INTRODUCTION

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Dosage compensation in mammals is achieved by a unique mechanism that leads to transcriptional silencing of almost all genes present on one of the two X chromosomes in female cells, a process known as X-chromosome inactivation (X inactivation) (24). In undifferentiated cells, all X chromosomes are active and X inactivation is initiated at the onset of differentiation both in vivo and in vitro (31). The number of X chromosomes that are to be inactivated is determined relative to the ploidy of the cell, and X inactivation is initiated only when the number of X chromosomes exceeds one in a diploid nucleus. Each cell then makes the epigenetic choice to keep one X chromosome active and to inactivate all supernumerary X chromosomes (the n - 1 rule, with n the total number of X chromosomes in the cell, and n - 1 the number of X chromosomes that are being inactivated). One model suggests the presence of a blocking factor in limited quantities that can only protect one X chromosome from inactivation per diploid set of chromosomes (2). X-chromosome choice is random in the embryonic lineage of the mouse and other mammals but is influenced by a number of epigenetic and genetic factors. In metatherian mammals, such as kangaroos (8), and in the extraembryonic tissues of some eutherian mammals, including mice (43), the paternal X chromosome is imprinted and always undergoes inactivation. In addition, the choice in embryonic tissues of mice is influenced by the X-controlling element (Xce) such that X inactivation is skewed toward the chromosome possessing the weaker Xce allele (29). It has been shown that at least four alleles exist in mice: Xce^a, Xce^b, Xce^c, and Xce^d, with Xce^a being the weakest and Xce^d being the strongest allele.

X-autosomal translocations and transgenic studies in mice have defined a master switch required for X inactivation, the Xic (X-inactivation center), a multifunctional locus carrying elements for choosing and silencing (14, 20, 21, 36). One of these elements is the Xist gene, which produces a spliced and polyadenylated untranslated RNA that coats the inactive X chromosome (4, 5, 6, 28). Targeted deletions have demonstrated that Xist is necessary for both the initiation of X inactivation and the silencing of X-linked genes (26, 34), as well as for X-chromosome choice (25). Xist RNA shows a distinctive expression pattern during differentiation as analyzed by fluorescent in situ hybridization (FISH). Prior to the onset of X inactivation, Xist is expressed in an unstable form from every X chromosome in male and female cells. Upon differentiation Xist transcription is downregulated in male cells and on the future active X in female cells. On the future inactive X the Xist transcript is stabilized and accumulates along the chromosome in cis (33, 41).

Recently, a new Xic element, Tsix, has been identified as a potential negative regulator of Xist expression. Tsix is an antisense gene to Xist whose major promoter is located 15 kb downstream of the Xist gene (18). The promoter overlaps with the previously defined DXPas34 locus (18), a 2.4-kb CpG island that is hypermethylated on the active X chromosome (as opposed to other CpG islands on the active X which remain hypomethylated) (9). The primary Tsix transcript completely overlaps Xist and has been shown to cover at least 40 kb (18). Recently, Tsix has been shown to undergo complex processing including alternative splicing and polyadenylation (39). According to this study the Tsix gene contains four exons, one of which (exon 4) overlaps with the highly conserved XCR. However, the functional significance and cellular localization of the spliced transcript remain to be characterized.

Like Xist, Tsix shows a characteristic expression pattern during X inactivation. In undifferentiated ES cells, Tsix is transcribed at low levels from all X chromosomes. After differentiation, Tsix transcription is shut off in adult tissues. Interestingly, at the onset of X inactivation Tsix expression becomes confined to one of the two X's and appears to specifically mark the future active X leading to the hypothesis that Tsix may regulate Xist expression during the initiation of X inactivation (10, 18, 30).

