Download Increased sex chromosome expression and epigenetic

JCS ePress online publication date 27 October 2009
Research Article
4239
Increased sex chromosome expression and
epigenetic abnormalities in spermatids from male
mice with Y chromosome deletions
Louise N. Reynard and James M. A. Turner*
Division of Developmental Genetics and Stem Cell Biology, MRC National Institute for Medical Research, London NW7 1AA, UK
*Author for correspondence ([email protected]).
Journal of Cell Science
Accepted 22 June 2009
Journal of Cell Science 122, 4239-4248 Published by The Company of Biologists 2009
doi:10.1242/jcs.049916
Summary
During male meiosis, the X and Y chromosomes are
transcriptionally silenced, a process termed meiotic sex
chromosome inactivation (MSCI). Recent studies have shown
that the sex chromosomes remain substantially transcriptionally
repressed after meiosis in round spermatids, but the
mechanisms involved in this later repression are poorly
understood. Mice with deletions of the Y chromosome long arm
(MSYq–) have increased spermatid expression of multicopy X
and Y genes, and so represent a model for studying post-meiotic
sex chromosome repression. Here, we show that the increase
in sex chromosome transcription in spermatids from MSYq–
mice affects not only multicopy but also single-copy XY genes,
Introduction
Spermatogenesis describes a series of complex, developmental
processes that are involved in the formation of mature haploid sperm
from diploid spermatogonial stem cells. In mice, spermatogenesis
occurs in the seminiferous epithelium of the testis and can be divided
into three phases: mitotic division of spermatogonia, meiosis, and
differentiation of round spermatids into sperm (spermiogenesis).
Genes on the sex chromosomes play an essential role in
spermatogenesis and, in mice, the Y chromosome encodes a limited
number of functions that appear to be restricted to spermatogenesis
and testis determination. Spermatogenesis-expressed genes are also
over-represented on the X chromosome, with a 15-fold enrichment
of spermatogonially expressed genes on the mouse X and a
threefold increase in sex- or reproduction-related genes on the
human X chromosome compared to the autosomes (Khil et al., 2004;
Lercher et al., 2003; Saifi and Chandra, 1999; Wang et al., 2001).
Furthermore, the mouse X chromosome is enriched in genes
expressed after meiosis, with at least 273 genes (18% of 1555
protein-coding genes) being expressed predominantly or exclusively
in spermatids (Mueller et al., 2008).
The transition from spermatogonium to mature sperm is
accompanied by dramatic changes in X and Y gene transcription.
During the spermatogonial divisions, the sex chromosomes are
transcriptionally active, with almost all of the genes on the Y short
arm (Yp) being transcribed. Although still transcriptionally active
during the leptotene and zygotene stages of meiosis, the sex
chromosomes are rapidly silenced at the zygotene-pachytene
transition; a phenomenon termed meiotic sex chromosome
inactivation (MSCI) (McKee and Handel, 1993). MSCI is a response
to the largely unsynapsed state of the XY pair (Baarends
et al., 2005; Turner et al., 2004; Turner et al., 2006; Turner et al.,
as well as an X-linked reporter gene. This increase in
transcription is accompanied by specific changes in the sex
chromosome histone code, including almost complete loss of
H4K8Ac and reduction of H3K9me3 and CBX1. Together, these
data show that an MSYq gene regulates sex chromosome gene
expression as well as chromatin remodelling in spermatids.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/22/4239/DC1
Key words: Spermatid, Sex chromosome, Post-meiotic sex chromatin,
Chromatin marks
2005) and is initiated when the tumour suppressor protein BRCA1
recruits the kinase ATR to the asynapsed axes of the X and Y
chromosomes (Turner et al., 2004). ATR translocates from the
axial elements to the chromatin loops, where it is hypothesised to
phosphorylate the histone H2A variant, H2AX, at serine 139
to form H2AX (Fernandez-Capetillo et al., 2003), instigating
heterochromatinisation of the sex chromosomes. The XY bivalent
is compartmentalised into a specialised chromatin domain known
as the sex body (Solari, 1974; Turner, 2007) and undergoes further
chromatin modifications, including histone H3 dimethylation,
histone H3 and H4 deacetylation (Khalil et al., 2004), H2A
ubiquitylation (Baarends et al., 1999), widespread replacement of
the histones H3.1 and H3.2 with the histone variant H3.3 (van der
Heijden et al., 2007) and incorporation of specific histone variants
such as macroH2A.1 (Hoyer-Fender et al., 2000).
Although BRCA1, ATR and H2AX dissociate from sex
chromosomes before the first meiotic division (Mahadevaiah et al.,
2001; Turner et al., 2004), the X and Y chromosomes remain
substantially transcriptionally repressed after meiosis (Namekawa
et al., 2006; Turner et al., 2006). Unlike MSCI, the maintenance of
XY silencing in spermatids is incomplete, with genes showing lowlevel reactivation (Mueller et al., 2008). There is a correlation
between X-linked gene copy number and escape from this postmeiotic repression, with the percentage of round spermatids
expressing the gene increasing with increasing copy number,
suggesting that gene amplification might compensate for the
repressive effects initiated by MSCI. Cytologically, the spermatid
X and Y form DAPI-dense heterochromatic domains known as postmeiotic sex chromatin (PMSC) (Namekawa et al., 2006), which
are located next to the centromeric heterochromatin. PMSC are
depleted in markers of transcription, including Cot1 and RNA
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polymerase II, and are enriched for several repressive chromatin
marks present on the sex body, including histone H3 dimethylated
at lysine 9 (H3K9me2) and CBX1 (chromobox protein homolog 1,
previously known as HP1) (Baarends et al., 2007; Greaves et al.,
2006; Khalil et al., 2004; Namekawa et al., 2006; Turner et al.,
2006). However, the X and Y chromosomes are continually
remodelled during the transition between meiosis and
spermiogenesis, and histone modifications associated with
transcriptionally active chromatin [e.g. histone acetylation and
histone H3 dimethylated on lysine 4 (H3K4me2)] are also enriched
on PMSC in round spermatids (Baarends et al., 2007; Greaves et
al., 2006; Khalil and Driscoll, 2006). These epigenetic changes could
explain why XY spermatid silencing is less complete than MSCI,
and might reflect a need to retain expression of some X- and Yencoded genes that are essential for spermiogenesis.
