Development and use of systems to study estrogen action

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DESCRIPTION (provided by applicant) To identify tissues and cells that contain functionally active estrogen receptors (ERs), a transgenic mouse, ERIN (estrogen receptor action indicator), was developed that functions as a reporter of ER activity by incorporating a transgene with an estrogen responsive promoter linked to the reporter gene beta-galactosidase. Initial analysis of ERIN mice demonstrated active ER in a variety of tissues, including uterus, pituitary, and liver. The ERIN mouse will be used to aid in the identification of 1) genes regulated in individual cells where ER-ligands, both estrogens and selective estrogen receptor modulators (SERMs), exert agonist activity and 2) novel ER comodulatory proteins. By following expression of the reporter gene in the presence of ER-ligands, cells containing activated ER will be isolated using fluorescence-activated cell sorting. First, RNA will be isolated from captured cells, and cRNA will be labeled and used to probe mouse oligonucleotide gene arrays to identify fingerprints of gene expression for specific classes of ER-ligands. Second, proteins will be isolated from sorted cells and subjected to affinity chromatography using specific ER-ligand complexes as bait, followed by proteome analysis using mixed peptide sequencing to identify proteins that interact directly with ER. It is anticipated that the identification of proteins important for the cell selective actions of different ER-ligands will partially explain ER-ligand cell specificity and facilitate the identification of the next generation SERMs by screening for compounds which facilitate one class of ER-cofactor interaction over another. Before this project was initiated, my mentor and I agreed that I could carry my work with the ERIN mice forward with me to a faculty position. Consequently, I selected this project as one around which I could develop an independent career. I feel that I am halfway toward accomplishing this goal. However, an additional period of training would allow me to extend this exciting research project into the new areas of genomics and proteomics. Duke University provides an excellent research environment to further these career and research objectives in the following specific ways: 1) the applicant's mentor, Dr. McDonnell, is an accomplished and productive researcher in the area of estrogen receptor action, 2) five collaborators with expertise in genomics, proteomics, and mammalian physiology and genetics have agreed to aid in the proposed research and training program, 3) a DNA microarray core facility and other key equipment are available on the Duke University campus, and 4) a new biweekly seminar series in genomics and proteomics will provide the latest information and a forum to discuss the proposed research project.