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First published online 5 December 2007
doi: 10.1242/dev.008490
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1 PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi,
Saitama, 332-0012, Japan.
2 Division of Human Genetics, National Institute of Genetics, Research
Organization of Information and Systems, 1111 Yata, Mishima, 411-8540,
Japan.
3 Department of Genetics, the Graduate University for Advanced Studies, 1111,
Yata, Mishima, 411-8540, Japan.
Author for correspondence (e-mail:
tsado{at}lab.nig.ac.jp)
Accepted 12 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: X inactivation, Antisense regulation, Chromatin modification, Gene targeting, Mouse embryos
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The noncoding Xist (X-inactive specific transcripts) gene
(Borsani et al., 1991
;
Brockdorff et al., 1991;
Brown et al., 1991) and its
antisense Tsix gene (Lee et al.,
1999) comprise one of the sense-antisense pairs that has been most
extensively studied so far. These two noncoding genes play a pivotal role in X
inactivation in mammals via a mechanism by which the dosage differences of
X-linked genes between males and females are equalized by inactivating one of
the two X chromosomes in females during early development
(Lyon, 1961). Although either
the paternal or maternal X chromosome is silenced in a basically random
fashion (random X inactivation) in the embryonic tissues, which give rise to
the fetus, X inactivation is imprinted in the extraembryonic tissues, from
which the placenta and a part of the extraembryonic membranes are derived, in
such a way that the paternally derived X is preferentially inactivated
(imprinted X inactivation) (Takagi and
Sasaki, 1975). At the onset of X inactivation, Xist
expression is induced on one of the two X chromosomes, and its 17-kb noncoding
transcripts associate in cis along the chromosome and make it
transcriptionally inactive by recruiting protein complexes involved in
heterochromatinization. Xist and Tsix are reciprocally
imprinted in the extraembryonic tissues so as to be expressed from the
paternal and the maternal X chromosome, respectively
(Kay et al., 1993;
Lee, 2000;
Sado et al., 2001). Since
Xist is essential for X inactivation to occur in cis, an
Xist-deficient X never undergoes inactivation in either embryonic or
extraembryonic tissues (Marahrens et al.,
1997; Penny et al.,
1996). Paternal transmission of the mutated X, therefore, results
in the failure of imprinted X inactivation and eventually a selective loss of
female embryos soon after implantation owing to the presence of two active X
chromosomes in the extraembryonic tissues
(Marahrens et al., 1997). By
contrast, Tsix deficiency, when maternally inherited, induces the
expression of the normally silent maternal copy of Xist and
subsequent inactivation of the mutated maternal X in the extraembryonic
tissues (Lee, 2000;
Sado et al., 2001). This ends
up causing functional nullisomy of the X chromosome in these particular
tissues of both male and female embryos, and eventually in death at the early
postimplantation stage. It therefore seems likely that at the onset of X
inactivation, while cessation of Tsix expression allows upregulation
of Xist and triggers the inactivation process on the future inactive
X, continued expression of Tsix is required for the future active X
to preclude the activation of Xist on it. This is consistent with the
fact that the Tsix-deficient X is inactivated in all cells of the
embryonic tissues or differentiating female ES cells heterozygous for the
mutation (Lee and Lu, 1999;
Sado et al., 2001).
Interestingly, mutant male embryos, even though they inherit the Tsix
deficiency from the mother, can survive if the wild-type extraembryonic
tissues are provided by an experimental reconstruction using wild-type
tetraploid embryos, implying the existence of a Tsix-independent
mechanism for Xist silencing in the embryonic tissues
(Ohhata et al., 2006). All
these facts taken together indicate that the extraembryonic tissues appear to
depend more on Tsix to regulate Xist than the embryonic
tissues do.
Although it is clear that Tsix is a negative regulator of
Xist effective only in cis, until recently the molecular mechanism of
antisense regulation by Tsix at the Xist locus remained
poorly understood. Recent analyses of the Tsix-deficient X in embryos
or ES cells, however, suggested that Tsix silences Xist
through modification of the chromatin structure
(Navarro et al., 2005;
Sado et al., 2005;
Sun et al., 2006).