Whereas the molecular events underlying MSCI are well
characterised, those involved in controlling sex-linked gene
expression in round spermatids are less clear. Mice lacking the
ubiquitin-conjugating enzyme HR6B have changes to the epigenetic
profile of the sex chromosomes in late meiotic and post-meiotic
cells, including increased enrichment of the active chromatin mark
H3K4me2 in diplotene spermatocytes and round spermatids
(Baarends et al., 2007). This is accompanied by increased expression
of several X-linked genes, suggesting that HR6B could be involved
in spermatid XY repression, possibly by controlling the histone
modifications associated with the sex chromosomes in late
spermatocytes and round spermatids.
In mice, deletions of the Y chromosome long arm (MSYq) are
associated with problems in sperm development and function.
Whole testis microarray analysis has shown that mice with MSYq
deletions (MSYq–) exhibit de-repression of several sex-linked
genes in spermatids, including Ube1x, which is also de-repressed
in Hr6b–/– spermatids (Baarends et al., 2007; Ellis et al., 2005).
This suggests that genes encoded by the Y long arm are involved
in spermatid sex chromosome repression. One MSYq candidate gene
is Sly, a multicopy gene that has been shown to interact with histonemodifying enzymes, including the histone acetyltransferase KAT5
(Reynard et al., 2009). However, only a small percentage of
spermatid-expressed sex-linked genes were reported to be derepressed in the MSYq– models, and it is unclear whether this derepression occurs at the transcriptional or post-transcriptional level.
Ellis and colleagues noted that X-linked gene de-repression
preferentially affected multiple-copy over single-copy genes, and
suggested that there might be something particular about the
chromosomal organisation of multicopy genes that is affected in
the MSYq deletion mice (Ellis et al., 2005). However, a recent study
has shown that in wild-type males, multicopy X genes exhibit
spermatid-specific expression, whereas single-copy X genes are
expressed abundantly before meiosis and at low levels in spermatids
(Mueller et al., 2008). This would make potential increases in singlecopy gene expression in spermatids from MSYq– mice more
difficult to detect by microarray analysis because these increases
would be masked by the abundant expression originating from
earlier cell types.
Here, we show that MSYq deletions cause increased expression
of sex-linked genes at the level of transcription. In addition, this
affects not only multicopy but also single-copy genes and an Xlinked transgene, demonstrating a global rather than a local defect
in the maintenance of XY silencing. In addition, mice with MSYq
deletions have specific abnormalities in the histone code of the round
spermatid sex chromosome and centromeric heterochromatin.
Results
Upregulation of X- and Yp-linked genes in the MSYq deletion
models is associated with increased nascent transcription
To determine whether the increased mRNA levels in MSYq deletion
models is the result of increased transcription of the affected genes,
we used RNA fluorescence in situ hybridisation (FISH) to monitor
nascent transcription of genes in round spermatids, the cell type of
interest. We analysed the expression of nine X, two Y and eight
randomly selected autosomally located genes in XY males compared
with two MSYq mutants, the first with a deletion of two thirds of
the Y chromosome (2/3MSYq–) and a second with complete
absence of MSYq (MSYq–). Round spermatid development occurs
normally in the two deletion models, with the relative proportion
of spermatids at each of the eight steps of round spermatid
development unchanged relative to the XY control (Burgoyne et
al., 1992; Conway et al., 1994) (L.N.R., unpublished observations).
For all eleven sex-linked genes, RNA FISH signals were observed
in a significantly (P<0.05) higher proportion of round spermatids
from MSYq– mice than from XY control mice, with the expression
increasing in proportion to the size of the deletion (Fig. 1A,B and
supplementary material Table S1). This effect was independent of
the copy-number, or location of the gene on the chromosome
(supplementary material Fig. S1), and included three genes (Slx,
4930527E24RIK and Vsig1) previously reported to have increased
testis mRNA levels in the MSYq– mice (Ellis et al., 2005), thus
substantiating the link between the percentage of expressing
spermatids and total mRNA levels. The most dramatic difference
was seen for Vsig1, with the number of RNA FISH positive
spermatids increasing from an average of 16.5% in XY mice to
38.2% in MSYq– mice. As well as an increase in the proportion of
expressing round spermatids, there was also an increase in the
number of RNA signals per spermatid for several of the sex-linked
genes, as shown by using a probe that detects both Ube1y1 and
Zfy1 transcripts (Fig. 1B,C and supplementary material Fig. S2AC). The mean number of Ube1y1/Zfy1 signals per spermatid more
than doubled, from 3.76 signals in the XY control (n50) to 8.3
signals in MSYq– spermatids (n50), with the maximum number
of RNA signals observed in a single nucleus increasing from 10 to
18.
For the eight autosomal control genes examined, there was no
significant difference between the three genotypes with respect to
the proportion of spermatids with RNA FISH signals or number of
RNA signals per cell (Fig. 1A), implying that autosomal
transcription remains unaffected in the MSYq deletion models and
that the relative proportion of round spermatids at each step of
development is unaffected. To further support this finding, dual RNA
FISH was performed on round spermatids from the XY and MSYq–
testes for an autosomal gene (Brca1 or Prdkc) together with either
an X-linked (Vsig1) or Y-linked gene (Zfy1). The percentage of
spermatids positive for a Brca1 or Prdkc RNA FISH signal did not
differ between the two genotypes, indicating that the same
populations of spermatids were analysed in the two models
(supplementary material Fig. S3). Within the Brca1-positive
population, the percentage of spermatids positive for Vsig1 (i.e.
