Dissection of Xist Functional Elements Involved in X-Chromosome Inactivation

Abstract

X-chromosome inactivation (XCI) is the epigenetic process of silencing one of the two X chromosomes in female mammals to balance X-linked gene dosage with that of males. This process is governed by the long noncoding RNA Xist. Xist is expressed from and coats one X chromosome in cis, leading to recruitment of various protein factors to silence gene expression. However, the functional RNA elements and molecular mechanisms involved in Xist coating and silencing remain ambiguous. The rationale for this work was thus to perform a systematic deletional analysis to identify functional motifs within the endogenous Xist locus in female cells. The screen identified regions important for correct splicing of the Xist transcript, such as the Repeat A region. Others, including Repeat F, were required for proper expression and/or RNA stability. Repeat E was crucial for restricting Xist localization to the inactive X (Xi) territory. Finally, Repeat B was necessary for recruitment of Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2) across the Xi, as well as for proper Xist coating. To gain mechanistic insight into Polycomb targeting and Xist localization, I identified protein trans factors associated with the Repeat B and E motifs. Repeat B function in Polycomb recruitment and Xist coating was shown to be mediated through direct interaction with the RNA-binding protein HNRNPK. Meanwhile, Repeat E function in Xist localization was found to be mediated through interaction with the nuclear matrix protein CIZ1. Just as Xist RNA is necessary to spread Polycomb complexes across the Xi, I found that Polycomb complexes are in turn necessary to properly spread Xist RNA. In addition, PRC1 and PRC2 occupancy on Xi was discovered to be mutually interdependent. Hence, Xist, PRC1, and PRC2 require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading has significant consequences, as deleting Repeat B during de novo XCI establishment causes failure of X-linked gene silencing and disrupts architectural reconfiguration of the X from and active to inactive chromosomal structure.