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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02747
1 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku,
Yokohama 230-0045, Japan.
2 Division of Stem Cell Research and Developmental Genetics, MRC National
Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA,
UK.
3 Laboratory of Veterinary Physiology, Tokyo University of Agriculture and
Technology, Fuchu, Tokyo 183-8509, Japan.
4 Nuclear Reprogramming Laboratory, Division of Gene Expression and Development,
Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK.
5 Department of Pediatrics, Shinshu University School of Medicine, Matsumoto,
Nagano 390-8621, Japan.
6 Research Institute of Molecular Pathology, The Vienna Biocenter, Dr Bohrgasse
7, A-1030 Vienna, Austria.
* Author for correspondence (e-mail: koseki{at}rcai.riken.jp)
Accepted 17 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mouse, Polycomb, Scmh1, Spermatogenesis, Apoptosis, XY body
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Paro, 1995;
Pirrotta, 1997).
Drosophila PcG gene products form large multimeric protein complexes
and are thought to act by changing the local chromatin structure, as suggested
by the synergistic genetic interactions between mutant alleles of different
Drosophila PcG genes
(Jürgens, 1985;
Franke et al., 1992;
Paro, 1995;
Pirrotta, 1997;
Shao et al., 1999). In
mammals, genes structurally and functionally related to Drosophila
PcG genes have been identified and mammalian PcG gene products form several
distinct complexes. Polycomb repressive complex-2 (Prc2), which contains the
product of Eed (the ortholog of the Drosophila extra sex combs gene),
Ezh2 (the ortholog of the Drosophila enhancer of zeste gene) and
Suz12, mediates trimethylation of histone H3 at K27 (H3-K27) by Ezh2 component
(Schumacher et al., 1996;
Laible et al., 1997;
van Lohuizen et al., 1998;
Sewalt et al., 1998;
van der Vlag and Otte, 1999).
The second complex, which is closely related to the Polycomb repressive
complex-1 (Prc1) in Drosophila, includes the products of the paralogs
of class 2 PcG genes (Levine et al.,
2002). This subset contains gene groups, namely Pcgf2
(also known as Rnf110 and Mel18, and hereafter referred to
as Rnf110) and Bmi1, Cbx2 (also known as M33),
Cbx4 (also known as MPc2) and Cbx8 (also known as
Pc3), Phc1 (also known as rae28), Phc2 and
Phc3, Ring1 and Rnf2 (also known as Ring1B)
(Levine et al., 2002). The
Prc1 complex is compositionally and functionally conserved between flies and
mammals (Shao et al., 1999;
Levine et al., 2002;
Gebuhr et al., 2000). In
mammals, chromatin binding of Prc1 involves its recognition of trimethylated
H3-K27 (Boyer et al., 2006;
Lee et al., 2006;
Fujimura et al., 2006). The
Prc1 complex has a significant impact on the control of not only
anteroposterior (AP) specification of the axis via Hox regulation, but also
the proliferation and senescence via regulation of the Ink4a/p53 pathway
(Jacobs et al., 1999).
Sex comb on midleg (Scm) gene is a member of
Drosophila PcG genes and, based on database comparison, its product
contains three separable functional domains
(Bornemann et al., 1996),
namely: a pair of N-terminal zinc fingers, two tandem 100-amino acid repeats,
called mbt repeats as they are also found in the fly tumor suppressor encoded
by the l(3)mbt [lethal(3) malignant brain tumor] gene, and
C-terminal homology domain of 65 amino acids, called the SPM domain. The SPM
domain is a self-binding protein interaction module and may mediate Scm
association to Prc1 and play a key role for PcG repression, although Scm
association to purified Prc1 is substoichiometric
(Levine et al., 2002). In
mammals, there are four paralogs for Drosophila Scm based on primary
sequence: Scmh1, Scml1, Scml2 and Sfmbt
(Tomotsune et al., 1999;
van de Vosse et al., 1998;
Montini et al., 1999;
Usui et al., 2000). The
mammalian Scmh1 protein has been shown to be a constituent of the mammalian
Prc1 (Levine et al., 2002),
which contains two highly conserved motifs, two mbt repeats in the N-terminal
region and an SPM domain in the C-terminal region, that are shared with its
Drosophila counterpart. The SPM domain of Scmh1 can mediate its
interaction with Drosophila polyhomeotic (Ph) and mammalian Phc1 and
Phc2, through their respective SPM domains
(Tomotsune et al., 1999). It
is also notable that tissue-specific Scmh1 mRNA levels in the testes
are the highest of all tissues analyzed and they increase during the
synchronous progression of first-wave spermatogenesis in parallel with
Phc1 (see Fig. S1A,B in the supplementary material). These
observations suggest a role of mammalian Prc1 during spermatogenesis.
Before the specialized cell division of meiosis, postmitotic spermatocytes
enter into an extended meiotic prophase, in which homologous autosomal
chromosomes pair and undergo reciprocal recombination. There is accumulating
evidence to suggest that the quality of this complex process is monitored by a
checkpoint to ensure spermatogenic success, as represented by the apoptotic
elimination of those spermatocytes with synaptic errors. During this period,
heteromorphic sex chromosomes pair only in a small pseudoautosomal region
(PAR) at their distal ends and undergo transcriptional inactivation, termed
meiotic sex chromosome inactivation (MSCI), by remodeling into
heterochromatin, thus forming the XY body
(Perry et al., 2001;
Odorisio et al., 1996;
Singer-Sam et al., 1990;
Turner et al., 2004;
Baarends et al., 1999;
Strahl and Allis, 2000;
Turner et al., 2000;
Hoyer-Fender et al., 2000;
Mahadevaiah et al., 2001;
Khalil et al., 2004).
