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First published online 5 December 2007
doi: 10.1242/dev.008490

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Crucial role of antisense transcription across the Xist promoter in Tsix-mediated Xist chromatin modification

Tatsuya Ohhata^1,2,*, Yuko Hoki^1,2, Hiroyuki Sasaki^2,3 and Takashi Sado^1,2,3,

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

View larger version (31K): [in this window] [in a new window]   Fig. 1. Tsix was truncated in the Xist gene by the introduction of a multiple polyadenylation sequence. (A) The genomic structure of the TsixpA allele is shown below the overall structure of the Xist/Tsix loci. The second intron of the human -globin gene, which harbors an mpA cassette in an antisense orientation with respect to Xist transcription, was introduced at the XhoI site (107228-107233 in GenBank Acc. No. AJ421479). SD, splicing donor; SA, splicing acceptor. (B) Targeting scheme for generating the TsixpA allele. Positions of the recognition sites of the relevant restriction enzymes and the probes used in C and D are shown. B, BamHI; E, EcoRI; H, HindIII; P, PvuII; X, XhoI. (C) Homologous recombination in ES cells was confirmed by Southern blotting. Probes and restriction enzymes used are shown below the blot. Appropriate recombination in the 5' region was confirmed using a 5'probe, Xist5-5, located outside of the fragment used for the short arm of the targeting vector. Since the recombination event in the 3' region could not be properly addressed with a single restriction enzyme, it was confirmed by two steps. Although use of probe XB verified the recombination event by the appearance of the expected 1.7 kb band upon BamHI digestion, the presence of a 5.6 kb band detected using probe 3' inner upon EcoRI digestion, which was also seen in parental R1 ES cells, verified that there was no other rearrangement in the fragment used for the long arm of the targeting vector. (D) Excision of the puromycin-resistance cassette by cre recombinase was confirmed by Southern blotting using tail DNA. PvuII digestion and subsequent hybridization with XB as a probe allowed differentiation of the wild-type, TsixpA2lox and TsixpA alleles.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Antisense transcription across the Xist promoter is diminished on XpA. (A) Positions of the primers used for cDNA synthesis (a,b,c indicated as arrows) and the region amplified by PCR (d,e,f,g,h) are shown relative to the introduced intron at the XhoI site. Primer a, AS90R2; primer b, R1910J; primer c, R371P1. PCR products were amplified using the following primer sets: d, Tsix2F/Tsix2R; e, Xist1175F/Xist1472R; f, 700P2/Xist-6(-)20; g, Xist (-540)F/8692R; h, 8111F/8418R. (B) The presence or absence of the antisense transcription in the region distal to the intron introduced at the XhoI site was examined by strand-specific RT-PCR in undifferentiated ES cells harboring each of the TsixpA and XistIVS alleles. Product e was amplified on cDNA primed by a. Product f was amplified on cDNA primed by either b or c. cDNA synthesis was carried out in either the presence (+) or absence (-) of reverse transcriptase. (C) The presence or absence of the elongation form of RNA pol II in the region distal to the mpA cassette was analyzed by ChIP assays using a monoclonal antibody (H5) against CTD phosphorylated at Ser2 in undifferentiated XY, XdcY, XpAY and XIVSY ES cells. The relative abundance of the chromatin immunoprecipitated by the antibody to the input was determined by real-time PCR. Although the elongation form of pol II was similarly distributed in the vicinity of the major transcription start site of Tsix (product d) among all types of ES cells, the level of the polymerase in the region downstream of the mpA cassette (product g and h) was significantly diminished in not only XdcY but also XpAY ES cells compared with wild-type and XIVSY ES cells. (D) RT-PCR was carried out on cDNA prepared from total RNA isolated from the placenta at E12.5. Although the proximal region of Tsix was amplified in all cases, the region distal to the mpA was barely amplified in XpAY and XpAX, indicating that antisense transcription in the Xist promoter region was efficiently attenuated by the mpA cassette in the placenta of the developing embryos, as expected.

