|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 15 November 2006
doi: 10.1242/dev.02670
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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.
3 PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi,
Saitama, 332-0012, Japan.
* Author for correspondence (e-mail: tsado{at}lab.nig.ac.jp)
Accepted 3 October 2006
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: X-inactivation, Antisense regulation, Xist, Tsix
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Kiyosawa et al.,
2003; Yelin et al.,
2003). The available evidence suggests that certain classes of RNA
are causally involved in not only gene regulation but also higher order
chromatin structure in various organisms
(Bernstein and Allis, 2005). In
addition, antisense transcription has often been implicated in either
transcriptional or post-transcriptional regulation of the sense partner or the
neighboring genes, although the exact molecular mechanisms have yet to be
determined (Katayama et al.,
2005).
It is known that X-inactivation in mammals, which ensures dosage
equivalence of X-linked genes between males and females
(Lyon, 1961), is regulated by
the noncoding Xist (X-inactive specific transcript) gene
(Brockdorff et al., 1992;
Brown et al., 1992) and its
antisense non-coding Tsix gene
(Lee et al., 1999) mapped in
the X chromosome inactivation center (Xic), a cytogenetically identified
master regulatory region of X-inactivation
(Avner and Heard, 2001).
Xist is essential for the initiation of X-inactivation to occur in
cis (Marahrens et al., 1997;
Penny et al., 1996), and its
expression is regulated by Tsix on the same chromosome in a negative
fashion (Lee, 2000;
Lee and Lu, 1999;
Sado et al., 2001). The
Xist/Tsix locus is probably one of the best-studied loci harboring a
sense-antisense pair of transcripts, and study of this locus should allow us
to obtain further insight into the molecular mechanisms underlying
antisensemediated gene regulation, which appears to be rather common in a
variety of systems.
The paternal X chromosome is selectively inactivated by imprinting in the
extra-embryonic tissues, which give rise to the placenta and part of the
extra-embryonic membranes (Takagi and
Sasaki, 1975), where the paternal copy of Xist is always
activated and the maternal one is never expressed
(Kay et al., 1993). Targeted
disruption of Tsix, when maternally inherited, induces ectopic
activation of the normally silent copy of Xist on the same X
chromosome in these tissues (Lee,
2000; Sado et al.,
2001). Subsequent inactivation of the mutated maternal X results
in functional nullisomy of the X chromosome in both males and females, and
eventual embryonic death at early postimplantation stages. Although the
targeted disruption of Tsix revealed that Tsix is a negative
regulator of Xist effective in cis, until recently little was known
about the molecular mechanisms underlying the antisense regulation at the
Xist locus. We recently showed that in the absence of Tsix,
the Xist locus fails to establish the repressive chromatin
configuration, and instead manifests the active modification in embryos,
suggesting that Tsix mediates Xist-silencing through
modification of the chromatin structure
(Sado et al., 2005). Others
also demonstrated Tsix-mediated chromatin effects using
undifferentiated and differentiating embryonic stem (ES) cells as an in vitro
model system (Navarro et al.,
2005; Sun et al.,
2006).
Tsix transcripts are subject to splicing and polyadenylation, like
common protein-coding RNAs (Sado et al.,
2001; Shibata and Lee,
2003). The major products of spliced Tsix RNA in
undifferentiated ES cells, which are about 2.7 kb and 4.3 kb in length on
northern blots, appear to be produced by differential usage of several polyA
signals found in exon 4 of Tsix
(Sado et al., 2001).
RT-PCR-based methods also revealed the presence of some variants produced by
alternative splicing (Sado et al.,
2001; Shibata and Lee,
2003). In addition, Tsix RNA exists in spliced and
unspliced forms, although the functional relevance of splicing remains
unknown. Because a significant fraction of the RNA is processed, and the
splicing variants identified so far all share exon 4, which is the only exon
possessing complementarity with the Xist sequence, it had been
postulated that the splicing products (especially the sequence in exon 4) play
an important role in the function of Tsix by masking the A-repeats in
Xist, a region known to be required for Xist-mediated
silencing (Wutz et al., 2002),
through base pairing (Shibata and Lee,
2003).
