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First published online 26 November 2008
doi: 10.1242/dev.026427
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1 Division of Human Genetics, National Institute of Genetics, Research
Organization of Information and Systems, 1111 Yata, Mishima 411-8540,
Japan.
2 Department of Genetics, The Graduate University for Advanced Studies
(Sokendai), 1111 Yata, Mishima 411-8540, Japan.
Author for correspondence (e-mail:
tsado{at}lab.nig.ac.jp)
Accepted 29 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: X-inactivation, Xist, Tsix, Gene targeting, Mouse embryo
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Several lines of
evidence indicate that the paternal X chromosome is preferentially inactivated
in the early preimplantation stage embryo
(Huynh and Lee, 2003;
Mak et al., 2004;
Okamoto et al., 2004). This
imprinted X-inactivation is maintained throughout development of the
extraembryonic tissues, such as the placenta and part of the extraembryonic
membranes (Takagi and Sasaki,
1975), which originate from the trophectoderm and primitive
endoderm of the blastocyst. In the epiblast lineage, a derivative of the inner
cell mass of the blastocyst, the previously inactivated paternal X transiently
restores transcriptional activity, and subsequently either the maternal or
paternal X undergoes inactivation in a basically random fashion as cells
differentiate (random X-inactivation).
It is known that the Xist (X-inactive specific transcript) gene
located in the X inactivation center (Xic), a cytogenetically identified
chromosomal region essential for X-inactivation to occur in cis, plays a
crucial role in both imprinted and random X-inactivation. Targeted disruption
of Xist renders the mutated X incompetent to undergo inactivation
(Marahrens et al., 1997;
Penny et al., 1996), and
therefore paternal transmission of Xist deficiency results in the
failure of imprinted paternal X-inactivation in the extraembryonic tissues
and, consequently, a selective loss of female embryos soon after implantation
owing to the extremely poor development of the extraembryonic tissues.
Xist encodes long non-coding transcripts as long as 17 kb in
length, which are subjected to splicing and polyadenylation like common
protein-coding RNAs (Brockdorff et al.,
1992; Brown et al.,
1992). The Xist RNA is peculiar in that it stays in the
nucleus and associates with the X chromosome, from which it is transcribed
(Brown et al., 1992;
Clemson et al., 1996), where it
eventually induces chromosomal silencing by unknown mechanisms. Although the
overall structure of the Xist gene is relatively conserved among
eutherian mammals, its nucleotide sequence diverges greatly
(Chureau et al., 2002;
Nesterova et al., 2001), which
is consistent with the presumed role of the Xist gene product as a
functional RNA. It is known, however, that several regions consisting of a
series of repeats are conserved between mouse and human
(Brockdorff et al., 1992;
Brown et al., 1992). Wutz et
al. previously showed that one of these repeats, known as the A-repeat, which
is located in the proximal part of the Xist RNA, is crucial for the
silencing function of the RNA (Wutz et
al., 2002). They demonstrated that the RNA transcribed from a
single copy Xist cDNA lacking the A-repeat driven by an inducible
promoter can accumulate on the X chromosome in male ES cells upon induction
but fails to initiate chromosomal silencing. The A-repeat contains 7.5 copies
of a conserved direct repeat unit, which harbors two short inverted repeats
that might fold into a secondary structure comprising two stem loops. These
findings imply that the stem loop structures might be the modules that
interact with the putative protein factors that are involved in chromosome
silencing.
