X chromosome inactivation is the process whereby one of the two X chromosomes in somatic cells of mammalian females is inactivated early in embryogenesis. While the essential features of X inactivation have been known for over 30 years, the genomic context within which this long-range chromosomal control mechanism operates remains unclear. The human X chromosome contains an estimated several thousand genes, and the genomic resources to support systematic and comprehensive analyses of gene expression on a chromosome scale are becoming increasingly available. We have developed two complementary approaches to examine the inactivation status of any X-linked gene that is expressed in fibroblast strains or in fibroblast-derived somatic cell hybrids. Our previous work has examined the inactivation status of over 200 X-linked genes and expressed sequence tags (ESTs), has revealed the existence of at least four distinct types, classified according to their response to X inactivation, and has provided strong evidence that the organization of the chromosome itself plays a significant role in determining the X inactivation profile of its genes. These data provide a framework for a systematic approach designed to examine the genomic and chromosomal basis for these differences and to integrate a comprehensive X inactivation profile of the X chromosome with the growing gene map and nucleotide sequence of the human X chromosome. The experiments described in this application have two specific aims: (i) to determine the expression of at least 1,500 X-linked genes or ESTs in a panel of mouse x human somatic cell hybrids containing active or inactive human X chromosomes; and (ii) to determine the expression of at least 500 X-linked genes or ESTs in a panel of human diploid fibroblasts demonstrating non-random X inactivation by evaluating the mono- or biallelic expression of a series of expressed polymorphisms. The proposed experiments should provide a comprehensive view of the expression of genes on the human X chromosome in the context of its reponse to the process of X inactivation. These genomic data, which we view as an essential part of the X chromosome genome project, will be of substantial value to those in the fields of human and medical genetics and will, in addition, provide the basis for future insights into the chromosomal, epigenetic and molecular mechanisms by which cis-limited control of X-linked gene expression is achieved.
