Human Molecular Genetics

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (41) Request Permissions Google Scholar Articles by Shibata, S. Articles by Lee, J. T. Search for Related Content PubMed PubMed Citation Articles by Shibata, S. Articles by Lee, J. T. Social Bookmarking          What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 2 125-136
© 2003 Oxford University Press

Characterization and quantitation of differential Tsix transcripts: implications for Tsix function

Shinwa Shibata and Jeannie T. Lee^*

Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114, USA

Received September 7, 2002; Accepted November 2, 2002

GenBank accession number: AF541962

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10–100-fold molar excess over Xist RNA. Second, only 30–60% of Tsix RNA is spliced at known exon–intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In mammals, either of the two X-chromosomes in females is inactivated to compensate for dosage (1), a phenomenon referred to as X-chromosome inactivation (XCI). The master switch for this long-range chromosomal silencing is termed the ‘X-inactivation center’ (Xic) (2,3) and is sufficient for chromosome counting, choice, initiation of XCI, and establishment of heterochromatin (4). Two unusual noncoding transcripts have been identified within the Xic. The 17 kb untranslated product of the Xist (X-inactive specific transcript) locus is unique in that it is transcribed only from inactive X chromosome (Xi) in female somatic cells and accumulates in cis along the Xi (5–7, for Xist reviews, refer to 8,9). Xist is indispensable for the silencing step of XCI (10,11). Its antisense counterpart, Tsix, originates 12 kb downstream of Xist and transverses the entire Xist locus, thus encompassing over 40 kb of the Xic (12). Prior to the onset of XCI, Tsix and Xist RNAs are co-localized to the Xic. Unlike Xist RNA, however, Tsix RNA has not been observed to paint the X (12). Indeed, its action is strictly limited to the Xic, where its expression represses the upregulation of Xist and designates the future active X (Xa). When Tsix expression is eliminated on one X in female cells, XCI occurs predominantly on the mutated X (13–15). In contrast, the augmentation of Tsix transcription blocks Xist RNA accumulation on the same chromosome (16,17). Current models propose that, prior to the onset of XCI (undifferentiated cellular state), the co-expression of Tsix along with Xist in cis prevents high level Xist expression and the initiation of XCI. At the onset of XCI, silencing of the future Xi can only proceed with the downregulation of Tsix in cis (12,14) and, conversely, the maintenance of the active state on the Xa depends on the persistent expression of Tsix on that chromosome (16,17).

What is the molecular basis of Tsix's action on Xist? Three classes of mechanisms have been proposed (8,9,12). First, Tsix's action may be independent of its transcription and RNA product. ‘Enhancer competition’ (18) between Xist and Tsix is one model that has been proposed for Tsix's role in determining X-chromosome choice (19). Two other classes of mechanisms include ‘transcription-dependent’ and ‘RNA-dependent’ models. In the former, antisense transcriptional action in itself provides the repressive force. For example, the opposing movement of the Tsix RNA polymerase complex could either result in topological constraints on Xist RNA production or lead to ‘counter-current collision’ of the converging Xist and Tsix polymerase machinery. Alternatively, antisense transcription across the Xist promoter (15,20) could impair the recruitment of transcriptional initiation machinery to Xist, perhaps by ‘promoter occlusion’. In contrast, the ‘RNA-dependent’ class of mechanisms postulates that Tsix works as a functional RNA. In one scenario, the Tsix transcript may anneal to and mask the functional domain of Xist RNA, thereby preventing Xist RNA from making a complex with silencing protein partners. The hybridization of the complementary RNAs may also enhance the degradation of sense and antisense RNAs. In this type of RNA-dependent action, the repressive mechanism is stoichiometric rather than catalytic. Thus, the regulatory antisense RNA would be expected to occur at a molar excess over Xist RNA. All of these models have yet to be evaluated experimentally.

Ultimately, the testing of each hypothesis will first require detailed characterization of Tsix RNA structure and quantitation of sense/antisense RNA abundance. Although the full-length antisense transcript spans >40 kb (12), several spliced isoforms have been described (15). One additional isoform (15) initiates approximately 16 kb upstream of the previously reported Tsix start site (12). An interesting feature of all these isoforms is that splicing eliminates virtually all of the complementarity to Xist, with the notable exception of Xist's 5' end. The relative abundance of spliced versus full-length transcript has not been determined. Furthermore, the functional relevance of spliced isoforms remains unclear.

Some understanding of the antisense mechanism may be shed by cross-species comparison. By strand-specific RT–PCR analysis, human TSIX transcription reportedly terminates within the 3' end of XIST (within XIST intron 4) so that there is only partial overlap between the two genes (21). This observation has one of three implications: first, it may imply that the functional domains of Tsix/TSIX lie in its 5' half. Second, it may instead suggest that the mechanism of antisense regulation differs completely at the mouse and human loci. Finally, the critical domain of murine Tsix may actually reside in its 3' terminus, a possibility consistent with one interpretation that human TSIX has lost function (22). A 3' functional end is appealing in light of the fact that Xist's silencing domain lies in a repeat sequence complementary to this 3' terminus of Tsix (23,24). Thus, to gain a better understanding of potential Tsix mechanisms, a more detailed structural and quantitative analysis of Tsix RNA is warranted and is achieved below.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Identification of a novel Tsix splice variant from an embryonic stem (ES) cell cDNA library
A striking feature of previously identified spliced Tsix RNAs is their relatively short lengths and their minimal overlap with Xist RNA (Fig. 1A). Notably, the region of complementarity lies exclusively at the 5' end of Xist (termed Tsix exon 4). Because the structural variations in Tsix RNA could have implications for the antisense mechanism, here we endeavored to determine if additional spliced variants existed, particularly longer isoforms which might have been missed by PCR-based methods such as rapid amplification of cDNA ends (RACE). We constructed a Tsix-specific cDNA library from wild-type male ES cells by placing a primer specific for the antisense transcript near the Xho I site within Xist exon 1 (Fig. 1A, arrow), a position that circumvented upstream repeats which may potentially interfere with first-strand synthesis. To select for higher molecular weight cDNAs, the inserts were size-fractionated prior to cloning. Positives were subsequently identified by colony hybridization using an Xist exon 1 probe (Fig. 1A, bi-directional arrow).

View larger version (18K): [in this window] [in a new window]   Figure 1. Identification of a novel Tsix splice variant. (A) The strategy of Tsix cDNA library construction. Tsix-specific RT primer is shown as an arrow. The DNA region used as a probe for colony hybridization is indicated as a bi-directional arrow. Open tall rectangles and smaller gray ones represent Xist and Tsix exons, respectively. Symbols xE1, xE2-6, and xE7 represent Xist exons and tE2, tE3, and tE4 are those of Tsix. B, Bam HI; X, Xho I restriction enzyme sites. (B) Resultant Tsix clones from library screening, shown as filled rectangles. Note the asterisk in clone B7, which represents newly identified splice donor at the 3' end of Tsix exon 2. The 5' end of these cDNA clones are: base pair 1938 (clone 8B), 2362 (12B), 2876 (10B) of Genbank sequence accession no. L04961; base pair 77697 (9B), 77687 (D37), 77674 (B7), 79118 (C7 and B43) of Genbank no. X99946. The intron of C7 and B43 starts from base pair 79839 of X99946. (C) PCR amplification of Tsix transcripts spanning DXPas34 locus and exon 4. M, 100 bp ladder marker.

