Regulation of imprinted X-chromosome inactivation in mice by Tsix

In mammals, X-chromosome inactivation is imprinted in the extra-embryonic l ineages with paternal X chromosome being preferentially inactivated. In thi s study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show t hat Tsix is transcribed from two putative promoters and its transcripts are processed, Expression of Tsix is first detected in blastocysts and is impr inted with only the maternal allele transcribed. The imprinted expression o f Tsix persists in the extraembryonic tissues after implantation, but is er ased in embryonic tissues. To investigate the function of Tsix in X-inactiv ation, we disrupted Tsix by insertion of an IRES beta geo cassette in the s econd exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic developmen t, inheritance of a disrupted maternal allele results in ectopic Xist expre ssion and early embryonic lethality, owing to inactivation of both X chromo somes in females and single X chromosome in males. Further, early developme ntal defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extr aembryonic tissues.