Applying peptide and protein domain microarrays to epigenetic research

将肽和蛋白质域微阵列应用于表观遗传学研究

• 批准号: 7879014
• 负责人: MARK T. BEDFORD
• 金额: $ 15.33万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879014
• 关键词: Amino Acid Motifs; Antibodies; Binding; Biological; Biological Assay; Cell physiology; Chemicals; Chromatin; Communities; Development; Disease; Epigenetic Process; Epitopes; Event; Generations; Goals; Health; Hereditary Disease; Histones; Homeostasis; Human; Immunologic Deficiency Syndromes; Knowledge; Lead; Libraries; Link; Lysine; Macromolecular Complexes; Methodology; Methylation; Microarray Analysis; Minority; Modeling; Modification; Molecular; Names; Nucleosomes; Other Genetics; Outcome; PHD Finger; Pathologic Processes; Peptide Library; Peptides; Physiological; Post-Translational Modification Site; Post-Translational Protein Processing; Protein Array; Protein Microchips; Proteins; Proteome; Proteomics; Reader; Reading; Research; Research Personnel; Role; Site; Software Tools; System; Technology; Technology Transfer; Tertiary Protein Structure; Testing; Therapeutic Intervention; Work; base; chromatin modification; combinatorial; experience; histone modification; human disease; insight; neoplastic; novel; programs; public health relevance; tool; tumor progression

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.

描述(由申请人提供)：组蛋白的动态翻译后修饰(PTM)由化学基团，如乙酰基、甲基基团和磷酸基团组成，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被称为“阅读器”，通过将染色质上的分子事件转换为生物学结果来定义特定修饰的功能后果。因此，组蛋白PTM阅读器的阐明对于理解染色质动态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展和进展至关重要。这项提议的长期目标是开发新的蛋白质阵列技术，用于蛋白质组范围的高通量发现和表观遗传事件的作者和读者的研究。在这项提议中要检验的一个普遍假设是，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广泛存在的染色质PTM的不同阅读器的能力。该提案将蛋白质甲基化作为一种典型的表观遗传学事件，计划了两个具体目标。在第一个目标中，将建立范例来验证和利用多重修饰的组蛋白多肽微阵列在使用蛋白质结构域文库(在Aim 2中开发)、纯化的染色质调节大分子复合体和抗组蛋白抗体的筛选中的高通量结合分析。在CHIP中，将进行酶分析，以研究多肽微阵列作为确定候选组蛋白修饰活性的平台的用途。在第二个目标中，将产生包含染色质相关结构域的全面文库的蛋白质结构域微阵列，并将其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，包括双重修饰多肽探针(在AIM 1中开发)和修饰特定的核小体探针。为实现这两个目标，将开发分析工具和软件并使之标准化，以促进将提案中开发的技术转让给表观遗传界。这项提议中新蛋白质组技术的产生允许快速和准确地发现染色质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应该被证明是非常有价值的。 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各种细胞功能。放置和读取表观遗传修饰的蛋白质调节失调经常与肿瘤疾病、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为表观遗传动力学如何在分子水平上发挥作用提供洞察力，并可能导致发现新的表观遗传学靶点，用于治疗各种人类疾病。

项目成果

Epigenome microarray platform for proteome-wide dissection of chromatin-signaling networks.

• DOI: 10.1371/journal.pone.0006789
• 发表时间: 2009-08-26
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Bua DJ;Kuo AJ;Cheung P;Liu CL;Migliori V;Espejo A;Casadio F;Bassi C;Amati B;Bedford MT;Guccione E;Gozani O
• 通讯作者: Gozani O

Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms.

Borrelidin 调节 VEGF 的选择性剪接，有利于抗血管生成亚型。

• DOI: 10.1039/c0sc00297f
• 发表时间: 2011
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Woolard,Jeanette;Vousden,William;Moss,StevenJ;Krishnakumar,Arjun;Gammons,MelissaVr;Nowak,DavidG;Dixon,Neil;Micklefield,Jason;Spannhoff,Astrid;Bedford,MarkT;Gregory,MatthewA;Martin,ChristineJ;Leadlay,PeterF;Zhang,MingQ;H
• 通讯作者: H