Development of technology for high resolution epigenetic profiling of chromatin

染色质高分辨率表观遗传分析技术的开发

• 批准号: 7570930
• 负责人: Alan Tackett
• 金额: $ 39.75万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-20 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570930
• 关键词: Affinity Chromatography; Antibodies; Automobile Driving; Base Pairing; Biological; Cell Differentiation process; Chromatin; Chromosomes; Complex; Cues; DNA Methylation; DNA Microarray Chip; DNA Microarray format; DNA Sequence; Development; Discrimination; Disease; Epigenetic Process; Eukaryota; Eukaryotic Cell; Event; Excision; Figs - dietary; Gene Expression; Gene Mutation; Genes; Genetic Transcription; Genome; Goals; Histone Code; Histones; Inherited; Laboratories; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Modification; Molecular; Nature; Nucleosomes; Organism; Output; Pathway interactions; Positioning Attribute; Post-Translational Protein Processing; Precipitation; Procedures; Proteins; Proteome; Proteomics; Protocols documentation; Public Health; RNA Interference; Range; Recruitment Activity; Regulation; Research Personnel; Resolution; Saccharomyces cerevisiae; Series; Signal Transduction; Stem cells; System; Techniques; Technology; Testing; Time; Transcriptional Regulation; Work; base; combinatorial; design; epigenomics; histone acetyltransferase; in vivo; new technology; programs; technology development

DESCRIPTION (provided by applicant): Chromosomes are separated into regions of distinct transcriptional activity. Key to the establishment of these regions is the presence of specific histone post-translational modifications (PTMs) on chromatin. These epigenetic marks provide signals that recruit proteins to activate or repress transcription. The precise marking of a given histone (or particular combination of markings) is key for driving the cellular program of gene expression. The mis-regulation of histone PTM addition or removal has been linked to diseases such as cancer. Current technologies used to study histone PTMs are largely limited by the analysis of a single histone PTM in context of chromosomal location, while it appears that the combinatorial nature of histone PTM addition is key for transcriptional regulation. We outline the development of a novel technology that will enable epigeneticists to isolate a given region of a chromosome (on the order of 5 nucleosomes), identify the contained and co-occupancy of histone PTMs, identify the proteome component of the particular region of chromatin and control for non-specifically interacting proteins/PTMs. This procedure will be referred to as ChAP-MS for chromatin affinity purification with mass spectrometry. The working hypothesis of our proposed work is: development of the ChAP-MS technology to specifically enrich small chromosome fragments for mass spectrometric analysis will enable epigeneticists to perform whole epigenome and chromosome proteome studies for the first time. Our objectives are 1) Development of a chromatin affinity purification (ChAP) technology that will permit efficient isolation of specific chromosomal fragments for use in epigenomic and proteomic profiling, 2) Devise an unambiguous readout strategy to be used in conjunction with ChAP analysis that permits discrimination between specific and non-specific protein/histone interactions with a defined chromosomal fragment, 3) Design microscale protocols to be used in conjunction with ChAP analysis for separating each core histone component of a given chromosomal fragment on the basis of PTM occupancy, thus allowing mass spectrometric profiling of histone PTMs, and 4) Use ChAP-MS to profile the epigenome and proteome of a histone acetyltransferase complex. PUBLIC HEALTH RELEVANCE: Understanding how histones are post-translationally modified will enable researchers to better understand the molecular mechanism of diseases such as cancer. Current technologies lack the ability to thoroughly analyze the combinatorial nature of these modifications in context of chromosomal location. We plan to develop a new technology that will provide a manner to specifically analyze these combinatorial modifications in the chromosomal context, thereby providing a new avenue for researchers to understand complex diseases.

描述（由申请人提供）：将染色体分为不同的转录活性区域。建立这些区域的关键是在染色质上存在特定的组蛋白后翻译后修饰（PTM）。这些表观遗传标记提供了募集蛋白质激活或抑制转录的信号。给定组蛋白的精确标记（或标记的特定组合）是推动基因表达细胞程序的关键。组蛋白PTM添加或去除的错误调节与癌症等疾病有关。用于研究组蛋白PTM的当前技术在很大程度上受到染色体位置中单个组蛋白PTM的分析的限制，而似乎组蛋白PTM添加的组合性质是转录调节的关键。我们概述了一种新型技术的开发，该技术将使表观遗传学家能够隔离染色体的给定区域（按5个核小体的顺序），识别组蛋白PTM的所包含和共占率，识别非特定相互作用蛋白/PTM的染色质和对照的蛋白质组成分。该过程将称为染色质亲和力纯度质谱法的CHAP-MS。我们提出的工作的工作假设是：开发Chap-MS技术，以专门富集小染色体片段进行质谱分析，这将使表观遗传学家首次进行整个表观遗传组和染色体蛋白质组研究。我们的目标是1）开发染色质亲和力纯化（CHAP）技术，该技术将允许在表观基因组和蛋白质组学分析中有效地隔离特定的染色体碎片，2）设计一个明确的读数策略，可以与CHAP分析相结合，以允许与特定的属性和非特异性蛋白质相互作用进行分析，以允许使用特定的蛋白质/非属性质量分析。结合CHAP分析，分析给定染色体片段的每个核心组蛋白成分基于PTM占用率，从而允许组蛋白PTM的质谱分析和4）使用Chap-Ms介绍了乙酰基转移酶复合酶的组蛋白蛋白蛋白的表观镜头和蛋白质组。 公共卫生相关性：了解后翻译后修饰的组蛋白如何使研究人员能够更好地了解癌症等疾病的分子机制。当前的技术缺乏在染色体位置彻底分析这些修饰的组合性质的能力。我们计划开发一种新技术，该技术将提供一种方式，以在染色体环境中专门分析这些组合修饰，从而为研究人员提供新的途径来了解复杂的疾病。