Formation of the XY body is conserved throughout the mammalian phylogenetic
tree and is therefore assumed to be essential for successful spermatogenesis
and the faithful segregation of sex chromosomes. Indeed, in mutants for the
gene encoding histone H2A.X and the tumor suppressor protein Brca1, failure to
form the XY body coincides with sterility due to the apoptotic elimination of
such mutant spermatocytes before completion of meiosis
(Fernandez-Capetillo et al.,
2003; Xu et al.,
2003). However, it has not been definitely demonstrated that
spermatogenic arrest in these mutants is because of failure to form the XY
body or due to some other reason. The condensation of the X and Y chromosome
to form the XY body is associated with post-translational modifications of
histones and the recruitment or exclusion of various chromatinassociated
proteins (Turner et al., 2001;
Hoyer-Fender et al., 2000;
Richler et al., 2000;
Mahadevaiah et al., 2001;
Khalil et al., 2004;
Baarends et al., 1999;
Baarends et al., 2005). Early
in the formation of the XY body, phosphorylated histone H2A.X (
H2A.X)
and ubiquitylated histone H2A (uH2A) are enriched at the XY body and then X
and Y chromosomes undergo sequential changes in their histone modifications,
which correlate with transcriptional status of sex chromosomes
(Mahadevaiah et al., 2001;
Baarends et al., 1999;
Baarends et al., 2005). The
functional involvement of these histone modifications at the XY body was
properly addressed for the first time in a study using Brca1 mutants,
in which H2A.X phosphorylation was shown to be essential to trigger MSCI
(Turner et al., 2004).
However, the roles of hyperubiquitylation of H2A on the X and Y chromosomes
have still not been addressed. Recent studies have revealed an Rnf2 component
of Prc1 to be an E3 component of ubiquitin ligase for histone H2A to link Prc1
with the XY body (de Napoles et al.,
2004; Baarends et al.,
1999; Baarends et al.,
2005).
In this study, we have generated a mouse line carrying a mutant Scmh1 allele that lacks the exons to encode an SPM domain. Axial homeotic transformations and premature senescence in mouse embryonic fibroblasts (MEFs) in the homozygotes indicated the role of Scmh1 as a PcG component. Approximately half the Scmh1-/- males were infertile, which correlates with an accelerated apoptosis of postmitotic pachytene spermatocytes. The present genetic study indicates the involvement of Prc1 during XY body maturation and the regulatory role of Scmh1 gene products in the exclusion of Prc1 from the XY body, which may in turn be required for the further progression of meiotic prophase.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Schematic representations of genomic organization and
targeting vector are shown in Fig. S2 in the supplementary material.
Scmh1 mutant mice were genotyped by PCR using the following
oligonucleotides: (a) 5'-GTCAGGTGTGCCCGCTACTGT-3' and (b)
5'-GATGGATTGCACGCAGGTTC-3' for the mutant allele; and (a) and (c)
5'-GGCCGACTAGGCCATCTTCTG-3' for the Scmh1 wild-type
allele. As Scmh1 and Phc2 loci were on chromosome 4 and
28x106 base pairs (bp) apart from each other, we first
generated recombinants in which Scmh1 and Phc2 mutant
alleles were physically linked. This double mutant allele was used to generate
Scmh1;Phc2 double homozygotes. Skeletal analysis was performed as
described previously (Kessel and Gruss,
1991). MEFs were maintained according to a 3T9 protocol as
described previously (Kamijo et al.,
1997).
In situ hybridization, RT-PCR and immunohistochemistry
In situ hybridization was performed as described previously (Yuasa et al.,
1996). The nucleotide sequences of the primers used for RT-PCR in this study
are listed in Table 1.
Immunohistochemistry was performed as described previously
(Hoyer-Fender et al.,
2000).
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Immunocytochemistry of spread spermatocytes
Meiotic prophase cell spreads and squashes were prepared as described
previously (Scherthan et al.,
2000). After washing with PBS for 3 minutes, slides bearing cell
spreads were processed for immunostaining using standard procedures. The
antibodies used for immunostaining in this study are listed in
Table 2. For the statistical
analyses, 300 spermatocytes derived from five mice with respective genotypes
were analyzed and the significance was further analyzed by
t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Although both male and female Scmh1-/-
mice were viable and grew normally to adulthood, homozygotes exhibited the
axial homeosis and premature senescence of MEFs in the homozygous mutants,
which was restored by the p19ARF or p53 mutation
(see Fig. S2F-K in the supplementary material). Therefore, Scmh1 is an
indispensable component of Prc1 in mice.
The expression and subcellular localization of Scmh1 during spermatogenesis
About half the homozygotes were sterile and had slightly smaller testes
than their wild-type littermates (Y.T., unpublished). Before studying the
pathogenesis of infertility in Scmh1 mutants, we examined Scmh1
expression during spermatogenesis by in situ hybridization and
immunohistochemical analysis. Scmh1 expression was seen in the
seminiferous tubules and interstitial cells
(Fig. 1Aa). In the seminiferous
tubules, morphological examination of the germ cell layers representing
meiotic spermatocytes (particularly those at the pachytene stage)
revealed that these germ layers were expressing the highest amount of
Scmh1, with the least amounts expressed in spermatogonia and round
spermatids (Fig. 1Ab). Sertoli
cells also expressed a significant amount of Scmh1. By using an
immunohistochemical technique, a light staining of the whole nucleus was
observed in the zygotene stage and in more advanced cells up to pachytene
spermatocytes (Fig. 1Ba). In
addition, focal localization of Scmh1 was seen in the chromocenter of round
spermatids (Fig. 1Bb).
Concordantly, Scmh1 expression in the testes correlated with
synchronous progression of the first-wave spermatogenesis (see Fig. S1B in the
supplementary material). From day 15 post partum (pp) onwards, the amount of
Scmh1 transcript progressively increased and reached a maximum level
by day 25 pp. Taken together, Scmh1 and its products are
predominantly expressed in postmitotic spermatocytes.
We went on to investigate subcellular localization of Scmh1 by using spread
meiotic spermatocytes. The synaptonemal complex protein Scp3, which is a
component of the axial element, was used to substage meiosis
(Xu et al., 2003). Scmh1
staining was seen in the nucleus as a diffused pattern from leptotene to early
pachytene spermatocytes (Fig.
1Ca-c and Y.T., unpublished). In late pachytene spermatocytes,
Scmh1 staining was significantly excluded from the XY chromatin domain
(Fig. 1Cd,e). Concordantly,
reciprocal localization of Scmh1 and H2A.X was seen in about 80% of
pachytene spermatocytes (Fig.
1Ce). Consistently, Scmh1 was excluded from the XY body in which
dimethylated histone H3 at K9 (H3-K9) was enriched
(Fig. 1Cf).
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H2A.X or uH2A. Reciprocal localization of these PcG
proteins and H2A.X or uH2A, within about 80% of spermatocytes,
indicated the exclusion of other PcG proteins from the XY body during the
pachytene stage, as well as Scmh1 (Fig.
