A Nucleosomal Biosensor for Identification and Isolation of Nuclear Hormone Recep

描述(申请人提供)：核激素受体(NHR)通过结合小的亲脂配体来调节转录，并对正常细胞功能和发育有深远的影响。NHR功能异常常见于癌症患者。这促使了针对NHR受体的治疗性配体的开发(例如，乳腺癌的他莫昔芬，早幼粒白血病的ATRA)。新的天然或合成的核受体配体很可能包括有用的抗癌剂。我们提出了一种创新的、高通量的方法来识别核受体的功能配体。我们假设体外基于核小体的生物传感器可以识别和区分激活或抑制核受体活性的配体。我们工作的总体目标是发展通过使用这种生物传感器快速筛选大型化学库的能力，以便为已知或孤立的核受体提供功能配体。我们开发了独特的工具来促进我们追求这些目标。这些包括试剂和仪器，使我们能够实时跟踪单分子和群体水平的核小体重塑。这个R21的应用集中在雌激素受体(ER)信号通路上，作为一个模型来展示这种分析的威力和敏感性。在第一个目标中，我们构建了一个核小体，在核小体中，含有雌激素反应元件(ERE)的荧光团标记的DNA包裹在组蛋白周围，从而将能量从供体荧光(Cy3)转移到受体荧光(Cy5)(荧光共振能量转移，或FRET)。在第二个目标中，我们测量雌激素受体激动剂特异性重塑含有ERE的核小体的能力，表现为FRET的丢失。核小体重塑将作为ERE序列、配体(激动剂与拮抗剂)和细胞提取物的组织来源的函数进行研究。还将开发一种仅使用重组蛋白的无细胞提取液生物传感器。在第三个目标中，我们将使核小体生物传感器适应于筛选文库的高通量格式，并使用ER激动剂、拮抗剂和非配体来验证该系统。在这个项目中开发的生物传感器应该能够快速筛选选定核受体的假定配体文库。此外，它将使快速剖析NHR途径的蛋白质或DNA成分成为可能。这种生物传感器应该比基于细胞的报告基因分析更快速、更通用，比只测量配体/受体结合的分析更能提供信息。

DESCRIPTION (provided by applicant): Nuclear hormone receptors (NHRs) modulate transcription by binding small lipophilic ligands and have a profound impact on normal cellular function and on development. Aberrant NHR function occurs frequently in cancers. This has prompted the development of therapeutic ligands targeting NHR receptors (e.g. tamoxifen in breast cancer, ATRA in promyelocytic leukemia). New natural or synthetic ligands for nuclear receptors are likely to include useful anticancer agents. We propose an innovative, high-throughput approach to identify functional ligands of nuclear receptors. We hypothesize that an in vitro nucleosome- based biosensor can identify and distinguish ligands that activate or inhibit nuclear receptor activity. The overall goal of our work is to develop the capacity to screen large chemical libraries rapidly for functional ligands to known or to orphan nuclear receptors through the use of such a biosensor. We have developed unique tools to facilitate our pursuit of these goals. These include reagents and instrumentation that enables us to follow nucleosomal remodeling at both the single molecule and population level in real-time. This R21 application focuses on the estrogen receptor (ER) signaling pathway as a model to demonstrate the power and sensitivity of this assay. In Aim One, we construct a nucleosome in which fluorophore-tagged DNA containing an estrogen response element (ERE) is wrapped around histones so that energy is transferred from the donor fluor (Cy3) to the acceptor fluor (Cy5) (fluorescence resonance energy transfer, or FRET). In Aim Two, we measure the ability of estrogen receptor agonists to specifically remodel ERE-containing nucleosomes, manifested as a loss of FRET. Nucleosome remodeling will be studied as a function of ERE sequence, ligand (agonist versus antagonist) and tissue source of cell extracts. A cell extract-free biosensor will also be developed utilizing only recombinant proteins. In Aim Three, we will adapt the nucleosomal biosensor to a high throughput format for screening libraries and validate the system using ER agonists, antagonists and nonligands. The biosensor developed in this project should enable rapid screening of libraries of putative ligands for chosen nuclear receptors. Moreover, it will enable rapid dissection of protein or DNA components of NHR pathways. This biosensor should be more rapid and versatile than cell-based reporter gene assays and more informative than assays that measure only ligand/receptor binding.