Human Molecular Genetics, 2003, Vol. 12, No. 2 125–136 DOI: 10.1093/hmg/ddg010 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; Revised and Accepted November 2, 2002 GenBank accession number: AF541962 Downloaded from https://academic.oup.com/hmg/article/12/2/125/608526 by guest on 27 September 2021 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 30 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 50 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 transcription blocks Xist RNA accumulation on the same chromosome (16,17). Current models propose that, prior to the In mammals, either of the two X-chromosomes in females is onset of XCI (undifferentiated cellular state), the co-expression inactivated to compensate for dosage (1), a phenomenon of Tsix along with Xist in cis prevents high level Xist expression referred to as X-chromosome inactivation (XCI). The master and the initiation of XCI. At the onset of XCI, silencing of the switch for this long-range chromosomal silencing is termed the future Xi can only proceed with the downregulation of Tsix ‘X-inactivation center’ (Xic) (2,3) and is sufficient for in cis (12,14) and, conversely, the maintenance of the active chromosome counting, choice, initiation of XCI, and establish- state on the Xa depends on the persistent expression of Tsix on ment of heterochromatin (4). Two unusual noncoding tran- that chromosome (16,17). scripts have been identified within the Xic. The 17 kb What is the molecular basis of Tsix’s action on Xist? Three untranslated product of the Xist (X-inactive specific transcript) classes of mechanisms have been proposed (8,9,12). First, locus is unique in that it is transcribed only from inactive X Tsix’s action may be independent of its transcription and RNA chromosome (Xi) in female somatic cells and accumulates in product. ‘Enhancer competition’ (18) between Xist and Tsix is cis along the Xi (5–7, for Xist reviews, refer to 8,9). Xist is one model that has been proposed for Tsix’s role in determining indispensable for the silencing step of XCI (10,11). Its X-chromosome choice (19). Two other classes of mechanisms antisense counterpart, Tsix, originates 12 kb downstream of include ‘transcription-dependent’ and ‘RNA-dependent’ mod- Xist and transverses the entire Xist locus, thus encompassing els. In the former, antisense transcriptional action in itself over 40 kb of the Xic (12). Prior to the onset of XCI, Tsix and provides the repressive force. For example, the opposing Xist RNAs are co-localized to the Xic. Unlike Xist RNA, movement of the Tsix RNA polymerase complex could either however, Tsix RNA has not been observed to paint the X (12). result in topological constraints on Xist RNA production or lead Indeed, its action is strictly limited to the Xic, where its to ‘counter-current collision’ of the converging Xist and Tsix expression represses the upregulation of Xist and designates the polymerase machinery. Alternatively, antisense transcription future active X (Xa). When Tsix expression is eliminated on across the Xist promoter (15,20) could impair the recruitment one X in female cells, XCI occurs predominantly on the of transcriptional initiation machinery to Xist, perhaps by mutated X (13–15). In contrast, the augmentation of Tsix ‘promoter occlusion’. In contrast, the ‘RNA-dependent’ class *To whom correspondence should be addressed. Email: [email protected] Human Molecular Genetics, Vol. 12, No. 2 # Oxford University Press 2003; all rights reserved 126 Human Molecular Genetics, 2003, Vol. 12, No. 2 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 stoichio- metric 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 quantita- Downloaded from https://academic.oup.com/hmg/article/12/2/125/608526 by guest on 27 September 2021 tion 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 50 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-specificRT–PCR analysis, human TSIX transcription reportedly terminates within the 30 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 50 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 30 terminus, a possibility consistent with one interpreta- tion that human TSIX has lost function (22). A 30 functional end is appealing in light of the fact that Xist’s silencing domain lies in a repeat sequence complementary to this 30 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. Figure 1. Identification of a novel Tsix splice variant. (A) The strategy of Tsix RESULTS 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 Identification of a novel Tsix splice variant from an bi-directional arrow. Open tall rectangles and smaller gray ones represent Xist and Tsix exons, respectively. Symbols xE1, xE2-6, and xE7 represent Xist embryonic stem (ES) cell cDNA library 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 A striking feature of previously identified spliced Tsix RNAs is rectangles. Note the asterisk in clone B7, which represents newly identified their relatively short lengths and their minimal overlap with splice donor at the 30 end of Tsix exon 2. The 50 end of these cDNA clones Xist RNA (Fig. 1A). Notably, the region of complementarity are: base pair 1938 (clone 8B), 2362 (12B), 2876 (10B) of Genbank sequence lies exclusively at the 50 end of Xist (termed Tsix exon 4). 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 Because the structural variations in Tsix RNA could have base pair 79839 of X99946. (C) PCR amplification of Tsix transcripts spanning implications for the antisense mechanism, here we endeavored DXPas34 locus and exon 4. M, 100 bp ladder marker. 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). may potentially interfere with first-strand synthesis. To select We constructed a Tsix-specific cDNA library from wild-type for higher molecular weight cDNAs, the inserts were size- male ES cells by placing a primer specific for the antisense fractionated prior to cloning. Positives were subsequently transcript near the Xho I site within Xist exon 1 (Fig. 1A, identified by colony hybridization using an Xist exon 1 probe arrow), a position that circumvented upstream repeats which (Fig. 1A, bi-directional arrow). Human Molecular Genetics, 2003, Vol. 12, No. 2 127 Downloaded from https://academic.oup.com/hmg/article/12/2/125/608526 by guest on 27 September 2021 Figure 2. 50 and 30 RACE to determine the structure of the novel splice variant.