Steroid Receptor Function and Nuclear Organization

描述（由申请人提供）：来自我们实验室和其他实验室的单细胞分析表明，基于类固醇受体的转录过程与活细胞内的时空动态密切相关。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种共调节因子，我们将验证动力学和区隔化的调节是转录激活所必需的假设。该测试将包括使用高定量成像方法在转录之前、期间和之后检查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：目标1将进一步表征活细胞和固定细胞中的ER/共调节因子组织、动力学和转换，在配体/非配体依赖的激活过程中使用光谱兼容的“GFP融合”。延时和多光谱成像和光漂白方法将通过脉冲追踪实验和使用FlAsH表位标记直接从活细胞进行超微结构比较来增强；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-阻遏物融合以及集成的lac-和tet-操作子DNA阵列来检查ER/共调节因子相互作用，并通过荧光报告基因关注局部染色质效应。Aim III将通过使用一系列er反应性催乳素（PRL）启动子关注包含整合转录单元的新细胞，这些启动子允许可视化整合位点并提供转录活性的生物传感器读数。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和共调节子是高度动态的（tl/2 =大约秒）。受体-共调节因子相互作用的直接时空检查现在是可能的，并将测试内质网激活，染色质调节和转录之间的联系；同时进行染色质免疫沉淀研究，从生化方法评估动力学。这些新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，允许同时对内质网功能的几个方面进行多重分析。第一次，受体激活的早期事件（秒/分钟）的机制和定量审查将有可能导致转录和直接跟随转录，并应导致开发新的药物治疗策略，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.