Although the above inducible expression assay in ES cells identified for the first time a likely functional domain in Xist RNA responsible for chromosomal silencing, its significance for X-inactivation taking place in the developing embryo has not yet been addressed. In this study, we introduced a mutant Xist allele lacking the A-repeat into the mouse and examined its effects on X-inactivation in the embryo. Our results clearly demonstrate that deletion of the A-repeat rendered the mutated X incompetent to undergo inactivation in embryos, which was consistent with the previous ES cell assay. However, the incompetence of the mutated X to undergo inactivation was apparently due to the lack of Xist expression on the mutated X chromosome. This finding suggests an unexpected essential role of the A-repeat as a genomic element for the appropriate regulation of Xist and subsequent X-inactivation in the mouse embryo.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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A was constructed so that a floxed HSV-tk and
PGK-neo fragment from pflox (a gift from En Li. location?) was flanked by
genomic fragments derived from Bac clone 333J22 containing the Xist
gene as shown in Fig. 1A (the
5' arm, nucleotides 97, 704-106, 412 in AJ421479; the 3' arm,
nucleotides 107, 228-113, 284 in AJ421479). J1 ES cells
(Li et al., 1992) were
electroporated with pXBA as previously described
(Sado et al., 2005), and
selection was started 24 hours later in the presence of 250 µg/ml G418. Of
254 selected colonies, four harbored the expected homologous recombination
(XistA2lox). Chimeric males
were generated and crossed with females heterozygous for a Tsix
deficiency (Sado et al., 2001)
to facilitate germ-line transmission in the same manner as previously
described (Sado et al., 2005).
Females heterozygous for
XistA2lox were crossed with
CAG-cre transgenic males to derive pups carrying
XistA. Excision of the
selection marker in ES cells was performed by transient expression of Cre
recombinase using pBS185 (Life Technology).
Histology
Deciduas dissected out from the uterus were fixed in Bouin's fixative.
Following dehydration, deciduas were embedded in Technovit 7100 (Kulzer),
sectioned at 2 µm, and stained with Hematoxylin and Eosin.
Genotyping blastocysts
Blastocysts were flushed from the uterus at E3.5 and the zona pellucida was
removed by acid tyroid treatment. Each blastocyst was transferred to 10 µl
of water and heated for 3 minutes at 95°C. Five microliters of this
solution was used for two-round PCR with semi-nested primer sets for
genotyping and sexing. The primers used for the first round amplification were
R700P2 (wild-type-specific), dA1F
(XistA-specific),
Xist1395R, Zfy1 and Zfy2. Subsequently, the wild-type Xist,
XistA and Zfy sequences
were individually amplified in a second round PCR using R700P2/F1063AS for
wild-type Xist, dA1F/F1063AS for
XistA and Zfy1/Zfy4 for
Zfy. Primer sequences used in this study are shown in
Table 1.Total RNA was extracted
from the remaining 5 µl using Trizol (Invitrogen) in the presence of 10
µg of E. coli tRNA.
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), using Xist2281F and Xist 2424R as primers
for Xist, Tsix2F and P422R for Tsix, and GapdF and GapdR2
for Gapd. The expression levels of Xist and Tsix
were normalized to Gapd levels as previously described
(Sado et al., 2006).
For allelic expression analysis of X-linked genes in the trophoblast and in
E7.5 embryos, cDNA was synthesized from 1 µg of total RNA using an oligo-dT
primer, and PCR was carried out using G6pdF4 and G6pdR4 as primers for
G6pd, and HprtF4 and HprtR3 for Hprt. The amplified products
of G6pd and Hprt were subsequently digested with
DraI and HinfI, respectively
(Sugimoto and Abe, 2007).
For real-time PCR of individual F1 blastocysts isolated from
XJF1XJF1 females crossed with either
XAY or wild-type XY males, each embryo was lysed in 10 µl
of distilled water by heating for 3 minutes at 95°C, and RNA was prepared
using Trizol (Invitrogen) in the presence of 10 µg of E. coli
tRNA. Following DNase I treatment, cDNA was synthesized using an oligo-dT
primer using the whole RNA sample in a reaction of 20 µl. One microliter of
cDNA was subjected to real-time PCR amplification of a region containing a
SacI polymorphism between JF1 and the laboratory strain in exon 7 of
Xist with XistEx7F31 and XistEx7R20. Because Xist and
Tsix are reciprocally imprinted in the blastocyst, SacI
digestion of the product allowed the confirmation of specific amplification of
Xist and not Tsix.