Brca1 Vsig1 double-positive spermatids) was more than doubled
in the MSYq– testis (44.3%) compared with the XY control
(21.5%). Additionally, the percentage of Prdkc-positive spermatids
also positive for a Zfy1 RNA FISH signal was increased in the
MSYq– testis relative to the XY control.
We also studied the expression of a tenth X-linked gene, Xiap,
which unlike the other sex-linked genes studied shows no
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Spermatid defects in MSY deletion mice
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Fig. 1. Increased transcription of sex-linked genes in round spermatids from mice with MSYq deletions. Gene-specific RNA FISH was performed on
spermatogenic cells from mice with two-thirds (2/3MSYq–) or complete (MSYq–) deletion of the Y long arm, with wild-type XY mice as a control. (A)Table
showing the average percentage of round spermatids expressing a given gene, on the basis of the presence of an RNA FISH signal. After angular transformation of
the individual percentages, an ANOVA test was used to determine whether the difference between the three genotypes was significant at the P0.05 level. sc, single
copy gene; mc, multicopy gene. More details on gene copy number are given in supplementary material Fig. S1. (B)RNA FISH on XY (left panel), 2/3MSYq–
(middle panel) and MSYq– (right panel) round spermatids using probes for Fmr1, Ddx3x, Slx and Ube1y1/Zfy1. DNA is stained with DAPI (blue) and the RNA
FISH signal is in red. Scale bar: 5m. For Slx and Ube1y1/Zfy1, there is an increase in the number of RNA FISH signals per spermatid in the 2/3MSYq– and
MSYq– deletion models compared with the XY control. (C)Quantification of the number of Ube1y1/Zfy1 RNA FISH signals per expressing round spermatid from
XY and MSYq– mice.
reactivation in XY spermatids (Mueller et al., 2008; Namekawa et
al., 2006). Xiap was silent in spermatids from the 2/3MSYq– and
MSYq– mice, suggesting that only genes normally transcribed in
round spermatids are affected by deletions of MSYq.
To investigate whether the increase in transcription of sex-linked
genes in MSYq mutants was restricted to spermiogenesis, we studied
expression of the X-linked Fmr1, Nxf2 and Scml2 genes, and the
Y-linked Uty gene during meiosis, when MSCI occurs. No
expression of these genes was detected by RNA FISH in late
pachytene spermatocytes from either XY or MSYq– testis
(supplementary material Fig. S2D). In addition, Cot1 RNA FISH
was performed on pachytene and diplotene cells from XY,
2/3MSYq– and MSYq– mice (n55 spermatocytes per genotype).
Cot1 DNA is enriched for repetitive sequences such as those found
in introns and 3⬘ untranslated regions, and thus nascent transcripts
hybridise to Cot1 probes. In XY spermatocytes, the H2AXpositive sex-body chromatin domain is negative for Cot1 RNA
(Namekawa et al., 2006; Turner et al., 2006), and Cot1 signals are
also absent from the sex-body chromatin domain in 2/3MSYq– and
MSYq– spermatocytes (supplementary material Fig. S2E),
indicating that MSCI occurs normally in Yq deletion mice.
Collectively, these findings indicate that the increased transcription
of X- and Y-encoded genes in mice with MSYq deletions is restricted
to the post-meiotic stages of germ cell development.
Taken together, these data show that the increased mRNA levels
of X- and Y-linked genes in spermatids from mice with Y long arm
deletions is associated with increased gene transcription.
Furthermore, this transcriptional upregulation affects single as well
as multicopy sex-linked genes that are distributed along the X and
Yp chromosomes. We do not rule out the possibility that autosomal
gene transcription is affected to some degree, but loss of MSYq
clearly has a more prominent effect on sex chromosome gene
expression.
Testis protein levels of upregulated sex-linked genes are
increased in the MSYq deletion models
The RNA FISH data presented above show that there is an increase
in transcription of genes located across the X chromosome,
specifically in round spermatids from mice with large deletions of
MSYq. To determine whether this effect was due to an intrinsic
property of sex-linked testis-expressed genes, we examined the
expression of an EGFP reporter transgene, which is inserted on the
distal part of the mouse X chromosome (Hadjantonakis et al., 2001;
Hadjantonakis et al., 1998). This transgene is transcribed in
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Fig. 2. Increased testicular levels of GFP and SLX in mice with partial deletions of the Yq. (A)Western blot analysis of testicular GFP levels from XY and
2/3MSYq– mice carrying an X-linked reporter GFP transgene. Membranes were hybridised with an anti-GFP antibody (top panel), then washed and reprobed for
actin (bottom panel) as a loading control. (B)Quantification of whole testis GFP levels from the western blot in A using ImageJ software. The level of GFP is
represented as the ratio of GFP to actin and is given an arbitrary value of one in the XY testis. Error bars represent the s.e.m. The difference in testis GFP levels
between the two genotypes was statistically significant (P<0.001, Student’s t-test). (C)GFP immunostaining of testis sections from XY (top panel) and 2/3MSYq–
(bottom panel) mice carrying an X-linked GFP transgene. The GFP protein is present in the cytoplasm of spermatogonia (asterisks), early meiotic cells, and
elongating spermatids (arrows). There is an increase in GFP levels in the spermatid cytoplasm in the 2/3MSYq– testis compared to the XY control, but the
spermatogonial levels of GFP are unchanged. (D)Whole testis western blot analysis of SLX levels in XY and 2/3MSYq– mice. Actin was used as a whole testis
loading control. (E)Quantification of SLX levels in the testis of XY and 2/3MSYq– males relative to actin from the western blot in D using ImageJ software. Error
bars represent the s.e.m. The difference in SLX levels between the two genotypes was found to be statistically significant (P<0.005, Student’s t-test). (F)SLX
immunostaining of spermatogenesis stage VIII seminiferous tubules from XY (top panel), 2/3MSYq– (middle panel) and MSYq– (bottom panel) mice. SLX is
present in the cytoplasm of round and elongating spermatids from all three genotypes, although there is an increase in SLX levels in the two Yq deletion models
compared with the XY control.
