Steroid Receptor Function and Nuclear Organization

描述(申请人提供)：来自我们实验室和其他实验室的单细胞分析表明，涉及基于类固醇受体的转录过程在活细胞内的时空动力学方面受到严格调控。利用荧光标记的雌激素受体-α(ER)和细胞培养模型系统中的几个辅助调节因子，我们将检验这一假设，即动力学和区间化的调节对于转录激活是必要的。这项测试将包括使用高定量成像I方法在转录前、转录期间和转录后检查ER功能。转录动力学的实时可视化将在三个具体目标中进行探索：目的I在配体/非配体依赖的激活过程中，利用光谱上I相容的GFP融合，进一步表征活细胞和固定细胞中ER/辅调节因子的组织、动力学和周转。延时和多光谱成像和光漂白方法将通过脉冲追逐实验和使用闪光表位标记直接从活细胞进行超微结构比较来加强；FRET研究将提供整个细胞核内蛋白质-蛋白质相互作用的视觉评估。AIM II将利用lac和tet抑制子的融合以及lac和tet操纵子的整合DNA阵列来检测ER/辅调节因子的相互作用，并利用荧光报告重点研究局部染色质的影响。AIM III将通过使用一系列ER-响应性催乳素(PRL)启动子来关注包含整合转录单位的新细胞，这些启动子允许可视化整合位点并提供生物传感器的转录活性读数。新的PRL阵列的实时光漂白数据显示，当结合整合的启动子时，ER和辅助调节子是高度动态的(tl/2=大约几秒)。受体-辅调节分子相互作用的直接时空检查现在是可能的，并将测试ER激活、染色质调节和转录之间的联系；同时将进行染色质免疫沉淀研究，以从生化方法评估动力学。这些新的方法将提供关于内质网功能的重要信息，以及它在活细胞内作用部位使用的机制，从而允许同时对内质网功能的几个方面进行多重分析。第一次，对受体激活的早期事件(秒/分钟)的机械和定量检查将可能导致和直接跟随转录，并将导致药物治疗的新策略的开发，特别是在激素反应性癌症中。

DESCRIPTION (provided by applicant): Single cell analyses from our lab and others suggest the processes involved in steroid receptor-based transcription are tightly regulated with respect to spatiotemporal dynamics within living cells. Using fluorescently-tagged estrogen receptor-alpha (ER) and several coregulators in cell culture model systems, we will test the hypothesis that regulation of both dynamics and compartmentalization are necessary for transcriptional activation. This test will include examination of ER function prior to, during, and after transcription using highly quantitative imaging I approaches. Real-time visualization of transcription dynamics will be explored in three specific aims: Aim I will further characterize ER/coregulator organization, dynamics and turnover, in living and fixed cells, using spectrally I compatible "GFP fusions during ligand/non-ligand dependent activation. Time-lapse and multi-spectral imaging, and photobleaching approaches will be augmented by pulse-chase experiments and ultrastructural comparison direct from live cells using FlAsH epitope tagging; FRET studies will provide visual protein-protein interaction assessment throughout the nucleus. Aim II will utilize lac- and tet-repressor fusions and integrated lac- and tet-operator DNA arrays to examine ER/coregulator interactions and focus upon local chromatin effects with a' fluorescent reporter. Aim III will focus upon new cells containing integrated transcription units by using a series of ER-responsive prolactin (PRL) promoters that allow visualization of the integration sites and provide biosensor readout of transcription activity. Real-time photobleaching data with new PRL arrays reveal ER and coregulator are highly dynamic (tl/2 = approximately seconds) when binding the integrated promoter. Direct spatiotemporal examination of receptor-coregulator interactions are now possible and will test the link between ER activation, chromatin modulation and transcription; concurrent chromatin immunoprecipitation studies will be performed to assess dynamics from a biochemical approach. These novel approaches will provide vital information regarding the function of ER, and mechanisms it uses at its site of action, within the living cell, allowing multiplex analyses of several aspects of ER function simultaneously. For the first time, mechanistic and quantitative scrutiny of early events (seconds/minutes) of receptor activation will be possible leading to and directly following transcription, and should lead to development of novel strategies for drug therapies, specifically in hormone-responsive cancers.