Targeted deletions that encompass the Tsix promoter, as well as the CpG island, were shown to lead to skewed Xist expression and nonrandom inactivation of the mutated X chromosome in ES cells. Introduction of the targeted alleles into mice demonstrated the importance of this locus for imprinted X inactivation in extraembryonic tissues (17, 39). Tsix expression in early blastocysts (3.5 days post coitum) is imprinted oppositely of Xist such that it is transcribed only from the maternal allele. Expression from both alleles is observed in late blastocysts at the time when the imprint is erased and both X's are primed for random X inactivation. Paternal inheritance of the deletion has no effect, but maternal inheritance leads to ectopic expression of maternal Xist in males and biallelic expression from both X chromosomes in female extraembryonic tissues. The majority of the embryos that inherit the deletion from the mother die during development due to placental defects. Lee proposed the presence of an imprinting center within the CpG-rich domain overlapping the Tsix promoter which responds to activating maternal and repressive paternal signaling to facilitate imprinted Tsix expression (17). Analysis of the DXPas34 locus and the Tsix promoter by bisulfite sequencing revealed that methylation does not represent the imprinting mark and is also unlikely to play a role in the regulation of Tsix transcription from this site (35). However, it cannot be excluded that the imprinting is associated with a methylation-independent mechanism. Taken together, in vitro and in vivo data suggest that the Tsix locus and/or transcript influences X-chromosome choice and imprinting of the X chromosome.

A number of genes have been identified whose imprinted expression is associated with the transcription of noncoding RNAs. For example at the H19/Igf2 locus, H19 encodes an untranslated RNA whose expression is imprinted oppositely to Igf2 (32). In many instances these noncoding RNAs are transcribed in the antisense direction, as is the case for the Igf2r gene and its antisense transcript Air (46). Antisense transcripts have also been identified at the UBE3A locus (38) and the Gnas locus (23). However, no evidence for the functionality of the transcripts exists in any of these cases. In fact the only noncoding transcript that has been analyzed for its functionality, H19, has been shown to play no role in the regulation of Igf2 (16, 40).

In the case of Tsix the role of antisense transcription also still remains to be established. Published results relied on targeted deletions that not only abolished Tsix transcription but also eliminated significant portions of genomic sequence such as portions of the DXPas34 locus that might harbor crucial regulatory elements (17, 19, 39).

Here we determined the role of Tsix transcription in random X inactivation by using embryonic stem (ES) cells as a model system for random X inactivation (19, 21, 34, 37). Two approaches were chosen to modulate Tsix transcription with minimal perturbation of genomic sequences. In the first approach, Tsix transcription was functionally inhibited by introducing a transcriptional stop signal into the transcribed region of Tsix. In the second approach, we created an inducible system for Tsix expression. We found that the truncation of the Tsix transcript recapitulates the phenotype of a targeted Tsix deletion, leading to complete nonrandom inactivation of the targeted X chromosome. Induction of Tsix transcription during ES cell differentiation, on the other hand, caused the targeted chromosome always to be chosen as the active chromosome. These results for the first time establish a function for antisense transcription in the regulation of X inactivation.

	MATERIALS AND METHODS

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Plasmid construction. The 5-kb SacII-SalI fragment of fragment 2 (cosmid MB4-14A, accession number U41395) cloned into pBluescript (Stratagene), pSacSal, was used for all targeting constructs. This fragment contains a unique HindIII site located 10,959 bp away from the end of the Xist cDNA (4) (GenBank accession number L04961) as an insertion site.

Cell culture and generation of transgenic ES cells. Undifferentiated ES cells were grown on mouse embryonic fibroblasts in Dulbecco modified Eagle medium (Gibco), 15% fetal calf serum (HyClone), and 1,000 U of leukemia-inhibiting factor (LIF)/ml. Differentiation of ES cells was induced in medium containing 10% fetal calf serum without LIF and with 40 ng of all-trans-retinoic acid (Sigma)/ml. Doxycycline (Sigma) was added to the medium at a final concentration of 1 µg/ml.

Southern probes. The 1-kb probe is a 986-bp PCR product specific for a region 2,207 to 3,193 bp downstream of the insertion site (relative to the direction of the Xist gene) and was created by using the primers indF (5'-AAGGCAGGGATTTTAGCGAT-3') and IndR (5'-TGCAGCCATTCTTTTCTGTG-3'). This probe was specific for a region outside the right arm of the targeting vector. The 3' internal probe is an 876-bp PCR product specific for a region 1,291 to 2,167 bp downstream of the insertion site (right arm of both constructs) obtained by using primers HS3intF (5'-AATCCGCATCAAAACCAAAG-3') and HS3intR (5'-GCAGAGCAGAGGTGATGAAA-3'). The 5' internal probe is a 183-bp PCR product specific for a region 743 to 926 bp upstream of the insertion site (left arm of the construct) created by using primers 5s and 5as (see below).

