The long-term goal of this work is to understand how RNA containing complexes modulate chromatin structure and gene expression. X chromosome inactivation is a particularly dramatic example of a change in chromatin structure that is regulated by RNA. The Xist gene regulates X-inactivation. Xist encodes a non-coding RNA that is transcribed exclusively from the Xi in female somatic cells. During initiation of X-inactivation Xist RNA spreads in cis to coat the chromosome that will become the Xi, and the spread of transcriptional silencing correlates tightly with the cis-spread of Xist RNA. My genetic, developmental and cytological analysis showed that Xist RNA is stabilized at the initiation of X-inactivation. These results lead to the hypothesis that a developmentally regulated Xist RNA - protein interaction facilitates stabilization and spread of Xist RNA on the Xi. As an independent investigator I have demonstrated that Xist RNA is part of an RNA-protein complex and that antisense affinity chromatography can be employed to purify this complex. Using a combination of powerful and complementary approaches, including biochemical purification, genetic manipulation of embryonic stem cells, and molecular cytogenetics, we propose to identify proteins that interact with Xist RNA. The mechanisms Xist uses to regulate X-inactivation are poorly understood and the identification of Xist-interacting proteins is the first step in analysis of the molecular basis of Xist RNA function. It is highly likely that RNA molecules are more generally used for chromatin remodeling and regulation of gene expression in eukaryotes since RNA is crucial for the functional organization of the nucleus and is an integral part of the dosage compensation apparatus in flies and mammals. Our understanding of the mechanisms by which RNA-protein complexes affect chromatin structure would be greatly facilitated by identification of additional Xist-like regulatory RNA molecules. We have developed a method to isolate chromatin associated RNAs and used this method to isolate an RNA that accumulates on mouse centromeres. We propose to characterize this novel RNA. The characterization of both the RNA and the protein components of Xist and other RNA-protein complexes that affect chromatin structure will provide insight into the role nuclear RNA plays in regulating gene expression.