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Spermatid defects in MSY deletion mice
spermatogonia, inactivated in meiotic cells during MSCI and
reactivated during spermiogenesis. As the increased transcription
of genes in the MSYq deletion mice is thought to lead to a
corresponding increase in their encoded proteins, we decided to
examine the expression of the EGFP transgene at the protein level.
Western blot analysis demonstrated that GFP levels are increased
by more than twofold in the 2/3MSYq– testes compared to XY
controls using both a whole testis (actin) or spermatid-specific
(DPP9) (Dubois et al., 2009) protein as a loading control (Fig. 2A,B
and supplementary material Fig. S4A,C). This indicates that the Xlinked EGFP transgene is also affected by X de-repression in the
MSYq deletion models. Immunofluorescence staining of testis
sections from GFP-transgenic XY and 2/3MSYq– males showed
that the pattern of GFP localisation is unaffected in the 2/3MSYq–
testis; however, the level of GFP is increased in 2/3MSYq– mice,
specifically in elongating spermatids (Fig. 2C, arrows), with
spermatogonial GFP levels remaining unchanged (Fig. 2C,
asterisks).
To further explore the effect of Yq deletions on sexchromosome-encoded protein levels, the expression of spermatidspecific X-encoded SLX protein (Reynard et al., 2007) was
examined in the 2/3MSYq– testis. Testis SLX levels are increased
by over 50% in the 2/3MSYq– testis relative to the XY control
(Fig. 2D,E and supplementary material Fig. S4B,D)
Immunostaining of XY, 2/3MSYq–, and MSYq– testis sections
revealed no change in the distribution and subcellular localisation
of SLX, with staining restricted to the cytoplasm of round and
early elongating spermatids (Fig. 2F and supplementary material
Fig. S4E). However, staining of spermatids was much stronger in
stage-matched seminiferous tubules from 2/3MSYq–, and MSYq–
mice compared with the XY control. Thus, the increased amount
4243
of SLX in the testis of the MSYq deletion models does not lead
to ectopic expression of SLX in other spermatogenic cell types,
but results in elevated protein levels in the cells that normally
express it.
Together, these RNA FISH and protein data show that many sexlinked genes expressed in round spermatids are upregulated in MSYq
deletion models, resulting in a corresponding increase in their
proteins, which potentially contribute to the phenotypes exhibited
by these mice.
The epigenetic profile of the spermatid sex chromosomes is
altered in mice with Yq deletions
The increase in sex-linked gene expression observed in the MSYq
deletion models might be accompanied by changes in the epigenetic
profile of the sex chromosomes. To explore this possibility, we
examined the sex chromosome structure and epigenetic marks in
round spermatids from XY, 2/3MSYq– and MSYq– mice.
Heterochromatic PMSC
To determine whether the sex chromosomes in spermatids from
MSYq deletion mice still form PMSC, spermatids from XY,
2/3MSYq– and MSYq– mice were stained with DAPI. PMSC was
cytologically visible as a DAPI-dense structure located next to the
chromocentre in 71.6% (514 out of 718) of XY round spermatids
and 65.4% of 2/3MSYq– round spermatids (Fig. 3A, arrows). PMSC
was discernable in 37.3% of round spermatids from MSYq– mice.
Chromosome painting revealed that PMSC is only present in Xbearing round spermatids from MSYq– mice, and that the Y*x
chromosome is not detected using DAPI (data not shown); this is
not surprising in light of the small size of this chromosome. These
results imply that the increased transcription of X-linked genes in
Fig. 3. The epigenetic marks associated
with PMSC are altered in MSYq–
spermatids. (A)The DAPI-dense
heterochromatic PMSC (arrows) still
forms in round spermatids from the
2/3MSYq– and MSYq– mice. (B)The
histone modification H48Ac is present
on PMSC in round spermatids from
XY (top row, arrows) and 2/3MSYq–
(middle row, arrows) mice, but is lost
from PMSC in the majority of round
spermatids from MSYq– mice (bottom
row, arrows). Far left: a stage I
seminiferous tubule stained for
H4K8Ac (red), DAPI (blue) and
H2AX (green). Left and right panel:
higher magnification of round
spermatids from a stage I tubule.
H4K8Ac is present throughout the
nucleus and is enriched on PMSC
(arrows) but excluded from the
chromocentre in spermatids from XY
and 2/3MSYq– mice. H4K8Ac is still
present in the nucleus of MSYq–
spermatids but is not enriched on
PMSC. Far right panel: H4K8Ac
immunostaining of surface-spread
round spermatids. H4K8Ac was
enriched on PMSC in 36.6% of
spermatids from XY mice but only
6.7% of MSYq– spermatids, where it is
reduced compared to the XY control
(see Table 1).
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Fig. 4. Decreased H3K9me3 and CBX1 enrichment on PMSC in spermatids from
MSYq– mice. (A)H3K9me3 is reduced on PMSC but not the chromocentre of
MSYq– spermatids. Far left panel: a stage VIII seminiferous tubule stained for
H3K9me3 (red), DAPI (blue) and the acrosomal protein DKKL1 (green). Left and
right panel: higher magnification of round spermatids from a stage VIII tubule.
H3K9me3 enrichment on the PMSC (arrows) is reduced in the MSYq– spermatids
(bottom row) compared to XY (top row) and 2/3MSYq– (middle row) spermatids.
