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First published online 15 November 2006
doi: 10.1242/dev.02670

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Tsix defective in splicing is competent to establish Xist silencing

¹ Division of Human Genetics, National Institute of Genetics, Research Organization of Information and Systems, 1111 Yata, Mishima, 411-8540, Japan.
² Department of Genetics, The Graduate University for Advanced Studies (Sokendai), 1111 Yata, Mishima, 411-8540, Japan.
³ PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama, 332-0012, Japan.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Generation of the splicing-defective Tsix allele by gene targeting. (A) Scheme for generating the TsixSA allele. Genomic structure of the Xist locus and the targeting vector are shown. Tsix exons (gray boxes) are also aligned in parallel with Xist exons (black boxes). Differential polyadenylation of the spliced Tsix transcripts occurs by recognizing one of the polyA signals (pA) shown in the distal region of exon 4. Positions of the probes used for Southern blotting in B and D are also indicated. P, PstI; Pm, PmaCI; R, EcoRI; S, SacI; Xb, XbaI. The PmaCI site destroyed in the cloning process is indicated as (Pm). (B) Homologous recombination was confirmed by Southern blotting. SA177 is one of the three ES lines harboring the correct targeting event. Genomic DNA digested with PstI (left) and SacI (right) was probed with PE0.24 and probe D (Sado et al., 2001), respectively. (C) Comparison of the TsixSA allele and the Xist1lox allele. The splicing acceptor site for exon 4 of Tsix, which is present in a 0.6-kb PmaCI-EcoRI fragment, is deleted in the TsixSA allele, but left intact in the Xist1lox allele. (D) The presence of the respective mutation in the mouse was confirmed by Southern blotting. Tail DNA digested with PstI was probed with XXh0.7.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Splicing products of Tsix are eliminated in ES cells. (A) The structure of the respective mutant allele is delineated with respect to the position of Tsix exons shown in gray. A proximal part of Tsix exon 4 shown in light gray indicates the region missing in the TsixSA allele. Tsix on Xdc chromosome is truncated by the insertion of IRES ßgeo (Sado et al., 2005). Positions of primers used for PCR and real-time PCR are also shown below the exons of Tsix. (R) and (Pm) are the EcoRI and PmaCI sites destroyed in the cloning process. (B) The absence of the splicing products of Tsix was confirmed by RT-PCR using cDNA prepared by random priming. The spliced form was detected using primer pair Xist1175F and 21b80F. The presence of the antisense transcription was monitored using primer pairs Tsix2F/Tsix2R and 8111F/8420R located in exon 2 and 4, respectively. (C) Antisense activity of Tsix was analyzed in each undifferentiated ES cell line by real-time PCR using primer sets Tsix2F/Tsix2R (exon 2) and 8111F/8420R (exon 4). (D) Northern blot analysis of polyA RNA isolated from ES cells carrying the respective mutation. Hybridization was serially performed using an RNA probe specific to Tsix (left) and Xist (middle), respectively, and a cDNA fragment of Gapd (right). The absence of the major splicing products is evident in TsixSA/Y ES cells. (E) DNaseI hypersensitive site assay using nuclei isolated from each ES cell line. Purified DNA was digested with HindIII and probed with Af0.4 (see Fig. 6). Neither of the known DNaseI hypersensitive sites (HS1 and HS5) found on the transcriptionally active Xist allele in somatic cells was observed in ES cells regardless of whether they harbor the mutation or not. A DNaseI hypersensitive site HS3 shown by an asterisk, which is known to be common to both the active and inactive X in somatic cells, was more prominent in XdcY than others. (F) Real-time PCR analysis of the transcripts from the Tsix/Xist locus in undifferentiated ES cells and 12-day embryoid bodies using primers R700P2 and Xist6(-)20 on strandspecifically prepared cDNA.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Spliced Tsix RNA is eliminated in the placenta. The absence of the splicing products of Tsix was confirmed in the E13.5 placenta carrying TsixSA in the same manner as in ES cells shown in Fig. 2A and B. It is evident that the splicing products are not detected in the placenta of TsixSA/+ and TsixSA/Y embryos despite the presence of the antisense transcription.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Xist is stably repressed in the absence of spliced Tsix RNA. Transcriptional activity of the Xist locus was analyzed by RT-PCR using RNA isolated from E13.5 male and female embryos carrying the respective mutation. Diagrams show the structure of each mutant allele and positions of primers used for PCR. Transcription of the Xist locus on the mutated X was examined using a primer set specific for EGFP.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Elimination of spliced Tsix RNA does not affect the methylation of the Xist promoter region. CpG methylation in the 5 ' region of Xist was analyzed in E13.5 embryos by Southern blotting. Diagrams of the promoter region of Xist show relevant restriction enzyme sites and the position of the probe used for this assay. CpG methylation was appropriately established even in the absence of spliced Tsix RNA in both sexes. B, BclI; H, HhaI; S, SacII.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Elimination of spliced Tsix RNA does not affect the establishment of closed chromatin structure. Chromatin structure in the 5 ' region of Xist was analyzed by the DNaseI hypersensitive site assay. The diagrams show the structure of the Xist/Tsix locus, and the positions of DNaseI hypersensitive sites identified on the respective mutated X chromosome as well as those of HindIII sites used for the digestion of purified DNA. The position of the probe (Af0.4) used for Southern hybridization is also indicated. Ectopic DNaseI hypersensitive sites detected on Xdc are not found in E13.5 embryos carrying TsixSA. White arrowheads indicate the fragment derived from HS3 on the mutated X; black arrowheads indicate those from HS1 and HS5 on the mutated X. H, HindIII.

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