For allelic expression analysis of Tsix in individual genotyped F1 blastocysts, two-round PCR was carried out using cDNA synthesized in a strand-specific manner with Tsix4R and GapdR. The first round PCR was carried out on whole cDNA produced using Tsix4F, Tsix4R, GapdF and GapdR (25 cycles). The second round PCR was carried out on one-twentieth of the first round reaction using Tsix4F and Tsix4R2 (33 cycles), or GapdF and GapdR2 (25 cycles). The amplified product of Tsix was subsequently digested with BsmAI.
RNA-FISH
An RNA probe was prepared by in vitro transcription with Cy3-UTP (Amersham
Pharmacia) and a plasmid (pBE1.5) containing a Xist cDNA fragment
(nucleotides 9829-11,335 in NR_001463 in GenBank) using T7 RNA polymerase.
Cytological preparations of blastocysts were made according to Okamoto et al.
(Okamoto et al., 2000).
Following examination of RNA-FISH, X- and Y-chromosome painting was carried out according to the manufacturer's instructions (Cambio) to determine the sex chromosome constitution.
RNA half-life assay
About 2.5x104 undifferentiated ES cells were seeded onto
each dish without feeder cells. The medium of each dish was replaced with that
containing 25 µg/ml of DRB (5,6-Dichloro-1-β-D-ribofuranosyl
benzimidazole, Calbiochem) on the following day and cells were collected every
three hours. One microgram of total RNA isolated at each time point was
converted into cDNA using an oligo-dT primer, and the level of Xist
and Tsix RNA was quantitated by real-time PCR using the primer sets
XistEx7F31/XistEx7R20 and Tsix4F/Tsix4R2, respectively. While
XistExF31/XistEx7R20 are located about 20-kb downstream from the 5' end
of Xist, Tsix4F/Tsix4R2 are located upstream of the transcription
start site of Xist. It was confirmed that there was no Tsix
cDNA in the reaction, which was long enough to serve as a template for
XistExF31/XistEx7R20. The abundance of the respective RNAs at each time point
was shown as the value relative to the abundance at 3 hours after the addition
of DRB.
Bisulfite sequencing
Bisulfite treatment of genomic DNA prepared from the trophoblast at E6.5
and sperm was carried out using a Bisulfast kit (TOYOBO), and DNA was purified
using an EZ kit (Zymo Research). Two-round PCR was carried out using
semi-nested primers. First round PCR was carried out with 25 cycles on whole
DNA prepared from the trophoblast or 300 ng of sperm DNA, and the second round
PCR was done with 30 cycles on one-twentieth of the reaction from the first
round PCR. Primer sequences will be provided upon request.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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A2lox) would functionally
disrupt the Xist gene, and that the mutated allele, when paternally
inherited, would result in a selective loss of female embryos soon after
implantation because of the failure of imprinted X-inactivation in the
extraembryonic lineages. We previously demonstrated, however, that this
female-specific lethality is sometimes rescued by the simultaneous presence of
a Tsix deficiency on the maternal X
(Ohhata et al., 2008;
Sado et al., 2005;
Sado et al., 2006).
Accordingly, the male chimeras were crossed with females heterozygous for the
Tsix deficient allele (Tsix)
(Sado et al., 2001) to
facilitate transmission of the
XistA2lox allele from fathers
to live female pups, as previously described. Consequently, females carrying
the Tsix allele and
XistA2lox allele on the
maternal and paternal X, respectively, were successfully recovered. They were
subsequently crossed with males expressing Cre recombinase ubiquitously to
derive XistA/+ females and
XistA/Y males. These animals
were apparently normal and fertile.
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A allele, we took advantage
of XistA/Y ES cells established
by transiently expressing Cre recombinase in
XistA2lox/Y ES cells (data not
shown). It is known that the Xist locus is transcribed at a very low
level in undifferentiated male ES cells, although this basal transcription is
eventually downregulated after differentiation. We examined the basal
transcription of Xist to determine whether it was affected by the
deletion of the A-repeat in undifferentiated
XistA/Y ES cells. Quantitative
RT-PCR with strand-specifically prepared cDNA revealed that, in
XistA/Y ES cells, the mutated
XistA RNA was expressed at a
level comparable to wild-type Xist RNA in the parental male ES cells
(Fig. 2A). In addition, the
XistA allele was downregulated
in the same manner as the wild-type allele after the induction of
differentiation (Fig. 2A).
