Steroid Receptor Function and Nuclear Organization

描述（由申请人提供）：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受到活细胞内时空动力学的严格调控。我们将在细胞培养模型系统中使用荧光标记的雌激素受体-α（ER）和几种辅调节因子，测试动力学和区室化调节对于转录激活是必要的这一假设。这项测试将包括检查ER功能之前，期间和转录后使用高度定量成像I方法。实时可视化的转录动力学将探讨在三个具体的目标：目的我将进一步表征ER/辅调节组织，动力学和营业额，在活细胞和固定的细胞，使用光谱I兼容的“GFP融合在配体/非配体依赖性激活。延时和多光谱成像，和光漂白的方法将增加脉冲追逐实验和超微结构比较直接从活细胞使用FlAsH表位标记; FRET研究将提供可视化的蛋白质-蛋白质相互作用评估整个细胞核。目的II将利用lac-和tet-阻遏蛋白融合和整合的lac-和tet-操纵基因DNA阵列来研究ER/辅调节因子的相互作用，并将重点放在局部染色质效应上。目的III将集中在新的细胞含有整合的转录单位，通过使用一系列的ER-响应催乳素（PRL）的启动子，允许可视化的整合位点，并提供生物传感器读出的转录活性。实时光漂白数据与新的PRL阵列揭示ER和辅调节是高度动态的（tl/2 =约秒）时，结合整合的启动子。直接时空检查受体coregulator的相互作用，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，同时染色质免疫沉淀研究将进行评估从生化方法的动态。这些新方法将提供有关ER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ER功能的几个方面进行多重分析。对于第一次，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，特别是在肿瘤反应性癌症中。

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.