To obtain further insight into this issue, we studied the functional significance of spliced Tsix RNA produced by antiparallel transcription through the endogenous Xist locus. Here we eliminated the splicing products of Tsix in embryos by disturbing the splicing between proximal exons and exon 4. Loss of spliced Tsix RNA, however, compromised neither the Tsix-mediated Xist silencing nor the establishment of the proper chromatin configuration in the Xist promoter region. Our findings suggest that chromatin modulation at the Xist locus is not mediated by spliced Tsix RNA but rather nascent/unspliced Tsix RNA or the antisense transcription per se.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) was
digested with PmaCI and SmaI to release a fragment harboring
the splicing acceptor site of Tsix exon 4, which was then replaced
with a fragment containing an IRES-EGFP and a puromycin resistance gene
similarly to how a targeting vector for the Xist2lox
allele was produced (Sado et al.,
2005). The targeting vector thus produced was electroporated into
R1 ES cells (Nagy et al.,
1993), and chimeras were produced as described previously
(Sado et al., 2005). Briefly,
out of 96 colonies, three harbored the expected homologous recombination
(Tsix
SA2lox), and they were
serially injected into blastocysts to generate chimeras. In this particular
case, two female chimeras crossed with C57Bl/6 males happened to transmit the
TsixSA2lox allele to both male
and female pups. Those females carrying
TsixSA2lox were crossed with
males that ubiquitously expressed cre recombinase to derive animals carrying
TsixSA.
RT-PCR and northern blotting
RT-PCR was carried out on cDNA prepared from 1 µg of total RNA using
either random primer or strand-specific primers. For strandspecific RT-PCR,
cDNA was synthesized using R1910J as a primer for Tsix and Xist7(-)20
as a primer for Xist. Subsequent amplification of a common sequence
in Tsix/Xist was performed using primers R700P2 and
Xist6(-)20. A Gapd-specific primer, GapdR, was also included in the
cDNA reaction to amplify a Gapd sequence using GapdF and GapdR2 for
normalization in real-time PCR analysis. Real-time PCR was performed on cDNA
thus produced using SYBR Premix Ex Taq (Takara) and a LightCycler (Roche)
according to the manufacturers' instructions. The expression levels of each
sequence were the value normalized by Gapd based on a mean value of
the respective sequence obtained from two or three independent experiments.
Primer sequences used in this study were as described previously
(Sado et al., 2005;
Sado et al., 2001) except for
the following: 8111F, ctg cca cct gct ggt tta tt; 8420R, cca cat gaa aga gat
cag ac; EGFP1, ctt ctt caa gga cga cgg ca; EGFP2, ttg tac agc tcg tcc atg cc;
Tsix2F, caa tct cgc aag atc cgg tga; Tsix2R, agt gga tgc agg act caa gat.
RNA extracted from ES cells harboring the respective mutation was subjected
to northern hybridization as described previously
(Sado et al., 2001). An RNA
probe was prepared from p10 and p10R for detection of Tsix and
Xist, respectively (Sado et al.,
2001). For Gapd, a cDNA fragment was used as a probe.
Methylation analysis and DNaseI hypersensitive site assay
For methylation analysis of the Xist promoter region, Southern
hybridization was performed in exactly the same manner as described previously
(Sado et al., 2005).
The DNaseI hypersensitive site assay was carried out as previously
described (Sado et al., 2005),
with the exception of a 0.4 kb AflIII genomic fragment (Af0.4) being
used as a probe.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). This allele was created so as not to delete the
splicing acceptor site for exon 4 of the overlapping antisense Tsix
transcript (Fig. 1C). Although
the Xist gene was disrupted by the replacement, primary Tsix
transcripts were still appropriately processed to yield the major 2.7-kb and
4.3-kb RNAs with a polyA tail (Fig.