RT-PCR and primers. RNA was prepared by using RNAzol B reagent (Teltest) and treated with RNase-free DNase I (Gibco) according to the manufacturer's instructions. Strand-specific reverse transcription-PCR (RT-PCR) was carried out essentially as described previously (18) by using 1 µg of total RNA per reaction. Control reactions that were carried out in the absence of reverse transcriptase were included in all cases. The following primers are described relative to the first bp of Xist cDNA: 1s (positions 272 to 294), 1as (positions 755 to 775), 2s (positions 10162 to 10183), 2sE7 (positions 11011 to 11030), and 2as (positions 11279 to 11308). The following primers are located relative to the end of Xist cDNA: 3s (positions 1131 to 1150), 3as (positions 1553 to 1572), 4s (positions 5118 to 5757), 4as (positions 6142 to 6161), 5s (positions 10033 to 10056), 5as (positions 10197 to 10216), 6s (positions 12250 to 12270), and 6as (positions 12714 to 12734). Primers 2s and 2as were used to amplify a product specific for the spliced Xist transcript (referred to as primer pair 2a). Primers 2sE7 and 2as were used to amplify the Tsix transcript.

Allele specific detection of Xist and Tsix The NcoI polymorphism in Xist exon 1 has been described previously (19). First-strand synthesis with primers specific for Xist (1as) or Tsix (1s), followed by PCR and detection, was as described previously (19). PCR was carried out for 28 cycles.

FISH. Cells were harvested by trypsinization and allowed to adhere on poly-L-lysine-coated microscope slides (Sigma) for 2 min before fixation in 4% formaldehyde-5% acetic acid-saline for 18 min. FISH was performed as previously described (44) with the following modification. After initial washes in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 37°C, unhybridized RNA was digested with 25 µg of RNase A/ml in 0.5 M NaCl-10 mM Tris (pH 7.5)-0.1% Tween 20 under a coverslip for 45 min at 37°C.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of ES cells expressing a truncated Tsix allele. In order to assess the functionality of the Tsix transcript, we created a truncated Tsix allele by introducing a transcriptional stop signal (triple polyadenylation site, i.e., tpA) (42) into a HindIII site 4 kb downstream of the major Tsix transcriptional start site (Fig. 1A). The presence of a strong splice acceptor (i.e., SA) (11) ensured trapping of any transcripts originating from all upstream promoters. In addition, the construct contained a HygroTK selectable marker and three loxP sites. After introduction of the construct into male and female ES cells by homologous recombination, the loxP sites facilitated the generation of two alleles after transient transfection with Cre-recombinase and selection for the loss of the TK gene as follows. (i) A 2 lox allele (stop allele) was created by deleting the selectable marker that was transcribed from the cytomegalovirus promoter. (ii) A 1 lox allele was generated by removal of both the selectable marker and the tpA/SA element. The 1 lox allele served as a control to ensure that effects observed in the presence of the stop allele did not simply arise from physical disturbance of the locus.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   (A) Scheme for the generation of the truncated Tsix (2 lox) and the control 1 lox alleles (see the text for details). The Tsix intron/exon structure, the two putative promoters, and regions deleted in previous studies (10, 19, 39) are indicated. (B) Southern analysis of targeted male and female cell lines after Cre-mediated recombination. DNA was digested with EcoRI and probed with the 5' internal probe (indicated in panel A) in order to detect the wild-type (1.7-kb), 1 lox (1.1-kb), and 2 lox (1.8-kb) alleles.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Presence of the 2 lox (stop) allele leads to expression of a truncated Tsix transcript, while leaving Xist expression unaffected. The 1 lox allele has no effect on Tsix transcription. (A) Strand-specific RT-PCR for Tsix and Xist was performed on wild-type and targeted undifferentiated male ES cells. The location of primer pairs is indicated as "PCR Positions." Primer pair 2a spans intron 6 and was used to amplify a product specific for the spliced Xist transcript. Primer pair 2b is located in exon 7 and was used to amplify the Tsix transcript. "No RT controls" refers to reactions carried out in the absence of reverse transcriptase. (B) Strand-specific RNA FISH for Tsix in undifferentiated ES cells. Male and female wild-type and targeted ES cells were probed with riboprobes specific for either the Tsix region overlapping Xist exons 4 to 7 (probe pBgl5K) or Tsix exon 4 (probe pTsixE4). The location of both probes is indicated in Fig. 1A.

