The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3, and its associated intracellular receptor are members of a unique family of ligand-inducible transcription factors. The primary interest of this investigator is determining the means by which the hormone-receptor complex alters transcriptional events, with an underlying emphasis on the endocrine, metabolic and immunologic abnormalities of patients with renal failure. This includes examination of DNA binding mechanisms, modifications of the receptor or its expression, and interactions with other proteins. The present application seeks to test the hypothesis that the vitamin D receptor binds in a qualitatively different manner to representative positive and negative DNA response elements. In addition, the orientation and nature of the complex will be defined on selected response elements. To carry out this work, the electrophoretic gel mobility shift assay will be utilized to identify receptor-DNA complexes using antibody and competition experiments. Once the identity of the receptor-DNA complexes is established, relative strengths of binding will be ascertained and absolute mobilities will be compared. Interference footprinting experiments in conjunction with the mobility shift assay will be used to identify the intimate sites of DNA contact. This will include examination of the DNA phosphate backbone as well as major groove interactions with individual bases, including guanine, thymidine and adenine residues. Comparisons between both positive and negative response elements will permit more rigorous determination of those contacts critical for high affinity interactions, and delineate differences that may influence receptor function. Because of the propensity of the vitamin D receptor to heterodimerize during DNA binding, a series of bromodeoxyuridine crosslinking experiments are planned to assess the nature of the bound complexes. Crosslinking of the receptor to bromodeoxyuridine substituted, radioactively labeled DNA response elements by ultraviolet light will covalently """"""""tag"""""""" the protein components of the complex in intimate contact with the DNA. Fractionation of the resultant mix by SDS-PAGE will permit an estimation of the size of linked protein(s), and, given the appropriate strategy, their orientation with respect to the DNA binding site. This work represents the first attempt to rigorously identify the points of DNA contact made by the vitamin D receptor to its response elements and should provide insight into the functional aspects of the protein.
Showing the most recent 10 out of 11 publications