 
After multiple rounds of screening, we isolated eight Tsix clones (Fig. 1B). Clones 8B, 12B and 10B are likely to be prematually terminated cDNAs because no promoter activity has been described in this region (No transcriptional initiation was observed in Tsix^CpG ES cells; see below). The clones 12B and 10B contained an additional 0.4 and 0.9 kb of proximal sequence and could therefore either represent prematurely truncated full-length Tsix or longer spliced isoforms. The spliced variants 9B, D37 and B7 appeared to be similar to cDNA clones previously described (15), each originating in the vicinity of the reported major transcription start site of Tsix (12). However, there also appeared to be some variation in splice junctions, as the 3' junction of exon 2 in clone B7 included an additional four bases (asterisk in Fig. 1B). This variability is consistent with the previous report that the 3' end of exon 2 could occur 17 bases upstream (exon 2') (15). We refer to this new exon 2 variant as exon 2''.

Finally, clones C7 and B43 were novel and were identical to each other. They differed from all previously described variants in two ways: first, they included the DXPas34 repeat (13), a region in which CTCF binding sites have been described (19). As in all prior Tsix isoforms, C7 and B43 spliced out almost all of the Xist gene body save 1.9 kb at Xist's 5'-most terminus (exon 4). The intron–exon structure of C7 and B43 obeyed the GT–AG rule. Second, the 5' ends of clones C7 and B43 lay in DXPas34, suggesting either premature termination of first-strand synthesis (possibly due to the repetitive nature of DXPas34) or potential promoter activity within DXPas34. To determine if this pattern of splicing occurred in vivo, we carried out RT–PCR of wild-type male ES cells using PCR primers in DXPas34 and Tsix exon 4. A product of the expected size was obtained (Fig. 1C), suggesting that the novel splice junctions are indeed utilized in vivo. Sequencing of the PCR product showed that it is identical to the corresponding region of clones C7 and B43 (data not shown). Thus, our attempts to isolate novel Tsix splice variants produced one new species.

5'- and 3'-RACE of the novel Tsix splice variant
In order to obtain the complete 5' structure of the novel species, we carried out 5'-RACE using a primer positioned downstream of DXPas34 (Fig. 2, right-directed arrowhead). We obtained three distinct clones (nos 19, 101 and 105). Clone no. 19 was truncated at the 5' end. The truncation may be due to an artifact of premature termination by reverse transcriptase or may reflect possible transcription initiation within DXPas34. The 5' ends of two other clones, nos 101 and 105, closely coincided with the known major Tsix start sites (12). Aside from the 5' termini, the sequences of these two clones were identical to each other and spanned all of the DXPas34 repeat. The repeat sequence differed slightly from the published genomic sequence (Genbank accession no. X99946) in having two gaps (asterisks in Fig. 2), most likely due to genomic sequence polymorphism rather than to RNA processing, because these gaps did not fit the GT–AG rule and coincided precisely with a single unit of the DXPas34 repeat. Nonetheless, some splicing activity was evident within DXPas34 (Fig. 2) and occurred in accordance with the GT–AG rule. Thus, in addition to the splicing patterns previously reported (15), our data suggested that at least one new splice variant includes the DXPas34 sequence. We name the novel DXPas34 exons of this new species ‘exon 1a’ and ‘exon 2a’ (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Figure 2. 5' and 3' RACE to determine the structure of the novel splice variant. A detailed map of the Tsix clone C7 at DXPas34 locus and the 3' and 5'-RACE products depicted as filled rectangles. The gene-specific primers used for RACE are shown as arrowheads. The 3' ends of 3'-RACE clones are: base pair 649 (clone no. 5), 1503 (no. 7) of Genbank sequence accession no. L04961; base pair 79991 (no. 14) of Genbank no. X99946. The hexanucleotide polyadenylation sequence (ATTAAA) starts from base pair 79959 of X99946. The 5' ends of 5'-RACE clones are: base pair 79428 (no. 19), 77777 (no. 101), 77675 (no. 105) of X99946. The single and double asterisks in the figure represent gaps of 34 bases (79311–79344) and 62 bases (78460–78521) each, which were found between the RACE clones and Genbank sequence X99946. The upstream intron is from base pair 78569 to 78942 of X99946. A, AgeI; B, BamHI; M, MluI; P, PstI; Sm, SmaI; Sl, SalI; X, XhoI restriction enzyme sites. Bars with dots on their top represents HpaII sites. Symbols xE1, xE2–6, and xE7 represent Xist exons. Symbols tE2, tE3, tE4, tE1a, and tE2a are Tsix exons.

 
To determine the 3' structure of the novel Tsix isoform, we performed 3'-RACE using two gene-specific primers positioned downstream of DXPas34 (Fig. 2, left-directed arrowheads). Three products were of the approximate sizes, 1.5 (no. 5), 0.7 (no. 7), and 0.4 kb (no. 14) (Fig. 2). Sequence analysis revealed that the splicing pattern of clone nos 5 and 7 was identical to that of C7 in that Tsix was spliced from DXPas34 to exon 4. Notably, the 3' ends of both clones occurred within blocks of A-rich sequence (bp 648–629 and bp 1502–1493 of Genbank sequence accession no. L04961), perhaps indicating mis-priming of the oligo-dT primer during first-strand synthesis (25). (This sequence is unlikely to be a true poly-A site because it does not conform to the poly-A consensus.) Clone no. 14 extended further 3' for 300 bp. A polyadenylation signal (ATTAAA) (26) occurs in this region, so the 3' end of clone no. 14 may actually represent one poly-A site for Tsix RNA. We termed this exon 2a variant exon 2a'.

How much of Tsix RNA is spliced?
As a first step towards understanding the significance of Tsix splicing, we next determined what fraction of Tsix is actually spliced by adapting the RNase protection assay (RPA). In the RPA technique, hybridization of a strand-specific RNA probe to target cellular RNA protects the annealed strands from RNase digestion, thereby providing information regarding both the length and the relative quantity of target RNA (27). By placing probes at known Tsix exon–intron boundaries, the amount of splicing at each exon–intron junction could be estimated by comparing the intensity of the bands corresponding to fully protected (unspliced) versus partially protected (spliced) probes. In each assay, we examined the extent of splicing in ES cells of four genetic backgrounds: wild-type male (129 strain), wild-type female (hybrid 129 x M.castaneus), mutant male carrying the Tsix^CpG null geno-type (14), and mutant female carrying the Tsix^EF1 gain-of-function genotype (17). The specificity of RPA bands for Tsix was inferred from their presence in wild-type ES cells and absence from the Tsix^CpG mutant, somatic cells and yeast.

