X chromosome dosage compensation in mammals is mediated by inactivating one X in female somatic cells. There is considerable evidence that some region of the X chromosome is required for its own inactivation. A search for such cis acting elements requires a more precise description of this region than is now available in any mammal. To learn more about the initial steps in dosage compensation and to facilitate identification of pertinent DNA sequences, we will localize and characterize the smallest segment of the inactive X chromosome essential for inactivation. This requires a good number od breakpoints that can be correlated with ability to inactivate. We will develop an extensive """"""""defined function"""""""" panel of somatic cell hybrids containing human X chromosomes with deletions in the Xq13 region considered to be the site of the putative X inactivation center (XIC). The deleted chromosomes will include those which retain the ability to undergo X inactivation, and others with impaired function, presumably by having lost the relevant sequences. With these mouse-human hybrid cells whose only human X is the deleted chromosome, and molecular probes, we will define a region invariably present in chromosomes that can inactivate, but absent or altered in those that do not. The dimensions of this region will be refined by identifying the minimal region of homology with mouse and marsupial X chromosomes. To further delineate the region we will look for genes within the conserved region which like XIST are transcribed exclusively off the inactive X and at the critical stage of embryogenesis when X inactivation is occurring; this transcriptional activity may have a direct role in X inactivation or may merely reflect changes in the chromatin close to sequences of interest, thereby revealing more precisely the location of the inactivation center, which itself may not be transcribed. Transcripts will be looked for by screening cDNA libraries from mouse embryos at stages just before the onset to just after inactivation is complete with mouse YAC clones containing regions of interest. These libraries also can be screened for candidate genes (like Xist) to determine if they are transcribed at the right time in development. We will analyze the region around candidate genes for differential DNA methylation and nuclease sensitivity as these chromatin features may also help us identify the region of interest. Any cDNAs identified in the embryonic library will be analyzed to determine if they come from the active or inactive X chromosome, and the locus considered a candidate for a role in X inactivation. Although the proposed studies may not definitively identify the nature and function of the XIC, they should bring us closer to doing so, by providing a more refined map of the region and a defined function hybrid panel which will be a unique resource for this purpose.