Far right panel: H3K9me3 immunostaining of surface-spread round spermatids
reveals that H3K9me3 enrichment on PMSC (arrows) is reduced in MSYq–
spermatids relative to the XY control. (B)Antibody staining for the
heterochromatin protein CBX1 (green) reveals that this protein is reduced on
PMSC of MSYq– spermatids (bottom row, arrows) compared to XY (top row,
arrows) and 2/3MSYq– spermatids (middle row, arrows).
the MSYq– spermatids is not associated with obvious X deheterochromatinisation.
Histone H4 acetylation
In light of the interaction between the Yq-encoded SLY1 protein
and the histone acetyltransferase KAT5 (Reynard et al., 2009), the
most obvious candidate histone modification to be affected in
2/3MSYq– and MSYq– mice was H4 acetylation. Acetylation of
H4K8 is associated with transcriptionally active regions of the
genome in somatic tissue, and is enriched on the sex chromosomes
in round spermatids (Greaves et al., 2006). Immunostaining of testis
sections from XY and 2/3MSYq– mice revealed that H4K8Ac is
present at low levels throughout the nuclei of round spermatids (Fig.
3B and supplementary material Fig. S5A), where it is excluded from
the chromocentre, but enriched on PMSC in a stage-specific
manner. By contrast, there was little or no enrichment of H4K8Ac
on the PMSC in round spermatids from MSYq– mice (Fig. 3B and
supplementary material Fig. S5A), although this modification was
still present throughout the nucleus and was excluded from the
chromocentre. Examination of surface-spread spermatids
demonstrated that the percentage of round spermatids with H4K8Ac
enrichment on the PMSC is significantly decreased (P<0.001, Chisquared test) in the two MSYq deletion models in proportion to
deletion size; from 36.6% in the XY control to 6.7% of round
spermatids from MSYq– males (Table 1). Furthermore, the level of
H4K8Ac is reduced on PMSC in those MSYq– spermatids that retain
it, compared to the XY control (Fig. 3B; compare top and bottom
rows). Analysis of testis sections indicated that there was no change
in the levels or distribution of H4K8Ac in germ cells prior to the
round spermatid stage (data not shown).
Next, we examined the localisation of H4K12 acetylation on
surface-spread round spermatids. In XY cells, three patterns of
H4K12 localisation were observed: enrichment of H4K12Ac on the
chromocentre (clustered centromeric heterochromatin), uniform
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Spermatid defects in MSY deletion mice
Table 1. Summary of the epigenetic marks enriched on the PMSC and chromocentre in round spermatids from XY, 2/3MSYq–
and MSYq– males
XY
2/3MSYq–
MSYq–
Chromatin mark
Spermatid staining pattern
%
n/total
%
n/total
%
n/total
DAPI
H4K8Ac
H4K12Ac
PMSC
PMSC
Chromocentre enrichment
Uniform nuclear staining
Chromocentre exclusion
PMSC
Chromocentre
Diffuse staining
PMSC and chromocentre
Chromocentre only
PMSC and chromocentre
Chromocentre only
71.6
36.6
16.6
50.6
32.8
66.6
57.1
23.0
76.6
23.4
82.1
17.9
514/718
71/194
51/308
156/308
101/308
211/317
181/317
73/317
232/303
71/303
307/374
67/374
65.4
11.4
7.9
32.8
59.3
40.0
43.3
47.9
67.3
32.7
78.0
22.0
399/610
21/185
24/305
100/305
181/305
122/305
132/305
146/305
212/315
103/315
295/378
83/378
37.3
6.7
0
32.7
67.3
27.9
39.6
51.3
41.4
58.6
49.1
50.9
265/710
13/193
0/303
99/303
204/303
88/316
125/316
162/316
125/302
177/202
185/377
192/377
H3K9me2
H3K9me3
CBX1
Journal of Cell Science
For each genotype, testis material from three individuals was used to make a single cell suspension and analysed. The number (n) of round spermatids with
enrichment of epigenetic marks on the PMSC is shown as a percentage of the total number of round spermatids counted (total). H4K8Ac is reduced on PMSC
from mice with MSYq deletions. H4K12Ac is reduced from the chromocentre in MSYq mutants. Fewer spermatids have H3K9me2 on both the chromocentre and
PMSC in MSYq mutants. The levels of H3K9me3 on PMSC is reduced in the MSYq mutants. The levels of CBX1 are reduced on PMSC in the MSYq mutants.
nuclear staining and exclusion of H4K12Ac from the chromocentre
(supplementary material Fig. S6A). These three patterns were also
observed in 2/3MSYq– spermatids, although there was no
enrichment of H412Ac on the chromocentre in MSYq– spermatids.
Furthermore, the percentage of round spermatids with H4K12Ac
excluded from the chromocentre was significantly increased in the
two Yq deletion models (P<0.001, Chi-squared test), and was
more than doubled in MSYq– mice compared with the XY control
(Table 1).
Histone H3 methylation
The transcriptional upregulation and loss of H4K8Ac might reflect
a global change in the conformation and activity of the PMSC in
round spermatids. To investigate this further, we examined
additional chromatin modifications associated with the PMSC.
Methylation of H3K9 is a marker of transcriptionally silent
chromatin.
Trimethylation of H3K9 is enriched on PMSC and chromocentre
or on the chromocentre only in round spermatids. On surface-spread
testis cells, localisation of H3K9me3 to PMSC was seen in 76.6%
of round spermatids from XY mice (Fig. 4A and Table 1), and 41.4%
of round spermatids from MSYq– mice. In MSYq– mice, H3K9me3
was only observed on PMSC in X-bearing round spermatids
(supplementary material Fig. S5C). Although there was no
difference in the intensity of H3K9me3 chromocentre staining
between the three genotypes, the extent of H3K9me3 enrichment
on PMSC was reduced in MSYq– spermatids relative to that in
normal males (Fig. 4A and supplementary material Fig. S5B).