These results demonstrated that neither the function of the Xist
promoter per se nor the mechanism for downregulating Xist on the
future active X was affected by the deletion.
We further analyzed the stability of
XistA RNA by treating cells
with DRB, an inhibitor of RNA polymerase II. Real-time PCR on cDNA prepared
from a series of DRB-treated cells demonstrated that there was no significant
difference in stability between wild-type and mutant Xist RNA
(Fig. 2B), indicating that the
deletion of the A-repeat did not impair the stability of the RNA.
Intriguingly, the expression level of Tsix was increased in the mutant male ES cells. The stability of Tsix RNA in the mutant ES cells was, however, comparable to that in wild-type ES cells (Fig. 2B). It seemed likely therefore that the higher expression of Tsix in the mutant was mediated not by an increased stability of the RNA (Fig. 2A), but by a higher level of transcription. The expression level of Tsix, however, declined once ES cells were induced to differentiate. This suggests that although the genetic alterations we introduced at the Xist locus somehow facilitated the transcription of Tsix on the mutated X, they did not affect the mechanism for downregulating Tsix upon differentiation.
Paternal transmission of XistA results in a selective loss of female embryos
The functional significance of the A-repeat in embryonic development was
first addressed by examining whether or not the mutated
XistA allele could be
transmitted to female pups from the father. Of 218 pups born to wild-type
females crossed with XistA/Y
males, 216 were male and 2 were female. One of the two females turned out to
be XO, where the X chromosome was maternal in origin, and the other female
inherited the XistA allele
(Fig. 3A), suggesting that most
female embryos had been lost in utero. In reciprocal crosses, the
XistA allele was transmitted to
apparently healthy female pups from the mothers at the expected ratio
(Fig. 3A). Thus, the selective
loss of females upon paternal transmission of the mutation was probably due to
defects in the imprinted X-inactivation in the extraembryonic lineages. When
embryos were dissected out at embryonic day (E) 6.5,
+/XistA (the maternal allele
precedes the paternal one by convention) female embryos, although found in a
reasonable number, were all stunted with an abnormal morphology that was
indistinguishable from that of female embryos carrying a dysfunctional
Xist allele derived from the father
(Fig. 3B). Histological
analysis revealed that the extraembryonic ectoderm was severely affected in
the morphologically abnormal embryos, which were most probably females
carrying the paternal XistA
allele (Fig. 3C). These embryos
were reminiscent of those carrying an extra-copy of the maternal X
(Goto and Takagi, 1998;
Tada et al., 1993) or the
paternally derived Xist-deficient X
(Marahrens et al., 1997).
These results strongly suggest that the deletion of the A-repeat severely
compromised the function of the Xist gene to initiate
X-inactivation.
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A) in the extraembryonic tissues. X-inactivation is
imprinted in the trophoblast, a derivative of the trophectoderm in the
blastocyst, in favor of the paternal X. The expression of X-linked genes was
analyzed using trophoblasts isolated from E6.5 embryos. Embryos were prepared
by crossing females carrying an X chromosome derived from JF1 (Mus m.
molossinus) with XAY males, so that the parental origin
of the X-linked gene transcripts could be addressed by the presence or absence
of restriction site polymorphisms between JF1 and the laboratory strains used
in this study (C57Bl/6 and 129). The regions harboring a polymorphism in the
X-linked G6pd and Hprt genes
(Sugimoto and Abe, 2007) were
amplified by RT-PCR and the products were subsequently digested with
DraI and HinfI, respectively. In the trophoblast of
wild-type female embryos (XJF1Xlab), the expression of
G6pd and Hprt was confined to the maternal alleles,
consistent with the imprinted paternal X-inactivation in this tissue
(Fig. 3D). By contrast,
expression of the paternal copy of these genes was evident in
XJF1XA females
(Fig. 3D). This result
demonstrated that genes on the paternally derived XA were,
at least in part, misexpressed in the trophoblast. It seemed likely therefore
that the paternal XA failed to undergo inactivation in the
extraembryonic lineages, where the paternal X is programmed to be
inactivated.