2D). We demonstrated that the function of Tsix was not
impaired by this alteration, and the repressive chromatin configuration was
properly established at the Xist promoter on the active mutated X
chromosome (Sado et al.,
2005). To elucidate the functional significance of spliced
Tsix RNA in the proposed Tsix-mediated chromatin regulation
at the Xist promoter, we attempted to disturb the splicing event by
deleting the splicing acceptor site for exon 4 of Tsix. Accordingly,
another targeting vector, which was very similar to the one used to derive the
Xist1lox allele, was created
(Fig. 1A). The difference
between these vectors was that the 5 ' arm either did or did not contain
a 0.6-kb PmaCI-EcoRI fragment harboring the splicing
acceptor site. Upon a correct targeting event, the splicing acceptor site was
successfully deleted on the single X chromosome in male ES cells by
replacement with the IRES-EGFP cassette
(TsixSA2lox)
(Fig. 1B,C). A puromycin
resistance gene was subsequently removed by transient expression of cre
recombinase in ES cells to generate the
TsixSA allele (data not
shown).
Splicing variants are not produced from the TsixSA allele in ES cells
We examined whether or not spliced Tsix RNA was eliminated in
TsixSA/Y ES cells by performing
RT-PCR using cDNA prepared by random priming. Unlike in XY and
Xist1lox/Y ES cells, a primer set spanning the introns
(Xist1175F and 21b80F) failed to amplify the product derived from spliced
Tsix RNA in TsixSA/Y
as well as in XdcY ES cells, which carry the
Xist1lox allele on a Tsix-deficient X chromosome
(Sado et al., 2005)
(Fig. 2A,B). The presence of
antisense transcripts was, however, evident in XY,
Xist1lox/Y, and
TsixSA/Y ES cells when another
primer set (8111F and 8420R) located in exon 4 was used, suggesting that the
antisense transcription itself was not disrupted. The relative abundance of
the antisense transcripts in each ES cell line was further analyzed by
real-time PCR using two primer sets designed to hybridize within either exon 2
(Tsix2F and Tsix2R) or exon 4 (8111F and 8420R)
(Fig. 2C). In XdcY,
antisense expression was detected in exon 2 at a level comparable to that in
XY but was barely detected in exon 4, which is consistent with the fact that
Tsix is truncated on Xdc
(Sado et al., 2005). On the
other hand, the level of antisense transcripts in
Xist1lox/Y and
TsixSA/Y turned out to be about
twice as high as that in wild-type XY ES cells in both regions. It is unclear
whether the transcription is upregulated or the stability of the antisense
transcripts is increased by the alteration introduced on the mutated X in
Xist1lox/Y and
TsixSA/Y. The absence of the spliced forms
in TsixSA/Y ES cells was
further confirmed by northern blotting using polyA RNA
(Fig. 2D). Although an RNA
probe specific to Tsix detected 2.7-kb and 4.3-kb bands in XY and
Xist1lox/Y ES cells, neither of these bands was detected
in TsixSA/Y or XdcY
ES cells. The hybridization signal observed in the high molecular weight
region in XY, Xist1lox/Y, and
TsixSA/Y, which was missing in
XdcY, probably represented unspliced Tsix RNA and/or
perhaps an incompletely processed one that contains the third intron. These
results confirmed that the
TsixSA allele was defective in
splicing between proximal exons and exon 4 as expected, with the antisense
expression activity being essentially unaffected.
|
SA/Y. Despite the
activation of the Xist locus, neither of the DNaseI hypersensitive
sites specifically found in the 5 ' region of the transcriptionally
active Xist allele in somatic cells (see
Fig. 6) was observed on the
mutated X in undifferentiated ES cells
(Fig. 2E). While the ectopic
activation of the Xist locus in XdcY was sustained even
after differentiation, expression of the chimeric transcripts in
Xist1lox/Y and
TsixSA/Y became downregulated
upon differentiation as embryoid bodies for 12 days
(Fig. 2F), suggesting that
developmental regulation of the Xist gene was compromised on the
Xdc chromosome, as previously described
(Sado et al., 2005) but not on
the mutated X in Xist1lox/Y and
TsixSA/Y. It is likely that the
repressive action of Tsix occurs after differentiation.
|
SA2lox were produced
through germline transmission from chimeric mice and subsequently crossed with
males expressing cre recombinase to derive animals carrying
TsixSA by excision of the
puromycin resistance gene (see Materials and methods)
(Fig. 1D). Since the
Xist gene on the mutated X was consequently destroyed, males failed
to transmit the TsixSA allele
to female offspring when crossed with wild-type females, as previously
described (Marahrens et al.,
1997; Sado et al.,
2005). The mutant allele was, however, transmitted to both male
and female pups from the mother at the expected ratio (data not shown).
|
|
SA, we studied the role of
spliced Tsix RNA in Tsix-mediated Xist silencing.
