Different theories exist to explain the molecular mechanisms of retinoid action in the control of epithelial differentiation, growth and tumorigenesis. One is centered on cellular retinoic acidbinding protein, CRABP, detected and characterized in our and other laboratories. This protein may mediate the biological activity of retinoic acid in a manner somewhat similar to steroid hormones. The proposed work will delineate the precise mechanism by which CRABP/retinoic acid may function at the genomic level. It is proposed that an immunization schedule for CRABP and a hybridoma antibody production technique be developed, and an immunological probe for detection, quantitation and purification of CRABP be accomplished. In an attempt to understand the functionality of the binding protein, such antibodies will be employed for immunolocalization of CRABP in chick embryo skin. A detailed study on the genomic regulation by retinoic acid will be undertaken in embryonic chick skin -- a tisue highly responsive to this retinoid. This will include the specific and nonspecific binding of retinoic acid to nuclei, nude nuclei, and chromatin, and the role of CRABP in the specific binding. Also proposed is to accomplish localization of CRABP and retinoic acid in nuclei by autoradiographic technique. A CRABP-mediated binding of retinoic acid to solubilized chromatin will also be undertaken. Binding sites of retinoic acid/CRABP within the nuclei will be explored with DNA as a possible target. A sucrose density gradient sedimentation technique will be standardized to determine this important acceptor site. Involvement of chromosomal protein fractions in the above critical interactions will be explored by reconstitution experiments. Also proposed is the study of the direct effect of retinoic acid/CRABP in DNA transcription. Nuclei isolated from vitamin A-deficient and normal cells will be utilized to study the specific effects produced by the binding protein in these transcriptional events by using a filter assay. We will also identify specific genes induced by exposure to retinoic acid by construction of a library of cDNA clones using RNA isolated from retinoic acid treated tissue. The role of retinoic acid and the retinoic acid-CRABP complex in controlling the expression of these genes will be characterized, and the possibility that retinoic acid acts at the top of a cascade regulation of gene expression will be examined. The proposed work thus attempts a broad and thorough examination on the genomic control mechanism of retinoic acid and retinoic acid/CRABP at cellular, nuclear, chromosomal, DNA, DNA-transcriptional, and gene induction and/or suppression levels by using multiple modern techniques.
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