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A could
undergo inactivation in the embryonic lineage, where the X chromosome imprint
is no longer effective, by using wild-type males carrying EGFP transgenes on
the single X (XGFP) (Nakanishi
et al., 2002) as the father. It has been shown that the expression
of the transgene reflects the activity of XGFP
(Ohhata et al., 2004). In
wild-type female embryos recovered at E7.5, GFP fluorescence was observed in
the embryonic tissue but not in the extraembryonic tissue, as expected
(Fig. 3E). This substantiates
that the transgene used here serves as a good reporter for addressing the
activity of the X bearing it at this stage of embryo development. Female
embryos heterozygous for XistA,
which were morphologically indistinguishable from their wild-type littermates,
were essentially negative for GFP throughout the embryo, suggesting that
XGFP was uniformly inactivated in the embryonic lineage, which is
normally subject to random X-inactivation
(Fig. 3E). Furthermore, allelic
expression analysis of G6pd and Hprt revealed that both
genes were expressed exclusively from XA, and that the
transcripts from XJF1 were barely detectable in
XAXJF1 heterozygotes
(Fig. 3F). These results
indicate that, as is the case with the X carrying the dysfunctional
Xist allele, XA is incompetent to undergo
inactivation in the embryonic lineage, as well as in the extraembryonic
lineages.
Expression of Xist is diminished on the mutated X in the preimplantation embryo
The above finding demonstrates that the A-repeat plays an essential role in
X-inactivation during mouse development. This is consistent with the previous
report by Wutz et al. that Xist RNAs lacking the A-repeat fail to
initiate X-inactivation in transgenic ES cells
(Wutz et al., 2002). In
particular, one of the mutated Xist RNAs tested by Wutz et al.
(SX), which lacks almost the same region as the
XistA RNA expressed from
XA, is defective in silencing despite its accumulation on
the X chromosome. This observation predicts that
XistA RNA coats
XA but fails to induce chromosomal silencing at the onset of
X-inactivation in the embryo. To examine whether this was the case, RNA-FISH
was carried out using an Xist-specific RNA probe in the
preimplantation embryo, in which only the paternal copy of Xist is
expressed and accumulated in cis (Kay et
al., 1993; Sheardown et al.,
1997). Embryos were recovered from wild-type females crossed with
XistA/Y males at the eight-cell
and blastocyst stages. All the female embryos should inherit the
XistA allele in this cross. The
sex of each embryo was identified by painting with X- and Y-specific probes
afterwards (data not shown). RNA-FISH demonstrated that, although the
expression of XistA was
detected in XXA eight-cell embryos, the hybridization signal
was very faint, essentially like a pinpoint
(Fig. 4), and eventually
disappeared at the blastocyst stage (Fig.
4). In control female embryos, accumulation of Xist RNA
was detected as an intense signal at the eight-cell and blastocyst stages
(Fig. 4). In agreement with
these observations, real-time RT-PCR on total RNA of individual blastocysts
demonstrated that the level of Xist RNA in XXA was
much lower than that in XX. The level in XXA was, in fact,
almost the same as that in XY, which was nearly below the detection limit
(Fig. 5A). This excluded the
possibility that XistA RNA,
although expressed in the blastocyst, failed to coat the mutated paternal X
chromosome, and is in contrast to human XIST RNA lacking the A-repeat
expressed in tumor cells by an inducible promoter, which does not coat the X
chromosome (Chow et al., 2007).
These results raised the unexpected possibility that the failure of
XA to undergo inactivation was primarily due to the lack of
XistA RNA coating the mutated
X.