We first asked whether the splicing products had been eliminated in mice as in
TsixSA/Y ES cells. It is known
that Tsix is imprinted to be expressed from the maternal allele in
the extraembryonic tissues such as the placenta
(Lee, 2000;
Sado et al., 2001). RNA
isolated from the placenta at embryonic day (E) 13.5 was subjected to RT-PCR.
While the presence of the spliced products was evident in both wild-type males
and females and those carrying maternal Xist1lox with
primers spanning exon 2 and exon 4 (Xist1175F and 21b80F), such products were
not observed in TsixSA/+
females or TsixSA/Y males
(Fig. 3). In contrast, the
expected fragment was amplified in all cases with a primer pair (8111F and
8420R) located in exon 4. These results suggest that
TsixSA impaired the splicing
event but not the antisense transcription per se during embryonic
development.
Xist is properly repressed in the absence of spliced Tsix RNA
In previous studies, loss of function mutation of Tsix caused
activation of Xist in cis (Lee,
2000; Sado et al.,
2001), which was attributed to the failure to establish the
repressive chromatin configuration at the Xist promoter
(Sado et al., 2005;
Sun et al., 2006). We first
examined if the elimination of spliced Tsix RNA abrogated the proper
regulation of Xist on the mutated X chromosome carrying the
TsixSA allele. In female
embryos heterozygous for the respective mutation, as shown in
Fig. 4, the mutated X stays
active in every cell because of Xist-deficiency on it. In our assay
system, therefore, the expression of chimeric RNA from the mutated active X
would demonstrate defects in the mechanism of silencing of Xist. As
previously shown, XdcX female embryos expressed chimeric RNA due to
functional deficiency of Tsix
(Fig. 4). Such ectopic
activation of the Xist promoter on the mutated X was not, however,
observed in either Xist1lox/+ or
TsixSA/+ embryos
(Fig. 4), suggesting that the
Xist promoter was appropriately repressed on the mutated X chromosome
carrying TsixSA. Similarly, the
expression of chimeric RNA was detected in XdcY but not in
TsixSA/Y. These results
demonstrated that the Xist locus was properly silenced even in the
absence of spliced Tsix on the mutated X.
Appropriate chromatin configuration was established at the Xist promoter on the X carrying TsixSA
It is known that the Xist promoter is differentially methylated in
female somatic cells, with the active allele on the inactive X being
unmethylated and the inactive allele on the active X being methylated
(Norris et al., 1994). Our
previous study demonstrated that functional loss of Tsix impairs
establishment of complete methylation at CpG sites in the Xist
promoter in cis. To examine whether CpG methylation was affected by loss of
the Tsix splicing products, we carried out Southern blot analysis.
Genomic DNA isolated from both male and female embryos carrying
TsixSA at E13.5 was digested
with methylation-sensitive HhaI or SacII in combination with
methylation-insensitive BclI. As shown in
Fig. 5, the Xist
promoter was completely methylated on the mutated active X carrying
TsixSA at a level comparable to
that on the active X carrying Xist1lox as well as the
wild-type allele in the embryonic and extra-embryonic tissues in both sexes.
This result suggests that CpG methylation is established even in the absence
of spliced Tsix RNA.
