on the Y regulation of X-linked genes in XY males (discussed in
or autosomes). This is surprising, considering higher eu- Lucchesi and Kuroda 2014). Crucially, the mechanism
karyotes are usually very intolerant of the loss of even a part that evolved in the fly is such that up-regulation occurs
of a chromosome; small chromosome deletions generally only in males, circumventing the problem of overexpres-
cause major deformities, whereas autosomal monosomies sion in XX females. Detailed exploration of Ohno’s hypoth-
are always lethal. How, then, do males survive with only one esis in mammals has had to wait for the advent of
X chromosome? methodologies that can accurately assay the expression of
In attempting to answer this question, it is important to large numbers of genes. The reason is that assaying tran-
realize that cells and organisms tolerate monoallelism for script levels of individual genes, however accurately, cannot
individual genes rather well. For example, a heterozygous determine whether the gene is up-regulated or not. Genes
individual with an allele encoding an inactive form of an vary enormously, one from another, in the level at which
enzyme, and thus with only half the normal level of active they are expressed, and even the same gene often changes
enzyme, is usually perfectly healthy, although homozygos- expression depending on the tissue or cell type in which it is
ity for the defective allele may be lethal. However, some housed, the stage of development, or even over time in the
genes are clearly more dose sensitive than others. Recent same cell. How, then, can we tell whether any particular
studies have suggested that a twofold dosage change in gene is twofold up-regulated? What is our baseline?

4 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

a b c d

a/2 b c d
No phenotype
Progressive allele loss
over evolutionary time
a/2 b/2 c d
No phenotype

a/2 b/2 c/2 d

Phenotypic effects

Selection for up-regulation

of enzymes a, b, c

Figure 2. The effects of progressive loss of X-linked alleles on a hypothetical signaling pathway. The diagram shows
successive components of the pathway as colored discs, each put in place by the actions of enzymes a, b, c, and d. The
size of the discs is proportional to the amount of each component (level 1). The model proposes that enzymes a, b,
and c are all encoded by genes on the X chromosome, and early in evolution were encoded by genes on the two
proto-X chromosomes. Alleles are lost as the proto-X progressively degenerates, eventually forming the gene-poor
Y chromosome (Fig. 1). A twofold reduction in the amount of an enzyme is likely to cause a reduction in its product,
although not necessarily twofold. The cell’s normal homeostatic mechanisms are likely to correct small disturbances
and there is likely to be little or no effect on subsequent steps in the pathway (level 2). Even the loss of two enzymes
may be corrected with no physiological effect (level 3). However, a stage will eventually be reached when the
cumulative effects of enzyme (gene) depletion cause key components to decrease below a critical level and trigger
an effect on phenotype (shown here on level 4). Selection pressure will be exerted to correct the phenotypic effect,
most readily by up-regulating expression of one or more of the remaining, single alleles of enzymes a, b, or c.

Recent technologies, first microarrays and now high- the weight of evidence strongly supports the up-regulated
throughput RNA sequencing, can assay the expression of expression of X-linked genes in both male and female cells,
large numbers of genes, allowing us to test the proposition the situation is complex, as is often the case with dosage
that, in any particular tissue or cell type, the “overall” ex- compensation. Although the X:autosome expression ratio
pression of X-linked genes in males (i.e., over the whole, is consistently .0.5, it does not reach 1.0 (Deng et al. 2011;
wide range of expression levels) is higher than that of au- Lin et al. 2011). The reason for this may be that up-regu-
tosomal genes. If the expression ratio of X-linked to auto- lation is not applied to all X-linked genes. Perhaps only the
somal genes in males, with one X and two copies of each more dosage-sensitive genes are up-regulated (Pessia et al.
autosome, is 0.5, then there is no up-regulation; that is, 2012), something that would fit with an evolutionary mod-
expression levels reflect gene copy number. On the other el in which up-regulation was determined on a gene-by-
hand, if it is 1.0, then there is complete, twofold up-regu- gene basis (Fig. 1).
lation and Ohno’s hypothesis is validated (Fig. 3). There are many questions that remain to be answered
Over the past 5 years or so, several studies using either concerning expression of X-linked genes, and the issues
microarray data (Nguyen and Disteche 2006; Lin et al. they raise are relevant to the much more intensively studied
2007) or, more recently, RNA sequencing (Deng et al. question of X-chromosome inactivation, with which the
2011) have addressed this issue providing strong evidence rest of this article is concerned. Perhaps most pressing is
for the up-regulation of X-linked genes in mammals. How- the need to decide whether genes are up-regulated by a
ever, the data requires careful interpretation. Complica- common, chromosome-wide mechanism, whether differ-
tions arise from the fact that the X chromosome contains ent genes have adopted different mechanisms, or whether
a higher proportion of tissue-specific genes (often involved both factors come into play. Similar issues have been ad-
in sexual development) than autosomes, with the result dressed in attempting to unravel the dosage compensation
that the proportion of X-linked genes in any given tissue mechanism in D. melanogaster, and although the male-
or cell type that is switched off (silenced) in both sexes is specific process that is used in flies is unlikely to operate
higher than for autosomes (Ellegren and Parsch 2007; Mei- in mammals, there are undoubtedly important lessons to
sel et al. 2012). This must be taken into account. Though be learned from this widely studied model organism.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 5

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

No compensation Full compensation

No up-regulation of X-linked genes Twofold up-regulation of X-linked genes

Ratio of medians = 0.5 Ratio of medians ~1.0

Number of genes

Number of genes
Gene activity Gene activity
X-chromosomal genes
Autosomal genes

Figure 3. Shift in median expression caused by selective up-regulation of X-linked genes. Measurement of the
expression of large numbers of genes on either the X chromosome (blue) or autosomes (red) by microarrays or
RNA sequencing shows that transcript levels are normally distributed with a range of expression levels spread over
several orders of magnitude. If expression of genes on the single male X (or the single active X in females) is not
compensated, then the X:autosome ratio of median expression levels should be 0.5, reflecting the difference in
(active) copies (left panel). Alternatively, if expression of X-linked genes is up-regulated twofold to compensate for
dosage differences, then the ratio of medians should be close to 1.0 (right panel).

1.3 Identification of an Inactive X in Mammalian tiple copies of the X (with karyotypes such as 47XXX or
Females 48XXXX), showed that all X chromosomes in excess of
one were inactivated. This has been generalized as the
In 1949, Barr and Bertram described the sex chromatin
“n – 1 rule,” which states that if an individual has n
body, a structure visible under the light microscope in
X chromosomes, then n – 1 will be inactivated (Ohno
the nuclei of only female cells in various mammalian spe-
1967). This rule explains the remarkably mild clinical
cies. The structure proved useful in studies of sexual abnor-
symptoms associated with X-chromosome aneuploidies.
malities, but it was not until 1959 that Ohno and colleagues
The X inactivation hypothesis has continued to provide
showed that this structure was derived from one of the two
an explanation for the peculiarities of X-linked gene ex-
female X chromosomes (see Ohno 1967). Shortly thereaf-
pression in female cells and has remained essentially un-
ter, in 1961, Mary Lyon described genetic experiments on
changed since first proposed. But the past 50 years or so
the expression of X-linked coat color genes in female mice.
have been spent trying to work out the molecular mecha-
To explain the patterns of inheritance for this variable
nisms by which it operates.
patchwork (mosaic) of coat color in individual female
mice, Lyon hypothesized that in each female cell one of
the two X chromosomes is stably inactivated early in 2 OVERVIEW OF X INACTIVATION
development (Lyon 1961). The sex chromatin body, now
2.1 X Inactivation Is Developmentally Regulated
known as the Barr body, is thus the cytological manifesta-
tion of the inactive X chromosome. Elegant experiments X inactivation in female mammals is developmentally reg-
using skin fibroblasts from females heterozygous for a poly- ulated. Both X chromosomes are active in the early zygote
morphism of the X-linked enzyme glucose-6-phosphate (Epstein et al. 1978), and inactivation then proceeds coin-
dehydrogenase, showed that only one of the two possible cident with cellular differentiation from the pluripotent
alleles was expressed in colonies grown from individual state. Normally there is an equal probability that cells
cells (clones), thereby demonstrating the heritability of will inactivate the X chromosome that is either derived
the inactive state from one cell generation to the next maternally (Xm) or paternally (Xp). Exceptions to this
(Davidson et al. 1963), and confirming the occurrence of are imprinted X inactivation that occurs throughout mar-
X inactivation in human females (Beutler et al. 1962). Fur- supials and in early preimplantation mouse embryos, in
ther studies of X inactivation in human females with mul- which it is always Xp that is inactivated. In the latter case,

6 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

Sperm
Xp active Oocyte
A Xm active
A
Germline
Xm active Two-cell stage
Fetus Xp active Xm active
Xm inactive A Xp active
Xp active A
or
Xm active
Xp inactive
R X inactivation
during the female
Xm inactive mouse life cycle Morula
Xp active Xm active
or Xp inactive
Xm active I
Xp inactive
R
Trophectoderm
Xm active
ICM Xp inactive
and epiblast I
Xm active
A - Active Xs Primitive endoderm
Xp active
Xm active
R - Random X inactivation A
Xp inactive
I - Imprinted X inactivation I

Figure 4. The cycle of X inactivation and reactivation. The X chromosome undergoes a cycle of X inactivation and X
reactivation during development. Red arrows indicate X inactivation steps and green arrows indicate X reactivation
steps. Inactivation first occurs in early preimplantation embryos (imprinted X inactivation) and subsequently in
cells of the epiblast at the time of gastrulation (random X inactivation). The inactive X is reactivated in ICM cells
when they are first allocated at the blastocyst stage, and also in the developing germ cells.

imprinted Xp inactivation is maintained in the first differ- they ensure stable propagation of the inactive X through
entiating lineages, namely, the extraembryonic trophecto- multiple rounds of cell division.
derm (TE) and primitive endoderm (PE) cells, but the
inactive X is reactivated in the inner cell mass (ICM) cells
that give rise to the embryo. Reversal of X inactivation also 2.3 Some Genes Escape X Inactivation
occurs in developing primordial germ cells (PGCs), ensur-
X inactivation affects most of the X chromosome, but some
ing the X chromosome is again active in the gamete.
genes escape silencing (Berletch et al. 2011). These include
Figure 4 illustrates the cycle of X inactivation and reactiva-
genes within a small region on the X chromosome that
tion in the female mouse.
pairs with the Y chromosome during male meiosis, referred
to as the PAR or XY pairing region (Fig. 1). Genes located in
this region do not require dosage compensation as two
2.2 Chromosome Silencing Involves Multiple
copies are present in both males and females.
Levels of Chromatin Modification
Other genes that escape X inactivation, both with
Silencing of the X chromosome is achieved at the level and without Y-linked homologs, have also been character-
of chromatin structure by modification of histone tails, ized. They total 15% of genes on the human X chromo-
incorporation or exclusion of variant histones, DNA some (Carrel and Willard 2005). Interestingly, many of
methylation of some CpG islands, and reorganization of these genes lie on the short arm of the chromosome (also
higher-order chromatin folding, all contributing to a stable referred to as the p arm), which, in evolutionary time,
facultative heterochromatic structure. There is redundancy is a recently acquired segment of the X chromosome.
built into the system and not all components are essential Studies in mouse indicate that some escapees can be inac-
for all aspects of silencing (e.g., Sado et al. 2004). The layers tivated in early ontogeny, with progressive reactivation
of chromatin modification are established progressively occurring during development (Sec. 4.6). In marsupials,
through ontogeny, as detailed in Section 4.4. Collectively, most genes studied have been found to escape X inactiva-

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 7

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

tion to some extent. This may reflect a failure to maintain Xist RNA provides the trigger for X-chromosome silencing
silencing through ontogeny, possibly related to the lack of (Lee et al. 1996; Penny et al. 1996; Wutz and Jaenisch 2000).
CpG island methylation on the inactive X (Xi) in these Studies to date indicate that this occurs, at least in part,
species. through Xist-mediated recruitment of chromatin modify-
ing complexes (Fig. 5A).
A second noncoding RNA, Tsix, is also located in the
2.4 X Inactivation Is Regulated by a Master Switch Xic region (Lee et al. 1999) and plays a key role in regulating
Locus: The X Inactivation Center (Xic) Xist expression. Tsix overlaps with the Xist gene, but is
Classical genetic studies showed that X inactivation is me- transcribed in the antisense direction; hence, its name is
diated by a single cis-acting master switch locus, referred to Xist spelled backwards.
as the X inactivation center (Xic). The Xic was shown to be Phylogenetic studies have revealed that the noncoding
required both for silencing the X chromosome in cis, and Xist RNA evolved from a protein-coding transcript, Lnx3
ensuring correct and appropriate initiation of random X (Duret et al. 2006). The Lnx3 gene has retained protein-
inactivation. More recent studies have characterized the Xic coding capacity in other vertebrate species and also in mar-
at the molecular level. The locus produces a large noncod- supial mammals. The latter finding was unanticipated as
ing RNA termed Xist (X-inactive-specific transcript) that obvious similarities in X inactivation in marsupials led to
has the unique property of binding in cis and accumulating the supposition that chromosome silencing is mediated by
along the entire length of the chromosome from which it a direct homolog of Xist. After many years of searching, a
is transcribed (Fig. 5) (Brown et al. 1991; Brockdorff et al. recent study has revealed that marsupials independently
1992; Brown et al. 1992). Coating of the chromosome with evolved a cis-acting noncoding RNA locus, Rsx (RNA on

Xist

Ontogeny

Xist at metaphase Xist at interphase Hypoacetylated X

Figure 5. Progressive chromosome-wide heterochromatinization induced by Xist RNA. (A) When the Xist gene is
expressed, the RNA binds to and coats the X chromosome from which it is transcribed (green dashed line). Xist RNA
is thought to trigger silencing of the chromosome by recruiting chromatin modifying activities (red and yellow
circles). The initial wave of silencing, in turn, leads to recruitment of additional layers of epigenetic modification
(white circles), further stabilizing the heterochromatic structure. Establishment of these different levels of epigenetic
silencing is achieved in a stepwise manner through development and ontogeny. (B) Localization of Xist RNA along
the X chromosomes is shown by in situ hybridization in both interphase and metaphase.

