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.