A Genetic Screen for Ecdysone Receptor Coactivators and Corepressors

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ABSTRACT The nuclear receptors are an ancient and widespread family of transcription factors that includes the receptors for the vertebrate steroid hormones, those for many other small lipophilic hormones (for example thyroid hormone and vitamin D), and many orphan receptors that may not be associated with hormones. The family also includes the polypeptides EcR and USP that together comprise the insect ecdysone receptor. A general picture of nuclear receptor function has emerged from extensive biochemical studies: Receptors bind to specific DNA target sequences where they may activate or repress transcription from a nearby promoter by binding to protein coactivators and corepressors that link the DNA-tethered receptor to the transcriptional apparatus. Binding of a ligand (steroid or other small lipophilic signaling molecule) to the receptor converts the receptor from one that binds corepressor to one that binds coactivator. The transcriptional activities regulated by the nuclear receptors are central to normal development. Moreover studies of nuclear receptor coactivators and corepressors are providing valuable clues to the rate-limiting steps in chromosomal gene activation and to processes that lead to tissue-specific regulation by hormones and other regulatory molecules. The ecdysone receptor in insects is a heterodimeric nuclear receptor composed of the protein EcR and USP. It mediates transcriptional responses to ecdysone, the steroid hormone that coordinates molting and metamorphosis in insects (and other arthropods). Many lines of evidence indicate that it is similar (though not identical) in both structure and function to the largest group of nuclear receptors, the type II or heterodimeric receptors and prior work has probed molecular aspects of the EcR/USP transcriptional response. Because of its well-developed genetics and because the Drosophila genome has been sequenced, the opportunity exists to begin to use genetic methods to identify proteins that interact with nuclear receptors. This project will develop a genetic screen using flies engineered to be sensitive to small fluctuations in the concentrations of proteins that interact with EcR/USP. The objective of the proposed experiments is the identification, by genetic means, of proteins that interact genetically (and physically) with EcR/USP. It is anticipated that the genes identified will include coactivators and co-repressors, chaperones, chromatin-remodeling factors, and other proteins that may be required for EcR/USP function. By distinguishing between global and tissue-specific interactions, the screen may provide insights into the roles of multiple, functionally similar corepressors and coactivators known from vertebrate studies. This screen has the potential to increase our understanding of gene expression and nuclear receptor function by: (1) Adding new molecules to the list of proteins that interact with nuclear receptors; (2) Identifying critical intermediates (coactivators and corepressors) in Drosophila where they can be integrated into on-going studies of gene regulation in development; (3) Applying a whole organism genetic approach to the analysis of a process (nuclear receptor interactions) that has heretofore been approachable only in cultured cells and by biochemistry; (4) Shedding light on tissue-and stage-specific aspects of coactivator and corepressor function