First, we examined the highly heterogeneous 5' end using RPA probes, E3SD and TxE2, which spanned sequence just downstream of the Tsix major starts and overlapped exons 2 and 3 (15) (Fig. 3A). When the TxE2 probe was used, we found that the majority of RNA was not spliced at this junction, as the fully protected band of 245 nucleotides (nt) was more prominent than any band corresponding to the spliced variants (170, 166 and 149 nt) (Fig. 3A, arrowheads). When the E3SD probe was used, we found that the unspliced form (501 nt) was again most abundant (Fig. 3A), while the spliced form was less detectable (110 nt). The minimal overlap between the probe and exon 2 probably made signals from exon 2 variants hard to detect. Additional bands between 200 and 400 nt might be attributed to RNA secondary structure or to sequence differences between 129 and M.castaneus (since some were observed only in the hybrid female line). However, they may also reflect as yet unidentified splice variants.

View larger version (46K): [in this window] [in a new window]   Figure 3. Determination of splicing efficiency by RNAse protection assays. (A) RPA at the 3' portion of Tsix exon 2 and exon 3. The probe TxE2 yields 245 nt signal for unspliced transcripts and 166, 149 or 170 nt signal for spliced ones corresponding to exon 2, 2' or 2'', respectively (arrowheads). The original transcript size of the probe is 332 nt. Probably, the probe E3SD yields 501 and 110 nt signals for unspliced and spliced transcripts, each (arrows). The latter corresponds to exon 3. The original size of E3SD riboprobe is 518 nt. Gray rectangles in the map represent Tsix exon 2 (tE2) and exon 3 (tE3). (B) RPA at the 3' part of DXPas34 locus. Gray rectangles in the map show newly identified Tsix exons 1a (tE1a) and 2a (tE2a). The C7PM probe yields 293 and 176 nt signals for unspliced and spliced transcripts, respectively. The original size of C7PM riboprobe is 343 nt. The position of uncharacterized third signal was shown as an arrowhead. (C) RPA at the 5' boundary of Tsix exon 4 (tE4). The probe TxE4 yields 186 nt signal for unspliced transcripts and 110 nt signal for spliced ones, while its original transcript size including multi cloning site of the vector is 273 nt. Various ES cell lines either undifferentiated (at day 0, d0) or partially differentiated (for 4 days, d4), and adult mouse liver RNA were examined. Yeast RNA with or without RNase digestion was used as controls (Y+ or Y-). The open larger rectangle and gray smaller one represent Xist exon 1 and Tsix exon 4, respectively. The faint band about 273 nt in size observed in a lane of TsixEF-1-female ES/d0 is likely to be a remnant of undigested probe. A, AgeI; B, BamHI; D, DraII; M, MluI; P, PstI; Sm, SmaI; Sl, SalI; X, XhoI restriction enzyme sites. Bars with dots on their top represents HpaII sites. CpG, TsixCpG; EF-1, TsixEF-1; WT, wild-type; M, 100 nt marker. (D) The estimation of the amount of unspliced (unspl) versus spliced (spl) Tsix transcripts at each exon–intron boundary. The signal volume of the bands marked as *1 to *4 (see C and A) was measured and the relative copy number was calculated and shown.

 
We next asked how much splicing takes place at the DXPas34 boundary (Fig. 3B). At this junction, the unspliced form (293 nt) was once again more abundant than the spliced variant (176 nt). The RPA also revealed a third band of 120 nt (arrowhead), suggesting yet more possible splicing variation at the boundary of DXPas34. To quantitate the extent of splicing, we determined the relative signal intensities of fully protected versus spliced bands by phosphorimaging and normalization for number of adenine bases within the target sequence. This analysis showed that there is at least two-fold more unspliced RNA (Fig. 3D). Based on relative band intensities by RPA (data not shown) and the results of cDNA cloning (Fig. 1B), splicing at this junction appeared to take place at a comparable frequency relative to exons 2 and 3, suggesting that the novel splice variant may be as abundant as the previously reported Tsix isoforms. These findings further argue for heterogeneity of splicing at the 5' end of Tsix.

It should be noted that even greater splicing variation may exist at the 5' end. Because our cloning strategy involved a size-selection step, splice variants of both lower and higher molecular weights may have been missed. Indeed, RNase protection analysis occasionally yielded specific bands that were not of predicted molecular weights (Fig. 3A and B), suggesting that additional splice junctions may have been utilized at the 5' end. Regardless, the results of RPA indicate that a large fraction of the antisense transcript remains unspliced at each known exon–intron boundary.

We finally examined the 3' end of Tsix using probes which lie at the upstream junction of exon 4 (Fig. 3C). Two specific signals were detectable in male and female ES cells (Fig. 3C), a 186 nt band corresponding to the unspliced RNA and a 110 nt band for the spliced variant. As their intensities were not so different, it was likely that there were comparable amounts of spliced and unspliced transcripts at this exon boundary. Quantitation by phosphorimaging demonstrated that there was either equal amounts of (wild-type female) or more (wild-type male) spliced RNA as compared with unspliced (Fig. 3D). Thus, Tsix RNA is more efficiently spliced at exon 4 than its 5' exons. Nevertheless, a significant fraction at the 3' end is also not processed. To determine whether the extent of splicing changes at the onset of XCI, we compared the RNA ratio between undifferentiated (day 0) and partially differentiated (day 4). We could not see any significant difference in the ratio of unspliced versus spliced signal intensity (Fig. 3C). These results suggest that splicing at exon 4 of Tsix RNA occurs more efficiently than at the 5' portion and that the extent of splicing does not change during differentiation.

The stoichiometry of Tsix and Xist RNA
At the onset of XCI on the future Xi, the downregulation of Tsix enables Xist to transition from a low to high expression state in cis. Current models of Tsix function invoke either an RNA-based mechanism or a mechanism based rather on transcriptional action. If Tsix functioned as an antisense RNA which titrates out sense RNA, its stoichiometry would be crucial to its function.

Here, we used real-time RT–PCR analysis to estimate Tsix RNA copy numbers in undifferentiated wild-type male, wild-type female, and Tsix^CpG male ES cells using primer pairs placed at 11 positions across the locus (Fig. 4A, Table 2). In this method, the starting template amount is quantitated against a synthetic control template whose copy number is determined by spectrophotometry. The results revealed a gradient of RNA abundance, with 10 times more transcript at the 5' portion as compared with the 3' end (Fig. 4B). This suggested that a significant portion of Tsix transcription terminated early before crossing the Xist gene body. Relevant to this, the transcript levels at positions 4–6 were low at steady state. Aside from transcription termination in the middle, the low copy number could reflect intronic RNA degradation since these positions occur in a Tsix ‘intronic’ region.

