Applying peptide and protein domain microarrays to epigenetic research

描述（由申请人提供）：化学部分（如乙酰基、甲基和磷酸基）对组蛋白的动态翻译后修饰（PTM）构成了影响基本生理和病理过程的关键表观遗传机制。识别不同组蛋白修饰的蛋白质和结构域，称为“阅读器”，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阅读器对于理解染色质动力学如何有助于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展和进展至关重要。该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表观遗传事件的作者和读者的研究。在这个提议中要测试的一般假设是，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广谱染色质PTM的不同读者的能力。该提案的重点是蛋白质甲基化作为一个典型的表观遗传事件，有两个具体的目标计划。在第一个目标中，将建立范式来验证和利用多重修饰的组蛋白肽微阵列，用于采用蛋白质结构域文库（在目标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的筛选中的高通量结合测定。将进行芯片酶测定以研究肽微阵列作为鉴定候选组蛋白修饰活性的平台的效用。在第二个目标中，蛋白质结构域微阵列包含一个全面的库染色质相关的结构域将被生成和表征为高通量的工具，表观遗传学研究。将开发多种类型的探针用于测试这些阵列，包括双修饰肽探针（在目标1中开发）和修饰特异性核小体探针。对于这两个目标，将开发和标准化分析工具和软件，以促进将提案中开发的技术转让给表观遗传学界。这一新的蛋白质组学技术的产生，允许快速和准确地发现染色质PTM读者应该证明是非常有价值的阐明表观遗传事件如何影响人类健康和疾病的建议。 公共卫生相关性：染色质的表观遗传事件调节对正常健康至关重要的多种细胞功能。放置和读取表观遗传修饰的蛋白质的失调通常与肿瘤疾病、免疫缺陷综合征和许多其他遗传疾病有关。我们提出的研究应该提供深入了解表观遗传动力学如何在分子水平上发挥作用，并可能导致发现新的表观遗传靶点，用于治疗多种人类疾病。

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.