8 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

the silent X), which fulfills the same function as Xist in some active (see Fig. 4). Nuclear transfer experiments
marsupial species (Grant et al. 2012). Like Xist RNA, Rsx showed that the repressive Xist imprint is established dur-
RNA is transcribed specifically from Xi and coats the length ing oocyte maturation (Tada et al. 2000). The molecular
of the X chromosome in cis. basis of the imprint is unknown, but DNA methylation is
not required, contrasting with many other imprinted genes
3 INITIATION OF X INACTIVATION (see Barlow and Bartolomei 2014 for details on genomic
imprinting).
3.1 Imprinted versus Random X Inactivation One theory for the preferential inactivation of Xp
The decision to inactivate an X chromosome needs to be in the zygote is that there is carryover of silencing of
tightly regulated. Male cells must avoid silencing their sin- the XY bivalent that is established during the pachytene
gle X chromosome, and female cells must avoid silencing stage of male meiosis (meiotic sex-chromosome inactiva-
both X chromosomes or keeping both X chromosomes tion, MSCI; Huynh and Lee 2003). Recent studies argue
active. Two different modes of regulation have been shown against this. First, MSCI has been shown to be a distinct
to operate. The imprinted mode of X inactivation silences and Xist-independent mechanism that is triggered in
the paternally derived X chromosome whereas the random pachytene by the presence of unpaired chromosomal re-
mode randomly inactivates either the maternal or paternal gions on both sex chromosomes and autosomes (Turner
X chromosome. Metatherian mammals (marsupials) use et al. 2006, and references therein). Second, expression
only the imprinted mode. Some eutherian ( placental) analysis of a number of X-linked genes in early zygotes
mammals (e.g., mouse) use the imprinted mode in extra- has shown that Xp silencing occurs de novo in response
embryonic lineages and the random mode in the embryo to zygotic Xp Xist expression (Okamoto et al. 2005, and
proper (Fig. 2). Other species, notably rabbit and human, references therein).
show only random X inactivation (Okamoto et al. 2011). The paternal Xist expression (and resultant Xp silenc-
This variation may be linked, in part, to differences in the ing) that begins at the onset of zygotic gene activation (at
timing of embryonic genome activation, which in mouse the two- to four-cell stage) indicates that the Xp Xist allele is
occurs relatively early, at the two-cell stage, compared with poised to express (Fig. 6). A region-specific demethylation
the four- to eight-cell stages in humans. of CpG sites in the Xist promoter occurs during spermato-
Important model systems for studying the initiation of genesis (Norris et al. 1994) and is thought to be important
X inactivation are early mouse embryos and embryonic for this.
stem (ES) cells derived from the ICM of these embryos. The Tsix gene, an antisense regulator of Xist, is required
XX ES cells are especially useful as they recapitulate initia- for imprinted X inactivation as deletion of the major pro-
tion of random X inactivation in vitro when cells are in- moter results in early embryo lethality when transmitted by
duced to undergo differentiation. There are currently no the maternal, but not the paternal, gamete (Lee 2000).
in vitro models that recapitulate initiation of imprinted X Lethality appears to be attributable to inappropriate Xm
inactivation. Xist expression (i.e., a failure to retain an active X chromo-
some both in XmYand XmXp embryos). It is not known if
expression of Tsix RNA is the primary imprint or functions
3.2 Regulation of Imprinted X Inactivation
only later to maintain the imprint.
Paternally imprinted X inactivation was first observed in
a marsupial (Sharman 1971). Subsequently, imprinted
3.3 Regulation of Random X Inactivation: Counting
X inactivation was shown to occur in the TE and PE of
mouse embryos (Takagi and Sasaki 1975). In imprinted In the random mode of X inactivation, cells use the n – 1
X inactivation, it is the parent of origin from which the rule described in Section 1.3, in which all X chromosomes
X derives that governs its status; that is, paternal, but except one are inactivated per diploid chromosome set. The
not maternal, X chromosomes are inactivated regardless process that senses the number of X chromosomes is often
of how many X chromosomes or chromosome sets are referred to as counting. Where more than one X chromo-
present. Note that the single X in XY males is always ma- some is present, the selection of active and inactive X chro-
ternally derived and therefore not inactivated in imprinted mosome—referred to as choice—is normally random.
tissues. However, there are factors that can bias this decision result-
What then is the nature of the imprint? Studies of Xist ing in nonrandom or skewed X inactivation. The process of
expression indicate that there is a repressive imprint on choice is considered separately in Section 3.4, but this di-
the Xm allele from morula stage mouse embryos. This vision is one of convenience as it is clear that counting and
imprint prevents Xist expression, keeping the X chromo- choice must be inextricably linked.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 9

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

Early/mid Late Egg

One- Two- Four- Eight- Morula
blastocyst blastocyst cylinder
cell cell cell cell
TE
PE
ICM

X inactivation
status: Active Imprinted Active Random
(only ICM cells)

Xp Xp Xp Xp or Xp
Xist:
Xm Xm Xm Xm Xm

Imprint Xist expression ICM repressor factor Blocking factor

Figure 6. Xist gene regulation in early development. The figure illustrates current knowledge and models for
imprinted and random Xist regulation in early XX mouse embryos. The Xm Xist allele arrives in the zygote with
a repressive imprint possibly mediated through the antisense Tsix locus (black square). The Xp Xist allele is primed to
be active and is expressed as soon as embryonic gene activation occurs at the two-cell stage. From the two- to four-
cell stage up until morula stage, Xp Xist is expressed in all cells (expression indicated by open rectangle and arrow at 5′
end). This pattern is maintained at the early blastocyst stage and subsequently in TE and PE cells and their fully
differentiated derivative (extraembryonic) tissues. In the late blastocyst, ICM Xist expression is extinguished,
possibly by an ICM-specific repressor factor (blue triangle). Xist expression then commences subsequently at the
time of gastrulation. Here, the blocking factor (black diamond) ensures that Xist expression cannot occur on one of
the two alleles (counting).

A number of different models for counting and choice repression, for example, by inducing Tsix, whereas the X
have been proposed (Fig. 7). Early models, developed be- chromosome produces Xist gene activators that compete
fore the discovery of Xist, invoked a single autosomally with the repressors. The resultant competition could create
encoded blocking factor present in limiting quantities suf- a probability for Xist gene activation that is modulated as
ficient to bind and repress a single X inactivation center cells begin to differentiate. The model predicts a stochastic
(Rastan 1983). In this model, X inactivation is a default probability of maintaining a single X active in all circum-
pathway that in diploid cells is inhibited on a single X stances, but also invokes checkpoint and feedback mecha-
chromosome (the active X). In cells with more than one nisms that ensure incorrect X inactivation patterns, notably
X chromosome, choice is determined by the probability inactivation of both or neither X chromosome in XX cells,
of the blocking factor binding to a given X inactivation- can be reset at an early stage. In part, this could be attrib-
center allele. utable to levels of X-linked activators becoming limiting
A related model invokes two factors: an autosomally once silencing has spread to the locus encoding the activa-
encoded blocking factor and an X-encoded competence tor on one of the two X chromosomes (Nora and Heard
factor (Gartler and Riggs 1983). The blocking factor is 2009).
suggested to be sufficient to disable the competence factor These models are not necessarily mutually exclusive
on a single X inactivation center. Other X inactivation cen- and, moreover, they continue to evolve to take into account
ters can be bound by competence factor leading to the new experimental findings. As things stand, none of the
onset of X inactivation. Although initially developed before models can be said to satisfactorily account for all available
the discovery of Xist, this model has been readopted to help data, but they nevertheless provide a useful framework both
explain experimental observations arising from the dele- for integrating current data and determining new experi-
tion of Tsix, the antisense regulator of Xist (Lee and Lu mental directions.
1999). Although a complete model for Xist regulation in ran-
A third and more contemporary model invokes a sto- dom X inactivation has not yet been found, there is a grow-
chastic process in which autosomal factors promote Xist ing consensus that there is a finely balanced competition

10 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

Blo
ockin
o n
ng
Blocking
factor
fa
actorr
ac
a

X
Xic or

B
Blo
occkin
ng
Blocking Competence
Co m
mpeten
n
nce
fa
acctorr
factor fac
c
ctor
factor

X
Xic or

Abort

Xic
Xic or
Xist
X
Xi Xist
Xist
re
eprresso
or
repressor activator
activator

Abort

Figure 7. Models for the regulation of random X inactivation. (A) Autosomally encoded blocking factor (yellow
shapes) is produced in sufficient quantities to occupy a single Xic. Binding of blocking factor to the Xic inhibits Xist
transcription, thus defining a single active X chromosome. Xist transcription occurs on any additional X chromo-
somes leading to X inactivation (dark green dots). Blocking factor binds to either the maternal (Xm) or paternal
(Xp) X chromosome with equal probability and in a cell-autonomous manner. (B) The two-factor model invokes
an X-encoded competence factor ( purple triangle) and an autosomally encoded blocking factor (yellow shapes).
Blocking factor titrates away competence factor ( purple triangle). In cells with a single X chromosome, there is
insufficient available competence factor to activate Xist, but in cells with additional X chromosomes, competence
factor can activate all X chromosomes except the single X chromosome bound by blocking factor. (C) The stochastic
model invokes that autosomally encoded repressors (yellow circles) and X-encoded activators ( purple circles)
compete with one another. All Xist alleles have an equal probability of being activated and this is increased in cells
with more than one X chromosome (higher levels of Xist activators). By chance, some cells with two X chromosomes
will initiate inactivation of either both or no X chromosomes. This may be dealt with by checkpoint mechanisms or
cell death.

between the pathways that repress Xist and those that acti- The antisense transcript spans the entire Xist locus, termi-
vate Xist. The decision to initiate and then maintain Xist nating immediately upstream of the major Xist promoter.
up-regulation on a given allele may then be reinforced by Antisense transcription across the Xist promoter region is
feedback and/or feedforward loops. required for Tsix-mediated repression (Ohhata et al. 2008).
Genetic studies have shown that the antisense gene, The detailed molecular mechanisms are not fully under-
Tsix, is an important Xist repressor. Tsix is transcribed on stood, but are thought to involve a switch in the histone
the X chromosome before and concordant with the onset modification state of the Xist promoter (Sado et al. 2005;
of Xist expression in random X inactivation. Tsix promoters Navarro and Avner 2010), and recruitment of the de novo
lie immediately downstream from the Xist locus (Fig. 8). DNA methyltransferase Dnmt3a (Sun et al. 2006).