View larger version (37K): [in this window] [in a new window]   Figure 4. Quantitation of Tsix transcripts by strand-specific real-time RT–PCR analyses. (A) The positions of amplicons examined. The symbol at each amplicon shows the type of template used, i.e. filled circles mean random primed cDNA and filled triangles represent strand-specific cDNA (antisense). The amplicons 1–3 for Tsix exon 1, 2, and the DXPas34 downstream exon (exon 2a), respectively, were placed inside each exon. Therefore, they allow amplification of both spliced and unspliced transcripts. The amplicon 7 spans across the 5' boundary of Tsix exon 4 and represents only unspliced transcript. The Xist copy number was examined using random primed cDNA at amplicon 12 (open circle), whose primers were placed in 3' of Xist exon 1 and in exon 3. pA, polyadenylation signal (AATAAA). The asterisk adjacent to one of the pA means there are three pA signals positioned closely. Large open rectangles, Xist exons (xE1, xE2–6, and xE7); middle gray rectangles, Tsix exons (tE1, tE2, tE3, and tE4); small filled rectangles, newly identified Tsix exons in DXPas34 locus (tE1a and tE2a). (B) The average copy number of Tsix and Xist transcripts in various ES cell lines. The mean number and standard deviations are presented in Table 2. The light gray round columns represents male TsixCpG ES cells, dark gray round columns male wild-type ES cells, and black square columns female wild-type ES cells. CpG, TsixCpG; WT, wild-type.

 

View this table: [in this window] [in a new window]   Table 2. Strand-specific primers, PCR primers, and template plasmid constructs used in real-time PCR analyses for Tsixa

 
To determine if Tsix RNA exists at a molar excess relative to Xist, we estimated Xist RNA abundance in undifferentiated ES cells, cells which have not undergone XCI. We found that undifferentiated ES cells contained less Xist RNA than Tsix (Fig. 4B, Table 1). (Note: the Xist copy number in female ES cells was slightly higher; we believe that much of this may be due to the inevitable presence of differentiating cells which would exhibit high-level Xist expression.) Thus, with respect to Tsix's 5' portion, there was a 100-fold molar excess of Tsix RNA over Xist RNA. With respect to Tsix's 3' end, there was a 10-fold excess. These results indicated that there is indeed a significant excess of Tsix RNA over Xist RNA.

View this table: [in this window] [in a new window]   Table 1. Quantitation of Tsix and Xist RNA at various genomic positions

 
Finally, we examined how Xist levels are altered in the absence of Tsix RNA. Previous studies have shown that, even in undifferentiated ES cells, the loss of Tsix expression resulted in increased steady-state Xist levels (14,28). Here, we first used real-time RT–PCR to more accurately quantitate the degree of upregulation in the mutant Tsix^CpG cell line and found a 10-fold increase in Xist RNA levels (Table 1, wild-type male versus Tsix^CpG male ES cells). However, despite this order of magnitude increase, Xist RNA neither spread along the X nor initiated silencing (14). We next asked if Xist levels further increased upon cell differentiation and the onset of XCI. We found that somatic female cells (liver) showed a further upregulation (about 100-fold, data not shown). This suggests the transition from low to high level state involves increase in steady-state Xist RNA amount, a result roughly consistent with the previous estimate (17,29). Taken together, the results of quantitative real-time RT–PCR demonstrated that, prior to the onset of XCI, Tsix RNA is indeed in great molar excess over Xist RNA and that its loss of expression correlates with Xist's transition to a high level state on the Xi.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
A synthesis of Tsix genetic studies has led to the ‘transcription-dependent’ and ‘RNA-dependent’ models of antisense regulation (8,9,12). Although not mutually exclusive, the two models differ in significant ways and make clear predictions. The transcription-dependent models invoke topological and steric constraints on Xist imposed by antisense transcriptional activity and do not require the antisense RNA product. In contrast, the RNA-dependent models postulate a repressive role for Tsix RNA and posit a stoichiometric titration of Xist RNA by the antisense transcript. As a step towards distinguishing between these potential mechanisms, we have characterized Tsix RNA structure and quantitated RNA abundance in vivo (Fig. 5). Several conclusions can be drawn from our results.

View larger version (25K): [in this window] [in a new window]   Figure 5. Summary of findings. (A) Deduced levels of antisense transcripts based on real-time PCR data in wild-type male ES cells. Targeting schemes and Xist functional domains in other studies are shown for comparison. +, 0–500; ++, 500–1000; +++, >1000 copies/50 ng of total RNA. The amount at 3' of DXPas34 exon and at 5' of exon 4 was based on both real-time PCR and RPA. The result in wild-type female ES cells is essentially the same. Large open rectangles, Xist exons; middle gray rectangles, Tsix exons; small filled rectangles, newly identified Tsix exons in DXPas34 locus. SA/pA, splice accepter and pA cassette; EF-1, elongation factor 1 promoter; Sc, ScaI; Sm, SmaI; Sl, SalI; M, MluI; H, HindIII. (B) Summary of Tsix splicing variants revealed by this study and others. (a) Previously reported Tsix exons (15). Note the two transcriptional start sites. Exon skipping is also observed. (b) DXPas34 variant of Tsix including exon 4 or (c) truncated form. (d) Unspliced Tsix. Open rectangles, Xist exons (xE1, xE2-6, and xE7); filled rectangles, Tsix exons. Numbers in the figure represent Tsix exons.

 
First, Tsix RNA occurs at a large molar excess over Xist RNA. Quantitative real-time RT–PCR analysis indicated that, in undifferentiated ES cells, Tsix RNA is present at >10–100-fold molar excess over Xist RNA per chromosome. Thus, if Tsix worked as an RNA entity, the local concentration of Tsix would be sufficiently high to bind and titrate out Xist RNA. At the molecular level, titration could involve enhancement of Xist RNA degradation through the Xist:Tsix double-stranded RNA (dsRNA) intermediate. The modulation of Xist RNA stability has been proposed to mediate the post-transcriptional regulation of Xist expression (30,31). This has led to much speculation about the potential involvement of RNA interference (RNAi) (32), an anti-viral and anti-transposon cellular defense mechanism in which aberrant dsRNAs are cleaved into 21–25 nt small interfering RNAs (siRNA) that then direct sequence-specific degradation of homologous RNAs (33). Recently, sense and antisense transcripts produced within centromeric repeats of Schizosaccharomyces pombe have been proposed to give rise to siRNAs which in turn regulate heterochromatic silencing at the centromere (34,35). However, at the Xist/Tsix locus, such siRNAs have not been found despite some effort (S. Shibata, unpublished observations). An alternative means by which Tsix RNA could titrate Xist RNA could be a masking of functional domains in Xist RNA. Recent work (24) demonstrated that Xist RNA contains a silencing domain located at the 5' terminus and a chromosome-binding domain more broadly distributed along the middle stretch of the 17 kb RNA. As will be further discussed later in this section, both the Tsix splicing patterns and its premature transcription terminations may provide additional clues to the molecular basis of Tsix action.

