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: 7688693
• 负责人: Alan Tackett
• 金额: $ 38.63万
• 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/7688693
• 关键词: Affinity Chromatography; Antibodies; Automobile Driving; Base Pairing; Biological; Chromatin; Chromosomes; Complex; Cues; DNA Methylation; DNA Microarray Chip; DNA Sequence; Development; Discrimination; Disease; Epigenetic Process; Eukaryota; 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; RNA Interference; 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; genome-wide; histone acetyltransferase; histone modification; in vivo; new technology; programs; public health relevance; stem cell differentiation; 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）：染色体被分成具有不同转录活性的区域。建立这些区域的关键是染色质上特定组蛋白翻译后修饰 (PTM) 的存在。这些表观遗传标记提供招募蛋白质来激活或抑制转录的信号。给定组蛋白（或特定标记组合）的精确标记是驱动基因表达的细胞程序的关键。组蛋白 PTM 添加或去除的错误调节与癌症等疾病有关。目前用于研究组蛋白 PTM 的技术在很大程度上受到染色体定位背景下单个组蛋白 PTM 分析的限制，而组蛋白 PTM 添加的组合性质似乎是转录调控的关键。我们概述了一项新技术的发展，该技术将使表观遗传学家能够分离染色体的给定区域（大约 5 个核小体），识别组蛋白 PTM 的包含和共占用，识别染色质特定区域的蛋白质组成分以及非特异性相互作用蛋白/PTM 的控制。该程序将被称为 ChAP-MS，用于通过质谱法进行染色质亲和纯化。我们提出的工作的工作假设是：开发专门富集用于质谱分析的小染色体片段的 ChAP-MS 技术将使表观遗传学家首次能够进行整个表观基因组和染色体蛋白质组研究。我们的目标是 1) 开发染色质亲和纯化 (ChAP) 技术，该技术将允许有效分离特定染色体片段，用于表观基因组和蛋白质组分析，2) 设计一种明确的读出策略，与允许区分的 ChAP 分析结合使用特定和非特定蛋白质/组蛋白与确定的染色体片段之间的相互作用，3) 设计与 ChAP 分析结合使用的微尺度方案，以分离每个核心组蛋白成分4) 使用 ChAP-MS 分析组蛋白乙酰转移酶复合物的表观基因组和蛋白质组。 公共健康相关性：了解组蛋白如何进行翻译后修饰将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的背景下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，提供一种在染色体背景下专门分析这些组合修饰的方法，从而为研究人员了解复杂疾病提供新途径。