The long-term objective of the research proposed in this application is to understand in molecular terms how nuclear hormone receptors regulate gene expression within the context of the processes of cell differentiation and growth control. The general strategy employed will be to attempt to describe how a specific trans-acting factor, the vitamin 1,25(OH)2D3 receptor (VDR), influences the proliferation and differentiation of cells of the immune system, while at the same time describing the receptor's structure and function utilizing in vitro and crystallographic approaches. Specifically, the goals are to: 1. Isolate and identify genes that are direct targets of VDR during 1,25(OH)2D3-induced differentiation of the human leukemic cell line U937. A variety of molecular cloning techniques, such as subtraction and expression cloning, will be used to isolate VDR primary response genes. 2. Characterize the mechanism of regulation of such genes by VDR and other potentially interacting factors, and how that regulation results in the cell's commitment to terminally differentiate. The genomic versions of relevant cDNAs will be cloned; how these genes are regulated during 1,25(OH)2D3-induced differentiation will then be investigated. 3. Describe structural and functional aspects of VDR using defined in vitro assays and protein crystallography. VDR will be overexpressed and purified, and its DNA binding and transcriptional regulatory activities examined in detail. Concurrently, an effort to solve the three- dimensional structure of the full-length VDR and smaller derivatives will be initiated in collaboration with two protein crystallography groups.
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