The proposed investigators deal with the mechanism by which oxysterol metabolites feedback to repress 3-hydroxy-3- methylglutaryl-CoA reductase (HMGR) and other genes of the cholesterogenic pathway. They focus upon the isolation, struc- ture and function of an oxysterol receptor which appears to mediate the inter action of oxysterols with the regulatory regions of these genes. Efforts to complete the isolation of the receptor from mouse L cells or mouse liver will involve conventional protein fractionation procedures with photoaffinity- labeled receptors and affinity chromatography with monoclonal and polyclonal antibodies or with an oxysterol affinity column. The amino acid sequence of the purified receptor will be determined and a portion of the corresponding DNA coding sequence synthesized to probe a mouse DNA library and to clone the receptor gene. Localization of the receptor in the nucleus or cytosol will be determined by enucleation of cells. Studied with the partially purified receptor will investigate conditions and mechanisms that lead to reversible and irreversible dissociation of heteromeric forms of the receptor. A factor(s) in cell cytosol that causes irreversible dissociation to the dimeric and trimeric receptor to a monomeric form will be identified. physical-chemical properties of the various forms of the receptor and their relative affinities for non-specific DNA and for oxysterol response elements from the promoters of regulated genes will be compared. The effect of the receptor in a HMGR transcriptional assay will be determined. Limited studies of invertebrates that do not synthesize sterols will be carried out to determine whether or not the receptor is present. Studies with mutant cell lines resistant to oxysterols will determine whether the mutations involve defects or deficiencies in the oxysterol receptor or the regulatory region of the HMGR gene. The results of these studies will increase our understanding of a regulatory system that is involved in cholesterol homeostasis and in cell growth, replication and differentiation.
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