2Aa-e). Consistently, Phc2 was excluded from the XY body in 77% of
spermatocytes, in which dimethylated H3-K9 was enriched
(Fig. 2Af). Taken together, PcG
complexes are excluded from the XY body at the late pachytene stage almost
concurrently with hyperdimethylation of H3-K9 at the XY body, whereas they are
continuously present in the autosomal regions.
Recent studies have repeatedly provided evidence indicating the engagement
of Prc1 by trimethylated H3-K27 mediated by Prc2
(Cao et al., 2002;
Kuzmichev et al., 2002). We
thus addressed whether the exclusion of Prc1 components from the XY body was
correlated with the degree of H3-K27 trimethylation at the XY chromatin
domain. Trimethylated H3-K27 was distributed throughout the nucleus as a
diffuse pattern from leptotene to zygotene stage spermatocytes despite the
fact that the signals were very dim (Fig.
2Ba and Y.T., unpublished). In early pachytene spermatocytes,
trimethylated H3-K27 staining was much stronger than in the earlier stages but
was significantly excluded from the XY chromatin domain
(Fig. 2Bb). In late pachytene
spermatocytes, its exclusion from the XY body was still maintained
(Fig. 2Bc). Therefore the
exclusion of trimethylated H3-K27 from the XY chromatin domain precedes those
of Prc1 components.
Impaired spermatogenesis in Scmh1-/- males
We first examined the histology of Scmh1-/- testes in
day 35 pp testes and revealed that about two-thirds were morphologically
altered to varying extents. The seminiferous tubules of
Scmh1-/- testes exhibited a reduction in the number of
spermatocytes and a lack of spermatids and mature spermatozoa
(Fig. 3Aa,b). Sertoli cells and
spermatogonia were morphologically and numerically normal. Mono- or
multinuclear large cells were sometimes seen. One-third of
Scmh1-/- testes were morphologically indistinguishable
from wild type. Therefore, spermatogenesis was variably affected in
Scmh1-/- testes.
We then examined Scmh1-/- testicular histology at various stages of first-wave spermatogenesis. Neither day 7 pp nor day 11 pp mutant mice exhibited any significant differences compared to wild type (Fig. 3B). The morphological changes in Scmh1-/- testes were observed in the seminiferous tubules as early as day 15 pp. At day 15 pp, most of the seminiferous tubules contained spermatogonia, Sertoli cells and several degenerating pachytene spermatocytes, whereas pre-leptotene to zygotene spermatocytes were seen rarely (see Fig. S3 in the supplementary material). Vacuoles were frequently seen in the luminal region. Based on these morphological parameters, days 15, 19, 25 and 30 pp testes were also examined. In conclusion, Scmh1-/- testes were progressively affected during first-wave spermatogenesis (Fig. 3B).
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Finally, the expression of stage-specific molecular markers were examined
by means of semi-quantitative RT-PCR analysis in wild-type and
Scmh1-/- testes at day 35 pp, in order to address which
stage of spermatogenesis was predominantly deleted in affected homozygous
mutants (Fig. 3D).
CyclinA1, calmegin, Bmp8a and CREM
genes were used as
markers for pachytene stage spermatocytes
(Sweeney et al., 1996;
Watanabe et al., 1994;
Zhao and Hogan, 1996;
Foulkes et al., 1992). These
were reduced more than threefold in Scmh1-/- when compared
with testes from wild type. In Scmh1-/- testes no change
was observed in the expression of A-myb, Dmc1, Mvh1 and
Scp3, which are expressed before the pachytene stage
(Mettus et al., 1994;
Habu et al., 1996;
Fujiwara et al., 1994;
Tanaka et al., 2000;
Klink et al., 1997). Taken
together, in Scmh1-/- testes, postmitotic spermatocytes
are predominantly depleted by apoptotic outbursts.
Apoptotic elimination of late pachytene spermatocytes occurs after synapsis of homologous chromosomes in Scmh1-/- testes
In order to further identify the meiotic substage at which
Scmh1-/- spermatocytes are predominantly affected,
immunolocalization studies were carried out in spread spermatogenic cells,
prepared from day 18 pp males, by using antibodies against uH2A, H2A.X
and Scp3. Accumulation of uH2A on the XY body was seen in pachytene
spermatocytes, whereas H2A.X demarcates the XY body from late zygotene
to diplotene stage (Baarends et al.,
1999; Baarends et al.,
2005; Mahadevaiah et al.,
2001; Fernandez-Capetillo et
al., 2003; Xu et al.,
2003). In particular, the degree of uH2A association to the XY
body was intriguing, as the Rnf2 component of class 2 PcG has been shown to be
an E3 component of ubiquitin ligase for histone H2A
(Wang et al., 2004;
de Napoles et al., 2004). We
did not see any significant difference between Scmh1-/-
and wild-type testes in the frequency of the spermatocytes, in which uH2A and
H2A.X localized on the XY bodies. This implies entry into pachytene
stage was not affected in Scmh1-/- (Y.T., unpublished).
Using Scp3 staining and morphology, we substaged further the spermatocytes, in
which uH2A was accumulated on the XY body, into early and late pachytene
stages (Fig. 4A). The frequency
of early pachytene spermatocytes was 37% in wild type and 66% in
Scmh1-/- (Fig.
4A). This suggests that Scmh1-/- spermatocytes
were incompletely depleted by late pachytene.
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). As
proper chromosome alignment and segregation in the first meiotic division are
ensured by recombination between homologous chromosomes, we examined the
localization of Mlh1, a mismatch-repair protein, that forms foci at sites of
meiotic crossover in mid- to late-pachytene spermatocytes
(Celeste et al., 2002). Mlh1
foci were distributed on the synaptonemal complexes in late pachytene
spermatocytes of both wild type and Scmh1-/-
(Fig. 4B). Consistent with this
observation, late pachytene spermatocytes remained in
Scmh1-/- testes exhibited normal Scp3 distribution,
including PAR of sex chromosomes (see Fig. S4 in the supplementary material).
Taken together, Scmh1 is dispensable for pairing and synapsis of homologous
chromosomes.
