Interaction of the Estrogen Receptor with DNA

描述（由申请人提供）：类固醇激素雌激素在女性生殖和乳腺组织的发育和维持中起关键作用，但也参与心血管、骨骼和神经功能的维持、乳腺癌细胞的增殖以及男性生殖道中精子的浓缩。 此外，雌激素和抗雌激素被广泛用于调节生育、缓解绝经后症状以及预防和治疗乳腺癌。雌激素的生物学效应是通过与细胞内雌激素受体（ER）结合并诱导ER与靶基因中的雌激素反应元件（ERE）结合而启动的。正是ER与ERE的这种相互作用，与多种共调节蛋白合作，导致基因表达的变化。在前一个奖项期间，我们证明，个别的ERE引起的ER构象的不同变化，这些ERE诱导的受体构象的变化影响辅激活剂的招聘，并最终改变基因表达。因此，DNA，像激素配体一样，作为受体构象的变构调节剂发挥作用。这项建议的长期目标是阐明参与调节雌激素反应基因的机制。我们建议分离和鉴定在不同ERE和激素存在下与ER相关的蛋白质，以表征这些蛋白质对受体结合并激活转录的能力的影响，并通过不同的ERE序列，并阐明这些蛋白质影响ER介导的反式激活的机制，使用分子和生化分析。我们将通过使用RNA干扰和染色质免疫沉淀测定来表征MCF-7细胞中已识别蛋白质的活性，测试我们的体外观察和瞬时转染测定的生理相关性。五种蛋白质的分离和初步表征产生了新的和令人兴奋的见解，以更好地了解这些ER相关蛋白质的功能作用，并提供了一个迷人的一瞥其活动的广度，其中包括组蛋白乙酰化，NA修复，肿瘤抑制和代谢处理。所获得的见解将有助于阐明雌激素和抗雌激素如何调节含有不同ERE序列的基因的转录，并增强我们对乳腺肿瘤细胞以及正常组织（如子宫和乳腺）对这些临床重要药物的细胞反应性的理解。

DESCRIPTION (provided by applicant): 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 the 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 proteins has produced new and exciting insights to better understand the functional roles of these ER-associated proteins 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.