Furthermore, our recent study demonstrated that splicing products of
Tsix RNA are dispensable for Tsix-mediated Xist
silencing in mouse embryos (Sado et al.,
2006). Therefore, we are particularly interested in the biological
significance of the antisense transcription across the Xist promoter.
To address this issue, in the present study we studied a mutation in mouse
causing termination of Tsix transcription in exon 4 before the
transcription proceeded across the Xist promoter region. This
mutation, which essentially eliminated antisense activity in the upstream
region of Xist, caused inappropriate activation of Xist and
aberrant chromatin modification in the 5' region of Xist, like
the loss of function mutation of Tsix previously reported
(Sado et al., 2005). These
findings demonstrate that the antisense transcription across the Xist
promoter plays a crucial role in Tsix-mediated silencing of the
Xist gene.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-globin gene (IVS2) was amplified by
PCR using primers Hu.Gl.IVS2-1(+)35SalI and Hu.Gl.IVS2-1(-)33SalI and cloned
into pBluescriptII-SK(-) to produce pIVS2. A fragment containing a
polyadenylation sequence that originated from the mouse Pgk1 gene and
a triple polyadenylation sequence (a multiple polyadenylation sequence, mpA)
amplified from the genome of a Rosa26R mouse using tpA-1(-)41NotI and
tpA-2(+)37XhoI as primers was placed 3' to a floxed puromycin-resistance
gene driven by the mouse Pgk1 promoter (PgkPuro cassette) to produce
pfloxPgkPuro-mpA. The floxPgkPuro-mpA cassette thus created was inserted at
the unique PvuII site present in the IVS2 fragment in an antisense
orientation with respect to IVS2. A 9.0 kb genomic
EcoRI-BamHI fragment (104,306-113,284 in GenBank J421479)
was cloned into pBluescriptII-SK (-) lacking an XhoI and a
SalI site (p
XS-EB9.0). The IVS2 fragment containing the
floxPgkPuro-mpA cassette was subsequently introduced into pXS-EB9.0 at
the unique XhoI site present in exon 1 of Xist to generate
pTsix-mpA. Similarly, the IVS fragment containing only the PgkPuro cassette
was inserted at the XhoI site in pXS-EB9.0 to generate
pCont-X.
Generation of mice and ES cells carrying each of the TsixpA and XistIVS alleles
Each of the targeting vectors (pTsix-mpA and pCont-X) was electroporated
into R1 ES cells (Nagy et al.,
1993), and selection with puromycin was carried out as previously
described (Sado et al., 2005).
The expected homologous recombination was found in 1 out of 192 colonies and 4
out of 240 colonies when pTsix-mpA or pCont-X was introduced, respectively.
Chimeric males were produced and serially crossed with females heterozygous
for the Tsix-deficient X
(XXTsix) to transmit the mutated
paternal X to female offspring as previously described
(Sado et al., 2005). The
floxed puromycin-resistance gene was subsequently removed by crossing females
heterozygous for either TsixpA2lox or
XistIVS2lox with males, which ubiquitously expressed cre
recombinase, to derive the TsixpA and
XistIVS allele, respectively. Removal of the floxed
puromycin cassette in ES cells was carried out by transfection of
cre-expressing plasmid pBS185.
Expression analyses
The visceral endoderm was separated from the yolk sac mesoderm according to
Dandolo et al. (Dandolo et al.,
1993). RNA was prepared from the embryo proper, visceral endoderm,
placenta and ES cells using RNeasy (Qiagen). Thermal conditions for all the
following PCRs will be provided upon request.
For the analysis of antisense transcription in the region distal to the mpA sequence in ES cells, cDNA was synthesized from 5 µg total RNA using Thermoscript (Invitrogen) at 56°C with each of the following three different primers: (1) AS90RS, (2) R1910J and (3) R371P1 for Tsix-specific priming in combination with GapdR for Gapd, and subsequently PCR was carried out on 1/25 of the cDNA thus prepared. Primers Xist1175F and Xist1472R were used for cDNA primed by (1) AS90RS, and primers R700P2 and Xist-6(-)20 were used for cDNA primed by (2) R1910J and (3) R371P1. A Gapd sequence was amplified as a control using GapdF/GapdR2.