The role of Scmh1 at the XY body in pachytene spermatocytes
Apart from homologous autosomes, the X and Y chromosomes pair along PAR and
undergo extensive and sequential remodeling into heterochromatin, thus forming
the XY body, which is associated with transcriptional inactivation. Failure to
form the XY body has been shown to coincide with male sterility and arrest of
spermatogenesis, although it is not yet definitely proven whether the XY body
is required for survival and fertility of male germ cells
(Fernandez-Capetillo et al.,
2003). Scmh1 and other PcG components were excluded at the
transition from early to late pachytene stage. Scmh1-/-
spermatocytes were affected at a stage that was temporally similar to that
concerning the exclusion of PcG proteins from the XY chromatin domain. These
observations prompted us to focus on whether spermatogenic arrest in
Scmh1-/- testes is accompanied by changes in chromatin
remodeling at the XY body. We first examined the degree of H3-K9 methylation,
acetylation and phosphorylated RNA pol II association to the XY bodies, which
have been shown to change during the pachytene stage
(Richler et al., 2000;
Khalil et al., 2004). In wild
type, 76 and 62% of the XY body marked by uH2A were hyperdi- and
hypermonomethylated at H3-K9, respectively, compared with 36 and 18%,
respectively, in Scmh1-/- testes
(Fig. 5Aa,b). Phosphorylated
RNA pol II was excluded from the XY body in 51% of the
Scmh1-/- spermatocytes compared with 90% of wild type
(Fig. 5Ac)
(Richler et al., 2000;
Khalil et al., 2004). These
results suggest that elimination of late pachytene spermatocytes in
Scmh1-/- testes is coincidental with the stage at which
the XY body undergoes chromatin remodeling. It is also noteworthy that
underacetylation of H3-K9 at the XY body was observed to a similar extent
between the wild-type and Scmh1-/- spermatocytes
(Fig. 5Ad). Changes in such
specific chromatin modifications at the XY body of
Scmh1-/- spermatocytes imply that they may not solely
represent their developmental arrest at late pachytene stage. We therefore
postulated a regulatory role for Scmh1 in sequential chromatin modifications
of the XY body.
|
H2A.X in Scmh1-/-
spermatocytes was examined. In 79% of wild-type spermatocytes, Phc1 and Phc2
were excluded from the XY body demarcated by H2A.X
(Fig. 5Ba,b). In
Scmh1-/- spermatocytes, the frequency of spermatocytes in
which Phc1 and Phc2 were excluded from the XY body was reduced to 40 and 27%,
respectively (Fig. 5Ba,b).
Similarly, trimethylated H3-K27 was excluded from the XY body demarcated by
uH2A in 88% of wild-type spermatocytes compared with 39% in
Scmh1-/- spermatocytes
(Fig. 5Bc). Therefore meiotic
spermatocytes, in which the sequential exclusion of trimethylated H3-K27 and
Prc1 components from the XY body had failed, may be predominantly depleted in
Scmh1-/- testes. As the exclusion of trimethylated H3-K27
from the XY body is shown to precede the exclusion of PRC1 components in wild
type, this result could be interpreted as recurrence of H3-K27 trimethylation
in the late pachytene stage. We therefore substaged the spermatocytes in which
trimethylated H3-K27 was excluded from the XY body into early and late
pachytene stages by using Scp3 staining and morphology. In early pachytene
stage, trimethylated H3-K27 was excluded from the XY body to a similar extent
between the wild type and Scmh1-/-
(Fig. 5Bd). In contrast, the
frequency of late pachytene spermatocytes, in which trimethylated H3-K27 was
excluded, was significantly reduced in Scmh1-/- testes
compared to wild type (Fig.
5Bd). This suggests that Scmh1 is required to maintain the
exclusion of trimethylated H3-K27 from the XY body in late pachytene
spermatocytes but not in early pachytene.
We went on to examine the localization of monomethylated histone H3 at K4
(H3-K4) and dimethylated histone H4 at K20 (H4-K20) because the mbt repeats,
which are also found in the Scmh1 N-terminal, have been shown to exhibit
specific binding to mono- and dimethylated H3-K9, monomethylated H3-K4 and
mono- and dimethylated H4-K20 (Kim et al.,
2006; Klymenko et al.,
2006). Neither hypermonomethylation of H3-K4 nor
underdimethylation of H4-K20 at the XY body were significantly different
between wild-type and Scmh1-/- spermatocytes
(Fig. 5Ca,b). It is
particularly noteworthy that H4-K20 underdimethylation at the XY body, which
was exclusively seen in the late pachytene spermatocytes in wild type, was not
affected in the mutants (Y.T., unpublished). Taken together, these results
show that apoptotic elimination of late pachytene spermatocytes in
Scmh1-/- testes is preceded by failure in hypermethylation
of H3-K9, exclusion of phosphorylated RNA pol II and Prc1 components and
undermethylation of H3-K27 at the XY body, whereas it is not accompanied by
changes in H3-K9 acetylation or methylation of H3-K4 or H4-K20. These results
support the idea that changes in chromatin modifications at the XY body of
Scmh1-/- spermatocytes are not simply a consequence of
apoptotic elimination of late pachytene spermatocytes. Instead, Scmh1 was
suggested to play the regulatory role for the sequential changes in chromatin
modifications of the XY body.
Phc2 mutation alleviates spermatogenic defects in Scmh1-/- spermatocytes
We postulated that Scmh1 functions via its direct interaction with Prc1 in
pachytene spermatocytes, as Scmh1 has been identified as a constituent of Prc1
components because, in general, mutant interactions of PcG alleles
have been shown to modify the respective phenotypes in mammals as well
(Bel et al., 1998;
Akasaka et al., 2001;
Isono et al., 2005). We have
generated Scmh1;Phc2 double mutants (dko) as Phc2 protein binds to
Scmh1 via its SPM domain and the homozygous mutants were viable and fertile
(Isono et al., 2005) (Y.T.,
unpublished). dko mice were viable and born according to the principles of
Mendelian inheritance, although some of them exhibited growth retardation
(Y.T., unpublished). The fertility of ten normal-sized dko and
Scmh1-/- males was tested by natural mating to
approximately 10-week-old C57BL/6 females. Strikingly, all the dko males were
fertile, whereas half the Scmh1-/- males were sterile
(Fig. 6B). Histological
inspections revealed that all the dko testes were morphologically
indistinguishable from wild type at day 35 pp and the frequency of apoptotic
outbursts in dko was significantly reduced in comparison with littermate
Scmh1-/- testes (Fig.
