Female eutherian mammals have evolved a mechanism which equalizes the dosage of functional X-linked genes in each somatic cell to that of males. This dosage compensation is accomplished by transcriptionally inactivating genes on one of the two X chromosomes in females. Thus, for most X-linked genes in female somatic cells, an active and inactive allele reside within the same nucleus but are differentially regulated and expressed. The process of X chromosome inactivation is developmentally regulated in female embryogenesis and involves the coordinate and chromosome-wide silencing of X-linked genes in cis. The long-term goal of this project is to determine the molecular mechanism of X chromosome inactivation. This proposal will investigate the molecular basis for establishing and maintaining the differential expression of a single X-linked gene, the hypoxanthine phosphribosyltransferase (HPRT) gene, on the active and inactive X chromosomes. We postulate that transcriptional regulation of the HPRT gene by X inactivation will involve a complex hierarchy of inter- dependent regulatory mechanisms, from long-range effects of higher order chromatin structure, to local nucleosome structure within the promoter and other regulatory regions, to individual sequence-specific DNA-protein interactions, each of which is crucial to the establishment and maintenance of differential expression of the HPRT gene on the active and inactive X chromosomes. We further postulate that the process of X chromosome inactivation will function through this hierarchy of mechanisms that regulate transcription of individual genes on the X chromosome. Thus, we will undertake a detailed study of the full range of mechanisms that influence HPRT gene transcription. The experiments proposed in this application will investigate two aspects of regulation of the HPRT gene by X chromosome inactivation. First, we will continue our detailed analysis of the HPRT promoter region on the active and inactive X chromosomes, and the effects of DNA methylation, local chromatin structure, and sequence- specific DNA-protein interactions on promoter function. Secondly, we will begin a long-term investigation of the structure and role of the HPRT chromatin domain in regulating expression of the HPRT gene on the active and inactive X chromosomes. This will involve defining the borders of the domain, then identifying and characterizing elements within the domain that regulate its chromatin structure. Understanding the mechanisms that regulate transcription of an individual X-linked gene by X inactivation should provide insights and experimental approaches to investigate the chromosomal and developmental aspects of this unique genetic process.
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