Photoreactive histone deacetylase probes for chromatin immunoprecipitation in can

描述(由申请人提供)：识别转录复合体的成分及其相关/调控的基因是所有现代发现的核心。组蛋白脱乙酰酶(HDAC)是染色质免疫沉淀法(CHIP)分析最多的蛋白质之一，具有重要的表观遗传功能。为此，芯片是一种成功的和广泛使用的技术来识别与感兴趣蛋白质相互作用的DNA区域。然而，当前的芯片技术有几个限制，这些限制经常构成严重的挑战。作为基于抗体的芯片分析的替代，我们建议开发一种新的技术，其中我们将使用我们的新型小分子HDAC光反应探针(PRPS)来检测HDAC与染色质的结合。在许多优点中，这项技术能够以HDAC异构体选择性或非选择性的方式询问转录复合体的作用，并且它具有很好的潜力来克服表位的可及性、假阴性和需要多种昂贵的抗体。我们最近的研究表明，高效的HDAC异构体选择性PRPS可以成功地用于在无细胞和基于细胞的实验中检测其靶标，并确定脱乙酰酶复合体的其他成分的身份和接近程度。我们假设，我们的高效双功能光反应型HDAC探针可以用于在转录复合体中识别HDAC，并补充HDAC抗体在芯片分析中的使用。为了解决这一假设，我们提出如下建议：目的1)我们将设计和鉴定化学支架、光反应基团(PRGS)、用于连接包含表位基团的手柄(IpHandle)和用于免疫沉淀的二级识别基团(TAGI)，以在与染色质相关的DC复合体的交联中获得强大的敏感性、异构体选择性和重复性。为芯片实验中的优化和验证确定最佳I类HDAC选择性和非选择性PRP的优先级。目的2)我们将评估和优化不同的HDAC异构体选择性和非选择性PRP，它们要么已经可用，要么将在AIM 1中开发用于芯片分析。我们将使用定义明确的雌激素受体(ER)结合位点，已知这些结合位点可以招募HDAC作为读数。将使用非光反应性HDAC抑制剂进行竞争实验，以验证基于PRP的芯片分析的结果。对HDAC、PRPS和HDAC抗体进行比较。HDAC-PRPS的特异性将使用siRNA方法进行评估。在完成拟议的研究后，我们希望开发出一种新的方法和新的工具来研究与染色质DNA相关的HDAC。总之，在本申请中开发的prp芯片将作为未来研究的基础，以了解表观基因组的重新编程如何与hdac抑制剂的有利和不利影响的潜在机制相关联，从而允许hdac抑制剂的全部临床潜力。 被揭开。我们的最终目标是利用这一项目的发现来加快基于HDAC的癌症安全疗法的开发。

DESCRIPTION (provided by applicant): Identification of the components of transcription complexes and the genes associated/regulated by them are at the heart of all modern discoveries. As key epigenetic players with essential biological functions, histone deacetylases (HDACs) are among the most frequently analyzed proteins by chromatin immunoprecipitation (ChIP). To this end, ChIP is a successful and widely used technique to identify regions of DNA interacting with the proteins of interest. However, current ChIP technologies have several limitations that frequently pose serious challenges. As an alternative to antibody-based ChIP assays, we propose to develop a novel technology where we will use our novel small molecule HDAC photoreactive probes (PRPs) to interrogate HDAC binding to chromatin. Among many advantages, this technology is capable of interrogating the role of transcription complexes both in HDAC isoform- selective or non-selective manner, and it has excellent potential to overcome the problem of accessibility of epitopes, false negatives, and necessity for multiple expensive antibodies. Our recent studies have demonstrated that highly potent HDAC isoform-selective PRPs can be successfully used to detect their targets in cell-free and cell-based experiments and to determine identity and proximity of other components of deacetylase complexes. We hypothesize that our highly potent bi-functional photoreactive HDAC probes can be used to "recognize" HDACs in transcription complexes and complement the use of HDAC antibodies in ChIP assays. To address this hypothesis, we propose the following: Aim 1) We will design and identify the chemical scaffolds, photoreactive groups (PRGs), handles for attachment of an epitope containing group (ipHandle), and the secondary recognition groups for immunoprecipitation (TagI) to achieve robust sensitivity, isoform selectivity, and reproducibility in crosslinking to chromatin-associated DC complexes. Prioritize best class I HDAC selective and non-selective PRPs for the optimization and validation in ChIP experiments. Aim 2) we will evaluate and optimize different HDAC isoform- selective and non-selective PRPs that are either already available or will be developed in Aim 1 for use in ChIP assays. We will use well-defined estrogen receptor ¿ (ER) binding sites that are known to recruit HDACs as a readout. Competition experiments will be conducted using non-photoreactive HDAC inhibitors to validate the outcomes of the PRP-based ChIP analysis. Comparisons between HDAC PRPs and HDAC antibodies will be made. Specificity of HDAC-PRPs will be evaluated using siRNA approaches. Upon completion of the proposed studies, we expect to have developed a novel approach and novel tools for studying HDACs associated with chromatin DNA. Altogether, PRP-ChIP developed in this application will serve as a foundation for future studies to understand how re-programming of the epigenome is linked to the mechanisms underlying the beneficial and adverse effects of HDAC inhibitors, thereby allowing the full clinical potential of HDAC inhibitors to be uncovered. Our ultimate goal is to use the findings of this project to speed up the development of safe HDAC-based therapeutics for cancer.