6A,C). Significant restorations of late pachytene spermatocytes
were also revealed by substaging spermatocytes using antibodies against di-
and monomethylated H3-K9, the phosphorylated form of RNA pol II and Phc1
(Fig. 6D-F and Y.T.,
unpublished). Defects in spermatogenesis were also significantly alleviated in
Scmh1-/-Phc2+/- albeit to a lesser
extent than dkos (Y.T., unpublished). Therefore Phc2 mutation
coincidentally restored aberrant chromatin modifications seen in the XY body
of Scmh1-/- spermatocytes and their developmental arrest
at the late pachytene stage. Taken together, this evidence suggests that Scmh1
is a regulatory component of Prc1 that mediates exclusion of Prc1 from the XY
body at the pachytene stage of meiosis. It is likely that the lack of Phc2
components may accelerate this exclusion irrespective of Scmh1.
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|
; Turner et al.,
2004; Turner et al.,
2005). Average expression levels of genes located on the autosomes
and sex chromosomes were compared between the wild type and
Scmh1-/- after conventional normalization. No significant
changes were found in the expression of autosomal and sex chromosomal genes in
Scmh1-/- spermatocytes (see Fig. S5A in the supplementary
material). Concordant with this result, the XY chromatin domain enriched in
H2A.X was negative for Cot-1 RNA in Scmh1-/-
spermatocytes as well as the wild type (see Fig. S5B in the supplementary
material). In conclusion, sequential chromatin modifications mediated by Scmh1
are not required to maintain MSCI. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Based on the present observations, we postulate that Scmh1 could primarily
promote the exclusion of Prc1 components from the XY body in the pachytene
spermatocytes because Scmh1 itself is a functional component of Prc1. By
contrast, failure to maintain exclusion of trimethylated H3-K27 and to undergo
H3-K9 methylation at the XY body in Scmh1-/- spermatocytes
may occur secondarily to the failure to exclude Prc1 from the XY body. At many
loci, epistatic engagement of Prc1 by Prc2 has been shown to be essential for
the mediation of transcriptional repression
(Lee et al., 2006;
Boyer et al., 2006;
Fujimura et al., 2006).
Preceding exclusion of trimethylated H3-K27, which represents Prc2 actions,
for Prc1 exclusion from the XY body, is consistent with epistatic roles of
Prc2 for Prc1 at the XY body. Therefore, Scmh1 may affect H3-K27
trimethylation at the XY body through the Prc1-Prc2 engagement. It is
noteworthy that H3-K27 trimethylation has been shown to be regulated by Prc1
at the XY body. This may imply that Prc1-Prc2 engagement is a reciprocal
rather than epistatic process at the XY body. This possibility should be
addressed by using conditional mutants for Prc2 components. We also
hypothesize a functional correlation between Prc1 exclusion and H3-K9
methylations at the XY body because the indispensable H3-K9 methyltransferase
complex, composed of G9a and GLP, is constitutively associated with E2F6
complexes, which share at least Rnf2 and Ring1 components with Prc1. Moreover,
several components of respective complexes are structurally related to each
other (Ogawa et al., 2002;
Trimarchi et al., 2001).
Intriguingly, although Prc1 components, apart from Rnf2, have been shown to be
excluded from the XY body at late pachytene stage, components of E2F6
complexes including Rnf2, RYBP, HP1 and G9a are retained (Y.T., K.I.
and H.K., unpublished). The most attractive scenario would be that exclusion
of Prc1 is a prerequisite for the functional manifestation of E2F6 complexes
to mediate the hypermethylation of H3-K9 at the XY body. We thus propose that
Scmh1-mediated exclusion of Prc1 from the XY body might be a prerequisite for
maintaining appropriate chromatin structure to undergo subsequent sequential
chromatin remodeling of the XY chromatin in pachytene spermatocytes.
We also suggest that sequential changes in chromatin modifications of the
sex chromosomes in the pachytene spermatocytes might be monitored by some
meiotic checkpoint mechanisms. This is supported by the temporal concurrence
of Prc1 exclusion from the XY body and apoptotic depletion of meiotic
spermatocytes, their coincidental restorations by Phc2 mutation, and
normal oogenesis and fertility in Scmh1-/- females (Y.T.
and H.K., unpublished). In addition, defects in the XY body formations have
been shown to correlate with apoptotic depletion of meiotic spermatocytes by
studies using H2A.X and Brca1 mutants, although
developmental arrests occurred by early pachytene stage
(Fernandez-Capetillo et al.,
2003; Xu et al.,
2003). However, this link has not been substantially
demonstrated.
Although Scmh1 has been shown to act together with Prc1, the role of Scmh1
for Prc1 might be modified in a tissue- or locusspecific manner because
spermatogenic defects by Scmh1 mutation are restored by Phc2
mutation, whereas premature senescence of MEFs is enhanced mutually by both
mutations (Y.T. and H.K., unpublished). This is supported by an
immunofluorescence study revealing the co-localization of Scmh1 with other
class 2 PcG proteins in subnuclear speckles in U2OS cells, whereas in female
trophoblastic stem (TS) cells it is excluded from the inactive X chromosome
domain, which is intensely decorated by Rnf2, Phc2 and Rnf110 (see Fig. S6A,B
in the supplementary material) (Plath et
al., 2004; de Napoles et al.,
2004). It may be possible to postulate some additional factors
that modify the molecular functions or subnuclear localization of Scmh1.
Indeed, most of the soluble pool of SCM in Drosophila embryos is not
stably associated with Prc1, although SCM is capable of assembling with the
Polyhomeotic protein by their respective SPM domains in the Polycomb core
complex (Peterson et al.,
2004). As the SPM domain is shared, not only by
polyhomeotic homologs, but also by multiple paralogs of the
Drosophila Scm gene, namely Scml1, Scml2, Sfmbt, l(3)mbt3
and others in mammals, these structurally related gene products may
potentially interact with Scmh1 and modulate its functions. Conservations of
crucial amino acid residues required for the mutual interaction of SPM domains
and multiple mbt repeats in these proteins may further suggest functional
overlap with Scmh1. It is notable that phenotypic expressions of
Scmh1 mutation are quite variable during spermatogenesis and axial
development even after more than five times backcrossing to a C57Bl/6
background. This incomplete penetrance might involve multiple paralogs of the
canonical Scm proteins, which may act in compensatory manner for
Scmh1 mutation, as revealed between Rnf110 and Bmi1
or Phc1 and Phc2 (Akasaka
et al., 2001; Isono et al.,
2005).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/579/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Akasaka, T., Kanno, M., Balling, R., Mieza, M. A., Taniguchi, M.
and Koseki, H. (1996). A role for mel-18, a Polycomb
group-related vertebrate gene, during the anteroposterior specification of the
axial skeleton. Development
122,1513
-1522.[Abstract]
Akasaka, T., van Lohuizen, M., van der Lugt, N.,
Mizutani-Koseki, Y., Kanno, M., Taniguchi, M., Vidal, M., Alkema, M., Berns,
A. and Koseki, H. (2001). Mice doubly deficient for the
Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for
maintenance but not initiation of Hox gene expression.