Tsix transcription is a negative regulator of Xist expression. Recent work has shown that deletion of the major Tsix transcriptional start site, including portions of the surrounding CpG island, leads to primary nonrandom inactivation of the mutant X in differentiated female ES cells, as well as in the epiblast lineages of the embryo proper (17, 19, 39). However, it remained unresolved whether Tsix action was mediated by a DNA element that had been deleted in the mutant locus or whether the observed phenotype was caused by loss of the antisense transcript and/or transcription through the region. We reasoned that, if the phenotype was caused by loss of Tsix transcription rather than by deletion of a crucial DNA element, then the 2 lox (stop) allele would lead to skewed X inactivation in differentiated female ES cells. In order to assess this hypothesis, DNA and RNA were isolated from undifferentiated ES cells, as well as from ES cells that were induced to differentiate with all-trans-retinoic acid in the absence of LIF (15) for 6 days. The presence of two X chromosomes of different parental origin in the female cell lines as confirmed by Southern analysis provided a way to analyze allele-specific expression of Xist by strand-specific RT-PCR. Two restriction fragment length polymorphisms in exon 1 (NcoI polymorphism) (19) and exon 7 (PstI polymorphism), respectively, were analyzed (see Fig. 4A). In wild-type male cells, Xist was expressed in undifferentiated cells and undetectable in differentiated male cells (see Fig. 4A, compare lanes 1 and 2), in agreement with Xist expression being silenced in male cells upon differentiation. Control female brain cells showed expression from both Xist alleles (see Fig. 4A, lane 3), because either X chromosome in these cells can be stochastically chosen for X inactivation. However, Xist expression from the 129 allele carrying the lower-strength Xce (Xce^a) relative to that of M. castaneus (Xce^c) (3) was more abundant. A similar pattern of Xist expression was observed in differentiated 1 lox cells (see Fig. 4A, lane 5) and differentiated wild-type ES cells (data not shown), indicating that random X inactivation was not affected by the presence of the loxP site. In contrast, cells heterozygous for the 2 lox (stop) allele showed complete skewing of Xist expression toward the targeted 129 allele which does not express Tsix (see Fig. 4A, lane 7).

Generation of an inducible Tsix allele. To generate an experimental system that allows regulated expression of Tsix RNA in response to doxycycline addition to the medium, we inserted a tetracycline-responsive promoter (12) into the HindIII site analogous to the insertion site of the stop construct (tetop, Fig. 3A). In addition, the construct contained a HygroTK selectable marker flanked by loxP sites. By using homologous recombination, the construct was introduced into a male (J1) ES cell line and into the 129 Xist allele of a female (F1-2.1) ES cell line carrying the nls-rtTA cDNA encoding a doxycycline-controlled transcriptional activator at the ubiquitously expressed ROSA 26 locus (45). Targeted male and female cell lines were transfected with Cre-recombinase and selected for loss of the TK gene. Male lines M.ind1a and M.ind1b and female lines F.ind1a and F.ind1b containing the inducible promoter were identified by Southern blotting (Fig. 3B).

