This proposal uses a variety of approaches to define, in molecular terms, how retinoids influence development and physiology by controlling patterns of gene expression. Proteins interacting with RARs and RXRs will be identified by molecular approaches with emphasis on yeast two-hybrid screens. Cell lines expressing His-tag receptors will be used to purify and identify proteins bound to receptors in vivo. A substantial effort will be made to characterize the properties of a recently described co-repressor termed SMRT as it relates to hormonal signalling. Antibodies will be prepared, interactions will be confirmed by co-immunoprecipitation and nuclear localization, and functional properties will be determined by cotransfection into mammalian cells as well as by complementation and in vitro transcription systems. In addition to biochemical dissection, important structural domains for the receptors will be overproduced and their three-dimensional structure determined via X-ray crystallography. The role of heterodimer formation in modulating ligand binding and transcriptional activation properties of the receptor will be determined, as the further modulation of these properties via the formation of a ternary complex with SMRT will be examined. Functional roles of the RARs/RXRs during embryonic development will be assessed by creating and characterizing loss-of-function gene mutations in the mouse germ line for RARs and SMRT.
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