Integrated microplate platform for epigenetic analysis

描述(由申请人提供)：表观遗传过程以细胞类型和细胞外环境决定的模式解释基因组序列。表观遗传信息通过组蛋白和DNA的共价修饰、核小体位置和组蛋白变体的替换来编码。表观遗传学是当今生物学研究最深入的领域之一，代表了疾病病理生理学、诊断和治疗的新范式。为了推进表观遗传学研究，我们引入了一种新型的染色质免疫沉淀平台--矩阵芯片，它利用96孔板中表面固定的抗体，在同一板上完成从染色质沉淀到聚合酶链式反应就绪的DNA纯化的整个过程，而不需要转移样本。这种高通量方法允许并行分析染色质和转录事件的数量比以前可能的多一个数量级。在检测染色质修饰剂等含量较低的蛋白质的DNA-蛋白质相互作用时，基质芯片比传统方法更灵敏。芯片分析包括三个独立的步骤：(1)样品采集/染色质裂解，(2)免疫沉淀和(3)DNA纯化和分析(qPCR/测序)。虽然在Matrix芯片中，步骤(2)和(3)大大简化，允许一次运行数百次分析，但样品采集/染色质碎裂是在试管中进行的，效率低，劳动强度高。该项目提出了一种创新的多学科方法(Matula博士，超声/工程学，Denisenko博士，分子生物学，Bomsztyk博士，染色质生物学/生物技术/仪器)，以开发一个平台，PIXUL-CHIP，它将把高效的样本采集和染色质剪切与微板中的免疫沉淀相结合，用于高通量表观遗传学分析。提出了以下目标。目的#1.建立一种用于高通量快速染色质剪切的像素化超声(PIXUL)处理器。目的#2.将样品采集、染色质剪切和免疫沉淀结合到一个用于表观遗传学分析的集成微板平台--PIXUL-CHIP。目的#3.测试PIXUL-CHIP作为研究药物介导的人胚胎干细胞(HESC)更新和分化相关基因座表观遗传学变化的平台。表观遗传学是一个快速增长的领域，但尽管如此，仍有大量未得到满足的需求需要进一步的技术进步，以促进表观遗传学过程的探索，并在翻译研究和临床应用中利用这些知识。作为一项高效的技术，PIXUL-CHIP将提供强大的手段，在广泛的系统中同时研究基因位点上的多个染色质修饰和修饰物，因此将成为研究表观遗传过程、发现表观遗传生物标志物和测试新药及其组合的宝贵平台。

DESCRIPTION (provided by applicant): Epigenetic processes interpret the genomic sequences in a cell-type and extracellular environment-dictated mode. Epigenetic information is encoded through covalent modifications of histones and DNA, nucleosome position and substitution by histone variants. Epigenetics is one of the most intensively studied fields of biology today and represents a new paradigm for the pathophysiology, diagnosis and treatment of diseases. To advance epigenetic studies, we have introduced a novel chromatin immunoprecipitation platform, Matrix ChIP, that utilizes surface-immobilized antibodies in a 96-well plate, where the entire procedure from chromatin precipitation to PCR-ready DNA purification is done on the same plate without sample transfers. This high- throughput method allows for parallel profiling of an order of magnitude greater number of chromatin and transcription events than was previously possible. Matrix ChIP is more sensitive than traditional approach to detect DNA-protein interactions of less abundant proteins such as chromatin modifiers. ChIP assay consists of three separate steps: (1) sample harvesting/chromatin fragmentation, (2) immunoprecipitation and (3) DNA purification and analysis (qPCR/sequencing). Although in the Matrix ChIP, steps (2) and (3) are greatly simplified allowing one to run hundreds of assays at a time, sample harvesting/chromatin fragmentation is done in test tubes with low efficiency and high labor intensity. This project proposes an innovative multidisciplinary approach (Dr. Matula, ultrasound/engineering, Dr. Denisenko, molecular biology, and Dr. Bomsztyk, chromatin biology/biotechnology/instrumentation) to develop a platform, PIXUL-ChIP, that will integrate highly efficient sample harvesting and chromatin shearing with immunoprecipitation in microplates for high-throughput epigenetic analysis. The following aims are proposed. Aim#1. To build a pixelated ultrasound (PIXUL) processor for high-throughput fast chromatin shearing. Aim#2. To combine sample harvesting and chromatin shearing with immunoprecipitation into an integrated microplate platform for epigenetic analysis, PIXUL-ChIP. Aim#3. To test PIXUL-ChIP as a platform for studies of epigenetic changes at selected gene loci associated with drug-mediated human embryonic stem cell (hESC) renewal and differentiation. Epigenetics is a fast growing field, but nonetheless, there are significant unmet needs that require further technological advances to facilitate exploration of epigenetic processes and exploit this knowledge in translational research and clinical applications. A highly efficient technology, PIXUL-ChIP will provide powerful means to simultaneously study multiple chromatin modifications and modifiers at gene loci in a wide range of systems, and as such will be a valuable platform for studying epigenetic processes, discovering epigenetic biomarkers, and testing novel drugs and their combinations.