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 11

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

Xist Jpx Ftx Xpr Rnf12

Tsix Cnbp2 25 kb

OCT4 RNF12
SOX2
KLF4 RNF12
C-MYC
OCT4 PRC2 REX1 Jpx RNA
YY1
SOX2
CTCF REX1
NANOG Ftx RNA
Xist

Xite Tsix
Tsix RNA 5 kb

Figure 8. Genes and regulatory elements in the X inactivation-center region. The key region on the mouse X
chromosome comprising known elements involved in Xist gene regulation is illustrated, showing noncoding
RNA (ncRNA) genes and protein-coding genes. The Xpr region, several hundred kilobases upstream of Xist, has
been implicated in trans-interaction of Xic alleles in XX cells and as such is thought to be important for initiation of
X inactivation. The expanded view illustrates the intron/exon structure of the Xist and Tsix loci, including the Xite
elements that function as Tsix enhancers. The network of protein factors (boxes) and ncRNAs (ovals) implicated in
Xist gene regulation is shown with arrows and bars indicating repressor and activator function, respectively. Note
that RNF12 mediates degradation of REX1, which functions both as a Xist repressor and a Tsix activator.

Tsix is thought to play a role in counting based on the in XX ES cells (Monkhorst et al. 2008). This study led
observation that Xist is strongly up-regulated in Tsix null directly to the identification of the first known Xist activa-
XY ES cells undergoing differentiation. However, not all tor, the ubiquitin E3 ligase Rnf12 (Jonkers et al. 2009).
Tsix null ES cell lines show this effect (the reasons for the Overexpression of Rnf12 promotes Xist expression in XY
discrepancy remain unclear) and, moreover, Tsix deletion ES cells, whereas Rnf12 deletion delays Xist up-regulation in
leads to overt Xist gene up-regulation in undifferentiated differentiating XX ES cells. The location of the Rnf12 gene
XY ES cells. These observations indicate that other path- immediately upstream of Xist supports the idea of a feed-
ways contribute to Xist repression. Recent work has high- back loop in which the spread of X inactivation on one X
lighted an important role for transcription factors linked to chromosome represses Rnf12, reduces levels of Rnf12 pro-
pluripotent cell circuitry, notably Oct4, Nanog, Sox2, and tein, and thereby serves to reduce the probability of inap-
Rex1 (Donohoe et al. 2009; Navarro et al. 2010). These propriately inactivating remaining X chromosomes. Rnf12
repressive effects are mediated by promoting Tsix expres- functions as an E3 ligase for ubiquitin-mediated degrada-
sion, directly repressing Xist, and also by suppressing levels tion of the pluripotency factor Rex1, found at the promoter
of the Xist activator, Rnf12 (see below). Because levels of regions of both Xist and Tsix genes. Thus, Rnf12-mediated
pluripotency factors decline rapidly at the onset of differ- degradation of Rex1 may simultaneously activate Xist and
entiation, the regulatory balance at the Xist locus will be repress Tsix (Gontan et al. 2012).
tipped toward activation, providing a plausible explanation Although it is clear that Rnf12 plays an important role in
for the link between the onset of Xist expression and cell Xist gene activation, the fact that Rnf12 null XX ES cells are
differentiation. able to up-regulate Xist, albeit with some delay, suggests the
Polycomb family repressors (elaborated in Secs. 4.4 and presence of additional activators, also possibly encoded
4.5) are also implicated in Xist repression based on ob- on the X chromosome. Such a role has been ascribed to a
served synergistic effects in cells with mutations affecting ncRNA, Jpx, which is located immediately upstream of the
both Tsix and the PRC2 Polycomb pathway (Shibata et al. Xist locus (Tian et al. 2010). The mechanism of Jpx RNA
2008). It is not yet known if this is linked to the pluripo- function is not yet fully understood although there is recent
tency factor pathway or represents a third independent Xist evidence that it evicts the silencing protein CTCF from the
repression pathway. Xist locus (Sun et al. 2013). Similarly, another ncRNA pro-
The existence and location of X chromosome-encoded duced by the Ftx locus, immediately upstream of Jpx, is
activators of Xist expression was predicted through the implicated in Xist gene activation (Chureau et al. 2011).
analysis of polyploid ES cells and a deletion encompassing The loci and pathways known to participate in Xist repres-
known Xist regulatory elements on a single X chromosome sion and Xist activation are summarized in Figure 8.

12 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

3.4 Regulation of Random X Inactivation: Choice X inactivation is initiated. Specifically, three-dimensional

(3D) fluorescence in situ hybridization (FISH; Xu et al.
In XX cells, the choice of which X chromosome to inacti- 2006; Augui et al. 2007) and locus tagging experiments
vate in a given cell is normally random. However, animals (Masui et al. 2011) have shown frequent associations of
that are heterozygous for certain Xic alleles can show a bias Xic alleles mediated by contacts close to the Tsix promoter
toward one allele or the other. Before discussing these, it is and a region located several hundred kilobases upstream of
important to first make the distinction between primary Xist, termed Xpr. Current models posit that Xpr is required
and secondary nonrandom X inactivation. Primary non- to establish initial trans-interactions, thus facilitating con-
random X inactivation relates to a bias in the choice step tacts in the Tsix region. These trans-interactions have been
that occurs at the time random X inactivation is initiated. proposed to mediate exchange of factors from one allele to
Secondary nonrandom X inactivation, on the other hand, another, providing an opportunity to break symmetry (see
occurs as a consequence of selection against cells that have Fig. 7 of Dekker and Misteli 2014).
nominated a given X chromosome as the inactive X. This
latter process does not strictly relate to choice, but occurs
3.5 Switching Modes of X Inactivation Regulation
commonly when mutations on one X chromosome confer
in Early Embryogenesis
a selective disadvantage to cells that inactivate the wild-type
allele. Examples include the secondary nonrandom X in- How do early mouse embryos instigate the switch from the
activation seen in carriers of serious genetic disorders such imprinted to the random mode of regulation (see Fig. 4)?
as Duchenne muscular dystrophy. The observed cell selec- Until recently, it was thought that the initiation of imprint-
tion effects can occur at the level of the whole developing ed and random X inactivation were both linked to cellular
embryo or in specific tissues or cell types, depending on the differentiation (Monk and Harper 1979). Thus, trophecto-
function of the mutant protein. derm and primitive endoderm lineages were thought to
The first documented example of primary nonrandom inactivate their Xp in response to the parental imprints
X inactivation came in classical genetic studies that identi- on Xist when they first differentiate. The three germ line-
fied variants at the “X controlling element” (Xce) in differ- ages that give rise to the embryo proper by gastrulation were
ent mouse strains (Cattanach 1974, and references therein). thought to first erase the Xist imprint and then undergo
Xce was mapped to the approximate location of the Xic random X inactivation (Fig. 6). More recent data, however,
on the mouse X chromosome, suggesting the two loci could show that Xp inactivation occurs before the onset of cellu-
be synonymous. This has been confirmed in more con- lar differentiation in cleavage stage embryos, and it occurs
temporary studies that locate Xce to a region immediately in all cells, including the precursors of the ICM (Fig. 6)
downstream from Xist. The molecular basis of the variation (Mak et al. 2004; Okamoto et al. 2004). Thus, imprinted X
underlying nonrandom X inactivation, however, remains inactivation in trophectoderm and primitive endoderm is a
to be determined. relic of the X inactivation pattern established in early cleav-
Primary nonrandom X inactivation has also been ob- age embryos. ICM cells must instigate a program to reverse
served in animals heterozygous for various Xic alleles, es- this initial wave of imprinted Xp inactivation, thereby set-
tablished in gene targeting experiments aimed at the ting the scene for subsequent random X inactivation. The
functional dissection of Xist and Tsix. A common theme basis for the reversal of Xp inactivation is unknown, but
emerging from these studies is that alleles that increase the may involve an ICM-specific program that represses Xp
probability of being selected for X inactivation are associ- Xist expression (see Sec. 5.1).
ated with mutations that either reduce levels of antisense
Tsix transcription or increase levels of sense Xist transcrip-
3.6 Evolutionary Variation in X Inactivation
tion (Lee and Lu 1999; Nesterova et al. 2003). There is also
Regulatory Mechanisms
recent evidence that choice is linked to an Xist RNA splicing
switch (Royce-Tolland et al. 2010). In the context of models To date, the laboratory mouse has been the primary model
for regulation of random X inactivation, biased choice can system for studies on the initiation of X inactivation. How-
be viewed as tipping the balance between Xist repressors ever, recent work suggests that the lessons drawn from such
and Xist activators on a given allele. studies may not be as broadly applicable as once thought
What then is the symmetry-breaking event that results (Okamoto et al. 2011). Analysis of human preimplantation
in the counting/choice machinery ultimately associating embryos has shown that XIST is up-regulated from the X
with only a single allele when there is equivalent access to chromosome in male embryos and both X chromosomes
both alleles? One idea that has emerged is that this may in female embryos during early preimplantation develop-
involve trans-interactions between Xic alleles at the time ment. This pattern is resolved in late blastocysts, in which

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 13

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

males extinguish XIST expression and females express it Experiments with an inducible Xist expression system in
from only one X chromosome. The early XIST expression mouse ES cells, in which the functions of Xist RNA mole-
in human embryos is not linked to chromosome silencing, cules carrying defined deletions could be tested, showed
consistent with the requirement for expression of X-linked that silencing can be attributed to a conserved repeat se-
genes. A further variation was revealed through experi- quence, the A-repeat, located at the 5′ end of the molecule,
ments with rabbits, in which female preimplantation em- whereas coating of the X is mediated by sequences scattered
bryos expressed Xist from both alleles in 25% of cells. In throughout the rest of the molecule (Wutz et al. 2002).
this case, Xist expression is linked to chromosome silenc- These observations show that Xist RNA spreading and chro-
ing, suggesting either a checkpoint mechanism that can mosome-silencing functions are mechanistically separable.
reverse and correct inappropriate biallelic Xist expression,
or cell selection events resulting in the loss of XX cells that
4.2 Spreading of Xist RNA and Chromosome
silence both X chromosomes (Okamoto et al. 2011). An
Silencing
important goal for the future will be to determine if there
are conserved features in these apparently diverse systems The association of Xist RNA with Xi is selective. It is not
as these would likely point to the fundamental underlying found along the PAR, which remains active and euchro-
mechanisms. matic, or at constitutive (centric) heterochromatin. More-
over, analysis of metaphase chromosomes shows a banded
localization that appears to correlate with gene-rich G-light
4 PROPAGATION AND MAINTENANCE bands (Duthie et al. 1999). In dividing cells, Xist RNA
OF THE INACTIVE STATE association with Xi is lost when cells enter anaphase. It is
then rapidly resynthesized in daughter cells at the onset of
4.1 Xist RNA, Gene Silencing, and Heterochromatin
G1. Xist RNA is tightly associated with the nuclear matrix
Assembly
in the interphase nucleus and localization is retained fol-
There is strong evidence that the Xist gene and its RNA lowing removal of chromatin by micrococcal nuclease, sug-
product provide both the switch that initiates X inactiva- gesting that Xist RNA does not contact underlying DNA
tion in cis and the means by which silencing spreads across sequences directly (Clemson et al. 1996).
the chromosome. The evidence comes from experiments, There are approximately 2 × 103 molecules of Xist
indicating that (1) Xist is unique in being expressed only RNA in a female somatic cell (Buzin et al. 1994). The tran-
from Xi, (2) Xist RNA levels increase dramatically in pre- script is relatively stable with an estimated half-life of 6–8 h
implantation embryos at the time of X inactivation, (3) Xist in dividing cells, a figure that is consistent with turnover
up-regulation precedes X inactivation and appears to be an occurring predominantly as a consequence of dissociation
absolute requirement for it to occur, (4) Xist RNA coloc- through mitosis. However, it may be that Xist RNA turn-
alizes with Xi in interphase nuclei and is distributed along over is inhibited in the presence of transcriptional inhi-
one of the two metaphase X chromosomes (see Fig. 5B), bitors, as a recent study using GFP-tagged Xist transcripts
and (5) Xist-containing transgenes, when inserted into au- in living cells determined a relatively fast turnover rate for
tosomes, can coat the autosome in cis with Xist RNA and Xist RNA (Ng et al. 2011).
initiate the adoption of a heterochromatin-like, transcrip- What then is known of the factors regulating Xist RNA
tionally silent chromatin structure. These findings suggest localization? One interesting link has come from analysis
that Xist RNA is both necessary and sufficient to trigger of the protein SAF-A/hnRNPU. Originally identified as an
heterochromatin formation and transcriptional silencing. hnRNP protein and a major component of the insoluble
However, continuing Xist expression is not required for the nuclear scaffold, SAF-A/hnRNPU was first linked to X in-
“maintenance” of X inactivation. For example, silencing of activation through cell imaging studies demonstrating
human X-linked genes is maintained in human:rodent so- enrichment of the protein over the interphase Xi territory
matic cell hybrids, in which Xist expression is lost on the in XX somatic cells (Pullirsch et al. 2010). This observation
retained human Xi chromosome (Brown and Willard is consistent with the association of Xist RNA with the
1994). This issue is discussed further in Section 5. nuclear matrix. More recent data suggest that genetic dis-
The mechanism(s) by which Xist RNA associates with ruption of SAF-A/hnRNPU causes dispersal of Xist RNA
and spreads along the Xi in cis and the mechanisms that through the nucleoplasm, indicating a role in Xist RNA
bring about changes in chromatin structure and gene silenc- localization (Hasegawa and Nakagawa 2011). Moreover,
ing are still not understood in detail. We do know that biochemical analysis suggests that the RNA binding do-
different regions of the Xist RNA molecule are responsible main of hnRNPU/SAFA interacts directly with Xist RNA
for gene silencing and spreading along the X chromosome. (Fig. 9A).

