Applying peptide and protein domain microarrays to epigenetic research

描述（由申请人提供）：组蛋白的动态翻译后修饰（PTM）是一种影响基本生理和病理过程的关键表观遗传机制，如乙酰、甲基和磷酸基团。识别不同组蛋白修饰的蛋白质和结构域被称为“读取器”，通过将染色质上的分子事件转导为生物学结果来定义特定修饰的功能后果。因此，阐明组蛋白PTM读取器对于理解染色质动力学如何促进表观遗传程序，以及染色质稳态的破坏如何从根本上影响癌症和其他疾病的发生和进展至关重要。本提案的长期目标是开发新的蛋白质阵列技术，用于蛋白质组范围内的高通量发现和研究表观遗传事件的作者和读者。在本提案中要测试的一个一般假设是，蛋白质阵列技术将彻底改变表观遗传学研究人员发现人类表观基因组中存在的染色质PTMs的广谱不同读者的能力。该提案侧重于蛋白质甲基化作为一个典型的表观遗传事件，计划有两个具体目标。在第一个Aim中，将建立范例来验证和利用多重修饰的组蛋白肽微阵列进行高通量结合分析，使用蛋白结构域文库（在Aim 2中开发），纯化的染色质调节大分子复合物和抗组蛋白抗体。在芯片酶分析将被执行，以调查多肽微阵列作为识别候选组蛋白修饰活性的平台的效用。在第二个目标中，包含染色质相关结构域的蛋白质结构域微阵列将被生成并表征为表观遗传学研究的高通量工具。将开发多种类型的探针来测试这些阵列，包括双重修饰肽探针（在Aim 1中开发）和修饰特异性核小体探针。为了这两个目标，将开发和标准化分析工具和软件，以促进提案中开发的技术向表观遗传社区的转移。这项新提出的蛋白质组学技术允许快速准确地发现染色质PTM读取器，对于阐明表观遗传事件如何影响人类健康和疾病具有很高的价值。

DESCRIPTION (provided by applicant): Dynamic post-translational modification (PTM) of histone proteins by chemical moieties such as acetyl-, methyl- and phospho- groups constitutes a key epigenetic mechanism that impacts on fundamental physiologic and pathologic processes. The proteins and domains that recognize distinct histone modification, named "readers", define the functional consequences of specific modifications by transducing molecular events at chromatin to biological outcomes. Thus, the elucidation of histone PTM readers is critical for understanding how chromatin dynamics contribute to epigenetic programs, and how disruption of chromatin homeostasis fundamentally impacts on the development and progression of cancers and other diseases. The long-term goal of this proposal is to develop novel protein array technologies for proteome-wide, high- throughput discovery and study of the writers and readers of epigenetic events. A general hypothesis to be tested in this proposal is that protein array technology will revolutionize the ability of epigenetic researchers to discover diverse readers for the broad spectrum of chromatin PTMs present in the human epigenome. The proposal focuses on protein methylation as a prototypical epigenetic event, with two specific Aims planned. In the first Aim, paradigms will be established to validate and utilize multiplex modified histone peptide microarrays for high-throughput binding assays in screens employing protein domain libraries (developed in Aim 2), purified chromatin-regulatory macromolecular complexes and anti-histone antibodies. In chip enzymatic assays will be performed to investigate the utility of peptide microarrays as a platform for identifying candidate histone-modifying activities. In the second Aim, protein domain microarrays containing a comprehensive library of chromatin-associated domains will be generated and characterized as high-throughput tools for epigenetic research. Multiple types of probes will be developed for testing these arrays, including dual modification peptide probes (developed in Aim 1) and modification specific nucleosomal probes. For both Aims, analytic tools and software will be developed and standardized to facilitate transfer of the technology developed in the proposal to the epigenetic community. The generation in this proposal of new proteomic technologies that allow for rapid and accurate discovery of chromatin PTM readers should prove highly valuable for elucidating how epigenetic events influence human health and disease. PUBLIC HEALTH RELEVANCE: Epigenetic events at chromatin regulate diverse cellular functions critical for normal health. Dysregulation of the proteins that place and read epigenetic modifications are frequently linked to neoplastic disease, immunodeficiencies syndromes and numerous other genetic disorders. Our proposed studies should provide insight into how epigenetic dynamics functional at the molecular level, and potentially lead to the discovery of new epigenetic targets for therapeutic intervention in diverse human diseases.