We went on to study the chromatin structure at the Xist locus by
DNaseI hypersensitive site assays. Several DNaseI hypersensitive sites have
been found in the Xist promoter region
(Sado et al., 2005;
Sheardown et al., 1997). It is
known that HS1 and HS5 are specific to the inactive X, and HS3 is common to
both the active and inactive X. In a previous study, we showed that functional
deficiency of Tsix results in the failure to establish the closed
chromatin structure in the Xist promoter region. As shown in
Fig. 6, neither of the ectopic
DNaseI hypersensitive sites seen on the Xdc was detected on the X
carrying TsixSA in either male
or female embryos at E13.5. This result indicates that the closed chromatin
structure is established at the Xist promoter even in the absence of
spliced Tsix RNA.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
Tsix transcripts are peculiar in that although a fraction of them
remain unspliced, various patterns of splicing all remove most of the region
complementary to Xist RNA from the 40-kb primary transcript, except
for a 1.9-kb region present in the proximal end of Xist, to yield the
major processed products of length 2.7 kb and 4.3 kb
(Sado et al., 2001;
Shibata and Lee, 2003). In
addition, the distal part of the processed Tsix RNA thus produced
covers the region up to 1.8-kb upstream of the Xist transcription
start site, where no transcription has been detected in the sense orientation.
These features of the antisense transcripts at the Xist locus imply
that the 1.9-kb region common to Tsix RNA and Xist RNA is
relevant to the effect of Tsix on the antisense transcription. It has
been shown that the function of Tsix is impaired by truncation of
Tsix in the downstream region of Xist without deleting any
genomic elements (Luikenhuis et al.,
2001; Shibata and Lee,
2004). In addition, Shibata and Lee failed to rescue the
loss-of-function phenotype of Tsix caused by the truncation even
though a Tsix minigene was introduced immediately downstream of
Xist so as to be transcribed in the same orientation as Xist
under the control of an exogenous constitutively active promoter
(Shibata and Lee, 2004). These
studies suggest that Tsix action is not merely derived from the
processed transcripts but requires concurrent antiparallel transcription
through Xist.
|
|
;
Lee and Lu, 1999;
Sado et al., 2001) or
transcriptional attenuation of Tsix
(Luikenhuis et al., 2001;
Shibata and Lee, 2004), loss
of spliced Tsix RNA compromised neither the Tsix-mediated
Xist silencing nor the establishment of proper chromatin
configuration in the Xist promoter region. These results indicate
that the splicing products of Tsix RNA are unexpectedly dispensable
for the chromatin modulation in the Xist promoter region. It is
possible that unspliced Tsix RNA on its own is sufficient for
mediating the establishment of the repressive chromatin configuration in the
Xist promoter region. Alternatively, the RNA products are not
required and the antisense transcription per se, especially across the
Xist promoter region, is responsible for the bona fide effect
mediated by Tsix. Another Tsix mutant allele, which
prematurely terminates the antisense transcription in exon 4 of Tsix
before it runs across the Xist promoter, appears to be defective in
Tsix function despite the fact that more than 90% of the entire
Tsix transcription unit is still transcribed to produce truncated RNA
(T. Ohhata, Y.H., H.S. and T.S., unpublished). The antisense transcription
across the Xist promoter region, therefore, seems to be crucial for
Tsix-mediated Xist silencing. Considering all these findings
taken together, we currently speculate that the antisense transcription is
more important for the Tsix-mediated regulation than the RNA
products. It is still possible, however, that the distal region of unspliced
Tsix RNA covering the Xist promoter region is involved in
the establishment of the appropriate chromatin conformation, perhaps through
the formation of a secondary structure. Further study will help to clarify the
role of RNA-antisense transcription in the antisense regulation by
Tsix at the Xist locus.