On surface-spread testicular cells, three patterns of H3K9
dimethylation were identified in round spermatids from normal
males: chromocentre and PMSC staining, PMSC staining only, or
diffuse nuclear staining (Fig. S6B). These three staining patterns
were also observed in round spermatids from 2/3MSYq– and
MSYq– mice. There was a significant reduction in the number of
round spermatids with H3K9me2 chromocentre staining, from
57.1% in XY spermatids to 43.3% and 39.6% in 2/3MSYq– and
MSYq– spermatids, respectively (P<0.001, Chi-squared test). The
level of H3K9me2 enrichment on PMSC in XY spermatids was
comparable to that on the PMSC of 2/3MSYq– and MSYq–
spermatids (supplementary material Fig. S6B), but the percentage
of round spermatids with enrichment of H3K9me2 on the sex
chromosome was decreased in the two Yq deletion models, in
proportion to the extent of the deletion. No difference in the
localisation or relative enrichment of H3K9 di- and trimethylation
was observed in meiotic cells between the MSYq– testis and XY
control (not shown).
Finally, we looked at the active histone modifications H3K36me3
and H3K4me3 in round spermatids from XY, 2/3MSYq– and
MSYq– mice. There was no difference in the localisation of either
histone mark between the three genotypes, with these modifications
being excluded from both PMSC and chromocentre in round
spermatids (supplementary material Fig. S7).
CBX1
The heterochromatin-associated protein CBX1 (also called M31 and
HP1) is recruited by H3K9 methylation (Lachner et al., 2001) and
is considered a marker of inactive chromatin. In light of the changes
in H3K9 tri- and dimethylation on the sex chromosome and
chromocentre in MSYq– spermatids, respectively, we next analysed
CBX1 in round spermatids. On surface-spread spermatogenic cells,
CBX1 was present on the chromocentre of all round spermatids
analysed from XY, 2/3MSYq– and MSYq– mice. In XY mice, CBX1
was enriched on the DAPI-dense X or Y PMSC in 82.1% of round
spermatids (Fig. 4B and Table 1). In round spermatids from MSYq–
mice, CBX1 localised to the sex chromosome domain in 49.1% of
spermatids examined; chromosome painting confirmed that these
were the X-bearing spermatids. As seen with H3K9me3, CBX1
staining was markedly reduced on the PMSC in MSYq– round
spermatids compared to XY round spermatids, despite there being
no difference in the intensity of chromocentre staining (Fig. 4B;
compare top and bottom panels).
In conclusion, although the X chromosome still forms a DAPIdense PMSC structure in round spermatids from 2/3MSYq– and
MSYq– testes, the epigenetic marks associated with PMSC are
altered compared to the XY control. These changes include reduced
enrichment of H3K9me3 and CBX1, and, most strikingly, almost
complete loss of H4K8Ac. There is also a significant reduction in
the proportion of round spermatids with chromocentre H3K9me2
and H4K12Ac staining in MSYq– males compared with the XY
control. A summary of these changes is given in Table 1.
Journal of Cell Science
4246
Journal of Cell Science 122 (22)
Discussion
In this study, we have established that mice with deletions of the
Y chromosome long arm have a global increase in transcription of
sex chromosome genes specifically in round spermatids, leading to
increased levels of their encoded proteins. Coupled with this, we
report that loss of the mouse Y chromosome long arm affects histone
modifications associated with the sex chromosomes and
chromocentre in round spermatids, in particular exerting an effect
on H4K8 acetylation and H3K9 methylation.
H3K9 methylation is a marker of transcriptionally inactive
chromatin and so the finding that H3K9me3 is reduced on the PMSC
in MSYq– spermatids is not unexpected given the increased
transcription of sex-linked genes in these cells. By contrast,
H4K8Ac is associated with active chromatin and has been
hypothesised to play a role in transcription of the X chromosome
in spermatids (Khalil et al., 2004). The reduction of H4K8Ac on
PMSC in spermatids with MSYq deletions is therefore surprising,
although H4K8Ac might be involved in replacement of histone
variants such as H2A and macroH2A with H2A.Z (Greaves et al.,
2006), which might be disturbed in these spermatids. Another
unexpected finding is that there is loss of H3K9me2 and H4K12Ac
histone modifications on the chromocentre in round spermatids from
mice with Yq deletions. This suggests that the maintenance of
epigenetic marks on the chromocentre and PMSC in round
spermatids are linked, although it is not known whether the changes
to the chromocentre histone modifications affects the transcriptional
repression of centromeric repeats.
A similar phenotype to that described here occurs in mice deficient
for the ubiquitin-conjugating enzyme HR6B (Baarends et al., 2007).
HR6B appears to play a role in controlling histone modifications on
the XY chromatin in late spermatocytes and round spermatids; Hr6b–/–
round spermatids also have reduced levels of H3K9me2 on the
centromeric heterochromatin. HR6B is involved in sex chromosome
silencing during the transition from the meiotic and post-meiotic
stages of spermatogenesis. By contrast, MSYq deletions only affect
gene transcription and epigenetic markers associated with the sex
chromosomes and centromeric heterochromatin in round spermatids.
The cause-effect relationship between the altered sex
chromosome histone code and the increase in expression of sexlinked genes in MSYq– spermatids has not been explored. The
increased expression in MSYq deletion mice might result from
altered sex chromosome chromatin conformation caused by changes
in their epigenetic profile, allowing increased access to the
transcriptional machinery. For example, overexpression of the S.
pombe Epe1 protein reduces the levels of H3K9me2 at
heterochromatic domains, increasing transcription from these
regions (Zofall and Gewal, 2006). Alternatively, increased
transcription of the sex chromosomes might lead to the replacement
of heterochromatin markers such as H3K9me3 with ‘active’
modifications and proteins, although H3K4me3 and H3K36me3
remain excluded from the PMSC in 2/3MSYq– and MSYq–
spermatids. The correlation between increased sex chromosome
expression and changes to the sex chromosome epigenetic profile
in mice with MSYq deletions implicates an MSYq-linked gene(s)
in the establishment and maintenance of sex chromosome
transcriptional repression during spermatid differentiation. This is
the first gene(s) to have a role in sex chromosome repression that
acts specifically in spermatids rather than during MSCI or during
the meiotic to post-meiotic transition.