The quantitative analysis demonstrated a strong inverse correlation between Tsix and Xist levels. In cells deleted for the antisense gene, Xist RNA levels increased by 10-fold. Interestingly, this increase is not accompanied by XCI (14). In female somatic cells that have undergone XCI, Tsix becomes undetectable while Xist RNA increases by approximately two orders of magnitude. These results suggest that the repression of Tsix is only sufficient to partially upregulate Xist. Additional events at the onset of cell differentiation must occur to fully stimulate Xist expression and initiate XCI.

A second conclusion of this work is that Tsix RNA is heterogeneously spliced at the 5' end. By employing a cDNA cloning strategy aimed at identifying larger splice variants, we discovered one new species which contains novel exons 1a and 2a. Interestingly, these novel exons span DXPas34, a repeat sequence that was recently shown to contain binding sites for the transcription factor and chromatin insulator, CTCF (19). Quantitative analyses by RPA suggested that this variant is not so different in abundance from isoforms containing exons 2 or 3 (15). We also showed that the pattern of splicing to exon 4 does not obviously change upon the onset of XCI. The possibility of additional splicing variation at the 5' end is not excluded by our study. In fact, low levels of specific but unaccountable bands in RPA analysis very likely suggest greater variability in splice site usage than presently measurable. The heterogeneity of splicing must be factored into models of Tsix mechanism.

Third, although the functional relevance of splicing remains unknown, a most striking feature is that all of the spliced variants identified to date share exon 4, the only exon that occurs within the region of complementarity between Xist and Tsix (15). Interestingly, this region coincides with a region of Xist that is required for silencing and has been proposed to be a target of binding for chromatin-associated proteins (24). By virtue of its complementary to the Xist silencing domain, the spliced Tsix variant may be instrumental in masking this domain. In this model, one facet of Tsix's repressive action would result from direct base-pairing between antisense and sense RNAs. On the other hand, Tsix transcripts which are not spliced at exon 4 may participate in regulating other aspects of Xist action. Indeed, other domains within Xist RNA are necessary for its cis-localization to the Xi (24,36) and for targeting of the Xi-enriched protein, macroH2A (37). It is possible this localization is blocked by Tsix transcripts that remain full-length or even by transcripts that terminate early. This repression may be achieved by direct RNA base-pairing or by the indirect action of antisense transcriptional process. Further work will be required to distinguish between the ‘RNA-dependent’ and ‘transcription-dependent’ models.

Fourth, a significant fraction of Tsix RNA persists in the unspliced state. The results of RNase protection analyses revealed that only 30–60% of Tsix RNA is actually spliced at measurable exon–intron boundaries. It is certainly possible that splicing at other junctions was missed by our analysis—splice variants of large size would have been very difficult to clone and their splice site usage would not have been factored into our calculations. Nonetheless, the data showed clearly that only a fraction of total RNA is spliced at the exon 4 junction and more than half of Tsix transcripts remain unspliced at the 5' end. The persistence of unspliced Tsix RNA seems surprising given that other mammalian mRNAs are very efficiently spliced to the extent that very little unprocessed RNA is detectable at steady state. Therefore, this persistence may have significant implications for how Tsix works. If Tsix action requires its RNA product, the comparable abundance of spliced and unspliced forms leaves open possible roles for both forms in regulating Xist expression. Spliced and unspliced forms may in fact play different roles. For example, splicing to the invariant exon 4 junction may block the silencing activity at the 5' end of Xist RNA, while the unspliced variants (many of which terminate before reaching the 5' end of Xist RNA) may block Xist RNA localization to the X. We must also consider the possibility that the operational domain of Tsix lies exclusively in its 5' half, especially given a recent report that human TSIX terminates in the 3' exons of XIST (21).

Indeed, our findings in mice have possible implications for human TSIX structure and function. The identification of the human TSIX homolog has raised the possibility that human XIST expression is regulated by a similar antisense mechanism (21). However, the human gene has been reported to differ from murine Tsix in at least one significant way: strand-specific RT–PCR analysis showed that human TSIX RNA is detectable only as distally as XIST intron 4 (21). This result was interpreted to mean that TSIX terminates in XIST intron 4 and that full-length TSIX and XIST RNAs only partially overlap. This interpretation could have one of several implications. First, the critical domain of TSIX function may lie in the region of overlap with XIST exons 5–7. Second, TSIX may not work as a functional RNA but may instead exert its repressive action by antisense transcription per se. Finally, unlike mouse Tsix, human TSIX may have no function in regulating XIST (22).

However, relevant to these interpretations, our current study demonstrated a diminishing gradient in murine Tsix transcript copy numbers, with the highest level of expression at the 5' end (Fig. 4B). This suggests that a considerable fraction of total transcript terminates early without crossing the Xist gene body. Thus, it must be considered that human TSIX may also exhibit a gradient of expression and that the lack of RT–PCR detection beyond XIST intron 4 may reflect a relative decrease in steady-state RNA copy number. A quantitative approach to studying human TSIX expression will be critical to address whether the human X-inactivation center (XIC) is truly regulated differently from murine Xic. Indeed, human XIST and mouse Xist RNAs share the required silencing domain at the 5' terminus. If the complementary between mouse Xist and Tsix RNAs were crucial for regulation, a similar mechanism might be utilized by human TSIX. Thus, further examination of the 3' terminus of human TSIX will be of special interest in the future. As an increasing number of antisense genes has come to light in recent years (38–43), a clearer understanding of how Tsix/TSIX regulates Xist/XIST will have implications beyond the control of X-chromosome inactivation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Construction and screening of the cDNA library for novel Tsix variants
A Tsix-specific cDNA library was prepared using the SuperScript Choice System (Life Technologies) according to the manufacturer's instruction. Briefly, the first strand was synthesized using a Tsix-specific primer (5'-GTG TGA GTG AAC CTA TGG-3') from 20 µg of total RNA extracted from J1 male ES cells (44). For efficient first-strand elongation, the reverse transcriptase reaction was carried out at 50°C for 4 h, adding 200 units of the enzyme every hour. Subsequently, the second strand was synthesized and the cDNA ligated to Eco RI adapters. The cDNA pool was then selected on a size-exclusion column, from which the first nine fractions (highest molecular weight fractions) were chosen for further work. These fractions were dominated by cDNA clones of 1.0 kb or greater in size. Eco RI-adapted cDNA molecules were ligated to Eco RI-digested pBluescript vector and electroporated into E.coli competent cells (Electromax DH10B cells, Life Technologies). Tsix cDNA clones were isolated by colony hybridization screenings using random-primed Xho I-Bam HI 1 kb fragment from Xist exon 1 as a probe. Sequencing was carried out by the DNA sequencing core facility at the Massachusetts General Hospital.