View larger version (33K): [in this window] [in a new window]   FIG. 3.   (A) Strategy for the generation of an inducible Tsix allele (see the text for details). (B) Southern analysis of targeted male and female cell lines after Cre-mediated recombination. XbaI-digested DNA was probed with the 5' internal probe (indicated in Fig. 1A). The wild-type allele gave a 3.8-kb band compared to the 1 lox (loop-out, 4.4-kb) and 2 lox (no loop-out, 7.7-kb) alleles.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Tsix transcription is a negative regulator of Xist expression. (A) Allele-specific RT-PCR for Xist expression was performed on control female brain cells, undifferentiated ES cells (undiff.), and ES cells that were induced for differentiated with all-trans-retinoic acid for 6 days (RA6). PstI indicates a polymorphism in Xist exon 7 that was detected after PstI digestion of a PCR product amplified with primer pair 2a (28 cycles). First-strand synthesis was performed with primer 2as. NcoI indicates a polymorphism in Xist exon 1 that has been described previously (19). Amplification was carried out with the primer pair 1s/as (28 cycles) after first-strand synthesis with primer 1as. No bands were detected in control reactions performed in the absence of reverse transcriptase (not shown). The polymorphic bands are indicated: "129" corresponds to the targeted 129 derived X chromosome, whereas "cast" corresponds to the nontargeted M. castaneus X. (B) Wild-type and targeted male ES cells were induced to differentiate with all-trans-retinoic acid for 2 days. Strand-specific RNA FISH for Xist was performed with a riboprobe specific for Xist exons 4 to 7 (probe pBgl5K, Fig. 1A). The proportion of cells expressing ectopic Xist signals is indicated below each panel. (C) Allele-specific expression of Tsix in the presence (+dox) and absence (dox) of the inducing agent doxycycline in female ES cells. Cells were either undifferentiated (undiff.) or were differentiated for 6 days in all-trans-retinoic acid (RA6). To detect the PstI polymorphism, strand-specific RT-PCR for Tsix was carried out with primer 2sE7 for first-strand synthesis, followed by amplification with primer pair 2b (30 cycles) and digestion of the PCR product with PstI. "129" corresponds to the Tsix allele derived from the 129 X chromosome that harbors the inducible promoter; "cast" refers to the nontargeted M. castaneus-derived X. (D) Tsix signals in induced cells are indistinguishable from the signals in uninduced and wild-type cells. Cells were either undifferentiated (undiff.) or differentiated with all-trans-retinoic acid for 6 days (RA6) and grown in the presence (+dox) or absence (dox) of doxycycline. Strand-specific RNA FISH was performed with a riboprobe specific for the Tsix region overlapping Xist exons 4 to 7 (probe pBgl5K, Fig. 1A). (E) Allele-specific expression of Xist in female ES cells. The X chromosome that constitutively expresses Tsix during differentiation is always chosen to remain active. Xist expression was skewed almost completely toward the nontargeted M. castaneus allele (cast) when Tsix was constitutively expressed from the 129 allele (129) (compare lanes 3 [dox] and 4 [+dox]). In undifferentiated cells induction of Tsix expression had no effect on Xist expression (compare lanes 1 [dox] and 2 [+dox]). Strand-specific RT-PCR was performed as described above.

Induction of Tsix transcription prevents the inactivation of an X chromosome in cis. We have shown that the X chromosome lacking Tsix transcription was always chosen for X inactivation. If the role of Tsix transcription was to block the accumulation of Xist, then the overexpression of Tsix should protect the X chromosome in cis from inactivation. To test this hypothesis, we compared Xist expression patterns from cells that were grown in the presence or absence of doxycycline. In both induced and uninduced undifferentiated cells, Xist was expressed from both alleles (Fig. 4E, lanes 1 and 2). This result was in agreement with the finding that Tsix overexpression from the 129 allele was not detectable in undifferentiated cells (Fig. 4C and D).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we generated two Tsix alleles in order to assess the role of Tsix transcription in the regulation of X-chromosome choice during X inactivation. We used, as a model system, ES cells which are known to undergo random X inactivation when induced to differentiate. In the first approach, a transcriptional stop signal was introduced into the transcribed region of Tsix flanked by a strong splice acceptor. The splice acceptor ensured trapping of all transcripts arising from upstream promoters, including the major promoter initially identified by Lee et al. (18) and the minor promoter recently characterized by Sado et al. (39). In the second approach, we created a doxycycline-inducible allele that enabled us to ectopically activate Tsix expression in a regulated manner. The objective was to functionally modify Tsix expression, while leaving potentially important regulatory DNA elements intact. This approach differs from previously published studies in which targeted deletions of Tsix not only abolished Tsix transcription but, in addition, deleted major parts of the DXPas34 locus (10, 19, 39), a CpG island that has been shown to be differentially methylated on the active and inactive X chromosome (9).

The results described here clearly establish for the first time the importance of active transcription for Tsix function. Loss of Tsix transcription leads to nonrandom inactivation of the X chromosome, with the mutant X being always inactivated (Fig. 4A), whereas constitutive expression of Tsix causes the mutant X chromosome to remain active (Fig. 4E). This unequivocally demonstrates that Tsix transcription through the Xist locus is crucial for preventing X-chromosome inactivation and confirms the role of Tsix as a negative regulator of Xist.

