The process by which transcription is regulated in eukaryotic cells is of fundamental importance in understanding biological mechanisms which control cell differentiation and growth. While these mechanisms are not yet completely understood, much information has been gained by studying DNA-binding proteins which influence transcription. The vitamin D3 receptor (VDR), a member of the steroid hormone receptor family which binds 1,25-dihydroxyvitamin D3, is implicated in the regulation of many proteins involved in cell differentiation, particularly those critical to calcium and phosphate metabolism. Vitamin D3 mediates its effects via a mechanism in which the vitamin:receptor complex is internalized into the nucleus, and interacts with responsive elements in genes. The purpose of this research proposal is to further characterize the interaction of VDR with its responsive element in DNA. In initial experiments, VDR binding to the promoter region of the osteocalcin gene will be used as a model system. Osteocalcin is the major noncollagenous protein found in bone, and has vitamin D3 responsive elements located in its gene promoter region. The osteocalcin gene promoter has been obtained from another laboratory, and the receptor will be purified from chicken intestine (work in progress). Binding of the vitamin D3 receptor to DNA will be studied using both nitrocellulose filter binding and gel retardation experiments. The goal of the initial phase of the research project proposed is to establish the optimum conditions which promote specific binding of receptor to its DNA binding site. Subsequent experiments will determine if VDR binds in a cooperative manner to DNA, as the trimeric sequence motif found in the responsive elements suggests. Finally, synthetic fragments containing the responsive sequence will be used in binding experiments to determine the effect of multiple responsive elements in binding of VDR. It is felt that the results from these experiments may be critical to defining the mechanisms involved in VDR gene regulation, and to understanding DNA regulatory proteins in general. Furthermore, similar experiments can be used to determine if vitamin D3 analogues alter VDR:DNA complex formation, as potential therapeutic agents for diseases such as osteoporosis, which are related to vitamin D3 deficiency.
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