Tsix is ectopically activated on the paternal XA in the blastocyst
Available evidence suggests that Tsix, the expression of which
becomes detectable as early as the eight-cell to morula stage (Y.H. and T.S.,
unpublished) and is confined to the maternal allele in the preimplantation
embryo, prevents the upregulation of Xist on the maternal X during
the process of imprinted X-inactivation
(Lee, 2000;
Sado et al., 2001). Given this
negative effect of Tsix on Xist expression, it was of
interest to explore whether the expression of Tsix was affected on
the paternal XA at the blastocyst stage. Accordingly, RNA
fractions of single genotyped blastocysts recovered from
XJF1XJF1 females crossed with XAY
males were individually converted into cDNA using gene-specific primers and
subjected to two-round PCR. The parental origin of the Tsix
transcripts was subsequently addressed by restriction digestion with
BsmAI, the recognition site of which is present only on the
laboratory strain-derived X chromosome
(Sugimoto and Abe, 2007).
Intriguingly, the paternal copy of Tsix, which is normally silent at
the blastocyst stage, was ectopically expressed from the mutated paternal
XA in XXA. Such ectopic activation of
Tsix was not observed in blastocysts that inherited either the
wild-type X or the X chromosome carrying another Xist mutant allele,
Xist1lox (Sado et al.,
2005), from the father (Fig.
5B; data not shown). This finding raised an interesting
possibility: that the unexpected silencing of Xist in
XXA embryos during preimplantation development might be
ascribed to the ectopic activation of the normally silent paternal copy of
Tsix on XA. Given the proposed function of
Tsix in the establishment of repressive chromatin in the
Xist promoter region (Navarro et
al., 2005; Sado et al.,
2005; Sun et al.,
2006), it is reasonable to assume that Tsix ectopically
activated on XA attracts repressive chromatin modifications,
which are not normally associated with the paternal allele, onto the
Xist promoter region in cis in the tissues where X-inactivation is
imprinted. Accordingly, the methylation status of 19 CpG sites in the
Xist promoter (McDonald et al.,
1998) was examined on the paternal XA in the
trophoblast isolated from XJF1XA embryos at E6.5.
The methylation profile of the maternal JF1-type allele could not be addressed
here because the trophoblast samples inevitably contained maternal tissues
with two XJF1 chromosomes. Bisulfite sequencing revealed that
whereas the paternal allele in XJF1XB6 embryos was
essentially unmethylated, as previously described
(McDonald et al., 1998),
significant methylation was evident on the paternal XA in
XJF1XA (Fig.
5C). It seems possible therefore that the ectopic expression of
Tsix facilitates CpG methylation at the Xist promoter on the
paternal XA in the trophoblast.
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A are not methylated in sperm).
Bisulfite sequencing revealed that the Xist promoter in sperm
exhibited even lower methylation in mutant XAY males than in
wild-type males (Fig. 6A). The
significance of this difference in the methylation levels is not known at
present, but this result indicates that the aberrant methylation found in the
Xist promoter on the paternal XA in the trophoblast
arises during embryogenesis.
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).
One of these domains, HS6, in Xite has been relatively well
characterized by bisulfite sequencing and has been shown to be heavily
methylated in sperm. It was postulated that if the methylation of this region
in sperm was causally involved in the repression of paternal Tsix in
the preimplantation embryo, it might be abolished in sperm isolated from
XAY males. We therefore assessed the methylation status of
this region in sperm of the mutant males. As shown in
Fig. 6B, this region was
heavily methylated on XA as on the wild-type X, suggesting
that the methylation status of HS6 in the Xite region was not
directly involved in the ectopic activation of Tsix on the paternal
XA chromosome. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). This
finding was further corroborated by the functional assay of Xist RNA
lacking the A-repeat expressed from the endogenous locus by the inducible
promoter in male ES cells (Wutz et al.,
2002). Although that study highlighted for the first time the
possible functional domain of Xist RNA that is crucial for inducing
chromosome-wide silencing, the importance of the A-repeat for X-inactivation
occurring in the mouse embryo had not been addressed until this study. The
XistA allele was created so
that it would produce a transcript nearly the same as the one lacking the
A-repeat expressed from the endogenous Xist locus upon induction in
the study by Wutz et al. (Wutz et al.,
2002). Genetic and molecular analyses of female embryos
heterozygous for XistA clearly
demonstrated that the deletion of the A-repeat rendered the mutated X
incompetent to undergo inactivation, indicating the crucial role of the
A-repeat in X-inactivation during mouse development. The presence or absence
of the A-repeat, however, does not seem to be directly involved in the primary
choice of X-inactivation in the embryonic lineage, as different deletions in
the Xist gene, although they retain the A-repeat, have resulted in
primary non-random X-inactivation similar to that observed in this study
(Marahrens et al., 1998;
Sado et al., 2005).
