| Within each cell of a female mammal, one X-chromosome is transcriptionally inactivated to compensate for the unequal dose of X-linked genes in females and males. This inactivation is triggered by Xist, an X-linked gene whose noncoding RNA accumulates along and coats one X-chromosome. At the outset of this work, the mechanisms governing Xist accumulation were vague, but the description and characterization of Tsix, a gene antisense to Xist, suggested that it might inhibit this accumulation. Here, I describe three lines of experimentation pertaining to Xist, Tsix, and the regulation of X-inactivation.;First, I have collaborated with others to re-evaluate a prominent hypothesis, in which a switch between alternate Xist promoters underlies the initiation of X-inactivation. We describe a previously unrecognized pseudogene upstream of Xist, and show that this pseudogene and Tsix transcripts confound the core of this hypothesis, the description of a novel upstream Xist promoter. Together with functional tests, these findings indicate that the promoter switch hypothesis is unlikely to be correct, and suggest instead that other mechanisms control the initiation of X-inactivation.;Second, I describe a gain-of-function manipulation in which a constitutive promoter is inserted into one Tsix allele in female embryonic stem cells. This alteration augments the transcriptional activity of Tsix and prevents Xist upregulation and X-inactivation in cis. However, it does not affect X-inactivation of the wild-type X-chromosome, nor does it seem to alter the X-chromosome choice mechanism that designates active and inactive X-chromosomes. Thus, Tsix expression regulates Xist upregulation, but may lie genetically downstream of X-chromosome choice.;Finally, I have identified and characterized Tsix cis-regulatory elements using a luciferase reporter gene. These experiments define the minimal promoter region of Tsix and demonstrate that it is constitutively active. They also define distal regulatory elements, one of which stimulates the Tsix promoter specifically in undifferentiated cells. The properties of this element suggest that it may regulate Tsix in vivo. These experiments also define flanking upstream and downstream sequences that cooperatively stimulate the Tsix promoter, in a manner that is unusually dependent on the context and orientation of these sequences. |