14 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

hnRNPU
YY1
YY1 (DNA bound)
Xist RNA
Xist locus

B Me
Me
x
Me Me Me
Me Me Me
Me
Me

Ub Ub

Enriched Depleted

Me
Me
Me
H3K27me3 (PRC2) Me H4K20me1 (PR-SET7?) H3ac, H4ac, H2Aac, H2Bac
Ub H2AK119ub (PRC1) macroH2A H3K4me3
Me
Me H3K9me2 Me CpG island methylation H2Abb

Figure 9. Factors involved in X inactivation spread. (A) The organization of LINE-1-rich (gray-shaded) and gene-
rich (blue-shaded) domains is important in defining the extent of Xist RNA spreading as illustrated by the obser-
vation that large gene-rich domains attenuate Xist RNA spreading (barred arrows). YY1 is implicated in tethering
Xist RNA (green wavy line) at the site of synthesis (brown circle) and interacting with Xist RNA to facilitate spreading
(red circle). hnRNPU/SAFA also plays a role in localization of Xist RNA in cis, binding to Xist RNA directly. (B)
Summary of characterized DNA methylation, histone modification, and histone changes at a silent gene on Xi.

A second factor implicated in Xist RNA localization is of Xist RNA and associated gene silencing, at least in some
the transcription factor YY1, which is proposed to be a cases (Popova et al. 2006). In other cases, limited silencing
bifunctional protein capable of binding both DNA ele- appears to result from aberrant long term maintenance of
ments at the Xic and Xist RNA (Fig. 9A) (Jeon and Lee silencing on autosomes following efficient spreading at the
2011; Thorvaldsen et al. 2011). Binding elements in the onset of X inactivation (Cattanach 1974).
Xist gene are suggested to function as nucleation sites Attenuated silencing on autosomes led to the idea that
that mediate docking of Xist RNA particles with Xi. there are sequences on the X chromosome, originally re-
ferred to as “way stations” or “booster elements” (described
in Gartler and Riggs 1983), that serve to amplify or enhance
4.3 Booster Elements Facilitate Spreading
the spread/maintenance of X inactivation. More recently, it
of X Inactivation
has been proposed that a common dispersed repeat family,
Although Xist RNA transgenes function on autosomes, the L1 long-interspersed repeats (LINE-1), are good can-
autosomal silencing is less efficient than silencing of the didates for the “way station” elements (Lyon 2003). These
X chromosome. The relative insensitivity of autosomes to repeat sequences are common in the human and mouse
Xist-mediated silencing was first reported in classical ge- genomes, but are particularly frequent along the X chro-
netic studies that analyzed gene silencing in X:autosome mosome. Further, LINE-1 elements are most common in
translocations. Specifically the spreading of silent chroma- the more condensed, gene-poor, G-banded regions of the
tin along the autosomal chromosome arm was found to be human and mouse genomes, suggesting that they may, in
variable between translocations and limited in extent. This some way, favor a chromatin conformation associated with
is attributable to autosomes resisting the initial spreading transcriptional silencing. In support of this idea, analysis of

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 15

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

gene silencing meditated by Xist transgenes located on au- human and mouse cells is depleted in acetylated histone
tosomes and in X autosome translocations indicates that H4 (Jeppesen and Turner 1993), and in this way resem-
large gene-rich LINE-1 depleted chromosomal domains bles constitutive, centric heterochromatin. This was the
are relatively refractory to the spreading of X inactivation first demonstration that the inactive X chromosome was
(Popova et al. 2006; Chow et al. 2010; Tang et al. 2010). marked by a specific type of histone modification. (His-
A common idea is that way station elements are synon- tone modifications and their functions are described in
ymous with Xist RNA-binding sites. In the case of L1 LINE Allis et al. 2014.) Subsequent experiments in several labo-
elements, this seems improbable as Xist RNA is concentrat- ratories confirmed these observations and further showed
ed in gene-rich chromosomal domains, reciprocal to L1 that acetylated isoforms of all four core histones (H2A,
LINE distribution. Interestingly, the high density of L1 H2B, H3, and H4) were depleted in both constitutive
LINEs on the X chromosome has fragmented gene-rich and facultative heterochromatin in interphase and meta-
domains such that they are, on average, much smaller phase cells (Fig. 5B). In particular, both centric hetero-
than on autosomes. Thus, one idea is that L1 LINE do- chromatin and Xi are depleted in H3 di- and trimethylated
mains influence the higher-order topological folding of at lysine 4 (H3K4me2 and H3K4me3; O’Neill et al. 2008).
the chromosome in such a way as to favor efficient in cis Like acetylation, H3K4me2/me3 are generally thought to
spreading of Xist RNA within gene-rich domains (Popova be markers of transcriptionally active, or potentially active,
et al. 2006; Tang et al. 2010). It is important to remember chromatin.
that the efficiency of Xist RNA spreading from its site of Other chromatin modifications that are enriched on
synthesis (i.e., the Xist locus) through the X-chromosome Xi include the histone modifications H3 trimethylated
territory will depend on both the overall configuration of at lysine 27 (H3K27me3) and H2A monoubiquitinated at
that territory in three dimensions and how the Xist locus is lysine 119 (H2AK119ub1; Fig. 9B). These modifications are
positioned within the territory. catalyzed by Polycomb repressor complexes (see Grossni-
RNA FISH analysis has shown that there is a major klaus and Paro 2014 for further discussion). There is also
burst of L1 LINE transcription maintained specifically on enrichment of H3K9me3, normally associated with con-
the inactive X chromosome at the time that X inactivation stitutive pericentric heterochromatin, in humans and some
is first established (Chow et al. 2010). L1 transcription has other species. In mouse, H3K9me2, and not H3K9me3,
been directly linked to silencing through a mechanism in- enrichment has been observed. Because distinct histone
volving overlapping transcripts and the production of lysine methyltransferases (KMTs) are associated with these
short RNAs, but may also play a role in facilitating spread modifications, this appears to represent a fundamental
of X inactivation in cis. difference in Xi heterochromatin in the different species.
Knowledge of Xist RNA binding sites beyond that Elevated H4K20me1, a histone modification associated
provided by cytological analysis is at present lacking, and with chromosome condensation, has also been observed
the development of a method to map binding sites at on Xi (Kohlmaier et al. 2004). Finally, in addition to spe-
near nucleotide resolution is an important goal for the cific histone modifications, a variant histone, macroH2A, is
future. enriched in Xi heterochromatin (Costanzi and Pehrson
1998), and conversely a different variant, [Link], is spe-
cifically depleted on Xi (Chadwick and Willard 2001).
4.4 Heterochromatic Structure of the Inactive X:
A careful analysis of the distributions of histone mod-
The Link with Chromatin Modifications
ifications across Xi in human cultured cells has provided
Since the very earliest light microscopical studies, it has insights into the complexity of the system (Chadwick and
been realized that Xi shares properties with heterochroma- Willard 2003). H3K9me3 and H3K27me3/H2AK119ub1
tin. Like the constitutive heterochromatin found at and are enriched at defined, but nonoverlapping, regions across
around centromeres, Xi remains visible and apparently Xi. Thus, unlike loss of histone acetylation, enrichment in
condensed throughout interphase (as the Barr body), and these modifications is a regional, not an overall, property of
its DNA is usually replicated late in S-phase. Xi is said to Xi. Intriguingly, those regions enriched in H3K27me3 are
consist of “facultative” heterochromatin. also enriched in Xist RNA and the variant histone macro-
Further parallels between Xi and constitutive hetero- H2A1.2. Conversely, those regions of Xi that are enriched in
chromatin have come from the use of indirect immuno- H3K9me3 also show enhanced levels of heterochromatin
fluorescence microscopy to study the distribution of protein HP1 (known to bind to methylated H3K9) and
histone modifications and variants both along metaphase H4K20me3 (also a mark associated with constitutive, cen-
chromosomes and in interphase nuclei. The facultative tric heterochromatin). Importantly, immunostaining of the
heterochromatin of the inactive X chromosome in both Barr body in interphase and the inactive X in metaphase

16 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

cells showed the same costaining patterns, suggesting that tion of H3K4 are as yet unknown. We know more about
the different domains are retained through the cell cycle. the enzymes responsible for putting histone modifications
Constitutive centric heterochromatin is enriched in in place. H2AK119u1 and H3K27me3 are deposited on Xi
methylated DNA, primarily 5′ -methylcytosine at CpG di- by the Polycomb repressive complexes PRC1 and PRC2,
nucleotides (see Li and Zhang 2014). This is consistent with respectively (Silva et al. 2003; de Napoles et al. 2004; de-
its low level of transcriptional activity. Perhaps surprisingly, tailed in Grossniklaus and Paro 2014). Recruitment of
the level of CpG methylation on Xi is not, overall, signifi- PRC2 to Xi is Xist dependent and it has been suggested
cantly higher than the rest of the genome. However, CpG that this is mediated by direct interaction of PRC2 com-
islands associated with silenced genes are highly methylat- ponents with the A-repeat element of Xist RNA (Zhao
ed and experimental evidence suggests that DNA methyl- et al. 2008). Direct interaction of PRC2 with A-repeat el-
ation plays an important role in the stabilization of the ements is unlikely, however, to be the whole story as PcG
inactive state. The overall reduction in CpG methylation recruitment to Xi does not occur during early mouse pre-
on Xi is apparently attributable to reduced methylation in implantation development (Okamoto et al. 2004), and
introns, intergenic regions, and possibly at common repeat transgenic Xist RNA from which the A-repeat element is
elements. deleted can still recruit PRC2, albeit inefficiently (Kohlma-
In most cases, chromatin features of the inactive X ier et al. 2004).
chromosome have been identified by immunofluores- Recruitment of the PRC1 complex to Xi is, in part,
cence analysis of either metaphase chromosomes or the attributable to the interaction of the chromodomain of
Barr body in interphase cells. However, localized changes the core protein CBX2/4/7 with H3K27me3, a histone
in histone modifications may also play important roles in modification put in place by the PRC2 complex (see Fig.
the various stages of the X inactivation process. Such 5 of Grossniklaus and Paro 2014). There is also a PRC2-
changes can be identified by high-resolution microscopy, independent pathway that recruits variant RYBP-PRC1
or by chromatin immunoprecipitation (ChIP) approach- complexes in which CBX proteins are replaced by the
es. ChIP analysis of chromatin surrounding the Xist locus RYBP subunit (Tavares et al. 2012).
has identified a region extending .340 kb 5′ of the Xist Specific KMT enzymes catalyzing H3K9me3 and
gene that is characterized by enrichment in methylated H3K9me2 on Xi in human and mouse, respectively, have
H3K9 and H3K27 in undifferentiated ES cells (Rougeulle not yet been formally identified, although the enzyme sys-
et al. 2004). Hypermethylation diminishes as the cells tems that establish these modifications at other sites in the
differentiate and X inactivation proceeds. Sites within genome are well described (Cheng 2014). DNA methyla-
this region are enriched in acetylated H3 and H4 (O’Neill tion of CpG islands on Xi requires the de novo methyl-
et al. 1999). Ongoing investigations will reveal to what transferase Dnmt3b, whereas Dnmt3a and the accessory
extent these localized histone modifications in the Xic protein, Dnmt3L, are dispensable (Gendrel et al. 2012).
region are early, causative events driving the X inactiva- An additional factor required for DNA methylation at
tion process, or downstream events that are ( possibly many Xi CpG islands is the protein Smchd1, an atypical
essential) components of an ongoing chromatin remod- member of the SMC (structural maintenance of chro-
eling process. mosomes) superfamily that includes components of the
An important recent innovation—high-throughput condensin and cohesin complexes with roles in chromo-
sequencing applied to chromatin fragments obtained by some organization and dynamics (Blewitt et al. 2008).
ChIP (ChIP-seq)—has been coupled with the use of sin- Smchd1 protein is enriched on Xi and homozygous mutant
gle-nucleotide polymorphisms (SNPs) in interspecific mice show female-specific embryo lethality attributable
mouse crosses to determine high-resolution maps of spe- to incomplete silencing of X-linked genes. However, the
cific modifications/factors on the inactive (Xi) relative to mechanism by which Smchd1 influences CpG island meth-
the active (Xa) X chromosome. Using this approach it was ylation and silencing remains to be determined.
shown that H3K27me3 on Xi occurs in large blocks, dis-
tributed broadly over promoters, gene bodies, and inter-
4.6 Higher-Order Chromatin Structure on Xi
genic regions (Marks et al. 2009).
Although Xi chromatin is often described as “condensed,”
careful microscopic analysis and 3D reconstruction of Xa
4.5 The Enzymology of Chromatin
and Xi chromosomes labeled with X-specific DNA probes
Modifications on Xi
suggests that the difference between them is more a matter
The enzymes responsible for the deacetylation of core his- of shape than the amount of chromatin per unit volume
tones (HDACs) during X inactivation or the demethyla- (Eils et al. 1996; Splinter et al. 2011). The position of Xi

