Direct detection of epigenetic modifications in their native chromatin contexts

直接检测天然染色质环境中的表观遗传修饰

• 批准号: 7570482
• 负责人: Jacob David Jaffe
• 金额: $ 32.88万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570482
• 关键词: Adopted; Affinity; Affinity Chromatography; Amino Acids; Antibodies; Area; Biochemical; Biological Assay; Biotin; Cancer Etiology; Cell Culture Techniques; Cells; Chemicals; Chromatin; Code; Complex; Cultured Cells; DNA; Detection; Development; Elements; Epigenetic Process; Fluorescent in Situ Hybridization; Gene Expression; Genetic; Genomics; Goals; Histones; Human Genome Project; Hybrids; Immunoprecipitation; Label; Laboratories; Learning; Mass Spectrum Analysis; Metabolic; Methodology; Methods; Modification; Nucleic Acid Probes; Pathway interactions; Post-Translational Protein Processing; Procedures; Process; Proteins; Reagent; Regenerative Medicine; Regulation; Relative (related person); Reporting; Signal Transduction; Specificity; Stable Isotope Labeling; Staging; Stem cells; Techniques; Technology; Temperature; Time; base; cell growth regulation; chromatin immunoprecipitation; crosslink; design; histone modification; locked nucleic acid; melting; novel; pluripotency; protein complex; public health relevance; research study; transmission process

DESCRIPTION (provided by applicant): Epigenetic modifications have been recognized as important factors in modification of gene expression levels, specification of cellular state and developmental potential, and etiology of cancer. In particular, post- translational modifications to histone proteins have been shown to be a key carrier of epigenetic information. Traditional techniques of studying these epigenetic modifications, such as chromatin immunoprecipitation (ChIP), have generally focused on examination of one mark at a time. However, it is likely that multiple epigenetic signals act together to form a complex code of transcriptional and cellular regulation. Mass spectrometry is uniquely suited to studying combinations of histone modifications by directly observing them together on protein molecules or in protein complexes. In this proposal, we will adopt some strategies used in ChIP to enable multiplex recognition and quantification of epigenetic modifications to histones. As part of these efforts, we will also assess the specificity of commonly used histone modification antibody reagents for ChIP by directly probing the modifications of the histones recovered. We will extend these techniques to the study of specific loci by developing a novel hybridization-based method of selective enrichment of targeted genomic regions. Throughout these studies, the use of cross-linking (as in ChIP) will maintain the native chromatin context of the cell so that co-occurrence of histone modifications can be discovered. We expect these technologies to be applicable to detailed studies of histone modifications present at developmentally important loci, especially those required for pluripotency. PUBLIC HEALTH RELEVANCE: The study of the transmission and regulation of epigenetic information is both the natural extension of and equally important to the Human Genome Project. Furthering our understanding in this area will help us learn about the developmental potential of stem cells and enable advances in regenerative medicine.

描述（由申请人提供）：表观遗传修饰已被认为是基因表达水平改变、细胞状态和发育潜力规范以及癌症病因学的重要因素。特别地，组蛋白的翻译后修饰已被证明是表观遗传信息的关键载体。研究这些表观遗传修饰的传统技术，如染色质免疫沉淀（ChIP），通常集中在一次检查一个标记。然而，很可能是多个表观遗传信号共同作用，形成一个复杂的转录和细胞调控密码。质谱法是唯一适合于研究组蛋白修饰的组合，通过直接观察它们在蛋白质分子或蛋白质复合物中的组合。在这个提议中，我们将采用一些ChIP中使用的策略，以实现对组蛋白表观遗传修饰的多重识别和定量。作为这些努力的一部分，我们还将通过直接探测回收的组蛋白的修饰来评估常用组蛋白修饰抗体试剂对ChIP的特异性。我们将通过开发一种新的基于杂交的选择性富集靶基因组区域的方法，将这些技术扩展到特定基因座的研究。在这些研究中，使用交联（如在ChIP中）将保持细胞的天然染色质背景，以便可以发现组蛋白修饰的共同出现。我们期望这些技术适用于详细研究发育重要位点的组蛋白修饰，特别是多能性所需的组蛋白修饰。 公共卫生相关性：对表观遗传信息的传递和调控的研究既是人类基因组计划的自然延伸，也同样重要。进一步了解这一领域将有助于我们了解干细胞的发育潜力，并促进再生医学的发展。