Investigation of X-inactivation using Tsix induction system

X chromosome inactivation (XCI) in mammals achieves dosage compensation of X chromosomal genes between XX females and XY males. One of the two female X chromosomes becomes transcriptionally inactivated early in development, such that in both male and female embryos one X chromosome is active. While the choice of which X chromosome to be inactivated in the embryonic lineage is random (random XCI), the paternally derived X chromosome is preferentially inactivated in the extra-embryonic lineages in mouse (Imprinted XCI). In this XCI process, firstly the numbers of X chromosomes are counted (counting), subsequently one of the two X chromosomes is chosen as inactive X (choice), and finally chosen X chromosome will be inactivated. X inactivation is regulated by a single locus - the Xic - on the X chromosome. Recently, pairing of the two Xic loci has been observed during the early state of differentiation in female ES cells. This suggests that Xic/Xic pairing may be correlated with counting and choice. Within the Xic several molecular elements have been identified. The long non-protein-coding Xist RNA is transcribed from the Xic on the future inactive X (Xi), attaches to Xi chromatin and accumulates over the chromosome triggering transcriptional silencing. Tsix is an antisense transcript acts as a negative regulator of Xist and regulates choice in XCI. However, the mechanism of Xic/Xic pairing and how does Tsix repress the Xist in cis has been unclear. In this proposal, to address the questions that if Tsix RNA regulates Xic/Xic pairing (project 1), Tsix transcription is dispensable for normal choice (project 2), and "XCI reprogramming" can be achieved in adult mice (project 5), gene targeting approach will be performed. Moreover, the function of Tsix in repressing Xist expression will be studied sequentially during ES cell and TS cell differentiation with Tsix induction system (project 3 and 4). These results will contribute to the understanding of the mechanisms of antisense regulations of Tsix and counting/choice in XCI.