View larger version (37K): [in this window] [in a new window]   Fig. 3. The Xist gene on XpA is dysfunctional. (A) The number of male and female pups born to wild-type females crossed with males hemizygous for the TsixpA allele. The sex ratio was extremely biased toward males. One of two females turned out to be XO. (B) The number of male and female embryos recovered at E7.5 from wild-type females crossed with XpAY males. Two of the embryos were genotyped as XXpAY. (C) Gross morphology of the typical XXpA embryo recovered at E7.5 is shown together with that of a male littermate. This phenotype is quite similar to that of females that inherit an Xist-deficient X from the father. e, embryonic ectoderm; ec ectoplacental cone; rm, Reichert's membrane. Scale bar: 0.5 mm. (D) The numbers of male and female pups born to females heterozygous for TsixpA crossed with wild-type males are shown. The TsixpA allele is transmitted to both male and female pups at the expected ratio. (E) Females heterozygous for TsixpA were crossed with XGFPY males and embryos recovered at E8.5 were examined for GFP expression. Although GFP fluorescence is uniformly observed in wild-type XXGFP embryos because cells that did not select XGFP as the inactive X are distributed throughout the body, XpAXGFP embryos are substantially negative for GFP, indicating that XGFP is invariably inactivated. This demonstrates that the Xist gene on XpA is dysfunctional. ys, yolk sac; am, amnion; al, allantois. Scale bar: 0.8 mm.

View larger version (36K): [in this window] [in a new window]   Fig. 4. The Xist locus is ectopically activated on the active XpA. (A) Total RNA prepared from E13.5 embryos was analyzed by northern blotting. Ectopic expression of Xist was evident from the single active X in XpAY male embryos. (B) Allelic expression of Xist was analyzed by RT-PCR in the embryo proper and the visceral endoderm. Females heterozygous for either TsixpA or XistIVS were crossed with JF1 males (XJF1Y), and RNA prepared from the embryo proper and visceral endoderm at E13.5 was subjected to RT-PCR on cDNA primed by Xist7(-)20 in a strand-specific manner. PvuII digestion of the amplified product revealed that in spite of the fact that XpA stayed active in essentially every cell in XpAXJF1 female embryos, Xist was expressed from the active XpA as well as the inactivated XJF1 in both the embryo proper and the visceral endoderm (upper panel). Similar activation of the Xist locus on XpA was also seen in both tissues of XpAY males. Such ectopic activation was never observed on XIVS in either tissue in either sex (lower panel). cDNA synthesis was carried out in the presence (+) or absence (-) of reverse transcriptase. (C) The levels of ectopic expression of Xist in the embryo proper and the visceral endoderm of XpAY males (XpAY-1 and XpAY-2) were compared with those of Xist in wild-type XX and XY embryos by real-time PCR using cDNA synthesized in B. Xist and Gapd sequences were amplified using primer set R700P2/Xist6(-)20 and GapdF/Gapdr2, respectively, and the expression level was normalized by the value for Gapd. Values are means ± s.d.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Methylation level of CpG sites in the 5' region of Xist is reduced on XpA. (A) The genomic structure of the 5' region of Xist on the wild-type X and XpA is shown along with recognition sites of the methylation-sensitive restriction enzymes and positions of probes used in this assay. (B-D) The methylation level in the 5' region of Xist was examined by Southern blotting in the embryo proper and the visceral endoderm of E13.5 embryos recovered from XpAX females crossed with wild-type males. Genomic DNA was digested with one of the four methylation-sensitive restriction enzymes shown in A in combination with BclI and probed with either HS0.7 or BE0.6. BclI digestion produced an 8.4 kb (black arrowhead shown on the right of the blot) and a 10.0-kb (white arrowhead shown on the right) band from the wild-type X and XpA, respectively. Although a decrease in CpG methylation was observed on XpA in both tissues of both male and female embryos, it was more prominent in the visceral endoderm. Since the restriction fragments derived from XpA digested by HhaI and SacII, when probed with HS0.7, migrated together with those from the wild-type X, the reduction of CpG methylation on XpA was less clear in the embryo proper of XpAX on the blot probed by HS0.7 (B), the same blot was reprobed with BE0.6 to visualize the fragments derived from XpA (indicated by asterisks), which appear only when the relevant restriction sites are unmethylated (C). Probing with HS0.7 was sufficient to show hypomethylation of the locus in the visceral endoderm (D). Hh, HhaI; S, SacII; Hp, HpaII; and P, PmaCI.

View larger version (8K): [in this window] [in a new window]   Fig. 6. The Xist locus on XpA fails to establish the repressive histone modification. Chromatin of the visceral endoderm was used for ChIP assays with the antibodies against the respective histone modifications. The immunoprecipitated chromatin was quantified by real-time PCR using Xist1F and Xist101R as primers, which amplify the first 101 bp sequence of Xist exon 1. The value for each modification, which is shown as relative abundance of the chromatin immunoprecipitated by each antibody compared with the input, was obtained from two or three independent experiments. Values are means ± s.d.

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