PCR and RACE
PCR amplification of the Tsix clone C7 was performed with primers rt50R (5'-GGA GAG CGC ATG CTT GCA ATT CTA-3') and TRXp (5'-AAA GCG TTC AAT AAG CCT GGC GTG-3') using random-primed cDNA from J1 cells as template. The amplification condition was 94°C, 9 min; (94°C, 30 s; 66°C, 30 s; 72°C, 1 min)x39 cycles. 5'- and 3'-RACE were carried out using the GeneRacer Kit (Invitrogen, CA, USA). Briefly, for 5'-RACE total RNA from J1 cells was reverse-transcribed with a random primer or a Tsix-specific primer (rt50R) and amplified by nested PCR. In the first round, the template was amplified by touch-down PCR with TFXp2 primer (5'-ACG CCA GGC TTA TTG AAC GCT TTG-3') and the 5' primer (supplied by kit) using the following conditions: 94°C, 7 min; (94°C, 30 s; 72°C, 30 s; 72°C, 3 min)xthree cycles; (94°C, 30 s; 70°C, 30 s; 72°C, 3 min)xthree cycles; (94°C, 30 s; 68°C, 30 s; 72°C, 3 min)xthree cycles; (94°C, 30 s; 66°C, 30 s; 72°C, 3 min)x36 cycles using AmpliTaqGold Taq DNA polymerase (Roche). In the second round, the first PCR product was diluted 1/100 and amplified with TFXp2 primer and the 5' nested primer (kit) using the same conditions. For 3'-RACE, the oligo-dT primed cDNA was amplified with the 3' primer (kit) and TRXp primer using the following conditions: 94°C, 9 min; (94°C, 30 s; 72°C, 30 s; 72°C, 3 min)xthree cycles; (94°C, 30 s; 70°C, 30 s; 72°C, 3 min)xthree cycles; (94°C, 30 s; 68°C, 30 s; 72°C, 3 min) x three cycles; (94°C, 30 s; 66°C, 30 s; 72°C, 3 min)x26 cycles. Subsequently the PCR product was diluted 1/100 and amplified again with the 3' nested primer (kit) and TRXn primer (5'-AGT TAA GGG CGT GAC TTG TAG CAG-3') as follows: 94°C, 9 min; (94°C, 30 s; 66°C, 30 s; 72°C, 3 min)x25 cycles. The final PCR products were purified by gel-electrophoresis, ligated to the plasmid vector, and sequenced.

RNase protection assay
Total RNA was extracted using Trizol reagent (Life Technologies) from wild-type male ES cells (J1), wild-type female ES cells (16.6) (12), mutant male ES cells carrying the Tsix^CpG allele (CG7) (14), and mutant female ES cells carrying a gain-of-function Tsix^EF-1 allele (2A1.H12) (17). Hereafter, the CG7 and 2A1.H12 ES cells are abbreviated as Tsix^CpG and Tsix^EF-1 ES cells, respectively. In the differentiation condition, ES cells were cultured without feeder cells in the media lacking LIF. The assay was performed using RPA III kit (Ambion). A 10 µg aliquot of total RNA was hybridized with 5x10⁴ cpm of riboprobe in each reaction. The plasmid constructs utilized for probe preparation were: Nco I-digested pGEM/TxE4 (5' boundary of Tsix exon 4, base pair 1830–2014 of Genbank sequence accession no. L04961) (6), Nco I-digested pGEM/TxE2 (3' boundary of Tsix exon 2, base pair 77759–78003 of Genbank accession no. X99946) (45), Bam HI-digested pBl/E3SD (Tsix exon 3 and adjacent sequence, base pair 77848–78349 of X99946), and Nco I-digested pGEM/C7PM (3' boundary of DXPas34 in relation to Tsix start site, base pair 79663–79954 of X99946). The former two plasmids were obtained by TA cloning following PCR, and the latter two were generated by subcloning SmaI–EcoO109I 0.5 kb fragment and PstI–MluI 0.3 kb fragment into pBluescript or pGEM5 vector, respectively. The radioactive riboprobes were transcribed with either SP6 or T7 RNA polymerase using [-³²P] UTP and gel-purified thereafter. The signal intensity of protected bands was measured by Phosphoimager instrument (Molecular Dynamics). The relative copy number of unspliced versus spliced Tsix variants was calculated with the following formula: (volume of unspliced band)/(number of adenine residues in both exon and intron part of the corresponding probe sequence) versus (volume of spliced band)/(number of adenine residues in exon part of the corresponding probe sequence).

Real-time PCR
See Table 2 for strand-specific first-strand primers, PCR primers and template plasmid constructs for standard curve preparation. The random-primed cDNA templates for real-time PCR analyses were prepared from DNase I-treated total RNA extracted from the ES cell lines using the Supercript first-strand synthesis system for RT-PCR (Life Technologies). The strand-specific cDNA templates were prepared in the same way except the reaction was carried out at 50°C in order to avoid nonspecific priming. A mixture of oligonucleotides shown in Table 2 was used for strand-specific Tsix cDNA synthesis. The plasmid construct pBl/C7.E14.10B was made by introducing the inserts of library clone C7 (base pair 1112–2828 of Genbank accession no. AF541962), 10B (base pair 803–2876 of Genbank accession no. L04961), and PCR amplified DNA fragment including Tsix exon 1, 2, 3 and 5' end of exon 4 (base pair 1–732 of Genbank accession no. AF138745) into pBluescript vector. pB1/C7.E14.10B was digested with NotI and SpeI restriction enzyme to separate the three inserts for standard curve preparation. The amount of sample cDNA template used in each PCR reaction was an equivalent to that converted from 50 ng of total RNA. The PCR amplification was carried out with the SYBR® green PCR master mix (Applied Biosystems, Warrington, UK) using the iCycler iQ^TM real-time detection system (BIO-RAD Laboratories, Inc., Hercules, CA, USA) using the following conditions: 95°C, 9.5 min; (95°C, 30 s; 66°C, 30 s; 72°C, 1 min)x40 cycles. Since SYBR green was used to detect amplified products, the specificity of each PCR reaction was ensured by either melting curve profiles (in which a single curve demonstrated the presence of only one duplex DNA species) or by agarose gel electrophoresis of PCR products in test runs (which showed the amplification of a single correctly sized band). The concentration of plasmid DNA used as template was determined based on the spectrophotometric absorbance at 260 nm. The 1:10 serial dilution from 10⁷ to 10² copies/µl were prepared for each template in order to make the standard curve in which the threshold cycle showing the first cycle number to detect SYBR green fluorescence was plotted. The copy number of Tsix and Xist transcripts in wild-type and mutant samples was estimated by comparison to the standards described above and subsequently normalized to ß-actin. Every PCR reaction was triplicated and repeated at least twice.