Development 128,1587
-1597.[Abstract]
Atsuta, T., Fujimura, S., Moriya, H., Vidal, M., Akasaka, T. and
Koseki, H. (2001). Production of monoclonal antibodies
against mammalian Ring1B proteins. Hybridoma
20, 43-46.[CrossRef][Medline]
Baarends, W. M., Hoogerbrugge, J. W., Roest, H. P., Ooms, M.,
Vreeburg, J., Hoeijmakers, J. H. and Grootegoed, J. A.
(1999). Histone ubiquitination and chromatin remodeling in mouse
spermatogenesis. Dev. Biol.
207,322
-333.[CrossRef][Medline]
Baarends, W. M., Wassenaar, E., van der Laan, R., Hoogerbrugge,
J., Sleddens-Linkels, E., Hoeijmakers, J. H., de Boer, P. and Grootegoed, J.
A. (2005). Silencing of unpaired chromatin and histone H2A
ubiquitination in mammalian meiosis. Mol. Cell. Biol.
25,1041
-1053.
Bel, S., Core, N., Djabali, M., Kieboom, K., Van der Lugt, N.,
Alkema, M. J. and Van Lohuizen, M. (1998). Genetic
interactions and dosage effects of Polycomb group genes in mice.
Development 125,3543
-3551.[Abstract]
Bornemann, D., Miller, E. and Simon, J. (1996).
The Drosophila Polycomb group gene Sex comb on midleg (Scm) encodes a zinc
finger protein with similarity to polyhomeotic protein.
Development 122,1621
-1630.[Abstract]
Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T.,
Mederios, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M.
K. et al. (2006). Polycomb complexes repress developmental
regulators in murine embryonic stem cells. Nature
441,349
-353.[CrossRef][Medline]
Cao, R., Wang, L., Xia, L., Erdjument-Bromage, H., Tempst, P.
and Zhang, Y. (2002). Role of histone H3 lysine 27
methylation in Polycomb-group silencing. Science
298,1039
-1043.
Celeste, A., Petersen, S., Romanienko, P. J.,
Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O. A., Reina-San-Martin,
B., Coppola, V., Meffre, E., Difilippantonio, M. J. et al.
(2002). Genomic instability in mice lacking histone H2AX.
Science 296,922
-927.
Cohen, P. E. and Pollard, J. W. (2001).
Regulation of meiotic recombination and prophase I progression in mammals.
BioEssays 23,996
-1009.[CrossRef][Medline]
de Napoles, M., Mermoud, J. E., Wakao, R., Tang, Y. A., Endoh,
M., Appanah, R., Nesterova, T. B., Silva, J., Otte, A. P., Vidal, M. et
al. (2004). Polycomb group proteins Ring1A/B link
ubiquitylation of histone H2A to heritable gene silencing and X inactivation.
Dev. Cell 7,663
-676.[CrossRef][Medline]
Fernandez-Capetillo, O., Mahadevaiah, S. K., Celeste, A.,
Romanienko, P. J., Camerini-Otero, R. D., Bonner, W. M., Manova, K., Burgoyne,
P. and Nussenzweig, A. (2003). H2AX is required for chromatin
remodeling and inactivation of sex chromosomes in male mouse meiosis.
Dev. Cell 4,497
-508.[CrossRef][Medline]
Foulkes, N. S., Mellstrom, B., Benusiglio, E. and Sassone-Corsi,
P. (1992). Developmental switch of CREM function during
spermatogenesis: from antagonist to activator. Nature
355, 80-84.[CrossRef][Medline]
Franke, A., DeCamillis, M., Zink, D., Cheng, N., Brock, H. W.
and Paro, R. (1992). Polycomb and polyhomeotic are
constituents of a multimeric protein complex in chromatin of Drosophila
melanogaster. EMBO J.
11,2941
-2950.[Medline]
Fujimura, Y., Isono, K., Vidal, M., Endoh, M., Kajita, H.,
Mizutani-Koseki, Y., Takihara, Y., Van Lohuizen, M., Otte, A. P., Jenuwein, T.
et al. (2006). Distinct roles of Polycomb group gene products
in transcriptionally repressed and active domains of Hoxb8.
Development 133,2371
-2381.
Fujiwara, Y., Komiya, T., Kawabata, H., Sato, M., Fujimoto, H.,
Furusawa, M. and Noce, T. (1994). Isolation of a DEAD-family
protein gene that encodes a murine homolog of Drosophila vasa and its specific
expression in germ cell lineage. Proc. Natl. Acad. Sci.
USA 91,12258
-12262.
Gebuhr, T. C., Bultman, S. J. and Magnuson, T.
(2000). Pc-G/trx-G and the SWI/SNF connection: developmental gene
regulation through chromatin remodeling. Genesis
26,189
-197.[CrossRef][Medline]
Habu, T., Taki, T., West, A., Nishimune, Y. and Morita, T.
(1996). The mouse and human homologs of DMC1, the yeast
meiosis-specific homologous recombination gene, have a common unique form of
exon-skipped transcript in meiosis. Nucleic Acids Res.
24,470
-477.
Hoyer-Fender, S., Costanzi, C. and Pehrson, J. R.
(2000). Histone macroH2A1.2 is concentrated in the XY-body by the
early pachytene stage of spermatogenesis. Exp. Cell
Res. 258,254
-260.[CrossRef][Medline]
Isono, K., Fujimura, Y., Shinga, J., Yamaki, M. O., Wang, J.,
Takihara, Y., Murahashi, Y., Takada, Y., Mizutani-Koseki, Y. and Koseki,
H. (2005). Mammalian polyhomeotic homologues Phc2 and Phc1
act in synergy to mediate polycomb repression of Hox genes. Mol.
Cell. Biol. 25,6694
-6706.