Several multicopy genes have been identified on the Yq that could
potentially have a role in modulating the epigenetic marks and
transcriptional activity of the sex chromosomes in round spermatids.
These include the Ssty gene family, Asty, Orly and Sly, all of which
are transcribed specifically in the testis and are absent in the MSYq–
testis (Conway et al., 1994; Toure et al., 2005; Ellis et al., 2007).
A good candidate gene is Sly, which encodes a protein (SLY1) that
is present in the both the nucleus and cytoplasm of round and early
elongating spermatids (Reynard et al., 2009). This protein is
reduced in the 2/3MSYq– testis and absent in the MSYq– testis,
and so the level of SLY1 in the Yq deletion models correlates with
the severity of the spermatid sex chromosome de-repression.
SLY1 contains a COR1 domain, which is thought to facilitate
chromatin binding, and this protein interacts with the
acetyltransferase and chromatin remodelling protein KAT5 in round
spermatids (Reynard et al., 2009). The transcription of Slx, the Xlinked homologue of Sly, is increased in proportion to the decrease
in Sly mRNA levels in the two MSYq deletion models, suggesting
that there might be regulatory interactions between Sly and Slx in
normal males (Ellis et al., 2005; Touré et al., 2005). It has been
hypothesised that Sly and Slx might have evolved to reciprocally
repress the expression of sex chromosome genes by their effect on
sex chromatin (Ellis et al., 2005). However, Slx encodes a
cytoplasmic protein present from step 2 of spermiogenesis (Reynard
et al., 2007) and at present there is no evidence that SLX interacts
with SLY1, KAT5 or other SLY1-interacting proteins (L.N.R.,
unpublished observations). In addition, reduction of H4K8Ac on
the PMSC in MSYq– spermatids is seen in step 1 round spermatids,
before Slx is transcribed and translated. Therefore, although Slx
upregulation might contribute to the phenotypes exhibited by the
MSYq deletion mice, it is not the primary cause of the increased
expression and epigenetic changes to the sex chromosomes in round
spermatids from these mice.
Another candidate gene is the Ssty family, which is present in at
least 200 copies on the Yq and is composed of two distinct members,
Ssty1 and Ssty2. Whereas both Ssty1 and Ssty2 are transcribed in
round spermatids, only a subset of Ssty1 transcripts is thought to
be translated, and no SSTY2 protein has been identified to date
(Toure et al., 2004a). The subcellular localisation and function of
the SSTY1 protein in spermatids is unknown, although the levels
of this protein are increased by approximately twofold in the
2/3MSYq– testis (Toure et al., 2004b). The biological role of the
Asty and Orly genes are also unknown, but might act as non-coding
RNAs as these genes have no identifiable open reading frame (Touré
et al., 2005; Ellis et al., 2007).
It seems counterintuitive that a Y-encoded gene would have
evolved a role in repressing expression of sex-linked genes
(presumably including itself) in round spermatids. However, the
silencing of the mammalian sex chromosomes in the male germline
by MSCI has been proposed to act as a genomic defence
mechanism against sex-ratio distorters (SRDs; an allele or gene
located on a sex chromosome that increases its own transmission
to the next generation) by silencing these genes during and after
meiosis (Ellis et al., 2005; Tao et al., 2007; Turner et al., 2006).
This therefore maintains a 1:1 male to female sex ratio, the only
evolutionary stable strategy (Fisher’s principle) (Fisher, 1930;
Hamilton, 1967). Several sex chromosome genes expressed during
spermiogenesis are thought to have important functions in sperm
maturation and fertility, and could evolve into SRDs. These SRDs
reduce male reproductive fitness by reducing the number of
functional sperm as well as by causing a deviation in the sex ratio;
thus, there is strong selection for sex-linked or autosomal
suppressors that ameliorate the deleterious effects of the distorter
Journal of Cell Science
Spermatid defects in MSY deletion mice
(Carvalho et al., 1997; Hamilton, 1967; Hurst, 1992; MontchampMoreau, 2006). However, new sex-ratio distorters might evolve
that are unaffected by existing suppressor elements, allowing sexratio meiotic drive to continue ad infinitum. By evolving a function
in sex chromosome silencing in spermatids, a Yq-linked suppressor
element can suppress multiple sex-linked SRD elements at once
rather than suppress only one.
In support of this hypothesis, a mild sex-ratio distortion in favour
of females is present in the offspring of 2/3MSYq– mice, and this
is hypothesised to be a consequence of a disruption in the
equilibrium between an X-linked meiotic driver and a suppressor
gene located on the Yq (Ellis et al., 2005). Unbalancing of the
Drosophila Dox and Ste X-linked distorter genes from their
suppressor results in a shortage of, or reduced fertilising ability of,
Y-bearing sperm by an unknown mechanism and leads to a sex
ratio in favour of females (Aravin et al., 2001; Tao et al., 2007a).
Furthermore, gross overexpression of the X-linked distorter might
be the cause of sterility in the MSYq– mice because unrepressed
drivers (e.g. Stellate in D. melanogaster) can lead to sterility rather
than drive.
In conclusion, the data we present in this paper provide evidence
for the role of an MSYq encoded factor(s) in the regulation of histone
modifications and transcription from the sex chromosomes in round
spermatids. Analysis of MSYq– mice carrying transgenes or targeted
mutations of the various MSYq-encoded genes will be invaluable
in investigating which gene or genes are responsible.