Another unexpected observation in this study was the activation of the
Xist locus on the mutated X chromosome in undifferentiated
Xist1lox/Y and
TsixSA/Y ES cells. Unlike in
XdcY ES cells, however, Xist becomes downregulated upon
differentiation in these mutant cells. This is consistent with the finding in
the mutant embryos that Tsix's function is compromised on
Xdc but not affected on the mutated X chromosome carrying
Xist1lox and
TsixSA. Nonetheless, the
activation of the Xist locus in the undifferentiated state in
Xist1lox/Y and
TsixSA/Y as well as in
XdcY ES cells suggests that in these mutant cells, the genetic
alteration introduced at the Xist locus impaired the silencing
mechanism at the Xist locus, which would be independent of
Tsix function, in undifferentiated ES cells. It is possible that the
IRES-EGFP fragment introduced at the mutated locus somehow induces the
Xist promoter, although the chromatin structure as revealed by the
DNaseI hypersensitive site assay showed no difference between the wild-type
and mutated alleles. Alternatively, the deleted region in these mutant alleles
may contain a cis regulatory element required for Xist-silencing in
undifferentiated cells. It has been shown that Xist that has been
expressed from the paternal allele since the 2-4 cell stage, becomes
downregulated in a cell that has contributed to the inner cell mass (ICM) at
the blastocyst stage (Mak et al.,
2004; Okamoto et al.,
2004). Tsix is expressed only from the maternal allele,
most probably in the trophectoderm, at this stage
(Lee, 2000;
Sado et al., 2001) and,
therefore, repression of the paternal Xist in the ICM should be
mediated by a Tsix-independent mechanism. It is tempting to speculate
that the putative cis element mentioned above, if any, might be involved in
the silencing of the hitherto active Xist in undifferentiated ICM
cells. It will be of interest to see if the Xist locus on the
paternally derived mutated X chromosome carrying either
Xist1lox or
TsixSA is activated at the 2-4
cell stage and subsequently downregulated in the ICM cells at the blastocyst
stage.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Avner, P. and Heard, E. (2001). X-chromosome
inactivation: counting, choice and initiation. Nat. Rev.
Genet. 2,59
-67.[CrossRef][Medline]
Bernstein, E. and Allis, C. D. (2005). RNA
meets chromatin. Genes Dev.
19,1635
-1655.
Brockdorff, N., Ashworth, A., Kay, G. F., McCabe, V. M., Norris,
D. P., Cooper, P. J., Swift, S. and Rastan, S. (1992). The
product of the mouse Xist gene is a 15 kb inactive X-specific transcript
containing no conserved ORF and located in the nucleus.
Cell 71,515
-526.[CrossRef][Medline]
Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G.,
Xing, Y., Lawrence, J. and Willard, H. F. (1992). The human
XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved
repeats and is highly localized within the nucleus.
Cell 71,527
-542.[CrossRef][Medline]
Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.
C., Maeda, N., Oyama, R., Ravasi, T., Lenhard, B., Wells, C. et al.
(2005). The transcriptional landscape of the mammalian genome.
Science 309,1559
-1563.
Katayama, S., Tomaru, Y., Kasukawa, T., Waki, K., Nakanishi, M.,
Nakamura, M., Nishida, H., Yap, C. C., Suzuki, M., Kawai, J. et al.
(2005). Antisense transcription in the mammalian transcriptome.
Science 309,1564
-1566.
Kay, G. F., Penny, G. D., Patel, D., Ashworth, A., Brockdorff,
N. and Rastan, S. (1993). Expression of Xist during mouse
development suggests a role in the initiation of X chromosome inactivation.
Cell 72,171
-182.[CrossRef][Medline]
Kiyosawa, H., Yamanaka, I., Osato, N., Kondo, S. and
Hayashizaki, Y. (2003). Antisense transcripts with FANTOM2
clone set and their implications for gene regulation. Genome
Res. 13,1324
-1334.
Lee, J. T. (2000). Disruption of imprinted X
inactivation by parent-of-origin effects at Tsix. Cell
103, 17-27.[CrossRef][Medline]
Lee, J. T. and Lu, N. (1999). Targeted
mutagenesis of Tsix leads to nonrandom X inactivation.
Cell 99,47
-57.[Medline]
Lee, J. T., Davidow, L. S. and Warshawsky, D.
(1999). Tsix, a gene antisense to Xist at the X-inactivation
centre. Nat. Genet. 21,400
-404.[CrossRef][Medline]
Luikenhuis, S., Wutz, A. and Jaenisch, R.
(2001). Antisense transcription through the Xist locus mediates
Tsix function in embryonic stem cells. Mol. Cell.
Biol. 21,8512
-8520.