	ACKNOWLEDGEMENTS

 
We thank N. Stavropoulos and Y. Ogawa for plasmid constructs and helpful advice. We are also grateful to M. Donohoe, K. Huynh and Y. Ogawa for critical reading of the manuscript. S.S. is supported by the fellowship program from the Japan Society for Promotion of Science. J.T.L. is an assistant investigator of the Howard Hughes Medical Institute. This work was supported by NIH grant RO1GM58835.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Email: lee{at}frodo.mgh.harvard.edu

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Lyon, M. (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature, 190, 372–373.[CrossRef][Medline]

2. Rastan, S. and Brown, S.D. (1990) The search for the mouse X-chromosome inactivation centre. Genet. Res., 56, 99–106.[Web of Science][Medline]

3. Brown, C.J., Lafreniere, R.G., Powers, V.E., Sebastio, G., Ballabio, A., Pettigrew, A.L., Ledbetter, D.H., Levy, E., Craig, I.W. and Willard, H.F. (1991) Localization of the X inactivation centre on the human X chromosome in Xq13. Nature, 349, 82–84.[CrossRef][Medline]

4. Lee, J.T., Strauss, W.M., Dausman, J.A. and Jaenisch, R. (1996) A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell, 86, 83–94.[CrossRef][Web of Science][Medline]

5. Brown, C.J., Ballabio, A., Rupert, J.L., Lafreniere, R.G., Grompe, M., Tonlorenzi, R. and Willard, H.F. (1991) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature, 349, 38–44.[CrossRef][Medline]

6. 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][Web of Science][Medline]

7. Clemson, C.M., McNeil, J.A., Willard, H.F. and Lawrence, J.B. (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol., 132, 259–275.[Abstract/Free Full Text]

8. Boumil, R.M. and Lee, J.T. (2001) Forty years of decoding the silence in X-chromosome inactivation. Hum. Mol. Genet., 10, 2225–2232.[Abstract/Free Full Text]

9. Avner, P. and Heard, E. (2001) X-chromosome inactivation: counting, choice and initiation. Nat. Rev. Genet., 2, 59–67.[CrossRef][Web of Science][Medline]

10. 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]

11. Marahrens, Y., Panning, B., Dausman, J., Strauss, W. and Jaenisch, R. (1997) Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Devl., 11, 156–166.[Abstract/Free Full Text]

12. 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][Web of Science][Medline]

13. Debrand, E., Chureau, C., Arnaud, D., Avner, P. and Heard, E. (1999) Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression. Mol. Cell. Biol., 19, 8513–8525.[Abstract/Free Full Text]

14. Lee, J.T. and Lu, N. (1999) Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell, 99, 47–57.[CrossRef][Web of Science][Medline]

15. 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]

16. 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.[Abstract/Free Full Text]

17. Stavropoulos, N., Lu, N. and Lee, J.T. (2001) A functional role for Tsix transcription in blocking Xist RNA accumulation but not in X-chromosome choice. Proc. Natl Acad. Sci. USA, 98, 10232–10237.[Abstract/Free Full Text]

18. Bartolomei, M.S., Webber, A.L., Brunkow, M.E. and Tilghman, S.M. (1993) Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Devl., 7, 1663–1673.[Abstract/Free Full Text]

19. Chao, W., Huynh, K.D., Spencer, R.J., Davidow, L.S. and Lee, J.T. (2002) CTCF, a candidate trans-acting factor for X-inactivation choice. Science, 295, 345–347.[Abstract/Free Full Text]

20. 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][Web of Science][Medline]

21. Migeon, B.R., Chowdhury, A.K., Dunston, J.A. and McIntosh, I. (2001) Identification of TSIX, encoding an RNA antisense to human Xist, reveals differences from its murine counterpart: implications for X inactivation. Am. J. Hum. Genet., 69, 951–960.[CrossRef][Web of Science][Medline]

22. Migeon, B.R., Lee, C.H., Chowdhury, A.K. and Carpenter, H. (2002) Species differences in TSIX/Tsix genes reveal the roles of these genes in X-chromosome inactivation. Am. J. Hum. Genet., 71, 286–293.[CrossRef][Web of Science][Medline]

23. Allaman-Pillet, N., Djemai, A., Bonny, C. and Schorderet, D.F. (2000) The 5' repeat elements of the mouse Xist gene inhibit the transcription of X-linked genes. Gene Expr., 9, 93–101.[Web of Science][Medline]

24. 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][Web of Science][Medline]

25. Nam, D.K., Lee, S., Zhou, G., Cao, X., Wang, C., Clark, T., Chen, J., Rowley, J.D. and Wang, S.M. (2002) Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription. Proc. Natl Acad. Sci. USA, 99, 6152–6156.[Abstract/Free Full Text]

26. Sheets, M.D., Ogg, S.C. and Wickens, M.P. (1990) Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucl. Acid Res., 18, 5799–5805.[Abstract/Free Full Text]

27. Gilman, M. (1987) Ribonuclease protection assay. In Ausubel, F.M., Brent, R., Kingston, R.E., More, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (eds), Current Protocols in Molecular Biology. John Wiley, Hoboken, NJ, Vol. 1, pp. 4.7.1–4.7.8.

28. Morey, C., Arnaud, D., Avner, P. and Clerc, P. (2001) Tsix-mediated repression of Xist accumulation is not sufficient for normal random X inactivation. Hum. Mol. Genet., 10, 1403–1411.[Abstract/Free Full Text]

29. Buzin, C.H., Mann, J.R. and Singer-Sam, J. (1994) Quantitative RT–PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies. Development, 120, 3529–3536.[Abstract]

30. Panning, B., Dausman, J. and Jaenisch, R. (1997) X chromosome inactivation is mediated by Xist RNA stabilization. Cell, 90, 907–916.[CrossRef][Web of Science][Medline]

31. Sheardown, S.A., Duthie, S.M., Johnston, C.M., Newall, A.E., Formstone, E.J., Arkell, R.M., Nesterova, T.B., Alghisi, G.C., Rastan, S. and Brockdorff, N. (1997) Stabilization of Xist RNA mediates initiation of X chromosome inactivation. Cell, 91, 99–107.[CrossRef][Web of Science][Medline]

32. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.[CrossRef][Medline]

33. Zamore, P.D., Tuschl, T., Sharp, P.A. and Bartel, D.P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101, 25–33.[CrossRef][Web of Science][Medline]

34. Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I. and Martienssen, R.A. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science, 297, 1833–1837.[Abstract/Free Full Text]

35. Reinhart, B.J. and Bartel, D.P. (2002) Small RNAs correspond to centromere heterochromatic repeats. Science, 297, 1831.[Free Full Text]

