Developmental Xist Induction is Mediated by Enhanced Splicing

Dosage compensation in female placental animals is accomplished by silencing one of the two X-chromosomes. X-chromosome inactivation (XCI) is initiated early in development by preferentially upregulating the lncRNA Xist expression from the future inactive X-chromosome. The work presented in this dissertation aims to understand the transcriptional regulation of Xist during XCI induction Xist has long been identified as being both required and sufficient to induce X-chromosome inactivation when expressed in cis from the future inactive X-chromosome. Xist has 8 exons, is alternatively spliced, 3' polyadenylated, and 5' capped similar to protein coding genes but is retained in the nucleus. Multiple studies have identified that one of the earliest events of XCI is upregulation of the Xist transcript. However post-transcriptional regulation of Xist hasn't been well characterized. The role of splicing of lncRNAs is currently unknown. Splicing of Xist has possibly been overlooked historically because Xist was first cloned as spliced RNA and the spliced form is sufficient to induce XCI therefore most studies have focused on the spliced form. In Chapter 2 I show that differentiation induces splicing and that this is post-transcriptionally regulated after upregulation of Xist during the initiation of XCI. New technologies have allowed identification of a significant amount of potential binding partners, suggesting more regulation of Xist exists than was previously known. In Chapter 3, I show a new role for known splice regulator PTBP1 in regulating splicing of Xist. My data shows that PTBP1 is required for efficient splicing of Xist and suggests that this interaction is indirect and may involve additional trans acting factors. In Chapter 4, I show that splicing efficiency may be associated with the choice of which X-chromosome to silence during induction of XCI. In summary the work presented in this dissertation suggests that splicing of Xist is an essential step of Xist induction from the inactive X-chromosome during XCI. This work suggests that inefficient splicing of Xist prevents expression of Xist, providing potential insight to how ES cells choose which chromosome to silence and potential binding partners involved in this choice