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A. and van
Lohuizen, M. (1999). The oncogene and Polycomb-group gene
bmi-1 regulates cell proliferation and senescence through the ink4a locus.
Nature 397,164
-168.[CrossRef][Medline]
Jürgens, G. (1985). A group of genes
controlling spatial expression of the bithorax complex in Drosophila.
Nature 316,153
-155.[CrossRef]
Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing,
J. R., Ashmun, R. A., Grosveld, G. and Sherr, C. J. (1997).
Tumor suppression at the mouse INK4a locus mediated by the alternative reading
frame product p19ARF. Cell
91,649
-659.[CrossRef][Medline]
Kessel, M. and Gruss, P. (1991). Homeotic
transformations of murine vertebrae and concomitant alteration of Hox codes
induced by retinoic acid. Cell
67, 89-104.[CrossRef][Medline]
Khalil, A. M., Boyar, F. Z. and Driscoll, D. J.
(2004). Dynamic histone modifications mark sex chromosome
inactivation and reactivation during mammalian spermatogenesis.
Proc. Natl. Acad. Sci. USA
101,16583
-16587.
Kim, J., Daniel, J., Espejo, A., Lake, A., Krishna, M., Xia, L.,
Zhang, Y. and Bedford, M. T. (2006). Tudor, MBT and chromo
domains gauge the degree of lysine methylation. EMBO
Rep. 7,397
-403.[Medline]
Klink, A., Lee, M. and Cooke, H. J. (1997). The
mouse synaptosomal complex protein gene Sycp3 maps to band C of chromosome 10.
Mamm. Genome 8,376
-377.[Medline]
Klymenko, T., Papp, B., Fischle, W., Köcher, T., Schelder,
M., Fritsch, C., Wild, B., Wilm, M. and Müller, J.
(2006). A Polycomb group protein complex with sequence-specific
DNA-binding and selective methyl-lysine-binding activities. Genes
Dev. 20,1110
-1122.
Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P.
and Reinberg, D. (2002). Histone methyltransferase activity
associated with a human multiprotein complex containing the Enhancer of Zeste
protein. Genes Dev. 16,2893
-2905.
Laible, G., Wolf, A., Dorn, R., Reuter, G., Nislow, C.,
Lebersorger, A., Popkin, D., Pillus, L. and Jenuwein, T.
(1997). Mammalian homologues of the Polycomb-group gene Enhancer
of zeste mediate gene silencing in Drosophila heterochromatin and at S.
cerevisiae telomeres. EMBO J.
16,3219
-3232.[CrossRef][Medline]
Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G.,
Levine, S. S., Kumar, R. M., Chevalier, B., Johnstone, S. E., Cole, M. F.,
Isono, K. et al. (2006). Control of developmental regulators
by Polycomb in human embryonic stem cells. Cell
125,301
-313.[CrossRef][Medline]
Levine, S. S., Weiss, A., Erdjument-Bromage, H., Shao, Z.,
Tempst, P. and Kingston, R. E. (2002). The core of the
polycomb repressive complex is compositionally and functionally conserved in
flies and humans. Mol. Cell. Biol.
22,6070
-6078.
Mahadevaiah, S. K., Turner, J. M., Baudat, F., Rogakou, E. P.,
de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W. M. and
Burgoyne, P. S. (2001). Recombinational DNA double-strand
breaks in mice precede synapsis. Nat. Genet.
27,271
-276.[CrossRef][Medline]
Mettus, R. V., Litvin, J., Wali, A., Toscani, A., Latham, K.,
Hatton, K. and Reddy, E. P. (1994). Murine A-myb: evidence
for differential splicing and tissue-specific expression.
Oncogene 9,3077
-3086.[Medline]
Miyagishima, H., Isono, K., Fujimura, Y., Iyo, Y., Takihara, Y.,
Masumoto, H., Vidal, M. and Koseki, H. (2003). Dissociation
of mammalian Polycomb-group proteins, Ring1B and Rae28/Ph1, from the chromatin
correlates with configuration changes of the chromatin in mitotic and meiotic
prophase. Histochem. Cell Biol.
120,111
-119.[CrossRef][Medline]
Montini, E., Buchner, G., Spalluto, C., Andolfi, G., Caruso, A.,
den Dunnen, J. T., Trump, D., Rocchi, M., Ballabio, A. and Franco, B.
(1999). Identification of SCML2, a second human gene homologous
to the Drosophila sex comb on midleg (Scm): a new gene cluster on Xp22.
Genomics 58,65
-72.[CrossRef][Medline]
Odorisio, T., Mahadevaiah, S. K., McCarrey, J. R. and Burgoyne,
P. S. (1996). Transcriptional analysis of the candidate
spermatogenesis gene Ube1y and of the closely related Ube1x shows that they
are coexpressed in spermatogonia and spermatids but are repressed in pachytene
spermatocytes. Dev. Biol.
180,336
-343.[CrossRef][Medline]
Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M. and
Nakatani, Y. (2002). A complex with chromatin modifiers that
occupies E2F- and Myc-responsive genes in G0 cells.
Science 296,1132
-1136.
Paro, R. (1995). Propagating memory of
transcriptional states. Trends Genet.
11,295
-297.[CrossRef][Medline]
Perry, J., Palmer, S., Gabriel, A. and Ashworth, A.
(2001). A short pseudoautosomal region in laboratory mice.
Genome Res. 11,1826
-1832.
Peterson, A. J., Mallin, D. R., Francis, N. J., Ketel, C. S.,
Stamm, J., Voeller, R. K., Kingston, R. E. and Simon, J. A.
(2004). Requirement for sex comb on midleg protein interactions
in Drosophila polycomb group repression. Genetics
167,1225
-1239.
Pirrotta, V. (1997). PcG complexes and
chromatin silencing. Curr. Opin. Genet. Dev.
7, 249-258.[CrossRef][Medline]
Plath, K., Talbot, D., Hamer, K. M., Otte, A. P., Yang, T. P.,
Jaenisch, R. and Panning, B. (2004). Developmentally
regulated alterations in Polycomb repressive complex 1 proteins on the
inactive X chromosome. J. Cell Biol.
167,1025
-1035.
Richler, C., Dhara, S. K. and Wahrman, J.