We initially postulated that the failure of X-inactivation could be
ascribed to the defect in XistA
RNA, which should be capable of coating the mutated X based on the inducible
expression assay in ES cells (Wutz et al.,
2002). Intriguingly, it was found that the level of
XistA RNA was greatly reduced
in XXA preimplantation embryos compared with that of
wild-type Xist RNA in XX embryos. This could be due to a reduction
either in the stability of the mutated RNA or in the expression level per se.
Quantitative RT-PCR demonstrated, however, that the stability of Xist
RNA detected in undifferentiated male ES cells was comparable regardless of
the presence or absence of the A-repeat, suggesting that the latter
possibility was more favorable. It is likely therefore that the reduction in
the level of Xist RNA in preimplantation embryos is primarily due to
the transcriptional silencing of Xist. Our result demonstrates that
the region encoding the A-repeat plays a crucial role as a regulatory element
in the appropriate regulation of Xist in vivo.
Intriguingly, the lack of Xist RNA in the blastocyst was
accompanied by an ectopic activation of the normally silent paternal copy of
Tsix on the same X chromosome. Furthermore, the Xist
promoter on the mutated paternal X in the trophoblast at E6.5 was aberrantly
methylated at CpG sites that are normally unmethylated on the paternal X.
These findings raised an interesting possibility that the transcriptional
silencing of Xist in preimplantation embryos is triggered by
ectopically expressed Tsix, which subsequently promotes CpG
methylation in the Xist promoter region on the mutated paternal X. In
this scenario, the deleted region in the
XistA allele, most probably the
A-repeat, is crucial for the appropriate repression of Tsix on the
paternal X at the onset of imprinted X-inactivation. Given the fact that the
transcription of Tsix is initiated 40 kb downstream from the
A-repeat, it is tempting to speculate that the A-repeat exerts its effect on
Tsix through a long-range chromatin conformation. However, the
opposite scenario is also possible: the upregulation of Tsix was
somehow caused by the primary silencing of Xist that resulted from
the loss of some crucial regulatory element located within the deleted region.
Because the aberrant methylation of the Xist promoter appears to be
established during embryogenesis, it might be expected that the mutated
XistA RNA would be transcribed
from the paternal XA in the early preimplantation embryo.
This was not the case, however, and the expression of paternal
XistA was diminished from the
very early stages. This observation may favor the later scenario that the
silencing of Xist is the primary event. These two possibilities
cannot be distinguished between on the basis of current data and the further
experimentation is certainly required. The simplest way to address this issue
is to terminate Tsix on XA and see whether
XistA is expressed or not. We
are currently trying to produce mice carrying a Tsix deficiency on
XA through second gene targeting in A2lox211 ES
cells.
Although the targeted deletion of the A-repeat did not allow us to address the functional significance of the A-repeat as an element in the Xist RNA because of the unexpected lack of expression from the mutated X, this study clearly demonstrates that the region encoding the A-repeat is essential as a genomic element for X-inactivation in the mouse embryo. Further attempts to identify the factors that interact with the DNA sequence harboring the A-repeat should provide further insight into the molecular mechanisms of Xist/Tsix regulation and the random choice of X-inactivation.
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Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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