A Nucleosomal Biosensor for Identification and Isolation of Nuclear Hormone Recep

描述（由申请人提供）：核激素受体（NHRs）通过结合小亲脂配体调节转录，对正常细胞功能和发育有深远影响。异常的NHR功能在癌症中经常发生。这促进了靶向NHR受体的治疗性配体的发展（例如用于乳腺癌的他莫昔芬，用于早幼粒细胞白血病的ATRA）。新的天然或合成的核受体配体很可能包含有用的抗癌剂。我们提出了一种创新的、高通量的方法来鉴定核受体的功能配体。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通过使用这种生物传感器，开发快速筛选已知或孤儿核受体的功能配体的大型化学文库的能力。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，使我们能够在单分子和群体水平上实时跟踪核小体重塑。本R21应用程序侧重于雌激素受体（ER）信号通路作为模型，以证明该检测的能力和敏感性。在Aim 1中，我们构建了一个核小体，其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，以便能量从供体荧光（Cy3）转移到受体荧光（Cy5）（荧光共振能量转移，或FRET）。在目的二中，我们测量了雌激素受体激动剂特异性改造含ere核小体的能力，表现为FRET的丧失。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功能进行研究。利用重组蛋白的无细胞提取物生物传感器也将被开发出来。在目标3中，我们将使核小体生物传感器适应高通量格式，用于筛选文库，并使用内质网激动剂、拮抗剂和非配体验证系统。本项目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NHR途径的蛋白质或DNA成分的快速解剖成为可能。这种生物传感器应该比基于细胞的报告基因检测更快速、更通用，比仅测量配体/受体结合的检测更有信息量。

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.