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 17

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

relative to other nuclear structures may also be important. illumination microscopy that are extending the resolution
For example, it has often been observed that the Barr body limit of conventional fluorescence microscopy (Schermel-
localizes to the nuclear periphery and/or the periphery of leh et al. 2008).
the nucleolus.
Further insights have come from analysis of the 3D
4.7 The Order of Events That Leads to X Inactivation
organization of genes on the Xi chromosome relative to
Xist RNA territories in the interphase nucleus (Chaumeil Differentiating XX ES cells have provided an invaluable
et al. 2006). Xist RNA is found to describe a territory that model system for studying the dynamics of X-chromosome
includes common repeat sequences on the X chromosome inactivation. In undifferentiated cells both X chromosomes
and in which RNA polymerase II (Pol II) is depleted. Dur- are active and Xist and Tsix are expressed at low levels.
ing the establishment of X inactivation and in line with Increased levels of Xist RNA and its coating of one X chro-
gene silencing, X-linked genes are recruited from positions mosome are first detected in a high proportion of cells after
external to the Xist RNA territory to sites either at the 1–2 d of differentiation. This is followed by rapid depletion
periphery or within the territory. Silencing deficient Xist of RNA Pol II within Xist RNA territories and then deple-
RNA transgenes in which the A-repeat region is deleted can tion of H3K4me3 (O’Neill et al. 2008). Recruitment of PcG
form an Xist RNA territory but fail to recruit genes. That proteins with associated methylation of H3K27 and mono-
the Xist RNA territory has been found to correspond to the ubiquitination of H2A occur in a similar timeframe, along
location of common repeat elements on the X chromosome with deacetylation of H3K9 and loss of H3K4 methylation
is somewhat paradoxical given that on metaphase chromo- (Silva et al. 2003; de Napoles et al. 2004; Rougeulle et al.
somes Xist RNA shows a reciprocal localization relative to 2004; O’Neill et al. 2008). Global histone deacetylation and
LINE-1 elements (Sec. 4.3). Further studies are needed to the accumulation of H3K9me2 on Xi are established with
resolve this point. some delay, occurring at days 3–5 in the majority of cells
An important role for higher-order chromosome or- (Fig. 10) (Keohane et al. 1996). The delayed appearance of
ganization in X inactivation is also suggested by the iden- some modifications implies that they are likely to be in-
tification of proteins potentially involved in chromosome volved in the maintenance/stability of the inactive state,
architecture, notably Smchd1 (see Sec. 4.5) and hnRNPU/ rather than its establishment. This interpretation assumes
SAFA (Sec. 4.2). An additional nuclear scaffold factor, that patterns of acetylation and methylation at the promot-
SATB1, has also been implicated in conferring competence ers of individual genes undergoing inactivation reflect
for Xist-mediated silencing (Agrelo et al. 2009), although those determined by immunofluorescence analysis of the
its role has recently been questioned because of the lack of whole chromosome, or large domains. Initial studies by
an X inactivation defect in SATB1 knockout mice (Necha- ChIP suggest that this is indeed the case (O’Neill et al.
nitzky et al. 2012). 2008), but further experimentation, using ChIP-seq, for
New methodologies to analyze 3D chromosome topol- example, is necessary.
ogy are shedding further light on higher-order chromo- Accumulation of the variant macroH2A1.2 histone on
some organization on Xi. The 4C method described in Xi occurs much later during XX ES cell differentiation
Dekker and Misteli (2014) and illustrated in their Fig. 5, (Mermoud et al. 1999). This variant histone has more
which quantifies the frequency of contact between defined than 200 additional amino acids in its carboxy-terminal
positions on the chromosome, has been used in conjunc- tail and several amino acid substitutions throughout the
tion with SNPs to discriminate Xi and Xa alleles in XX molecule. Interestingly, Xist RNA expression is required to
somatic cells demonstrating that preferred long-range con- retain macroH2A on Xi in somatic cells (Csankovszki et al.
tacts involving loci on Xa are lost on Xi (Splinter et al. 1999), but is not sufficient to recruit macroH2A in early
2011). These interactions are partially restored by deletion differentiation stages (Mermoud et al. 1999; Wutz et al.
of the Xist locus, despite the fact that Xist deletion does 2002).
not result in reactivation of X-linked genes (see Sec. 4.1). The late recruitment of three other factors to Xi has now
Similarly, the 5C method has been applied to study the been reported: hnRNPU/SAFA, Ash2l (Pullirsch et al.
regulatory landscape of the Xic during the onset of X inac- 2010), and Smchd1 (Gendrel et al. 2012). These observa-
tivation demonstrating that Xist and Tsix lie within separate tions indicate that the establishment of silent chromatin on
topological domains (Nora et al. 2012; see Fig. 7 of Dekker Xi occurs in a sequential manner with at least two clearly
and Misteli 2014). The application of these new method- separated phases (Fig. 10).
ologies will facilitate important advances in our under- Selective DNA methylation of Xi CpG islands in ES cells
standing of Xi structure, in particular, when coupled to accumulates slowly during differentiation (Gendrel et al.
advanced microscopy approaches such as 3D structured 2012). This is consistent with early studies showing that

18 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

100
Xist RNA domains
RNA Pol II depletion

Maximal levels (%)

H3K9ac and H3K4me3 depletion
H3K27me3 and H2AK119ub deposition
H4ac depletion
50
macroH2A deposition
Smchd1 recruitment
Ash2l and hnRNPU recruitment
CpG island methylation
CpG island methylation (Smchd1 independent)
0
0 1 2 3 4 5 6 7 8 9 10
Days of differentiation

Figure 10. The order of events in differentiating XX ES cells. The diagram summarizes the order in which different
silencing pathways are integrated during establishment of X inactivation in differentiating XX ES cells. Early events,
depletion of RNA Pol II, loss of H3K4me3/H3K9Ac, and deposition of Polycomb-associated modifications occur
coincident with the onset of Xist RNA expression. H4 hypoacetylation and a transition to late replication in S-phase
occur slightly later. Enrichment for macroH2A, Smchd1 Ash2l, and hnRNPU/SAF-A occur in a defined temporal
window relatively late in the differentiation time course. Accumulation of DNA methylation over CpG island occurs
slowly following recruitment of Smchd1, although a subset of CpG islands acquire DNA methylation more rapidly
and in a Smchd1-independent manner.

methylation of the Hprt promoter on Xi occurs relatively Xist-coated chromatin is dependent on the continued pres-
late in the developing embryo (Lock et al. 1987), a finding ence of Xist RNA (Csankovszki et al. 1999), but does not
that led to the idea that DNA methylation is responsible for require transcriptional silencing, as it is seen also in chro-
stabilization, or locking, of the inactive state rather than in mosomes coated with a mutant Xist RNA lacking regions
initiation and spreading. In differentiating XX ES cells, a necessary for silencing (Wutz et al. 2002). Thus, X inacti-
large proportion of CpG islands acquire little or no DNA vation can be seen as the end result of a series of parallel
methylation before day 7 of differentiation, attributable to processes, only some of which are interdependent.
the absence of Smchd1 on Xi before this time (see Sec. 4.5). It should also be noted that a different order of events
A proportion of CpG islands acquire methylation earlier may occur during the establishment of imprinted X inac-
and at a faster rate, and in these cases methylation is tivation in preimplantation embryos. Notably enrichment
Smchd1 independent (Gendrel et al. 2012). Thus, the pic- of H3K27me3 is not detected until the 16-cell stage, con-
ture that emerges is of a coordinated and carefully regulated siderably later than the onset of Xist expression (two- to
sequence of events by which chromatin changes on the four-cell stage; Mak et al. 2004; Okamoto et al. 2004). This
Xi are put in place as development proceeds (summarized may indicate a requirement for specific developmentally
in Fig. 10). It is remarkable that some of these changes, regulated cofactors to recruit the PRC2 PcG complex to Xi.
such as histone deacetylation and DNA methylation, take The relationship between the various chromosome-
place after the cells have started to progress down various wide modifications and gene silencing that occur on the
different pathways of differentiation. It seems that the pro- inactivating X in female ES cells is by no means clear.
gram responsible for the completion of X inactivation pro- Recent data using microarrays (Lin et al. 2007) or RNA
ceeds independently of other cell differentiation programs. sequencing (Deng et al. 2011) to measure expression of
However, it is important to note that some aspects of ran- X-linked genes shows that individual genes are inactivated
dom X inactivation can proceed only after differentiation at various times during ES cell differentiation, with some
has begun. For example, switching on expression of Xist genes escaping inactivation altogether. It seems that, for
transgenes in “undifferentiated” ES cells triggers various most X-linked genes, silencing is triggered by conditions
histone modifications associated with heterochromatiniza- that occur at differing stages of ES cell differentiation.
tion, and also the transition to replication in late S-phase
(Wutz and Jaenisch 2000), but there is no detectable incor-
4.8 Xist-Mediated Silencing: “Belts and Braces”
poration of macroH2A; only after the cells have been in-
duced to differentiate does macroH2A colocalize with Xist Accumulating evidence illustrates multiple pathways con-
RNA on the chromosome containing the Xist transgene tributing to the establishment of gene silencing on the Xi.
(Rasmussen et al. 2001). Association of macroH2A with At the nucleosomal level there is a gain and loss of specific

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 19

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

Gene on Satb1
Gene off Smchd1 (?)
Regulatory element Repeat-rich regions
Xist RNA Nuclear/nucleolar
periphery

Figure 11. Factors involved in Xist-mediated silencing. Depiction of changes in higher-order chromosome archi-
tecture during the establishment of X inactivation. Xist RNA initially coats repeat rich chromosomal domains; genes
and other regulatory elements occupy an external position. As X inactivation proceeds, genes are internalized within
the Xist territory with consequent restriction in the mobility of chromosome loops. Establishment of X inactivation
is also linked to positioning of the chromosome on the nuclear and/or nucleolar periphery. Nuclear scaffold factors
(SATB1) and chromosome structure factors (Smchd1) may play a role in the reorganization of chromosome
architecture on Xi.

