UCLA

Despite decades of biochemical, proteomic and genetic characterizations of the Xist longnon-coding RNA, the master regulator of X-chromosome inactivation (XCI), the spatiotemporal kinetics of Xist expression and how it coordinates the dynamic recruitment of protein effectors to direct robust gene silencing across the X-chromosome remain unexplored. Furthermore, most studies of Xist are conducted in the mouse system, so its role in mediating X-chromosome dampening (XCD), a unique mode of X-chromosome dosage compensation in humans, is still an open question. Therefore, in this thesis, I sought to plug this lack of understanding by (i) elucidating the molecular mechanism by which the chromosome-wide silencing compartment on the inactive X-chromosome (Xi) is formed and maintained across XCI, (ii) determining if XIST is responsible for XCD in human embryonic development, and (iii) characterizing the link between the genomewide localization patterns of Xist and its effect on gene silencing. iii In Chapter 2, we employed sophisticated super-resolution microscopy and single particle tracking techniques in living mouse embryonic stem cells (ESCs) undergoing XCI to precisely interrogate the spatiotemporal kinetics of Xist upregulation and its association with key protein partners that direct various aspects of XCI, and found that Xist first forms distinct foci that are locally restricted to about 50 sites across the Xi. Each site contains two Xist molecules, which seed the spatial concentration of integral protein interactors into what we term as a supra-molecular complex (SMC), made up of CIZ1 and CELF1 for localization of Xist across the Xi, PCGF5 and other components of Polycomb Repressive Complex 1 (PRC1) for inducing heterochromatinization and Xi compaction, and the transcriptional repressor SPEN, which is aided by its intrinsically disordered regions (IDRs) to promote phase separation-mediated aggregation of more SPEN molecules for the faithful and robust maintenance of X-linked gene silencing. In Chapter 3, we demonstrated the requirement of XIST in regulating XCD, as CRISPR-Cas9-mediated ablation of XIST in various human pluripotent stem cell (hPSC) lines that model XCD in vitro resulted in the loss of transcriptional downregulation on the dampened X-chromosome (Xd). By applying a high throughput genomics method that maps the chromatin binding of XIST, we discovered increased XIST enrichment on the Xi relative to the Xd, suggesting that XIST may be unable to exert complete silencing due to its reduced accumulation on the Xd. Moreover, we characterized a novel function of XIST, which can surprisingly propagate to certain autosomal regions beyond the X-chromosome territory, as well as repress some of the autosomal genes that it targets, resulting in a sex iv imbalance of the XIST-enriched autosomal gene expression levels that is also observed in human pre-implantation embryos in vivo. In Chapter 4, we built on our findings from Chapter 3 and showed that like in hPSCs in which the Xd exhibits a lower enrichment of XIST binding, early differentiating mouse ESCs also display diminished levels of Xist accumulation on the Xi, accompanied by reduced X-linked gene silencing, compared to the higher levels of Xist enrichment in late differentiated cells with complete XCI. Additionally, we characterized the distribution of Xist to autosomes in the mouse system, and illustrated how Xist spreading to the A- and B-compartments of the genome can be used to predict its differential ability to trigger gene silencing. Furthermore, we highlighted the remarkable ability of Xist to enact partial Xlinked gene repression in a mouse somatic cell line, which has surpassed the narrow developmental window surrounding pluripotency that was previously established in the XCI field for Xist-mediated silencing. Collectively, our findings gathered from these research projects have greatly expanded on the current knowledge about the molecular landscape established by Xist in setting up the Xi compartment, revealed insights on how XIST controls both XCI and XCD, and examined the genome-wide localization of Xist/XIST and its consequent gene silencing dynamics. In addition to addressing several open questions in the XCI field, our knowledge gained from this body of work on the Xist lncRNA may be extended to characterize other lncRNAs that contribute to the establishment and/or maintenance of gene regulatory nuclear compartments.