Defining the sequence requirements for Xist function in X inactivation

Mammalian genomes encode thousands of long non-coding (lnc) RNAs, many with important functions including the regulation of gene expression, yet, how lncRNAs function remains largely unexplored. The lncRNA Xist provides a remarkable model to investigate the function of lncRNAs in gene regulation, as it spreads from its site of transcription on the X chromosome over the entire chromosome in cis to induce gene silencing, alter chromatin state, and modulate the three-dimensional chromosome architecture in the process of X-chromosome inactivation (XCI). XCI is fundamentally important for female mammalian development but, despite its critical role, the mechanisms by which Xist carries out the various tasks associated with XCI still remain largely unclear. Recently, our and other labs proposed that Xist fulfills its different roles during XCI, such as gene silencing, chromatin association, spreading, recruitment of repressive chromatin regulators, membrane-less compartment formation, through different RNA domains, which in turn recruit different proteins. The 17kb long Xist RNA consists of a series of conserved repeats, termed A-F, as well as intervening non-repeat regions. Both repeat and non-repeat regions have been demonstrated to bind proteins, so both types of sequences can form functional domains. However, except for the A-repeat, which is now known to mediate silencing by recruitment of the proteins SPEN and RBM15, the function of nearly all other Xist sequences is still unknown. We sought to determine the Xist domains required for the initiation and maintenance of XCI in female mESCs. In Chapter 2, we identified the Xist F-repeat DNA sequence as a critical regulatory element of Xist expression during initiation of XCI using CRISPR mediated deletional analysis of Xist sequences. We also identify E2F3 as a potential interactor of the Xist-F-repeat DNA sequence using an in vitro pulldown assay coupled with mass spectrometry analysis. In Chapter 3, we identified the Xist E-repeat as a functional domain of Xist RNA that is required for the formation of a membrane-less silencing compartment on the inactive X. The E-repeat enables this condensate formation through recruitment of PTBP1, MATR3, TDP-43, and CELF1 via its multivalent protein binding sites, resulting in higher order assemblies of these proteins. Collectively, our findings have expanded on our understanding of the diverse molecular mechanisms employed by Xist-protein interactions at both the DNA and RNA level, to reveal the means by which Xist integrates different functions through its domains. Our data has revealed new paradigms for regulation of gene expression by lncRNAs and uncovered important insights into the molecular regulation of XCI by Xist