posttranslational modifications, incorporation of different of Xi and associated pathways are secondary, occurring in
histone variants, and DNA methylation at CpG islands response to silencing established by an as yet uncharacter-
(Fig. 9B). At a higher-order level there are changes in the ized primary mechanism.
architecture of chromatin loops and the reorganization
of chromosome domains and chromosome position in
the nucleus, potentially mediated by chromosomal pro- 5 X-CHROMOSOME REACTIVATION
teins such as Smchd1, SATB1, and SAFA/hnRNPU (Fig. AND REPROGRAMMING
11). As such, Xi can be viewed as a “belts and braces” system
5.1 X Reactivation in Normal Development
in which different pathways play overlapping or redundant
roles. Within this framework it is likely that different path- Multiple layers of epigenetic modification contribute to the
ways are more or less important at specific times in devel- silencing of the inactive X chromosome. As a result, the
opment, an idea that is supported by the observation that, repressed state is generally highly stable and attempts to
in contrast to somatic cells, chromosome silencing in cells reverse it experimentally have been consistently unsuccess-
of the early embryo is dependent on ongoing Xist expres- ful. However, there are circumstances in the course of nor-
sion. Finally, it is becoming apparent that different path- mal development in which the entire X chromosome is
ways contribute differently to silencing of specific genes or reactivated. The best studied example is reversal of X inac-
subsets of genes on Xi. For example, female Smchd1 null tivation in developing PGCs. In mouse, PGCs are specified
embryos that fail in mid-gestation, show up-regulation of at about 7–8 days of development, shortly after gastrula-
only a small proportion of loci on Xi (Blewitt et al. 2008). tion. At this time, cells of the embryo have already under-
In this regard, it is important to note that there is signifi- gone random X inactivation. Subsequently, the developing
cant variability in the time that individual Xi genes are PGCs migrate along the hindgut region of the embryo and
silenced following the onset of Xist expression. Thus, to arrive at the genital ridges, the structures that give rise to the
some degree it may be necessary to consider the contribu- adult gonads. It is at this time that XX PGCs reactivate their
tion of different X inactivation pathways on a gene-by-gene Xi (Monk and McLaren 1981). This event occurs coinci-
basis. dent with a more general epigenetic reprogramming that
Although significant progress has been made toward includes erasure of parental imprints and genome-wide
identifying pathways involved in Xist-mediated silencing, DNA demethylation (see Fig. 5 of Barlow and Bartolomei
it is likely that key factors remain to be found. Notably, we 2014; see also Reik and Surani 2014 for more detail).
do not yet know the critical factors that interact with the A- X-chromosome reactivation in PGCs may indicate a
repeat region of Xist RNA to initiate the silencing process. specialized mechanism for reversing the multilayered het-
Thus, it remains a possibility that the known modifications erochromatic structure. Extinction of Xist RNA expression

20 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

has been seen to correlate with X reactivation, but given showed rapid reactivation of a marker gene on Xi in cleav-
that silencing is Xist independent in XX somatic cells, it is age stage nuclear transfer embryos. Despite this, the nucle-
not certain that this is causative. It is possible that PGCs fail us retained some memory of which X had been inactive
to establish all of the marks associated with silencing and because in cloned embryos the donor cell Xi was also the Xi
are, therefore, more susceptible to reactivation. Consistent in trophectoderm cells of the placenta. In contrast, cells of
with this is the evidence that CpG island methylation does the embryo proper showed random X inactivation (see Fig.
not occur on the Xi in developing PGCs in mouse (Grant 12). Presumably, X reactivation and reprogramming that
et al. 1992). occurs in the developing ICM gives the embryo a second
A second example of X reactivation is the reversal chance to reset epigenetic information from the donor nu-
of imprinted Xp inactivation in the ICM lineage of blasto- cleus (see Mekhoubad et al. 2012).
cyst stage embryos, discussed in Section 3.5, which again is Recent studies have shown that ectopic Xist activation
associated with wider genome reprogramming events. This makes a significant contribution to the inefficiency of re-
reactivation also correlates with extinction of Xist RNA and productive cloning in mice (Inoue et al. 2010). Using XY
a loss of epigenetic marks associated with silencing. Again, donor somatic nuclei, the single Xist allele is activated in
it is possible that pre-ICM cells fail to establish all of the cloned embryos. Similarly, using XX donor somatic nuclei,
marks associated with silencing and are, therefore, more both Xist alleles are activated. Use of donor cells in which
susceptible to X reactivation. Xist is deleted dramatically improves cloning efficiency.
Presumably, the host oocyte erases Xist gene repression
5.2 X Reactivation during Experimental present on the active X chromosome in XYand XX somatic
Reprogramming cells. It follows that embryos cloned using donor nuclei
with an intact Xist locus can only survive early preimplan-
X reactivation has also been observed under specific exper- tation development if reprogramming of somatic Xist re-
imental circumstances. It occurs during nuclear transfer pression fails. This could explain why, in surviving embryos
of somatic nuclei to unfertilized oocytes and following cloned from normal XX somatic cells, the donor cell Xi
fusion of somatic cells with totipotent cell types such as remains inactive in the extraembryonic lineages for which
ES, embryonic germ (EG), or embryonal carcinoma (EC) X inactivation patterns are normally determined at the
cells (e.g., see Tada et al. 2000). Finally, X reactivation oc- two- to four-cell stage (Fig. 6).
curs when XX somatic cells are converted to induced plu- Although X reactivation occurs during iPS reprogram-
ripotent stem (iPS) cells (Maherali et al. 2007). ming of mouse somatic cells, the situation in human iPS
Nuclear transfer embryos provide a particularly inter- cells appears more complex. Early iPS cultures retain an Xi,
esting example. Experiments in mice (Eggan et al. 2000) but X reactivation can occur subsequently, dependent on

Somatic Enucleated Early/mid Late

+ One-cell Two-cell Four-cell Morula
nucleus oocyte blastocyst blastocyst

A B A B A B A B A B A B A B
X inactivation
status: or

Somatic Xist Random X inactivation

erasure in ICM postimplantation

Figure 12. Regulation of X inactivation in cloned mouse embryos. The figure illustrates an XX donor cell with the
inactive X chromosome (A) coated with Xist RNA (green line). In this model, transcription from the donor nucleus,
including Xist RNA, is repressed by oocyte factors until the two-cell stage, resulting in X reactivation. Recommence-
ment of Xist expression then occurs at the two-cell stage. Xist is then reexpressed again from the inactive X allele from
the donor cell. This would be attributable to retention of a mark such as DNA methylation at the Xist promoter. This
pattern is maintained in cells allocated to the TE and PE lineages, but not in pluripotent epiblast in which Xist
expression is again extinguished, leading to a second reactivation event. In the ICM, erasure of the epigenetic marks
governing donor Xist expression allows for subsequent random X inactivation in the embryo proper.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 21

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

exact culture conditions. This is likely to relate to different ing to see if mechanistic links with Xist and marsupial Rsx
levels of pluripotency as observed also in comparing mouse ncRNAs emerge in future studies.
ES cells and epiblast stem cells (EpiSCs; Bao et al. 2009). There remain many unanswered questions. Although
Human ES cells (hESCs) are more similar to EpiSCs than to progress has been made in defining the cis-acting sequences
ES cells from mouse, and consistent with this, XX hESCs and trans-acting factors that regulate counting and choice,
retain an inactive X chromosome (Tchieu et al. 2010). their further elucidation provides an exciting challenge.
Similarly, although we now know some of the chromatin-
5.3 Lessons from Inducible Xist Transgenes modifying complexes involved in maintaining X inactiva-
tion, such as the Polycomb-group complexes, the signal for
A series of experiments using inducible Xist transgenes in establishing chromosome-wide silencing, triggered by Xist
ES cells has greatly increased our understanding of stability RNA, remains unknown. Similarly, the mechanistic links
versus reversibility of X inactivation. First, it was shown between chromosome-wide changes in Xi chromatin and
that Xist RNA can establish X inactivation in undifferenti- the silencing of individual genes remain elusive. Other key
ated ES cells and during very early stages of differentiation, questions are to understand how silencing spreads across
but not subsequently (referred to as the “window of op- the chromosome and what role, if any, way stations ( per-
portunity”; Wutz and Jaenisch 2000). Ectopic expression of haps LINE elements) play in this process and in the stabi-
the nuclear scaffold/matrix protein, SATB1, confers the lization/maintenance of the silent state. This may relate
ability to respond to Xist RNA in thymic lymphoma and to the intriguing question of how X inactivation is reversed
fibroblast cells (Agrelo et al. 2009), suggesting that this is at in some cell types and stages of development, but is essen-
least one component that is important for developmental tially irreversible in others. This latter question relates to
competence (but see also Nechanitzky et al. 2012). The the wider and crucially important issue of understanding
ability of cells to respond to Xist RNA broadly correlates genome plasticity and reprogramming through develop-
with reversibility of X inactivation. Thus, silencing was ment. Finally, the recent validation of an old hypothesis,
reversed when the transgene was switched off in ES cells namely, the up-regulated expression of genes on the single
or during early differentiation stages, but not in later dif- active X in males and females, has given us a new view of the
ferentiation or somatic cells. context in which X inactivation operates and a better un-
Returning to X reactivation and reprogramming, the derstanding of the true complexity of dosage compensation
inducible transgene data imply that in defined cellular in mammals.
environments, namely, undifferentiated ES cells, X reacti-
vation will occur when expression of Xist RNA is extin-
guished. If we consider that those cells in which X REFERENCES
reactivation has been documented to occur (i.e., PGCs, ∗ Reference is also in this collection.
ICM cells, iPS, EG, and EC cells) are all similar to ES cells
in terms of pluripotency and plasticity, then extinction of Agrelo R, Souabni A, Novatchkova M, Haslinger C, Leeb M, Komnenovic
Xist expression may underlie X reactivation in all cases. V, Kishimoto H, Gresh L, Kohwi-Shigematsu T, Kenner L, et al. 2009.
SATB1 defines the developmental context for gene silencing by Xist in
lymphoma and embryonic cells. Dev Cell 16: 507 – 516.
∗ Allis CD, Jenuwein T, Reinberg D. 2014. Overview and concepts. Cold
6 SUMMARY AND FUTURE DIRECTIONS Spring Harb Perspect Biol doi: 10.1101/cshperspect.a018739.
In recent years, there has been significant progress in our Augui S, Filion GJ, Huart S, Nora E, Guggiari M, Maresca M, Stewart AF,
Heard E. 2007. Sensing X chromosome pairs before X inactivation via
understanding of the molecular mechanism of X inactiva- a novel X-pairing region of the Xic. Science 318: 1632 –1636.
tion. To date, this progress has been fed by advances in Bao S, Tang F, Li X, Hayashi K, Gillich A, Lao K, Surani MA. 2009.
related fields of epigenetic research and has, in turn, stim- Epigenetic reversion of post-implantation epiblast to pluripotent em-
bryonic stem cells. Nature 461: 1292 – 1295.
ulated advances in other fields. An example of the latter is ∗ Barlow DP, Bartolomei MS. 2014. Genomic imprinting in mammals.
the growing evidence that some clusters of imprinted genes Cold Spring Harb Perspect Biol 6: 018382.
are regulated by cis-acting ncRNAs in much the same way Berletch JB, Yang F, Xu J, Carrel L, Disteche CM. 2011. Genes that escape
that Xist regulates the X chromosome (see Barlow and Bar- from X inactivation. Hum Genet 130: 237 – 245.
Beutler E, Yeh M, Fairbanks VF. 1962. The normal human female as a
tolomei 2014). Similarly, the independent evolution of mosaic of X-chromosome activity: Studies using the gene for C-6-PD-
ncRNA regulating X inactivation in marsupials further il- deficiency as a marker. Proc Natl Acad Sci 48: 9 – 16.
lustrates the potential generality of in cis silencing by Blewitt ME, Gendrel AV, Pang Z, Sparrow DB, Whitelaw N, Craig JM,
ncRNA. Conversely, other studies point to distinct types Apedaile A, Hilton DJ, Dunwoodie SL, Brockdorff N, et al. 2008.
SmcHD1, containing a structural-maintenance-of-chromosomes
of ncRNA that function in trans in gene silencing (e.g., hinge domain, has a critical role in X inactivation. Nat Genet 40:
HOTAIR [Rinn et al. 2007; Rinn 2014]). It will be interest- 663 – 669.

