The steroid hormone estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function, proliferation of breast cancer cells, and concentration of sperm in the male reproductive tract. Furthermore, estrogens and antiestrogens are widely used in regulating fertility, alleviating postmenopausal symptoms, and preventing and treating breast cancer. The biological effects of estrogen are initiated by binding to the intracellular estrogen receptor (ER) and inducing the ER to bind to estrogen response elements (EREs) residing in target genes. It is this interaction of ER with the ERE, in cooperation with multiple coregulatory proteins, that leads to changes in gene expression. During the previous award period, we demonstrated that individual EREs elicit distinct changes in ER conformation and that these ERE-induced changes in receptor conformation influence coactivator recruitment and ultimately alter gene expression. Thus, DNA, like a hormonal ligand, functions as an allosteric modulator of receptor conformation. The long term goal of this proposal is to elucidate mechanisms involved in regulating estrogen-responsive genes. We propose to isolate and identify proteins that associate with the ER in the presence of different EREs and hormones, to characterize Lhe effects of these proteins on the receptor's ability to bind to and activate transcription through different ERE sequences, and to elucidate mechanisms by which these proteins influence ER-mediated transactivation using molecular and biochemical analyses. We will test the physiological relevance of our in vitro observations and transient transfection assays by characterizing the activity of the identified proteins in MCF-7 cells using RNA interference and chromatin immunoprecipitation assays. The isolation and initial characterization of five _roteins has produced new and exciting insights to better understand the functional roles of these ER-associated _roteins and provided a fascinating glimpse of the breadth of their activities, which include histone acetylation, )NA repair, tumor suppression, and metabolic processing. The insights gained will help delineate how estrogens and antiestrogens regulate transcription of genes containing distinct ERE sequences and enhance our understanding of cellular responsiveness of mammary tumor cells, as well as normal tissues, such as the uterus and breast to these clinically important pharmaceutical agents.
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