For the analysis of allelic expression of Xist, cDNA was prepared from total RNA of E13.5 embryo proper and visceral endoderm in a strand-specific manner as described above using primers Xist-7(-)20 and GapdR, and PCR was carried out using primer sets R371P1 and Xist-6(-)20, and GapdF and GapdR2 for Xist and Gapd, respectively. An amplified product of Xist was digested with PvuII and electrophoresed on a 4% NuSieve 3:1 agarose gel.
For the analysis of Tsix expression in the placenta, RT-PCR was
carried out on cDNA prepared by random priming as previously described
(Sado et al., 2005). A
proximal and a distal part of Tsix were amplified using primer sets
Tsix2F and Tsix2R, and Xist (-540) and Xist-10(-)20, respectively
(see below).
A northern blot was prepared using 20 µg total RNA from the embryo
proper at E13.5 as previously described
(Sado et al., 2005) and
serially probed with pR97E1 for Xist
(Sado et al., 1996) and a cDNA
fragment for Gapd.
Methylation analysis
Genomic DNA isolated from the embryo proper and visceral endoderm at E13.5
was digested with BclI in combination with either
methylation-sensitive restriction endonuclease SacII, HhaI,
PmaCI or HpaII, electrophoresed on a 0.7% agarose gel,
blotted on a nylon membrane and hybridized with either HS0.7 or BE0.6 as a
probe (Sado et al., 2005).
Chromatin immunoprecipitation
About 2x106 undifferentiated ES cells were used as a
source of chromatin for ChIP assays using an antibody against the CTD of RNA
polymerase II large subunit phosphorylated at serine 2 (H5) (Abcam) and normal
mouse IgG (Sigma) as a mock control. Sonicated soluble chromatin was incubated
with 3 µg of either H5 or mouse normal IgG overnight at 4°C. Chromatin
precipitated with H5 was purified by incubating for another 3 hours at 4°C
with anti-mouse IgM antibody raised in goat (Sigma), which had been previously
bound to Dynabead-Protein-G (Invitrogen). Washing and elution of
immunoprecipitated chromatin and extraction of DNA were carried out as
previously described (Sado et al.,
2005).
The visceral endoderm separated from the yolk sac mesoderm was individually
suspended in 10 mM Tris-HCl, pH 7.4, 3 mM CaCl2, 2 mM
MgCl2 including proteinase inhibitor Complete (Roche), to which an
equal volume of 10 mM Tris-HCl, pH 7.4, 3 mM CaCl2, 2 mM
MgCl2,1% NP-40, Complete was added. Following crosslinking by
formaldehyde, cells were collected by centrifugation and resuspended in SDS
lysis buffer. Subsequent procedures for the preparation of soluble chromatin
were carried out in the same manner as previously described
(Sado et al., 2005). To avoid
potential variation in the efficiency of immunoprecipitation among different
lots of commercially available polyclonal antibodies, we used monoclonal
antibodies raised against H3K4me2, H3K4me3, H3K9me2 and H3K27me3 (gifts from
Hiroshi Kimura and Naohito Nozaki), each of which had been previously bound to
anti-mouse IgG-Dynabeads 280. Normal mouse IgG was used as a mock control.
Real-time PCR
For quantitative analysis, real-time PCR was carried out as previously
described (Sado et al., 2006).
The primers used were: Tsix2F/Tsix2R (for region d in
Fig. 2A), Xist (-540)F/8692R
(for region g in Fig. 2A),
8111F/8418R (for region h in Fig.
2A), and Xist1F/Xist101R (for
Fig. 6).