Materials and Methods
Mice
All XY and 2/3MSYq– mice were produced at the MRC National Institute for Medical
Research (London, UK) on a MF1 random bred background. XY mice carry an RIII
strain of Y chromosome and are the appropriate controls for the 2/3MSYq– and MSYq–
mice models. The 2/3MSYq– mice have an RIII strain Y chromosome with an
interstitial deletion removing approximately two-thirds of the MSYq and were derived
from a stock originating from the mice described by Conway and colleagues (Conway
et al., 1994). MSYq– mice [XSxraY*X mice (Burgoyne et al., 1992) and XY*xSxra
mice (Yamauchi et al., 2009)] lack the entire Y-specific (non-PAR) gene content of
MSYq, with the only Y-specific material provided by the Y short-arm-derived factor
Sxra. XSxraY*x males were produced by mating XY*X females (Burgoyne et al.,
1998; Eicher et al., 1991) to XYSxra males (Cattanach, 1987; Cattanach et al., 1971).
XY*xSxra mice were produced by Monika Ward (Institute for Biogenesis Research,
John A. Burns School of Medicine, University of Hawaii, Honolulu, HI) using ICSI
of sperm from XSxraY*x (Yamauchi et al., 2009). D4/XEGFP mice (Hadjantonakis
et al., 2001; Hadjantonakis et al., 1998) were obtained from Andras Nagy (Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON). Hemizygous EGFP
transgenic XX females were bred with either XYRIII or 2/3MSYq– MF1 males to
produce XY-GFP and 2/3MSYq-GFP mice, with non-transgenic littermates used as
controls. Animal procedures were in accordance with the United Kingdom Animal
Scientific Procedures Act 1986 and were subject to local ethical review.
RNA FISH
RNA fluorescence in situ hybridization (FISH) was performed on surface-spread
spermatogenic cells from adult XY, 2/3MSYq– and MSYq– testes as described
previously (Reynard et al., 2007) using the probes listed in supplementary material
Table S2. For each gene, RNA FISH was replicated three times using three different
mice per genotype, and the presence of an RNA FISH signal was examined in
approximately 100 round spermatids per mouse (supplementary material Table S1).
The percentage of expressing spermatids per mouse was converted into angles and
a one way analysis of variance (ANOVA) test performed comparing the three
genotypes. For dual RNA FISH, 200 round spermatids were counted per genotype
for the presence of an RNA signal.
Western blot analysis
Testis protein extraction and western blotting was performed as described previously
(Reynard et al., 2007) using the rabbit polyclonal anti-SLX antibody (Reynard et al.,
2007) at 1:2500, anti-GFP antibody at 1:5000 (Invitrogen), rabbit anti-DPP9 antibody
at 1:3000 (Abcam, ab42080) and mouse anti-actin antibody at 1:7000 (Sigma, A5441).
After incubating with the appropriate secondary antibody (anti-mouse IgG, anti-rabbit
IgG or anti-goat IgG) coupled to horseradish peroxidase (DAKO), the signal was
revealed by chemiluminescence (SuperSignal, Pierce) and recorded on X-ray film.
4247
Antibody staining and chromosome painting of surface spread
spermatids
A single cell suspension was made from the testis of three mice per genotype and
fixed for 5 minutes at room temperature (R/T) in 2.6 mM sucrose, 1.86% formaldehyde
solution. The cell suspension was then centrifuged at 112 g for 5 minutes at R/T, the
fixative removed and the cells resuspended in PBSA. Three drops of the cell
suspension were added to Superfrost Plus slides (BHD) and allowed to air dry at
R/T. The cells were permeabilised by adding 0.5% Triton X-100 to the slides for 10
minutes at R/T and then washed once in PBS for 5 minutes before being blocked in
blocking buffer (PBS, 0.15% BSA, 0.1% Tween-20) for 1 hour at R/T. The slides
were incubated overnight in the appropriate primary antibody diluted in blocking
buffer at 37°C, washed in PBS three times, and incubated in secondary antibody
diluted in PBS for 1 hour at 37°C. After washing the slides three times in PBS, the
cells were mounted in Vectorshield mounting media with DAPI and observed.
Chromosome painting was performed after antibody staining using FITC StarFISH
mouse X or Y chromosome paint (Cambio, Cambridge, UK) as described previously
(Reynard et al., 2007).
Preparation and immunostaining of testis section
After dissection, mouse testes were either fixed in 4% paraformaldehyde overnight
at R/T, or pre-fixed in 4% paraformaldehyde for 4 hours at R/T before being fixed
in dilute Bouin’s solution (0.675% picric acid, 9.25% formaldehyde, 4.9% glacial
acetic acid diluted in milliQ water) overnight at 4°C. Testes were then dehydrated,
embedded, and cut into 3 m sections as described previously (Reynard et al., 2007).
The sections were mounted onto Superfrost slides, rehydrated and subjected to antigen
retrieval by boiling in 0.01M sodium citrate solution pH 6.0. After boiling, the slides
were rinsed in PBS before continuing the antibody staining protocol described above
for surface-spread cells. A list of antibodies used in this study is given in supplementary
material Table S3.
We thank Prim Singh (Division of Immunoepigenetics, Department
of Immunology and Cell Biology, Research Center Borstel, Borstel,
Germany) for the gift of an antibody against CBX1, and Monika Ward
for providing XY*xSxra testis material. We are also grateful to Paul
Burgoyne for mouse breeding, help with statistical analysis and the
critical reading of this manuscript, Shantha Mahadevaiah for interesting
discussions and Andrew Ojarikre for help with mouse breeding. This
work was supported by the Medical Research Council. Deposited in
PMC for release after 6 months.
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