We found, in agreement with this, that the loss of Tsix transcription led to ectopic Xist expression in a subset of differentiating male cells (Fig. 4B), an effect that was specific for differentiating cells since Xist accumulation is detectable only in ca. 1% (n = 367) of undifferentiated cells. These results differ from previous studies that found ectopic Xist expression to be restricted to cells of extraembryonic tissues (17, 19, 39). In these tissues X inactivation is normally imprinted such that expression from the maternal Xist allele is silenced. Deletion of the Tsix promoter/DXPas34 locus erased this imprint, leading to ectopic Xist expression from the maternal allele in addition to upregulation of Xist expression from the paternal allele. However, it is possible that ectopic Xist upregulation still occurred in the epiblast lineage of these embryos. Cells that undergo aberrant dosage compensation are not viable and are progressively lost from the population. Since our findings suggest that only a small fraction of cells of the epiblast lineage are affected, the loss of these cells may not have an obvious effect on the overall cell population. Interestingly, Sado et al. (39) did observe Xist accumulation in a subset of mutant male ES cells grown under nondifferentiating conditions, a finding consistent with these cells representing a subpopulation that had already entered differentiation and initiated stochastic upregulation of Xist expression.

In addition, the phenotype described here differs from that of a 65-kb knockout that included the Tsix promoter (7). This knockout also resulted in nonrandom X inactivation of the deleted X in female ES cells. However, in cells carrying only one intact X chromosome (X0), ectopic Xist upregulation was observed in virtually all of the cells. In contrast, we see ectopic expression only in a subset of cells. An explanation for this difference may be provided by a model proposed by Lee and Lu (19). This model suggests that Xist expression is regulated by a complex interplay of positive and negative factors. A blocking factor, present in limited amount, may mark one X chromosome per diploid set of autosomes as the future active X. Our work indicates that this blocking factor most likely acts by regulating Tsix transcription and may perhaps consist of a complex of autosomal and X-linked factors that bind to the Tsix promoter. In addition, Lee and Lu suggest the presence of competence factors that are required to initiate Xist upregulation on the future inactive X once the active X is chosen. These competence factors are thought to be expressed only in cells that need to undergo dosage compensation, i.e., in cells that carry more than one X per diploid set of autosomes. Therefore, loss of Tsix transcription as the target of the blocking factor would not be sufficient to induce upregulation of Xist expression in male cells. However, the observation that stochastic ectopic Xist expression is still observed in some cells may indicate that competence factors are not strictly limited to cells that carry more than one X chromosome. Male cells might simply be less competent to upregulate Xist expression in the absence of the blocking factor because they are not poised for X inactivation and might therefore provide a less-permissive environment for Xist upregulation and stabilization.

Recent findings suggest that Tsix consists of four exons and is subject to complex alternative splicing (39). We found that the localization of these spliced forms most likely overlaps with the localization of the unspliced transcript, as indicated by FISH with a strand-specific probe specific for the common exon 4 (probe pTsixE4; Fig. 2B). In our study, both the transcriptional stop signal and the inducible promoter were inserted downstream of exons 1 through 3, leaving exon 4 as the only exon whose expression was modified. In view of our results two types of models for the mechanism of Tsix action can be proposed. (i) Tsix function could be mediated by a functional RNA. According to this model, exon 4 would be the only exon that is necessary and sufficient for Tsix function. Tsix RNA may interact directly with Xist DNA or local chromatin to interfere with Xist transcription. Alternatively, the formation of an Xist/Tsix RNA duplex may trigger RNA degradation or mask functional domains in Xist RNA. Interestingly, exon 4 overlaps with the Xist transcription unit and the XCR, a conserved region that is crucial for Xist function (1). (ii) It is possible that transcriptional activity of the locus per se, rather than the RNA itself, is important for function so that the processed form of Tsix would play only a minor role in the regulation of Xist expression. Antisense transcription might, for example, be involved in regulating the chromatin domain around the Xist locus. This mechanism has been shown to be crucial for the expression of the human -globin gene (13), where intergenic transcription was found to be required for the remodeling of chromatin structure to allow expression of this locus in a developmentally regulated manner. Similarly, Tsix transcription may directly modulate the chromatin structure through the Xist to allow access of developmentally regulated factors. Alternatively, transcriptional interference between Tsix and Xist might inhibit efficient transcription of the Xist gene, thereby preventing Xist accumulation.

It will be interesting to see by what mechanism Tsix transcription regulates Xist expression. In addition, upstream factors that control Tsix expression and therefore constitute the potential blocking factor still remain to be identified