22 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

Brockdorff N, Ashworth A, Kay GF, McCabe VM, Norris DP, Cooper PJ, Deng X, Hiatt JB, Nguyen DK, Ercan S, Sturgill D, Hillier LW, Schlesinger
Swift S, Rastan S. 1992. The product of the mouse Xist gene is a 15 kb F, Davis CA, Reinke VJ, Gingeras TR, et al. 2011. Evidence for com-
inactive X-specific transcript containing no conserved ORF and locat- pensatory upregulation of expressed X-linked genes in mammals,
ed in the nucleus. Cell 71: 515– 526. Caenorhabditis elegans and Drosophila melanogaster. Nat Genet 43:
Brown CJ, Willard HF. 1994. The human X-inactivation centre is not 1179– 1185.
required for maintenance of X-chromosome inactivation. Nature Donohoe ME, Silva SS, Pinter SF, Xu N, Lee JT. 2009. The pluripotency
368: 154– 156. factor Oct4 interacts with Ctcf and also controls X-chromosome pair-
Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi ing and counting. Nature 460: 128 – 132.
R, Willard HF. 1991. A gene from the region of the human X inacti- Duret L, Chureau C, Samain S, Weissenbach J, Avner P. 2006. The Xist
vation centre is expressed exclusively from the inactive X chromosome. RNA gene evolved in eutherians by pseudogenization of a protein-
Nature 349: 38– 44. coding gene. Science 312: 1653– 1655.
Brown CJ, Hendrich BD, Rupert JL, Lafreniere RG, Xing Y, Lawrence J, Duthie SM, Nesterova TB, Formstone EJ, Keohane AM, Turner BM,
Willard HF. 1992. The human XIST gene: Analysis of a 17 kb inactive Zakian SM, Brockdorff N. 1999. Xist RNA exhibits a banded localiza-
X-specific RNA that contains conserved repeats and is highly localized tion on the inactive X chromosome and is excluded from autosomal
within the nucleus. Cell 71: 527– 542. material in cis. Hum Mol Genet 8: 195– 204.
Buzin CH, Mann JR, Singer-Sam J. 1994. Quantitative RT-PCR assays Eggan K, Akutsu H, Hochedlinger K, Rideout W 3rd, Yanagimachi R,
show Xist RNA levels are low in mouse female adult tissue, embryos Jaenisch R. 2000. X-chromosome inactivation in cloned mouse em-
and embryoid bodies. Development 120: 3529– 3536. bryos. Science 290: 1578– 1581.
Carrel L, Willard HF. 2005. X-inactivation profile reveals extensive Eils R, Dietzel S, Bertin E, Schrock E, Speicher MR, Ried T, Robert-
variability in X-linked gene expression in females. Nature 434: Nicoud M, Cremer C, Cremer T. 1996. Three-dimensional reconstruc-
400 –404. tion of painted human interphase chromosomes: Active and inactive
Cattanach BM. 1974. Position effect variegation in the mouse. Genet Res X chromosome territories have similar volumes but differ in shape and
23: 291 – 306. surface structure. J Cell Biol 135: 1427 – 1440.
Chadwick BP, Willard HF. 2001. A novel chromatin protein, distantly Ellegren H, Parsch J. 2007. The evolution of sex-biased genes and sex-
related to histone H2A, is largely excluded from the inactive X chro- biased gene expression. Nat Rev Genet 8: 689– 698.
mosome. J Cell Biol 152: 375 – 384. Epstein CJ, Smith S, Travis B, Tucker G. 1978. Both X chromosomes
Chadwick BP, Willard HF. 2003. Chromatin of the Barr body: Histone function before visible X-chromosome inactivation in female mouse
and non-histone proteins associated with or excluded from the inac- embryos. Nature 274: 500– 503.
tive X chromosome. Hum Mol Genet 12: 2167– 2178. Gartler SM, Riggs AD. 1983. Mammalian X-chromosome inactivation.
Charlesworth B. 1996. The evolution of chromosomal sex determination Annu Rev Genet 17: 155 – 190.
and dosage compensation. Curr Biol 6: 149– 162. Gendrel AV, Apedaile A, Coker H, Termanis A, Zvetkova I, Godwin J, Tang
Charlesworth D, Charlesworth B. 2005. Sex chromosomes: Evolution of YA, Huntley D, Montana G, Taylor S, et al. 2012. Smchd1-dependent
the weird and wonderful. Curr Biol 15: R129– R131. and -independent pathways determine developmental dynamics of
Chaumeil J, Le Baccon P, Wutz A, Heard E. 2006. A novel role for Xist CpG island methylation on the inactive X chromosome. Dev Cell 23:
RNA in the formation of a repressive nuclear compartment into which 265– 279.
genes are recruited when silenced. Genes Dev 20: 2223– 2237. Gontan C, Achame EM, Demmers J, Barakat TS, Rentmeester E, van
∗ Cheng X. 2014. Structural and functional coordination of DNA and Ijcken W, Grootegoed JA, Gribnau J. 2012. RNF12 initiates X-chro-
histone methylation. Cold Spring Harb Perspect Biol 6: a018747. mosome inactivation by targeting REX1 for degradation. Nature 485:
Chow JC, Ciaudo C, Fazzari MJ, Mise N, Servant N, Glass JL, Attreed M, 386– 390.
Avner P, Wutz A, Barillot E, et al. 2010. LINE-1 activity in facultative Grant M, Zuccotti M, Monk M. 1992. Methylation of CpG sites of two X-
heterochromatin formation during X chromosome inactivation. Cell linked genes coincides with X-inactivation in the female mouse em-
141: 956– 969. bryo but not in the germ line. Nat Genet 2: 161– 166.
Chureau C, Chantalat S, Romito A, Galvani A, Duret L, Avner P, Rou- Grant J, Mahadevaiah SK, Khil P, Sangrithi MN, Royo H, Duckworth J,
geulle C. 2011. Ftx is a non-coding RNA which affects Xist expression McCarrey JR, VandeBerg JL, Renfree MB, Taylor W, et al. 2012. Rsx is a
and chromatin structure within the X-inactivation center region. Hum metatherian RNA with Xist-like properties in X-chromosome inacti-
Mol Genet 20: 705– 718. vation. Nature 487: 254 –258.
Clemson CM, McNeil JA, Willard HF, Lawrence JB. 1996. XIST RNA ∗ Grossniklaus U, Paro R. 2014. Transcriptional silencing by Polycomb-
paints the inactive X chromosome at interphase: Evidence for a novel group proteins. Cold Spring Harb Perspect Biol 6: a019331.
RNA involved in nuclear/chromosome structure. J Cell Biol 132: 259 – Hasegawa Y, Nakagawa S. 2011. Revisiting the function of nuclear scaf-
275. fold/matrix binding proteins in X chromosome inactivation. RNA
Costanzi C, Pehrson JR. 1998. Histone macroH2A1 is concentrated in Biol 8: 735– 739.
the inactive X chromosome of female mammals. Nature 393: 599 – Huynh KD, Lee JT. 2003. Inheritance of a pre-inactivated paternal X
601. chromosome in early mouse embryos. Nature 426: 857– 862.
Csankovszki G, Panning B, Bates B, Pehrson JR, Jaenisch R. 1999. Con- Inoue K, Kohda T, Sugimoto M, Sado T, Ogonuki N, Matoba S, Shiura H,
ditional deletion of Xist disrupts histone macroH2A localization but Ikeda R, Mochida K, Fujii T, et al. 2010. Impeding Xist expression from
not maintenance of X inactivation. Nat Genet 22: 323 – 324. the active X chromosome improves mouse somatic cell nuclear trans-
Davidson RG, Nitowsky HM, Childs B. 1963. Demonstration of fer. Science 330: 496 – 499.
two populations of cells in the human female heterozygous for glu- Jeon Y, Lee JT. 2011. YY1 tethers Xist RNA to the inactive X nucleation
cose-6-phosphate dehydrogenase variants. Proc Natl Acad Sci 50: 481 – center. Cell 146: 119– 133.
485. Jeppesen P, Turner BM. 1993. The inactive X chromosome in female
∗ Dekker J, Misteli T. 2014. Long-range chromatin interactions. Cold Spring mammals is distinguished by a lack of histone H4 acetylation, a cyto-
Harb Perspect Biol doi: 10.1101/cshperspect.a019356. genetic marker for gene expression. Cell 74: 281– 289.
de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Jonkers I, Barakat TS, Achame EM, Monkhorst K, Kenter A, Rentmeester
Nesterova TB, Silva J, Otte AP, Vidal M, et al. 2004. Polycomb group E, Grosveld F, Grootegoed JA, Gribnau J. 2009. RNF12 is an X-encoded
proteins Ring1A/B link ubiquitylation of histone H2A to heritable dose-dependent activator of X chromosome inactivation. Cell 139:
gene silencing and X inactivation. Dev Cell 7: 663– 676. 999– 1011.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 23

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
N. Brockdorff and B.M. Turner

Keohane AM, O’Neill LP, Belyaev ND, Lavender JS, Turner BM. 1996. X- Navarro P, Avner P. 2010. An embryonic story: Analysis of the gene
inactivation and histone H4 acetylation in embryonic stem cells. Dev regulative network controlling Xist expression in mouse embryonic
Biol 180: 618 –630. stem cells. Bioessays 32: 581– 588.
Kohlmaier A, Savarese F, Lachner M, Martens J, Jenuwein T, Wutz A. Navarro P, Oldfield A, Legoupi J, Festuccia N, Dubois A, Attia M, Schoor-
2004. A chromosomal memory triggered by Xist regulates histone lemmer J, Rougeulle C, Chambers I, Avner P. 2010. Molecular coupling
methylation in X inactivation. PLoS Biol 2: E171. of Tsix regulation and pluripotency. Nature 468: 457– 460.
Lee JT. 2000. Disruption of imprinted X inactivation by parent-of-origin Nechanitzky R, Davila A, Savarese F, Fietze S, Grosschedl R. 2012. Satb1
effects at Tsix. Cell 103: 17 – 27. and Satb2 are dispensable for X chromosome inactivation in mice. Dev
Lee JT, Davidow LS, Warshawsky D. 1999. Tsix, a gene antisense to Xist at Cell 23: 866 – 871.
the X-inactivation centre. Nat Genet 21: 400– 404. Nesterova TB, Johnston CM, Appanah R, Newall AE, Godwin J, Alexiou
Lee JT, Lu N. 1999. Targeted mutagenesis of Tsix leads to nonrandom X M, Brockdorff N. 2003. Skewing X chromosome choice by modulating
inactivation. Cell 99: 47 – 57. sense transcription across the Xist locus. Genes Dev 17: 2177– 2190.
Lee JT, Strauss WM, Dausman JA, Jaenisch R. 1996. A 450 kb transgene Ng K, Daigle N, Bancaud A, Ohhata T, Humphreys P, Walker R, Ellenberg
displays properties of the mammalian X-inactivation center. Cell 86: J, Wutz A. 2011. A system for imaging the regulatory noncoding Xist
83 – 94. RNA in living mouse embryonic stem cells. Mol Biol Cell 22: 2634 –
∗ Li E, Zhang Y. 2014. DNA methylation in mammals. Cold Spring Harb 2645.
Perspect Biol 6: a019133. Nguyen DK, Disteche CM. 2006. Dosage compensation of the active X
Lin H, Gupta V, Vermilyea MD, Falciani F, Lee JT, O’Neill LP, Turner BM. chromosome in mammals. Nat Genet 38: 47– 53.
2007. Dosage compensation in the mouse balances up-regulation and Nora EP, Heard E. 2009. X chromosome inactivation: When dosage
silencing of X-linked genes. PLoS Biol 5: e326. counts. Cell 139: 865– 867.
Lin H, Halsall JA, Antczak P, O’Neill LP, Falciani F, Turner BM. 2011. Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot
Relative overexpression of X-linked genes in mouse embryonic stem T, van Berkum NL, Meisig J, Sedat J, et al. 2012. Spatial partitioning of
cells is consistent with Ohno’s hypothesis. Nat Genet 43: 1169– 1170; the regulatory landscape of the X-inactivation centre. Nature 485:
author reply 1171– 1172. 381 – 385.
Lock LF, Takagi N, Martin GR. 1987. Methylation of the Hprt gene on the Norris DP, Patel D, Kay GF, Penny GD, Brockdorff N, Sheardown SA,
inactive X occurs after chromosome inactivation. Cell 48: 39– 46. Rastan S. 1994. Evidence that random and imprinted Xist expression is
∗ Lucchesi JC, Kuroda MI. 2014. Dosage compensation in Drosophila. Cold controlled by preemptive methylation. Cell 77: 41 –51.
Spring Harb Perspect Biol doi: 10.1101/cshperspect.a019398. Ohhata T, Hoki Y, Sasaki H, Sado T. 2008. Crucial role of antisense
Lyon MF. 1961. Gene action in the X-chromosome of the mouse (Mus transcription across the Xist promoter in Tsix-mediated Xist chroma-
musculus L.). Nature 190: 372 – 373. tin modification. Development 135: 227 –235.
Lyon MF. 2003. The Lyon and the LINE hypothesis. Semin Cell Dev Biol Ohno S. 1967. Sex chromosomes and sex linked genes. Springer-Verlag,
14: 313– 318. Berlin.
Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E. 2004. Epigenetic
M, Yachechko R, Tchieu J, Jaenisch R, et al. 2007. Directly repro- dynamics of imprinted X inactivation during early mouse develop-
grammed fibroblasts show global epigenetic remodeling and wide- ment. Science 303: 644 – 649.
spread tissue contribution. Cell Stem Cell 1: 55– 70. Okamoto I, Arnaud D, Le Baccon P, Otte AP, Disteche CM, Avner P,
Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, Otte AP, Heard E. 2005. Evidence for de novo imprinted X-chromosome inac-
Brockdorff N. 2004. Reactivation of the paternal X chromosome in tivation independent of meiotic inactivation in mice. Nature 438:
early mouse embryos. Science 303: 666 – 669. 369 – 373.
Marks H, Chow JC, Denissov S, Francoijs KJ, Brockdorff N, Heard E, Okamoto I, Patrat C, Thepot D, Peynot N, Fauque P, Daniel N, Diaban-
Stunnenberg HG. 2009. High-resolution analysis of epigenetic chang- gouaya P, Wolf JP, Renard JP, Duranthon V, et al. 2011. Eutherian
es associated with X inactivation. Genome Res 19: 1361 – 1373. mammals use diverse strategies to initiate X-chromosome inactivation
Marshall Graves JA, Shetty S. 2001. Sex from W to Z: Evolution of ver- during development. Nature 472: 370– 374.
tebrate sex chromosomes and sex determining genes. J Exp Zool 290: Oliver B. 2007. Sex, dose, and equality. PLoS Biol 5: e340.
449 – 462. O’Neill LP, Keohane AM, Lavender JS, McCabe V, Heard E, Avner P,
Masui O, Bonnet I, Le Baccon P, Brito I, Pollex T, Murphy N, Hupe P, Brockdorff N, Turner BM. 1999. A developmental switch in H4 acet-
Barillot E, Belmont AS, Heard E. 2011. Live-cell chromosome dynam- ylation upstream of Xist plays a role in X chromosome inactivation.
ics and outcome of X chromosome pairing events during ES cell EMBO J 18: 2897– 2907.
differentiation. Cell 145: 447– 458. O’Neill LP, Spotswood HT, Fernando M, Turner BM. 2008. Differential
Meisel RP, Malone JH, Clark AG. 2012. Disentangling the relationship loss of histone H3 isoforms mono-, di- and tri-methylated at lysine 4
between sex-biased gene expression and X-linkage. Genome Res 22: during X-inactivation in female embryonic stem cells. Biol Chem 389:
1255 – 1265. 365 – 370.
Mekhoubad S, Bock C, de Boer AS, Kiskinis E, Meissner A, Eggan K. Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N. 1996. Re-
2012. Erosion of dosage compensation impacts human iPSC disease quirement for Xist in X chromosome inactivation. Nature 379: 131 –
modeling. Cell Stem Cell 10: 595– 609. 137.
Mermoud JE, Costanzi C, Pehrson JR, Brockdorff N. 1999. Histone mac- Pessia E, Makino T, Bailly-Bechet M, McLysaght A, Marais GA. 2012.
roH2A1.2 relocates to the inactive X chromosome after initiation and Mammalian X chromosome inactivation evolved as a dosage-com-
propagation of X-inactivation. J Cell Biol 147: 1399– 1408. pensation mechanism for dosage-sensitive genes on the X chromo-
Monk M, Harper MI. 1979. Sequential X chromosome inactivation cou- some. Proc Natl Acad Sci 109: 5346– 5351.
pled with cellular differentiation in early mouse embryos. Nature 281: Popova BC, Tada T, Takagi N, Brockdorff N, Nesterova TB. 2006. Atten-
311 – 313. uated spread of X-inactivation in an X;autosome translocation. Proc
Monk M, McLaren A. 1981. X-chromosome activity in foetal germ cells Natl Acad Sci 103: 7706 – 7711.
of the mouse. J Embryol Exp Morphol 63: 75– 84. Pullirsch D, Hartel R, Kishimoto H, Leeb M, Steiner G, Wutz A. 2010.
Monkhorst K, Jonkers I, Rentmeester E, Grosveld F, Gribnau J. 2008. X The Trithorax group protein Ash2l and Saf-A are recruited to the
inactivation counting and choice is a stochastic process: Evidence for inactive X chromosome at the onset of stable X inactivation. Develop-
involvement of an X-linked activator. Cell 132: 410 – 421. ment 137: 935– 943.