36. Beletskii, A., Hong, Y.K., Pehrson, J., Egholm, M. and Strauss, W.M. (2001) PNA interference mapping demonstrates functional domains in the noncoding RNA Xist. Proc. Natl Acad. Sci. USA, 98, 9215–9220.[Abstract/Free Full Text]

37. Csankovszki, G., Panning, B., Bates, B., Pehrson, J.R. and Jaenisch, R. (1999) Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat. Genet., 22, 323–324.[CrossRef][Web of Science][Medline]

38. Wutz, A., Smrzka, O.W., Schweifer, N., Schellander, K., Wagner, E.F. and Barlow, D.P. (1997) Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature, 389, 745–749.[CrossRef][Medline]

39. Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. and Lalande, M. (1998) An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nat. Genet., 19, 15–16.[Web of Science][Medline]

40. Wroe, S.F., Kelsey, G., Skinner, J.A., Bodle, D., Ball, S.T., Beechey, C.V., Peters, J. and Williamson, C.M. (2000) An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc. Natl Acad. Sci. USA, 97, 3342–3346.[Abstract/Free Full Text]

41. Lee, Y.J., Park, C.W., Hahn, Y., Park, J., Lee, J., Yun, J.H., Hyun, B. and Chung, J.H. (2000) Mit1/Lb9 and Copg2, new members of mouse imprinted genes closely linked to Peg1/Mest(1). FEBS Lett., 472, 230–234.

42. Smilinich, N.J., Day, C.D., Fitzpatrick, G.V., Caldwell, G.M., Lossie, A.C., Cooper, P.R., Smallwood, A.C., Joyce, J.A., Schofield, P.N., Reik, W. et al. (1999) A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith–Wiedemann syndrome. Proc. Natl Acad. Sci. USA, 96, 8064–8069.[Abstract/Free Full Text]

43. Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. and Feinberg, A.P. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith–Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc. Natl Acad. Sci. USA, 96, 5203–5208.[Abstract/Free Full Text]

44. Li, E., Bestor, T.H. and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69, 915–926.[CrossRef][Web of Science][Medline]

45. Simmler, M.C., Cunningham, D.B., Clerc, P., Vermat, T., Caudron, B., Cruaud, C., Pawlak, A., Szpirer, C., Weissenbach, J., Claverie, J.M. et al. (1996) A 94 kb genomic sequence 3' to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx. Hum. Mol. Genet., 5, 1713–1726.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				J. T. Lee Lessons from X-chromosome inactivation: long ncRNA as guides and tethers to the epigenome Genes & Dev., August 15, 2009; 23(16): 1831 - 1842. [Abstract] [Full Text] [PDF]

				Y. Ogawa, B. K. Sun, and J. T. Lee Intersection of the RNA Interference and X-Inactivation Pathways Science, June 6, 2008; 320(5881): 1336 - 1341. [Abstract] [Full Text] [PDF]

				K. V. Prasanth and D. L. Spector Eukaryotic regulatory RNAs: an answer to the 'genome complexity' conundrum Genes & Dev., January 1, 2007; 21(1): 11 - 42. [Abstract] [Full Text] [PDF]

				T. Sado, Y. Hoki, and H. Sasaki Tsix defective in splicing is competent to establish Xist silencing Development, December 15, 2006; 133(24): 4925 - 4931. [Abstract] [Full Text] [PDF]

				A. M. Gussow, N. V. Giordani, R. K. Tran, Y. Imai, D. L. Kwiatkowski, G. F. Rall, T. P. Margolis, and D. C. Bloom Tissue-Specific Splicing of the Herpes Simplex Virus Type 1 Latency-Associated Transcript (LAT) Intron in LAT Transgenic Mice J. Virol., October 1, 2006; 80(19): 9414 - 9423. [Abstract] [Full Text] [PDF]

				S. Vigneau, S. Augui, P. Navarro, P. Avner, and P. Clerc An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation PNAS, May 9, 2006; 103(19): 7390 - 7395. [Abstract] [Full Text] [PDF]

				A. Werner and A. Berdal Natural antisense transcripts: sound or silence? Physiol Genomics, October 17, 2005; 23(2): 125 - 131. [Abstract] [Full Text] [PDF]

				S. Landais, R. Quantin, and E. Rassart Radiation Leukemia Virus Common Integration at the Kis2 Locus: Simultaneous Overexpression of a Novel Noncoding RNA and of the Proximal Phf6 Gene J. Virol., September 1, 2005; 79(17): 11443 - 11456. [Abstract] [Full Text] [PDF]

				M. Landers, M. A. Calciano, D. Colosi, H. Glatt-Deeley, J. Wagstaff, and M. Lalande Maternal disruption of Ube3a leads to increased expression of Ube3a-ATS in trans Nucleic Acids Res., July 18, 2005; 33(13): 3976 - 3984. [Abstract] [Full Text] [PDF]

				P. Navarro, S. Pichard, C. Ciaudo, P. Avner, and C. Rougeulle Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation Genes & Dev., June 15, 2005; 19(12): 1474 - 1484. [Abstract] [Full Text] [PDF]

				T. Sado and A. C. Ferguson-Smith Imprinted X inactivation and reprogramming in the preimplantation mouse embryo Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R59 - R64. [Abstract] [Full Text] [PDF]

				N. Stavropoulos, R. K. Rowntree, and J. T. Lee Identification of Developmentally Specific Enhancers for Tsix in the Regulation of X Chromosome Inactivation Mol. Cell. Biol., April 1, 2005; 25(7): 2757 - 2769. [Abstract] [Full Text] [PDF]

				S. Shirasawa, H. Harada, K. Furugaki, T. Akamizu, N. Ishikawa, K. Ito, K. Ito, H. Tamai, K. Kuma, S. Kubota, et al. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease Hum. Mol. Genet., October 1, 2004; 13(19): 2221 - 2231. [Abstract] [Full Text] [PDF]

				M. Landers, D. L. Bancescu, E. Le Meur, C. Rougeulle, H. Glatt-Deeley, C. Brannan, F. Muscatelli, and M. Lalande Regulation of the large (~1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn Nucleic Acids Res., June 29, 2004; 32(11): 3480 - 3492. [Abstract] [Full Text] [PDF]

				V.L. CHANDLER Poetry of b1 Paramutation: cis- and trans-Chromatin Communication Cold Spring Harb Symp Quant Biol, January 1, 2004; 69(0): 355 - 362. [Abstract] [PDF]

				T. B. Nesterova, C. M. Johnston, R. Appanah, A. E.T. Newall, J. Godwin, M. Alexiou, and N. Brockdorff Skewing X chromosome choice by modulating sense transcription across the Xist locus Genes & Dev., September 1, 2003; 17(17): 2177 - 2190. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (41) Request Permissions Google Scholar Articles by Shibata, S. Articles by Lee, J. T. Search for Related Content PubMed PubMed Citation Articles by Shibata, S. Articles by Lee, J. T. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics