UC Irvine

Long noncoding RNA (lncRNA) have been identified in all eukaryotes but the functions and mechanisms of many lncRNA are unknown, and remain challenging to study in vivo. Mammalian X-Chromosome Inactivation (XCI) is regulated by lncRNA and has emerged as a model system in which to study lncRNA function. The "master regulator" of XCI is lncRNA Xist, which coordinates epigenetic silencing one of the two X chromosomes in females. While the primary Xist activating factor has been under debate, the lncRNA Jpx has emerged as a proposed activator. However, Jpx's function and mechanism have been unclear due to conflicting findings in mouse embryonic stem cell models.

I hypothesized that Jpx activates Xist expression in vivo, and that it would do so using both trans and cis genetic mechanisms. To test this, I utilized two transgenes, Tg(Jpx) and Tg(Jpx, Xist), to develop a novel Jpx transgenic mouse model and study the function of Jpx in mouse XCI. I found that transgenic Jpx mice were viable and fertile, although transgenic male viability was lower in certain Tg(Jpx) lines. Using RT-qPCR and Fluorescence in situ Hybridization (FISH) in mouse embryonic fibroblasts (mEFs) and post-implantation embryos, I observed a dose-dependent relationship between Jpx copy number and Xist expression in transgenic mice. This suggests that Jpx is sufficient to activate Xist expression in vivo. Further, I analyzed Jpx's allelic origin with RNA and DNA FISH and observed Jpx using both trans and cis mechanisms to activate Xist expression. RT-qPCR showed reduced X-linked gene expression in male and female Tg(Jpx) mEFs, suggesting that ectopic Xist can induce XCI. The presence of XCI in the male may explain the reduction in transgenic male viability. Overall, I have demonstrated that Jpx is a trans- and cis-acting Xist activator in mice. In the discussion, I have provided suggestions for future studies, including live cell imaging of the Jpx transcript and development of a Jpx knockout mouse model. The work presented here provides a framework for lncRNA functional studies in mice, and will help us understand how lncRNA regulate eukaryotic gene expression.