24 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019406

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press
Dosage Compensation in Mammals

Quinn A, Koopman P. 2012. The molecular genetics of sex determination some adopts a unique three-dimensional conformation that is depen-
and sex reversal in mammals. Semin Reprod Med 30: 351 – 363. dent on Xist RNA. Genes Dev 25: 1371 – 1383.
Rasmussen TP, Wutz AP, Pehrson JR, Jaenisch RR. 2001. Expression of ∗ Strome S, Kelly WG, Ercan S, Lieb JD. 2014. Regulation of the X chro-
Xist RNA is sufficient to initiate macrochromatin body formation. mosomes in Caenorhabditis elegans. Cold Spring Harb Perspect Biol 6:
Chromosoma 110: 411 – 420. a018366.
Rastan S. 1983. Non-random X-chromosome inactivation in mouse X- Sun BK, Deaton AM, Lee JT. 2006. A transient heterochromatic state in
autosome translocation embryos—Location of the inactivation cen- Xist preempts X inactivation choice without RNA stabilization. Mol
tre. J Embryol Exp Morphol 78: 1 – 22. Cell 21: 617– 628.
∗ Reik W, Surani MA. 2014. Germline and pluripotent stem cells. Cold Sun S, Del Rosario BC, Szanto A, Ogawa Y, Jeon Y, Lee JT. 2013. Jpx RNA
Spring Harb Perspect Biol doi: 10.1101/cshperspect.a019422. activates Xist by evicting CTCF. Cell 153: 1537– 1551.
∗ Rinn JL. 2014. lncRNAs: Linking RNA to chromatin. Cold Spring Harb Tada T, Obata Y, Tada M, Goto Y, Nakatsuji N, Tan S, Kono T, Takagi N.
Perspect Biol 6: a018614. 2000. Imprint switching for non-random X-chromosome inactivation
Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Good- during mouse oocyte growth. Development 127: 3101 –3105.
nough LH, Helms JA, Farnham PJ, Segal E, et al. 2007. Functional Takagi N, Sasaki M. 1975. Preferential inactivation of the paternally
demarcation of active and silent chromatin domains in human HOX derived X chromosome in the extraembryonic membranes of the
loci by noncoding RNAs. Cell 129: 1311– 1323. mouse. Nature 256: 640 – 642.
Rougeulle C, Chaumeil J, Sarma K, Allis CD, Reinberg D, Avner P, Heard Tang YA, Huntley D, Montana G, Cerase A, Nesterova TB, Brockdorff N.
E. 2004. Differential histone H3 Lys-9 and Lys-27 methylation profiles 2010. Efficiency of Xist-mediated silencing on autosomes is linked to
on the X chromosome. Mol Cell Biol 24: 5475– 5484. chromosomal domain organisation. Epigenetics Chromatin 3: 10.
Royce-Tolland ME, Andersen AA, Koyfman HR, Talbot DJ, Wutz A, Tavares L, Dimitrova E, Oxley D, Webster J, Poot R, Demmers J, Bezstar-
Tonks ID, Kay GF, Panning B. 2010. The A-repeat links ASF/SF2- osti K, Taylor S, Ura H, Koide H, et al. 2012. RYBP-PRC1 complexes
dependent Xist RNA processing with random choice during X inacti- mediate H2A ubiquitylation at polycomb target sites independently of
vation. Nat Struct Mol Biol 17: 948 –954. PRC2 and H3K27me3. Cell 148: 664 – 678.
Sado T, Okano M, Li E, Sasaki H. 2004. De novo DNA methylation is Tchieu J, Kuoy E, Chin MH, Trinh H, Patterson M, Sherman SP, Aimiu-
dispensable for the initiation and propagation of X chromosome in- wu O, Lindgren A, Hakimian S, Zack JA, et al. 2010. Female human
activation. Development 131: 975– 982. iPSCs retain an inactive X chromosome. Cell Stem Cell 7: 329 – 342.
Sado T, Hoki Y, Sasaki H. 2005. Tsix silences Xist through modification of Thorvaldsen JL, Weaver JR, Bartolomei MS. 2011. A YY1 bridge for X
chromatin structure. Dev Cell 9: 159 – 165. inactivation. Cell 146: 11 – 13.
Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Tian D, Sun S, Lee JT. 2010. The long noncoding RNA, Jpx, is a molecular
Cardoso MC, Agard DA, Gustafsson MG, et al. 2008. Subdiffraction switch for X chromosome inactivation. Cell 143: 390 – 403.
multicolor imaging of the nuclear periphery with 3D structured illu- Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS. 2006.
mination microscopy. Science 320: 1332– 1336. Pachytene asynapsis drives meiotic sex chromosome inactivation and
Sharman GB. 1971. Late DNA replication in the paternally derived X leads to substantial postmeiotic repression in spermatids. Dev Cell 10:
chromosome of female kangaroos. Nature 230: 231 – 232. 521– 529.
Shibata S, Yokota T, Wutz A. 2008. Synergy of Eed and Tsix in the repres- Wutz A, Jaenisch R. 2000. A shift from reversible to irreversible X inac-
sion of Xist gene and X-chromosome inactivation. EMBO J 27: 1816 – tivation is triggered during ES cell differentiation. Mol Cell 5: 695– 705.
1826. Wutz A, Rasmussen TP, Jaenisch R. 2002. Chromosomal silencing and
Silva J, Mak W, Zvetkova I, Appanah R, Nesterova TB, Webster Z, Peters localization are mediated by different domains of Xist RNA. Nat Genet
AH, Jenuwein T, Otte AP, Brockdorff N. 2003. Establishment of his- 30: 167– 174.
tone h3 methylation on the inactive X chromosome requires transient Xu N, Tsai CL, Lee JT. 2006. Transient homologous chromosome pairing
recruitment of Eed-Enx1 polycomb group complexes. Dev Cell 4: marks the onset of X inactivation. Science 311: 1149– 1152.
481 –495. Zhao J, Sun BK, Erwin JA, Song JJ, Lee JT. 2008. Polycomb proteins
Splinter E, de Wit E, Nora EP, Klous P, van de Werken HJ, Zhu Y, Kaaij LJ, targeted by a short repeat RNA to the mouse X chromosome. Science
van Ijcken W, Gribnau J, Heard E, et al. 2011. The inactive X chromo- 322: 750 – 756.

Cite this article as Cold Spring Harb Perspect Biol 2015;6:a019406 25

 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press

Dosage Compensation in Mammals

Neil Brockdorff and Bryan M. Turner

Cold Spring Harb Perspect Biol 2015; doi: 10.1101/cshperspect.a019406

Subject Collection Epigenetics

Metabolic Signaling to Chromatin Epigenetic Determinants of Cancer

Shelley L. Berger and Paolo Sassone-Corsi Stephen B. Baylin and Peter A. Jones
Histone and DNA Modifications as Regulators of Maintenance of Epigenetic Information
Neuronal Development and Function Geneviève Almouzni and Howard Cedar
Stavros Lomvardas and Tom Maniatis
Histone Modifications and Cancer A Structural Perspective on Readout of Epigenetic
James E. Audia and Robert M. Campbell Histone and DNA Methylation Marks
Dinshaw J. Patel
Epigenetics and Human Disease The Necessity of Chromatin: A View in
Huda Y. Zoghbi and Arthur L. Beaudet Perspective
Vincenzo Pirrotta
Induced Pluripotency and Epigenetic Germline and Pluripotent Stem Cells
Reprogramming Wolf Reik and M. Azim Surani
Konrad Hochedlinger and Rudolf Jaenisch
Long-Range Chromatin Interactions Comprehensive Catalog of Currently Documented
Job Dekker and Tom Misteli Histone Modifications
Yingming Zhao and Benjamin A. Garcia
RNAi and Heterochromatin Assembly Epigenetic Regulation of Chromatin States in
Robert Martienssen and Danesh Moazed Schizosaccharomyces pombe
Robin C. Allshire and Karl Ekwall
Dosage Compensation in Drosophila Histone Variants and Epigenetics
John C. Lucchesi and Mitzi I. Kuroda Steven Henikoff and M. Mitchell Smith

For additional articles in this collection, see [Link]

Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved
 Downloaded from [Link] on December 3, 2023 - Published by Cold Spring Harbor Laboratory Press

For additional articles in this collection, see [Link]

Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved