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First published online 28 February 2007
doi: 10.1242/dev.000018
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Primer |
Division of Stem Cell Biology and Developmental Genetics, MRC NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK.
e-mail: jturner{at}nimr.mrc.ac.uk
SUMMARY
X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.
Introduction
Meiotic sex chromosome inactivation (MSCI) is the process of
transcriptional silencing of the X and Y chromosomes that occurs during the
meiotic phase of spermatogenesis. This silencing occurs at pachytene, when
maternal and paternal autosomal homologues have completed pairing, or
synapsis, and is mediated by large-scale chromatin remodelling of the X and Y
chromosomes. MSCI is found in the male germ line of almost all organisms
possessing differentiated sex chromosomes, but its importance to developmental
biologists, particularly to those studying germ cell development and
infertility, is only just beginning to be understood. MSCI has also received a
great deal of attention in the somatic X-inactivation field. Specifically, it
has been suggested that MSCI plays a role in establishing the silencing of the
paternally inherited X chromosome in preimplantation mouse female embryos
(Huynh and Lee, 2003
).
In this primer, I outline recent advances in our understanding of MSCI in mammals. MSCI is a manifestation of a general meiotic-silencing mechanism called meiotic silencing of unsynapsed chromatin (MSUC) (see Table 1), which is beginning to throw much-needed light on our understanding of meiotic-derived sterility. MSCI is initiated by several DNA-repair proteins and is maintained by virtue of histone modifications that are associated with transcriptional silencing in a wide variety of developmental contexts. New studies are beginning to challenge the dogma that MSCI is restricted to meiosis, because the X and Y chromosomes retain a repressed state throughout round spermatid development. These findings from studies of MSCI have far-reaching implications for our understanding of germ cell epigenetics, meiotic chromosome dynamics and, more generally, infertility.
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The transcriptional activity of the sex chromosomes varies considerably as
germ cells progress through the successive stages of spermatogenesis (see
Fig. 1). During the early
stages of spermatogenesis, spermatogonial stem cells divide by mitosis to
generate progressively more-differentiated progeny; here, genes on the X and Y
chromosomes are transcriptionally active. One study
(Wang et al., 2001) has
revealed that a disproportionately large number of spermatogonial-specific
transcripts originate from the X and Y chromosomes. This is in agreement with
the predictions of Fisher (Fisher,
1931) and Rice (Rice,
1984) that the X chromosome should accumulate sexually
antagonistic genes that are beneficial to one sex but harmful to the other
(Khil and Camerini-Otero,
2005). When a recessive mutation that provides a reproductive
advantage to males appears on the X chromosome, that advantage is immediately
apparent because the X chromosome is present in only a single copy (i.e. the
mutation is hemizygous). Thus, sexually antagonistic genes beneficial to males
(i.e. those involved in spermatogenesis) should accumulate on the X chromosome
(see Khil and Camerini-Otero,
2005).
Following spermatogonial divisions, germ cells enter meiosis. During the
earliest meiotic substage, leptotene, when the DNA double-strand breaks (DSBs)
that initiate meiotic recombination are formed, the X and Y chromosomes are
still transcriptionally active, and remain so throughout zygotene
(Fig. 1)
(Turner et al., 2005), when
DSBs are processed to form the single-stranded tails that drive homologous
synapsis (Box 1). However, shortly after the zygotene-to-pachytene transition,
when meiotic synapsis between autosomes is complete, the X and Y chromosomes
are rapidly silenced and compartmentalized into a peripheral nuclear subdomain
called the sex- or XY-body (Solari,
1974; McKee and Handel,
1993). MSCI then persists throughout the rest of pachytene and
diplotene.
Following diplotene, germ cells undergo two successive rounds of cell
division, when homologous chromosomes and their respective sister chromatids
separate (the paired homologues, each comprised of two sisters, are termed
bivalents). The resulting haploid daughter cells then undergo spermiogenesis,
during which the DNA of these cells undergoes increasing compaction,
facilitated by the replacement of histones with protamines. Protamines are
small arginine- and cysteine-rich proteins that facilitate the high level of
chromatin compaction required during sperm formation. Until recently, the
transcriptional status of the sex chromosomes during spermiogenesis was poorly
understood; few published studies focused on the activity of specific
sex-linked genes, but these studies did conclude that the transcription of
these genes was reactivated on the X and Y chromosomes
(Hendriksen et al., 1995;
Hendriksen, 1999;
Wang et al., 2005). It was
recently reported, however, that genes that are expressed in late
spermatogenesis are under-represented on the X chromosome
(Khil et al., 2004).
Subsequently, three further studies
(Namekawa et al., 2006;
Turner et al., 2006;
Greaves et al., 2006) have
found cytological evidence that gene silencing is maintained on the X and Y
chromosomes in spermatids, as described in more detail below. In summary,
these studies indicate that the X and Y chromosomes are transcriptionally
active during spermatogonial divisions and early meiotic stages, but become
transcriptionally repressed from pachytene onwards, through to the end of
spermatogenesis.
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MSCI was previously thought to be mediated in much the same way as somatic
X chromosome inactivation (XCI) (see Table
1). XCI is initiated by an X-encoded RNA called Xist
(inactive X specific transcript), which coats the X chromosome from which it
is transcribed (termed acting in-cis)
(Penny et al., 1996;
Marahrens et al., 1997).
Xist recruits an array of chromatin-modifying enzymes to the future
inactive X chromosome that induce gene silencing by catalysing methylation,
ubiquitylation and deacetylation of defined histone residues
(Heard and Disteche, 2006). In
males, Xist is expressed exclusively in the testis
(McCarrey and Dilworth, 1992;
Salido et al., 1992;
Richler et al., 1992;
Ayoub et al., 1997) and has
been reported to coat the sex body in a manner analogous to that in female
somatic cells. The presence of Xist on the Y as well as on the X
chromosome in the sex body spawned the `quasi-cis' model of MSCI, in which X
chromosome-derived Xist transcripts spread from the X chromosome to
the Y chromosome via the region of X-Y synapsis
(Ayoub et al., 1997). However,
subsequent studies have found that, although Xist is essential for
XCI (Penny et al., 1996;
Marahrens et al., 1997), it is
dispensable for MSCI (McCarrey et al.,
2002; Turner et al.,
2002). Attention has since focused on defining the molecular
events that lead to MSCI.
A central player in MSCI is the histone H2A variant H2AX (histone family,
member X) (Fig. 2). H2AX is
abundant in the mammalian testis, in comparison to other tissues
(Mahadevaiah et al., 2001),
and is a core component of the nucleosome of meiotic cells
(Fernandez-Capetillo et al.,
2003). H2AX plays an essential role in the DNA-damage response
(Celeste et al., 2002):
following DNA DSB-induction (Box 1), H2AX is rapidly phosphorylated at
serine-139 to form 
H2AX (Rogakou et
al., 1999) and recruits members of the DNA-repair machinery (e.g.
MDC1, mediator of DNA damage checkpoint 1) to the sites of breaks
(Stucki et al., 2005)
(Fig. 2 and
Table 1). H2AX phosphorylation
occurs in response to the formation of DNA DSBs during leptotene
(Mahadevaiah et al., 2001)
(Fig. 2A). However, an
additional wave of H2AX phosphorylation also takes place at the
zygotene-pachytene transition, when MSCI commences. This second wave of
phosphorylation occurs only on the chromatin of the X and Y chromosomes
(Mahadevaiah et al., 2001;
Turner et al., 2005)
(Fig. 2D). Because
post-translational histone modifications are known to control gene expression
by altering higher-order chromatin structure, Mahadevaiah et al.
(Mahadevaiah et al., 2001)
proposed a causative role for H2AX phosphorylation in MSCI. A subsequent study
revealed this to be the case - H2AX-null male mice display complete
meiotic arrest associated with MSCI failure
(Fernandez-Capetillo et al.,
2003). In order to demonstrate unequivocally that H2AX
phosphorylation is essential for the initiation of MSCI, a mouse with a point
mutation of serine-139 needs to be generated.
Two recent studies indicate that the DNA-repair protein ATR (ataxia
telangiectasia and Rad3 related) (see Table
1), a member of the PI3-like kinase family, phosphorylates H2AX
and is therefore required for MSCI (Turner
et al., 2004; Bellani et al.,
2005). In contrast to the other PI3-like kinases [ATM (ataxia
telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase, also known
as PRKDC - Mouse Genome Informatics)], ATR colocalises with H2AX on the
sex chromosomes once MSCI initiates until H2AX
dephosphorylation occurs at the diplotene-metaphase I transition. Mice with
an Atr mutation die early in embryogenesis and thus cannot be used to
directly address the role of ATR in MSCI
(Brown and Baltimore, 2000).
However, normal H2AX phosphorylation and MSCI occurs in Atm- or
DNA-PK-deficient mice, indicating that Atm and
DNA-PK do not normally contribute to MSCI
(Turner et al., 2004;
Bellani et al., 2005)
(Fig. 2). The correct targeting
of ATR to the X and Y chromosomes depends on the tumour suppressor protein
BRCA1 (breast cancer 1, early onset) (see
Table 1), which also localises
to the X and Y chromosomes during MSCI
(Turner et al., 2004)
(Fig. 3). Brca1 is
placed upstream of Atr, based on studies of mice with a deletion of
exon 11 of Brca1 (Xu et al.,
2003; Turner et al.,
2004). As in H2AX-null males, MSCI is defective in
Brca1 mutant mice, because H2AX phosphorylation does not take place
on the X and Y chromosomes. Instead, it occurs at ectopic sites throughout the
meiotic nucleus. This ectopic H2AX phosphorylation is the result of the
defective localization of ATR to the XY bivalent. Thus, recruitment of ATR to
the X and Y chromosomes depends on BRCA1, either directly or
indirectly.
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| Box 1. A simplified model of the formation and repair of DNA DSBs during
meiosis
DNA DSBs are the initiating events in meiotic recombination and are created
by the SPO11 enzyme. This results in phosphorylation of H2AX by ATM. The
resulting DNA DSB is 5'-to-3' resected by the MRE11-RAD50-NBS1
(MRN) complex (MRE11 is also known as MRE11A and NBS1 as NBN), and the
resulting tail is stabilised by RPA (also known as RPA1), before the loading
of RAD51, which enables strand invasion and drives synapsis. The resulting
intermediate can follow one of at least two fates: Holliday-junction
formation, which is in turn cleaved, giving rise to either crossovers or
non-crossovers; or strand displacement, giving rise to only non-crossovers.
For a detailed description of players in each stage of meiotic recombination,
see Krogh and Symington (Krogh and
Symington, 2004
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At or shortly after the initiation of MSCI, the X and Y chromosomes undergo
further post-translational modifications (reviewed in
Hoyer-Fender, 2003;
Handel, 2004)
(Fig. 2E and see below),
including H2A ubiquitylation (forming uH2A) (see
Table 1)
(Baarends et al., 1999),
deacetylation of histones H3 and H4
(Khalil et al., 2004),
dimethylation of H3 (forming H3K9me2)
(Khalil et al., 2004) and the
sumoylation of an, as yet, undefined target(s)
(Rogers et al., 2004;
Vigodner and Morris, 2005).
H2A ubiquitylation may depend on the E2-conjugating enzymes UBE2A and UBE2B
(see Table 1), which colocalise
with uH2A in meiotic cells (Baarends et
al., 2005). The methyltransferase that catalyses H3 lysine
dimethylation is unknown. One candidate, SUV39H2 (suppressor of variegation
3-9 homolog 2) (see Table 1),
localizes to the sex body, but the appearance of H3K9me2 is unperturbed in
SUV39H2-deficient mice (Peters et al.,
2001). Some of these post-translational modification products
(i.e. uH2A, deacetylated H3 and H4, and H3K9me2) may serve in the maintenance
of MSCI, because they remain associated with the X and Y throughout the
meiotic divisions and into spermiogenesis, long after H2AX dephosphorylation
has taken place.
MSCI may also depend on the incorporation of specific histone variants,
such as H2AFY (H2A histone family, member Y; previously MACROH2A1.2)
(Hoyer-Fender et al., 2000)
and H2AZ (H2A histone family, member Z; also known as H2AFZ - Mouse Genome
Informatics) (Table 1)
(Greaves et al., 2006).
MacroH2A1.2 is an unusually large histone variant comprised of a full-length
H2A domain together with a large non-histone domain, and has previously been
implicated in XCI (Costanzi and Pehrson,
1998). H2AZ plays an essential role in the maintenance of
heterochromatin and in chromosome segregation, and is unique in that it
associates with the X and Y chromosomes only after meiosis is complete
(Greaves et al., 2006). This
implies that H2AZ may function in the maintenance of MSCI (see below). The
chromodomain proteins CBX1 (chromobox homolog 1)
(Motzkus et al., 1999;
Metzler-Guillemain et al.,
2003) and CBX3 (chromobox homolog 3)
(Metzler-Guillemain et al.,
2003) have also been implicated in MSCI (see
Table 1).
MSCI: a consequence of synaptic failure
Over recent years it has become apparent that MSCI is in fact a
manifestation of MSUC, a more general meiotic-silencing mechanism
(Schimenti, 2005) (see
Table 1). In meiotic cells,
homologues synapse via a proteinaceous scaffold called the synaptonemal
complex (SC). The SC consists of two axial elements, which form during
leptotene between the sister chromatids, and of a central component, which
forms as synapsis takes place (de Boer and
Heyting, 2006). Meiotic DNA is arranged in loops that attach at
their base to these axial elements (see
Fig. 2C). As the X and Y
chromosomes only synapse via a homologous distal segment, such that much of
the X and Y axial elements are unsynapsed during pachytene, the proteins
involved in MSCI might be expected to localise to the chromatin of the arms of
the DNA loops, surrounding the axial element, where most genes reside. Indeed,
this is exactly where H2AX is found
(Turner et al., 2004).
However, prior to MSCI initiation, both BRCA1 and ATR associate exclusively
with the axial element of the X and Y chromosomes
(Turner et al., 2004)
(Fig. 2C). Shortly after, ATR
translocates from the axial element to the chromatin loops, concomitant with
the appearance of H2AX at those sites
(Fig. 2D). Based on the
association of BRCA1 and ATR with the unsynapsed X and Y chromosome axial
element, and on their absence from the distal regions of synapsed sex
chromosomes, it was proposed that MSCI and a lack of synapsis were intimately
linked (Turner et al., 2004).
It became apparent that BRCA1 and ATR were recognizing the axial elements of
the X and Y chromosomes simply because they were unsynapsed, rather than
because of some special feature of these chromosomes.
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) questioned whether unsynapsed autosomes would also attract
BRCA1, and whether this would ultimately lead to autosomal silencing. Using
T(X;16)16H male mice, in which an X-16 reciprocal translocation frequently
results in errors in chromosome 16 synapsis, it was demonstrated that regions
of unsynapsed chromosome 16 were indeed positive for BRCA1, ATR and
H2AX and were silenced (Turner et
al., 2005). Baarends et al.
(Baarends et al., 2005) also
reported similar findings in mice that contained a translocation between
chromosome 1 and 13, using uH2A as a marker of silencing. Both authors then
studied the localization of MSCI proteins in the XO female mouse
(Speed, 1986). These mice have
only one X chromosome instead of two, and the single X chromosome has no
homologous partner with which to synapse during meiosis. Both studies found
that the unsynapsed chromosomes were also silenced during female meiosis. In a
later study, Turner et al. (Turner et al.,
2006) tested whether MSCI could be prevented by providing the
normally unsynapsed X or Y chromosome with a synaptic partner. This proved to
be the case. For example, in XYY mice, in which the Y chromosome is provided
with an additional Y chromosome, fully synapsed YY bivalents were negative for
H2AX and evaded MSCI. These results are summarized in
Fig. 3B,C,E.
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) and
Neurospora crassa (Shiu et al.,
2001), in which they may function in genome defence
(Shiu et al., 2001). In C.
elegans, the single X chromosome of male meiotic cells is enriched in
H3K9me2, and, when autosomes fail to synapse, they acquire the same repressive
histone mark (Bean et al.,
2004). In Neurospora, DNA that is unsynapsed during
meiosis is silenced but, in contrast to the situation in mammals
(Okamoto et al., 2005), this
silencing affects all homologous DNA sequences, whether those sequences are
synapsed or not. For this reason, meiotic silencing in Neurospora has
been termed meiotic silencing by unpaired DNA (MSUD) (see
Table 1). MSUD is thought to
function post-transcriptionally, because it uses components of the RNAi
pathway, including the RNA-dependent RNA polymerase (RdRP) sad-1
(suppressor of ascus dominance 1)
(Shiu et al., 2001) and the
argonaute-like protein Sms-2 (suppressor of meiotic silencing 2)
(Lee et al., 2003). RdRPs
function in RNAi by converting single-stranded RNA precursors into
double-stranded RNA, which are then cleaved by Dicer to form short interfering
RNAs (siRNAs). These small RNA molecules induce destruction of homologous mRNA
via an argonaute-containing protein complex RISC (RNA-induced silencing
complex) (Dawe, 2004). The
data of Turner et al. (Turner et al.,
2005) and Baarends et al.
(Baarends et al., 2005)
indicate that MSUC operates at the transcriptional level, but this does not
preclude the possibility that RNAi is involved, because the core RNAi
machinery can silence genes at the transcriptional level
(Grewal and Jia, 2007).
Indeed, a recent study has found that maelstrom (MAEL), whose
Drosophila orthologue is implicated in RNAi
(Findley et al., 2003),
localises to the sex body (Costa et al.,
2006).
A curious unanswered question is why does MSUC/MSCI use proteins involved
in DSB repair? As already outlined, in mammals, meiotic DNA-DSB formation
precedes synapsis (see Box 1). When synapsis fails, the resulting unsynapsed
chromosome axes are replete with unrepaired DNA DSBs. Could it be that
unsynapsed axes are recognized as such through the presence of BRCA1-bound
DSBs, which act as nucleation centres for the later MSUC response? Two studies
have found that mice with a mutation in Spo11, which encodes an
enzyme responsible for meiotic DSB formation, have defective MSCI, indicating
a requirement for DSBs in meiotic silencing
(Bellani et al., 2005;
Barchi et al., 2005). However,
other data suggests that meiotic DSBs actually antagonize the MSCI response,
possibly by sequestering the MSCI machinery and thereby preventing its
relocation to the XY bivalent (Barchi et
al., 2005; de Vries et al.,
2005).
MSUC and failure of MSCI as causes of infertility
In mice and humans, errors in autosomal synapsis are usually associated
with meiotic arrest and impaired fertility, with the severity of the
impairment increasing in proportion to the degree of asynapsis
(Ashley, 2000). Because the
formation of DSBs required for recombination precedes synapsis in mammals,
autosomes that fail to synapse retain numerous unrepaired DNA DSBs. In yeast,
a recombination checkpoint halts meiosis in response to unrepaired meiotic
DSBs (Roeder and Bailis,
2000), and the existence of an equivalent checkpoint in female
mammals has recently been inferred (Di
Giacomo et al., 2005).
Theoretically, MSUC may also contribute to meiotic arrest when errors in
synapsis take place, by silencing `meiosis-critical' genes
(Shiu et al., 2001)
(Fig. 3B,E). This concept is
most easily understood in the context of the XO female mouse
(Fig. 3E). During normal XX
female meiosis, the two X chromosomes synapse completely; this permits gene
expression from both X chromosomes (Fig.
3D). However, in the XO mouse, the absence of a synaptic partner
should trigger an MSUC response (Baarends
et al., 2005; Turner et al.,
2005), which would ultimately result in the inactivation of the
whole X chromosome. Thus, all X-encoded genes essential to cell survival would
be silenced, with the obvious result being that the oocyte would perish. XO
mice are, nevertheless, fertile because a proportion of the oocytes
successfully complete meiosis; it has been hypothesized that these survivors
are those in which the single X chromosome forms a hairpin structure during
pachytene, effectively engaging in non-homologous self-synapsis
(Speed, 1986). Significantly,
this self-synapsis would allow the X chromosome to evade MSUC
(Baarends et al., 2005;
Turner et al., 2005)
(Fig. 3F), thereby allowing the
continued transcription of its genes.
As in XO females, the single X chromosome in normal (XY) males is silent
throughout meiosis, but, clearly, without ill-effect. Here, MSCI is tolerated
because male germ cells are equipped with an X-gene `back-up' system, in which
genes carrying out essential metabolic functions (e.g. Pgk1 and
Pdha1), integrate at autosomal sites by virtue of a
retroposon-mediated duplication event
(Wang, 2004). The expression
of these retroposed copies is male-specific and occurs at the initiation of
MSCI, thus compensating for the silencing of X-encoded products. The loss of
function of at least one of these retrogenes, Utp14b, has been shown
to cause spermatogenic arrest (Bradley et
al., 2004; Rohozinski and
Bishop, 2004), highlighting the importance of this back-up system
for male meiosis.
Somewhat counter-intuitively, it seems that the failure of MSCI, which
results in X and/or Y chromosome gene transcription, leads to spermatocyte
death during pachytene. Mutations in genes required for MSCI, including
H2ax (Fernandez-Capetillo et al.,
2003) and Brca1 (Xu
et al., 2003), cause meiotic arrest midway through pachytene;
however, in these instances it is difficult to determine whether the primary
cause of the meiotic arrest is due to MSCI failure, because these mutants also
have defective meiotic recombination and could therefore trigger a putative
recombination checkpoint. A better model for addressing the requirement for
MSCI in meiosis is the XYY mouse, because the Y chromosomes evade MSCI but
without defective meiotic recombination
(Fig. 3C). A quantitative
analysis of synaptic configurations has shown that, although cells with YY
bivalents are abundant during early pachytene, their numbers drop as pachytene
proceeds (Turner et al.,
2006). The only cells that reach late pachytene are those in which
all three sex chromosomes are unsynapsed at the zygotene-pachytene transition
and, thus, are completely silenced. These findings reveal how the escape of
the Y chromosome from MSCI is cell-lethal at some point between early and
mid-pachytene, presumably due to the toxic effects of the misexpression of one
or more Y chromosome genes during meiosis. Escape of the X chromosome from
MSCI, as occasionally seen in T(X;16)16H males, has an equally lethal effect
(Turner et al., 2006).
Post-meiotic transcriptional repression of the sex chromosomes
Recently, two studies have found that the paternally inherited X chromosome
of female-mouse pre-implantation embryos is silenced from a much earlier stage
than previously thought (Huynh and Lee,
2003; Okamoto et al.,
2004). One study (Huynh and
Lee, 2003) found evidence for paternal X chromosome inactivation
from the two-cell stage, whereas the other
(Okamoto et al., 2004) found
that inactivation begins at the four-to-eight-cell stage, when Xist
expression is initiated. In light of their findings, Huynh and Lee
(Huynh and Lee, 2003) have
suggested that the paternal X chromosome is already inactive at the point of
fertilization, and is therefore pre-inactivated during spermatogenesis, by
means of MSCI (Huynh and Lee,
2003). This model fitted well with data from C. elegans:
in XX offspring, the paternal X chromosome exhibits hallmarks of
transcriptional silencing that persist from fertilisation until the 10- to
15-cell stage, but this imprint is lost at an earlier embryonic stage in
hermaphrodites sired by males that lack MSCI
(Bean et al., 2004). In their
model, Huynh and Lee (Huynh and Lee,
2003) proposed that Xist functions to maintain this
pre-inactivated state. If MSCI were to underlie imprinted XCI, then it would
clearly have to be maintained beyond meiosis and throughout the rest of
spermatogenesis. The role of MSCI in imprinted XCI in mammals has been the
subject of controversy (Reik and
Ferguson-Smith, 2005). A recent study
(Okamoto et al., 2005) has
found that transgenes that contain Xist and its surrounding genes are
not subject to MSCI and can subsequently undergo imprinted XCI in female
embryos, demonstrating that Xist expression alone is sufficient for
imprinted XCI. The debate over the role of MSCI in imprinted XCI has
encouraged a re-examination of X and Y chromosome activity in the post-meiotic
period. Previous observations had suggested that transcriptional repression of
the sex chromosomes was restricted to the period of meiosis (hence MSCI) (see
Table 1). For example, some
sex-linked genes were found to be reactivated in round spermatids
(Hendriksen et al., 1995). In
addition, proteins functioning in the initiation phase of MSCI (BRCA1, ATR and
H2AX) were seen to disassociate from the XY bivalent prior to the first
meiotic division (Mahadevaiah et al.,
2001; Turner et al.,
2004), suggesting that MSCI was reversed at this stage.