(2000). Histone macroH2A1.2 is concentrated in the XY compartment
of mammalian male meiotic nuclei. Cytogenet. Cell
Genet. 89,118
-120.[CrossRef][Medline]
Scherthan, H., Jerratsch, M., Dhar, S., Wang Y. A., Goff, S. P.
and Pandita T. K. (2000). Meiotic telomere distribution and
Sertoli cell nuclear architecture are altered in Atm- and Atm-p53-deficient
mice. Mol. Cell Biol.
20,7773
-7783.
Schumacher, A., Faust, C. and Magnuson, T.
(1996). Positional cloning of a global regulator of
anterior-posterior patterning in mice. Nature
384, 648.[Medline]
Sewalt, R. G., van der Vlag, J., Gunster, M. J., Hamer, K. M.,
den Blaauwen, J. L., Satijn, D. P., Hendrix, T., van Driel, R. and Otte, A.
P. (1998). Characterization of interactions between the
mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of
different mammalian polycomb-group protein complexes. Mol. Cell.
Biol. 18,3586
-3595.
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C.
T., Bender, W. and Kingston, R. E. (1999). Stabilization of
chromatin structure by PRC1, a Polycomb complex. Cell
98, 37-46.[CrossRef][Medline]
Singer-Sam, J., Robinson, M. O., Bellve, A. R., Simon, M. I. and
Riggs, A. D. (1990). Measurement by quantitative PCR of
changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during
mouse spermatogenesis. Nucleic Acids Res.
18,1255
-1259.
Strahl, B. D. and Allis, C. D. (2000). The
language of covalent histone modifications. Nature
403, 41-45.[CrossRef][Medline]
Sweeney, C., Murphy, M., Kubelka, M., Ravnik, S. E., Hawkins, C.
F., Wolgemuth, D. J. and Carrington, M. (1996). A distinct
cyclin A is expressed in germ cells in the mouse.
Development 122,53
-64.[Abstract]
Tanaka, S. S., Toyooka, Y., Akasu, R., Katoh-Fukui, Y.,
Nakahara, Y., Suzuki, R., Yokoyama, M. and Noce, T. (2000).
The mouse homolog of Drosophila Vasa is required for the development of male
germ cells. Genes Dev.
14,841
-853.
Tomotsune, D., Takihara, Y., Berger, J., Duhl, D., Joo, S.,
Kyba, M., Shirai, M., Ohta, H., Matsuda, Y., Honda, B. M. et al.
(1999). A novel member of murine Polycomb-group proteins, Sex
comb on midleg homolog protein, is highly conserved, and interacts with
RAE28/mph1 in vitro. Differentiation
65,229
-239.[CrossRef][Medline]
Trimarchi, J. M., Fairchild, B., Wen, J. and Lees, J. A.
(2001). The E2F6 transcription factor is a component of the
mammalian Bmi1-containing polycomb complex. Proc. Natl. Acad. Sci.
USA 98,1519
-1524.
Turner, J. M., Mahadevaiah, S. K., Benavente, R., Offenberg, H.
H., Heyting, C. and Burgoyne, P. S. (2000). Analysis of male
meiotic "sex body" proteins during XY female meiosis provides new
insights into their functions. Chromosoma
109,426
-432.[Medline]
Turner, J. M., Burgoyne, P. S. and Singh, P. B.
(2001). M31 and macroH2A1.2 colocalize at the pseudoautosomal
region during mouse meiosis. J. Cell Sci.
114,3367
-3375.[Medline]
Turner, J. M., Aprelikova, O., Xu, X., Wang, R., Kim, S.,
Chandramouli, G. V., Barrett, J. C., Burgoyne, P. S. and Deng, C. X.
(2004). BRCA1, histone H2AX phosphorylation, and male meiotic sex
chromosome inactivation. Curr. Biol.
14,2135
-2142.[CrossRef][Medline]
Turner, J. M., Mahadevaiah, S. K., Fernandez-Capetillo, O.,
Nussenzweig, A., Xu, X., Deng, C. X. and Burgoyne, P. S.
(2005). Silencing of unsynapsed meiotic chromosomes in the mouse.
Nat. Genet. 37,41
-47.[Medline]
Usui, H., Ichikawa, T., Kobayashi, K. and Kumanishi, T.
(2000). Cloning of a novel murine gene Sfmbt, Scm-related gene
containing four mbt domains, structurally belonging to the Polycomb group of
genes. Gene 248,127
-135.[CrossRef][Medline]
van de Vosse, E., Walpole, S. M., Nicolaou, A., van der Bent,
P., Cahn, A., Vaudin, M., Ross, M. T., Durham, J., Pavitt, R., Wilkinson, J.
et al. (1998). Characterization of SCML1, a new gene in Xp22,
with homology to developmental polycomb genes.
Genomics 49,96
-102.[CrossRef][Medline]
van der Vlag, J. and Otte, A. P. (1999).
Transcriptional repression mediated by the human polycomb-group protein EED
involves histone deacetylation. Nat. Genet.
23,474
-478.[CrossRef][Medline]
van Lohuizen, M., Tijms, M., Voncken, J. W., Schumacher, A.,
Magnuson, T. and Wientjens, E. (1998). Interaction of mouse
polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate
Pc-G complexes. Mol. Cell. Biol.
18,3572
-3579.
Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst,
P., Jones, R. S. and Zhang, Y. (2004). Role of histone H2A
ubiquitination in Polycomb silencing. Nature
431,873
-878.[CrossRef][Medline]
Watanabe, D., Yamada, K., Nishina, Y., Tajima, Y., Koshimizu,
U., Nagata, A. and Nishimune, Y. (1994). Molecular cloning of
a novel Ca(2+)-binding protein (calmegin) specifically expressed during male
meiotic germ cell development. J. Biol. Chem.
269,7744
-7749.
Xu, X., Aprelikova, O., Moens, P., Deng, C. X. and Furth, P.
A. (2003). Impaired meiotic DNA-damage repair and lack of
crossing-over during spermatogenesis in BRCA1 full-length isoform deficient
mice. Development 130,2001
-2012.
Yuasa, S. (1996). Bergmann glial development in
the mouse cerebellum as revealed by tenascin expression. Anat.
Embryol. 194,223
-234.[Medline]
Zhao, G. Q. and Hogan, B. L. (1996). Evidence
that mouse Bmp8a (Op2) and Bmp8b are duplicated genes that play a role in
spermatogenesis and placental development. Mech. Dev.
57,159
-168.[CrossRef][Medline]
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