Primers
Primer sequences were as follows: Hu.Gl.IVS2-1(+)35SalI,
5'-GGGTCGACTCAAGGTGAGTCCAGGAGATGTTTCAG-3'; Hu.Gl.I -
VS2-1(-)33SalI, 5'-GGGTCGACAGGAGCTGTTGAGATGAAAGGAGAC-3';
tpA-1(-)41NotI, 5'-TTATATTAAGGCGGCCGCATCAGCTTGATGGGGATCCAGAC-3';
tpA-2(+)37XhoI, 5'-TTCTAACTCGAGCAGAAGCTTGCAGATCTGCGACTCT-3';
AS90R2, 5'-TGCGGGATTCGCCTTGATTT-3'; Xist1472R,
5'-AGGGCAGGTCACATGACTTC-3'; Xist-10(-)10,
5'-ACAAAATGGCTCCTTGGTTC-3'; 21b80F,
5'-CCTGCAAGCGCTACACACTT-3'; F748P1,
5'-CAGGTAGTGCAATAACTCACAA-3'; 8111F,
5'-CTGCCACCTGCTGGTTTATT-3'; 8418R,
5'-ACATGAAAGAGATCAGACAC-3'; Xist1F,
5'-TGTTTGCTCGTTTCCCGTGGAT-3'; Xist101R,
5'-CATAAGGCTTGGTGGTAGGG-3'; GapdF, GapdR, GapdR2, R1910J, R371P1,
Xist21F, Xist(-540)F, Xist1175F, Xist1554R, Xist-6(-)20, Xist-7(-)20, R700R2
and 8692R were previously described (Sado
et al., 2005). Tsix2F and Tsix2R were described by Sado et al.
(Sado et al., 2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-globin locus (IVS2) and
inserted them in exon 1 of Xist in an appropriate orientation in the
targeting vector, as shown in Fig.
1B. We assumed that the inserted fragment would be spliced out
upon transcription as an intron from the primary transcript of Xist
and that the resultant processed transcript would behave as a functional
Xist RNA. ES cells harboring this allele
(TsixpA2lox) were produced by gene targeting
(Fig. 1B,C). In parallel, we
also used gene targeting to generate another mutant allele,
XistIVS2lox, which harbored the same intron without the
mpA cassette at exactly the same position as the
TsixpA2lox allele (see Fig. S1 in the supplementary
material). The selection marker was subsequently removed from both mutated
alleles by transient expression of cre recombinase to generate
TsixpA and XistIVS, which were
subjected to further analyses.
Transcription of Tsix was prematurely terminated in the Xist gene
To test whether Tsix transcription was effectively terminated
before reaching the Xist promoter on the X chromosome carrying the
TsixpA allele (XpA) but not on the X carrying
the XistIVS allele (XIVS), strand-specific
RT-PCR was carried out in the respective mutant ES cells. As shown in
Fig. 2A,B, the expected
fragment (PCR product e in Fig.
2A) was amplified in all cases in which cDNA was synthesized with
primer a located proximal to the mpA cassette. By contrast, PCR product f was
barely detectable in ES cells carrying TsixpA when either
primer b or c located downstream of the mpA cassette was used for cDNA
synthesis. The antisense transcription was evident in the regions proximal and
distal to the inserted intron in ES cells harboring the
XistIVS allele (Fig.
2B), demonstrating that the presence of the intron itself did not
affect the transcriptional elongation of Tsix in the relevant region.
These results suggested that the antisense transcription beyond the inserted
mpA cassette was dramatically reduced on XpA.
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; Morris et al.,
2005). Immunoprecipitated chromatin prepared from ES cells was
subjected to PCR using a primer set located in the proximal region of exon 2
of Tsix (PCR product d) and primer sets located in the promoter
region of Xist (PCR products g and h)
(Fig. 2A,C). In addition to
wild-type XY, XpAY and XIVSY male ES cells, male ES
cells carrying Xdc, on which Tsix is truncated owing to
the insertion of an IRESβgeo cassette at the distal region of exon 2
(Sado et al., 2005;
Sado et al., 2001), were
included in the assay. The elongation form of RNA pol II was evenly
distributed in exon 2 of Tsix in all types of ES cells, whereas it
was significantly reduced in the promoter region of Xist in
XpAY to a level comparable with that in XdcY, which
essentially lacks Tsix transcription
(Fig. 2C). The distribution of
RNA pol II phosphorylated at Ser2 was higher in the distal region of
Tsix (region g and h) than in the proximal region (region d) in XY
and XIVSY ES cells, a pattern that is commonly observed on
transcriptionally active genes (Ahn et al.,
2004; Morris et al.,
2005). It therefore seems reasonable to conclude that the majority
of the antisense transcription beyond the mpA cassette was significantly
diminished as expected.
The TsixpA allele prematurely terminates antisense transcription in vivo
The TsixpA2lox and XistIVS2lox
alleles were introduced into mice through chimeric males crossed with females
heterozygous for Tsix deficiency, and the puromycin-resistance gene
was subsequently removed to generate the TsixpA and
XistIVS allele, respectively, as previously described
(Sado et al., 2005). We first
examined whether these alleles behaved in the same manner as in the mutant ES
cells. Since Tsix is imprinted in the extraembryonic tissues to be
expressed from the maternal allele in both sexes
(Lee, 2000;
Sado et al., 2001), we used
total RNA isolated from embryonic day (E) 12.5 placentas of male embryos
recovered from TsixpA/+ and XistIVS/+
females crossed with wild-type males for RT-PCR. Transcription of
Tsix was evident in the proximal region of Tsix on the
mutated X carrying either TsixpA or
XistIVS (Fig.
2D). Similarly, spliced products of Tsix were also
produced from these mutated X chromosomes (data not shown). When PCR was
performed using a primer set located in the promoter region of Xist,
by contrast, the expected fragment was readily detectable in XIVSY
as well as wild-type XY placenta, but was significantly reduced in
XpAY (Fig. 2D).
Similarly, antisense transcription was barely detected in the promoter region
of Xist in the placenta isolated from XpAX females. These
results indicate that the antisense transcription runs across the
Xist promoter on XIVS as on the wild-type X, but is
effectively attenuated on XpA in vivo as expected.
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). Of 176 offspring obtained from 43 litters, 174 were males
and 2 were females (Fig. 3A).
One of the two females was XO and the remaining one had inherited the mutated
X. The strong bias toward male pups suggested that Xist on the
mutated X was dysfunctional and therefore unable to induce X inactivation.
When examined at E7.5, female embryos, although found at a reasonable ratio,
exhibited abnormal morphology typical of those suffering from the failure of
imprinted X inactivation (Fig.
3B,C) (Marahrens et al.,
1997). In the reciprocal crosses, the mutated X was maternally
transmitted to both male and female pups with the Mendelian ratio
(Fig. 3D). Taking advantage of
an X-linked GFP transgene (XGFP), the X inactivation pattern was
examined in female embryos heterozygous for TsixpA. As
shown in Fig. 3E,
XpAXGFP females recovered at E8.5 were virtually
negative for GFP fluorescence, indicating that XGFP had been
uniformly inactivated even in the embryonic lineage, where X inactivation
should occur basically at random. Taking all these findings together, we
concluded that the insertion of the extra intron unexpectedly made the
Xist gene dysfunctional and that as a result, XpA was
essentially defective in undergoing inactivation. Similarly, paternal
transmission of XistIVS resulted in embryonic lethality
(data not shown), demonstrating that the function of Xist on
XIVS was also impaired.
Loss of Tsix transcription in the Xist promoter region results in ectopic activation of the Xist gene
Although Xist on these mutated X chromosomes turned out to be
dysfunctional, the TsixpA allele successfully terminated
the antisense transcription as expected. This allowed us to fully address the
effects brought about by the loss of Tsix transcription in the
Xist promoter region on Xist silencing by using a previously
described approach (Sado et al.,
2005; Sado et al.,
2006).