The view that MSCI is restricted to meiosis was first challenged by Khalil
et al. (Khalil et al., 2004),
who carried out a comprehensive immunoassay of histone modifications
associated with the active and inactive chromatin state on post-pachytene
spermatogenic cells. They revealed that some modification products associated
with heterochromatin, most notably H3K9me2, remained on the X and Y
chromosomes throughout the first and second meiotic divisions and in round
spermatids (Fig. 2E-H),
indicating that repressive chromosome marks persist on the X and Y chromosomes
post-meiotically. However, based on RNA polymerase II antibody staining, they
concluded that the X and Y chromosomes were, nevertheless, transcriptionally
reactivated. Shortly after this study, three other groups reported that the X
and Y chromosomes were heterochromatic, and therefore under-transcribed, in
round spermatids (Namekawa et al.,
2006; Turner et al.,
2006; Greaves et al.,
2006) (see Fig. 1).
In one of these studies (Namekawa et al.,
2006), this `post-meiotic sex chromatin' (PMSC;
Table 1), so-called to reflect
its inactive state, was detected at even later stages, during the period of
spermatid elongation. An examination of X chromosome transcriptional activity
on a gene-by-gene basis using microarray and quantitative reverse
transcriptase-PCR analysis revealed that approximately 87% of X chromosome
genes are relatively repressed in the post-meiotic period
(Namekawa et al., 2006).
The continued presence on the X and Y chromosomes from meiosis into
spermiogenesis of histone marks that are associated with a transcriptionally
repressed state suggests that post-meiotic sex chromosome repression (PSCR)
(see Table 1) is a direct
consequence of MSCI. Because MSCI is essentially X and Y chromosome-specific
MSUC, Turner et al. (Turner et al.,
2006) next investigated whether, in general, meiotic silencing
persists into spermiogenesis by studying the transcriptional fate of an
unsynapsed autosomal segment in spermatids using RNA FISH (fluorescence in
situ hybridization). This unsynapsed autosomal segment did indeed retain a
repressed state, brought about by a condensed chromatin structure. PSCR is
therefore dependent on MSUC, but it is less penetrant, because the
reactivation of genes that reside within the unsynapsed autosomal segment do
occur in a small population of spermatids. Thus, the emerging picture is that
the X and Y chromosomes are silenced during the pachytene stage of meiosis,
and that most sex-linked genes remain repressed post-meiosis. The fact that
the X chromosome is transcriptionally repressed during most of spermatogenesis
may, at face value, seem to contradict the prediction that spermatogenesis
genes accumulate on the X chromosome
(Fisher, 1931;
Rice, 1984). However, it is
clear that these genes are expressed prior to MSCI taking place, during the
spermatogonial divisions (Khil et al.,
2004). Nevertheless, a significant minority of X- and Y-linked
genes are transcribed during spermiogenesis, and some of these serve an
indispensable function in sperm differentiation. For instance, the loss of the
long arm of the mouse Y chromosome, which contains several spermatid-expressed
genes, including Ssty1 and Ssty2 (spermiogenesis specific
transcript on the Y 1 and 2, respectively) and Sly (Sycp3 like
Y-linked), is associated with sperm head abnormalities and male infertility
(Toure et al., 2004;
Toure et al., 2005). How these
genes evade the repressive effects of PSCR remains an intriguing question.
Conclusion
In summary, research into MSCI has proved insightful on many levels. It has
revealed unexpected links between the DNA DSB-repair proteins and chromatin
silencing, provided functional insight into the link between
chromosome-pairing failure and infertility, and may eventually have
far-reaching implications in our understanding of imprinting mechanisms. An
important avenue for future study will be how, if at all, DNA DSBs are
required for the initiation of MSCI and MSUC. If MSUC really contributes to
meiotic arrest in the face of chromosome asynapsis, it might also be possible
to genetically manipulate MSUC in order to rescue asynapsis-associated germ
cell loss, as seen in the XO female mouse. Although the role of MSCI in
imprinted XCI in eutherians remains controversial, the possibility that it
underlies imprinted XCI in marsupials, which do not have a Xist gene
(Duret et al., 2006), is an
attractive possibility, and could be addressed by examining whether MSCI and
PSCR operate in these organisms. Finally, an intriguing question is how are
some X and Y chromosome genes expressed during spermatid differentiation,
despite the repressive effects imposed by PSCR? These and other questions are
likely to keep those of us interested in the fascinating field of MSCI busy
for a long time to come.
ACKNOWLEDGMENTS
I thank Paul Burgoyne for critical reading of the manuscript and apologise to those authors whose work was not discussed due to word limitations.
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