The mouse Xist gene encodes an un-translated RNA required for dosage compensation. Xist RNA is expressed from the inactive X chromosome in female mouse cells and is required for the transcriptional silencing of virtually all other genes resident on the inactive X chromosome.Xist RNA specifically coats the inactive X chromosome. Regions of striking sequence conservation occur between mouse and human homologs of Xist acting factors that likely bind Xist and participate in the dosage compensation process have yet to be identified. A strategy to access the functional significance of conserved regions of Xist will be implemented and trans-acting factors that bind Xist RNA will be identified. Transgenic Xist genes containing precise deletion of the conserved regions will be expressed in a model cell-culture system, and the effects of these mutations will be determined by performing established assays for Xist function and RNA localization. In addition, there independent approaches designed to identify trans-acting factors that bind to conserved regions of Xist RNA are outlined. Following their identification, the biological significance of these trans-acting factors will be addressed by the developing antibodies to assess the intracellular locations of Xist binding proteins. Also, Xist RNA-protein interactions will be studied in vitro.