We first examined whether the Xist locus became ectopically activated by the truncation of Tsix in E13.5 male embryos. Northern blotting of total RNA demonstrated that although wild-type male embryos did not express Xist at all, a significant amount of Xist RNA was expressed in XpAY embryos (Fig. 4A). We also carried out strand-specific RT-PCR using RNA isolated from the embryos proper (embryonic lineage) and the visceral endoderm (extraembryonic lineage) in both sexes recovered at E13.5 from TsixpA/+ females crossed with males carrying a Mus. m. molossinus (JF1)-derived X chromosome (XJF1). A PvuII site polymorphism between the laboratory strains and JF1 allowed us to assess the allelic expression of Xist in females. As shown in Fig. 4B, Xist on the X chromosome of the laboratory strain (Xlab) was predominantly expressed in the embryo proper of wild-type females, probably owing to the Xce effect, which is known to cause some bias in the choice of chromosome for X inactivation, whereas the great majority of Xist detected in the visceral endoderm was derived from XJF1, in agreement with imprinted paternal X inactivation. In both XpAXJF1 and XIVSXJF1 females, the mutated X should remain active in every cell because of the inability of Xist to induce inactivation. Xist on the active X is usually repressed in somatic cells. However, although the Xist locus on the mutated X in XIVSXJF1 females was appropriately repressed, it was ectopically activated in XpAXJF1 females in both embryonic and extraembryonic tissues (Fig. 4B). This was also the case in XpAY male embryos (Fig. 4B). By contrast, such ectopic activation of Xist was not observed on XIVS in either embryonic or extraembryonic lineages in either sex (Fig. 4B). It should be noted that although Xist on the mutated XpA was incompetent for inducing chromosomal inactivation, as mentioned above, the introduced intron was spliced out from the primary transcript as expected (data not shown). Nonetheless, these results suggest that the premature termination of Tsix transcription compromises the Tsix-mediated Xist silencing.
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Loss of Tsix transcription across the Xist promoter region results in reduced CpG methylation in the 5' region of Xist
Next, we studied the effect of the premature termination of Tsix
in exon 4 on the chromatin in the Xist promoter region. It is known
that CpG sites in the Xist promoter of the transcriptionally active
allele on the inactive X are largely unmethylated, whereas the
transcriptionally inactive allele on the active X is highly methylated
(Norris et al., 1994). Our
previous study demonstrated that Tsix is required for the
establishment of CpG methylation in the Xist promoter region
(Sado et al., 2005). We
therefore examined whether this Tsix function is impaired by the
premature termination. The methylation profile of the Xist promoter
region was examined in the embryo proper and in the visceral endoderm at E13.5
by Southern blotting using four different methylation-sensitive restriction
enzymes in combination with methylation-insensitive BclI. Although
this region on XIVS, which stays active, was completely methylated,
like that on the wild-type active X, in both the embryo proper and visceral
endoderm (see Fig. S2 in the supplementary material), a reduction in
methylation was evident on XpA
(Fig. 5), which also stays
active, especially in the extraembryonic tissues. Intriguingly, the decrease
in CpG methylation was much more prominent in the visceral endoderm than in
the embryo proper in both sexes, which is consistent with the difference in
the level of Xist expressed ectopically in these tissues. These
results suggest that the loss of Tsix transcription across the
Xist promoter region diminishes the function of Tsix and
causes a reduction in CpG methylation in the 5' region of Xist,
as was seen on Xdc (Sado et
al., 2005).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This was
accompanied by reduced CpG methylation and aberrant histone modifications in
the 5' region of Xist, especially in the extraembryonic
tissues. By contrast, neither ectopic activation of Xist nor aberrant
chromatin modifications were observed on the active X bearing
XistIVS, which retains the antisense transcription across
the Xist promoter. These findings strongly suggest that it was the
transcriptional attenuation of Tsix, not the introduction of the
exogenous DNA sequences, that compromised the function of Tsix in
establishing the transcriptionally repressive chromatin configuration in the
5' region of Xist. However, we cannot completely exclude the
possibility that the observed phenotype was caused by disruption of not only
Tsix function but also Xist function.
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; Sado et al.,
2001). Shibata and Lee further suggested that
Tsix-mediated Xist silencing requires concurrent
antiparallel transcription across the Xist gene
(Shibata and Lee, 2004). Here
we have demonstrated that TsixpA, which still retained the
transcription of the proximal 93% of the region encoding the Tsix
gene, failed to establish Xist silencing, suggesting that
Tsix has to be transcribed across the Xist promoter to
establish silencing of Xist. Although Air and
Kcnq1ot1 noncoding antisense RNAs have also been suggested to be
involved in chromatin modification, truncation of these RNAs impairs not only
expression of the respective sense partner but also allelic expression of
neighboring imprinted genes in the respective imprinted domains
(Mancini-Dinardo et al., 2006;
Sleutels et al., 2002). This
implies that the actions of Tsix and these noncoding genes are
distinct in terms of mediating the chromatin modifications.
