Photoreactive histone deacetylase probes for chromatin immunoprecipitation in can

描述（由申请人提供）：转录复合物组分及其相关/调控基因的鉴定是所有现代发现的核心。组蛋白去乙酰化酶（HDAC）是染色质免疫沉淀技术（ChIP）研究的最多的蛋白质之一，具有重要的表观遗传学功能。为此，ChIP是一种成功且广泛使用的技术，用于识别与感兴趣的蛋白质相互作用的DNA区域。然而，目前的ChIP技术有几个限制，经常构成严重的挑战。作为基于抗体的ChIP测定的替代方法，我们建议开发一种新技术，其中我们将使用我们的新型小分子HDAC光反应性探针（PRP）来询问HDAC与染色质的结合。在许多优点中，该技术能够以HDAC同种型选择性或非选择性方式询问转录复合物的作用，并且其具有克服表位可及性、假阴性和需要多种昂贵抗体的问题的优异潜力。我们最近的研究表明，高效的HDAC亚型选择性PRP可以成功地用于检测其目标在无细胞和基于细胞的实验，并确定身份和接近脱乙酰酶复合物的其他组件。我们假设我们的高效双功能光反应性HDAC探针可用于“识别”转录复合物中的HDAC，并补充ChIP测定中HDAC抗体的使用。为了解决这一假设，我们提出以下建议：目的1）我们将设计和鉴定化学支架、光反应基团（PRG）、用于连接含表位基团的手柄（ipHandle）和用于免疫沉淀的二级识别基团（TagI），以实现与染色质相关DC复合物交联的稳健灵敏度、同种型选择性和再现性。优先考虑最佳I类HDAC选择性和非选择性PRP，用于ChIP实验中的优化和验证。目的2）我们将评估和优化不同的HDAC同种型选择性和非选择性PRP，这些PRP已经可用或将在目的1中开发用于ChIP测定。我们将使用已知招募HDAC的明确的雌激素受体（ER）结合位点作为读数。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ChIP分析的结果。将进行HDAC PRP和HDAC抗体之间的比较。将使用siRNA方法评价HDAC-PRP的特异性。在完成这些研究后，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-ChIP将作为未来研究的基础，以了解表观基因组的重编程如何与HDAC抑制剂的有益和不良作用的机制相关联，从而使HDAC抑制剂具有充分的临床潜力。 被发现我们的最终目标是利用该项目的发现来加速开发安全的基于HDAC的癌症治疗方法。

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.