Lyon, M. (1961). Gene action in the
X-chromosome of the mouse (Mus musculus L). Nature
190,372
-373.[CrossRef][Medline]
Mak, W., Nesterova, T. B., de Napoles, M., Appanah, R.,
Yamanaka, S., Otte, A. P. and Brockdorff, N. (2004).
Reactivation of the paternal X chromosome in early mouse embryos.
Science 303,666
-669.
Marahrens, Y., Panning, B., Dausman, J., Strauss, W. and
Jaenisch, R. (1997). Xist-deficient mice are defective in
dosage compensation but not spermatogenesis. Genes
Dev. 11,156
-166.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder,
J. C. (1993). Derivation of completely cell culture-derived
mice from early-passage embryonic stem cells. Proc. Natl. Acad.
Sci. USA 90,8424
-8428.
Navarro, P., Pichard, S., Ciaudo, C., Avner, P. and Rougeulle,
C. (2005). Tsix transcription across the Xist gene alters
chromatin conformation without affecting Xist transcription: implications for
X-chromosome inactivation. Genes Dev.
19,1474
-1484.
Norris, D. P., Patel, D., Kay, G. F., Penny, G. D., Brockdorff,
N., Sheardown, S. A. and Rastan, S. (1994). Evidence that
random and imprinted Xist expression is controlled by preemptive methylation.
Cell 77,41
-51.[CrossRef][Medline]
Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D. and Heard,
E. (2004). Epigenetic dynamics of imprinted X inactivation
during early mouse development. Science
303,644
-649.
Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. and
Brockdorff, N. (1996). Requirement for Xist in X chromosome
inactivation. Nature
379,131
-137.[CrossRef][Medline]
Sado, T., Wang, Z., Sasaki, H. and Li, E.
(2001). Regulation of imprinted X-chromosome inactivation in mice
by Tsix. Development
128,1275
-1286.[Abstract]
Sado, T., Hoki, Y. and Sasaki, H. (2005). Tsix
silences Xist through modification of chromatin structure. Dev.
Cell 9,159
-165.[CrossRef][Medline]
Sheardown, S. A., Newall, A. E., Norris, D. P., Rastan, S. and
Brockdorff, N. (1997). Regulatory elements in the minimal
promoter region of the mouse Xist gene. Gene
203,159
-168.[CrossRef][Medline]
Shibata, S. and Lee, J. T. (2003).
Characterization and quantitation of differential Tsix transcripts:
implications for Tsix function. Hum. Mol. Genet.
12,125
-136.
Shibata, S. and Lee, J. T. (2004). Tsix
transcription-versus RNA-based mechanisms in Xist repression and epigenetic
choice. Curr. Biol. 14,1747
-1754.[CrossRef][Medline]
Sun, B. K., Deaton, A. M. and Lee, J. T.
(2006). A transient heterochromatic state in Xist preempts X
inactivation choice without RNA stabilization. Mol.
Cell 21,617
-628.[CrossRef][Medline]
Takagi, N. and Sasaki, M. (1975). Preferential
inactivation of the paternally derived X chromosome in the extraembryonic
membranes of the mouse. Nature
256,640
-642.[CrossRef][Medline]
Wutz, A., Rasmussen, T. P. and Jaenisch, R.
(2002). Chromosomal silencing and localization are mediated by
different domains of Xist RNA. Nat. Genet.
30,167
-174.[CrossRef][Medline]
Yelin, R., Dahary, D., Sorek, R., Levanon, E. Y., Goldstein, O.,
Shoshan, A., Diber, A., Biton, S., Tamir, Y., Khosravi, R. et al.
(2003). Widespread occurrence of antisense transcription in the
human genome. Nat. Biotechnol.
21,379
-386.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  Y. Hoki, N. Kimura, M. Kanbayashi, Y. Amakawa, T. Ohhata, H. Sasaki, and T. Sado A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse Development, January 1, 2009; 136(1): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohhata, Y. Hoki, H. Sasaki, and T. Sado Crucial role of antisense transcription across the Xist promoter in Tsix-mediated Xist chromatin modification Development, January 15, 2008; 135(2): 227 - 235. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
