| Achoimre: | A prototype long non-coding RNA (lncRNA) is the Xist RNA (X-inactive specific transcript) in X chromosome inactivation (XCI), a mammalian form of dosage compensation, in which one of the two Xs in each female cell is inactivated to balance the X-linked gene dosage between males and females. The Xist RNA transcripts specifically coat the chromosome territory of the inactive X (Xi) to establish the chromosome-wide gene silencing. Besides this unique and fascinating behaviour, the working mechanism of Xist RNA remains largely elusive and hence is well worth of a detailed study in live cells. In this study, we explored three different approaches to label Xist in live cells: Spinach2 aptamer, sgRNA-dCas9-GFP and Pumilio homology domain (PUF). Using the Pumilio system, we successfully established a high-quality live-cell imaging system of Xist RNA. Two PUF binding sites (PBS), PBSb recruiting PUFb-EGFP to label Xist, and PBSa replacing the A-Repeat (a critical domain of Xist) to generate “ΔA mutant” and to recruit PUFa-effectors for functionally dissecting Xist, were applied to an X-linked, single-copy, full-length, inducible Xist transgene in male mouse embryonic stem cells. Upon induction, the emergence and dynamics of Xist signals (the “sunrise”) show interesting differences between undifferentiated and differentiating cells. Sunrise in undifferentiated cells is of late-onset, heterogeneous and slow-growing and exhibits milder gene-silencing effects compared to differentiating cells, showing that induced XCI in undifferentiated cells is counteracted by X chromosome reactivation (XCR). Intriguingly, our data suggest that Kat8 and Msl2, homologues of Drosophila dosage compensation proteins, are involved in mammalian XCR. Furthermore, ΔA Xist clouds are smaller and more compact than wild type clouds, demonstrating that during the onset of XCI, Xist first forms a silencing compartment and then spreads out to active genes in an A-repeat dependent manner. Recruiting Xist-binding proteins onto ΔA mutant reveals significant roles for Lbr and Ythdc1 in Xist spreading and gene-silencing, respectively. Besides live-cell imaging of Xist, in this thesis, I also summarise a pilot project that I conducted in my graduate research, which studies the roles of single-strand DNA breaks in X chromosome pairing.
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