The extraembryonic lineages primarily depend on Tsix to prevent Xist activation
The truncation of Tsix had distinct impact on the Xist
silencing mechanism in the embryo proper versus the visceral endoderm, which
belong to the embryonic and extraembryonic lineages, respectively. Real-time
PCR revealed that the level of ectopic expression of Xist was higher
in the visceral endoderm than in the embryo proper. Although a reduction in
CpG methylation in the 5' region of Xist was detected in both
lineages, it was more prominent in the visceral endoderm than in the embryo
proper in both sexes. Similarly, aberrant histone modifications were much more
evident in the visceral endoderm than in MEF (data not shown). It therefore
seemed likely that the truncation of Tsix had more profound effects
on the Xist silencing mechanism in the extraembryonic tissues than in
the embryonic tissues. We recently suggested that the embryonic tissues but
not the extraembryonic tissues possess a mechanism to shut off Xist
in a Tsix-independent manner if Xist becomes ectopically
expressed (Ohhata et al.,
2006). Since the majority of Xist RNA transcribed from
XpA is subject to the expected splicing of the intron introduced in
exon 1, this transcript is almost the same as that transcribed from the
wild-type Xist allele, except for the addition of some flanking
sequences of the intron. It is possible that the presence of such transcripts
activates the putative Tsix-independent Xist silencing
mechanism and induces some repressive modifications in the Xist
promoter region in the embryonic tissues. Since this mechanism is missing in
the extraembryonic tissues, the visceral endoderm would exhibit severer
effects on Xist silencing than the embryo proper or MEF as a result
of the truncation of Tsix.
The TsixpA allele renders the overlapping Xist gene dysfunctional
Although the truncation of Tsix inevitably altered the genomic
structure of the overlapping Xist gene, we assumed that the function
of Xist would be relieved if the exogenous sequence containing the
mpA cassette was removed by splicing. However, the TsixpA
allele accordingly designed did not act in the expected way. Paternal
transmission of XpA resulted in lethality of female offspring at
peri-implantation stages, probably owing to the failure of imprinted paternal
X inactivation in the extraembryonic lineages, suggesting that the
Xist on XpA was dysfunctional. Although the expected
splicing event was observed in both the mutant male ES cells and the visceral
endoderm isolated from the XpAY embryos (data not shown), it is
possible that the excision of this intron was not as efficient as we expected.
The splicing products were retained in the nucleus, like those transcribed
from the wild-type Xist allele, ruling out the possibility that the
exogenous intron derived from the protein-coding gene enhanced export of the
transcripts to the cytoplasm. Another possibility is that the ectopic
expression of Xist on XpA was not high enough to induce X
inactivation. In fact, the ectopic expression of Xist in the visceral
endoderm of XpAY embryos was about 30-40% of the level of
Xist expression in the visceral endoderm of wild-type female embryos,
and RNA fluorescent in situ hybridization (FISH) failed to detect accumulation
of Xist RNA in the mutant male MEFs (data not shown). Although we
cannot rule out the possibility that the antisense transcription across the
Xist promoter is not the sole event responsible for
Tsix-mediated Xist silencing, the incomplete activation of
Xist could be due to the structural alteration of a putative cis
element required for the full activation of Xist. The insertion of
the intron, in fact, increased the physical distance between the Xist
promoter and the YY1 binding sites recently reported to be necessary for
Xist expression (Kim et al.,
2006). This could have diminished the appropriate interaction
between them required to fully activate Xist. It is also formally
possible that the presence of the extra sequence left after the expected
splicing event has a negative impact on the function of Xist RNA.
Further analyses will shed light on the mechanism by which Xist
becomes fully activated and mediates the chromosomal silencing upon the
cessation of Tsix transcription on the same chromosome.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/227/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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