Identification of combinatorial histone post-translational modifications within t

描述(申请人提供)：表观遗传学是研究在没有DNA突变的情况下，环境线索如何改变基因组的解释以产生表型差异的研究。DNA和组蛋白尾部的修饰影响染色质的可遗传状态，并与转录、复制、修复、重组和基因沉默有关。组蛋白/表观遗传密码假说表明，组蛋白翻译后修饰(PTM)的组合性质在不同的生物输出中发挥作用，如基因表达的调节。Tackett实验室的长期目标之一是开发在染色体位置环境中识别组合组蛋白PTM的技术，并确定它们如何调节基因表达。目前的技术，如染色质免疫沉淀(CHIP)，允许研究人员仅在基因组上将单个组蛋白PTM定位于已知的DNA序列。在这份NIH博士后奖学金提案中，我们概述了分离染色体特定区域、鉴定该纯化染色体区域内单个组蛋白分子上的PTM组合、鉴定与该纯化染色体区域相关的非组蛋白蛋白质以及控制非特异性富含蛋白质的技术的发展。基因转录分析将被用来从功能上检验我们的发现。这套技术将被称为CHAP-MS，用于染色质亲和纯化的质谱分析。我们假设组蛋白PTM的特定模式提供了调节基因表达的关键调控蛋白的结合，而识别这一点的技术的发展将使表观遗传学家能够识别改变正常基因表达的疾病的靶点，如癌症。我们将使用酿酒酵母作为一个模型系统来确定特定基因位点上的组蛋白PTM的组合模式、相关蛋白和相关基因的表达。将追求以下目标：具体目标1：优化化学交联技术，以防止在分离和纯化特定染色体区域时发生动态染色质解离。具体目标2：设计一株酿酒酵母菌株，用于分离特定的染色体片段，该染色体片段将受到组合组蛋白PTM及其相关蛋白的质谱学鉴定。 公共卫生相关性：组蛋白翻译后修饰(PTM)激活或抑制转录，导致疾病和癌症。识别组合组蛋白PTM将使表观遗传学家能够识别癌症等改变正常基因表达的疾病的靶点。

DESCRIPTION (provided by applicant): Epigenetics is the study of how environmental cues can alter interpretation of the genome to yield phenotypic differences in the absence of a DNA mutation. Modifications to DNA and to histone tails influence the heritable state of chromatin and are associated with transcription, replication, repair, recombination, and gene silencing. The histone/epigenetic code hypothesis states the combinatorial nature of histone post- translational modifications (PTMs) plays a role in distinct biological outputs such as the regulation of gene expression. One of the long-term goals of the Tackett laboratory is to develop technologies for the identification of combinatorial histone PTMs in the positional context of the chromosome and determine how they regulate gene expression. Current technology such as chromatin immunoprecipitation (ChIP) allows researchers to genomically position only a single histone PTM to a known sequence of DNA. In this NIH postdoctoral fellowship proposal, we outline the development of technology for the isolation of a particular region of a chromosome, identification of combinations of PTMs on single histone molecules within this purified chromosomal region, identification of non-histone proteins associated with this purified chromosomal region and controlling for non-specifically enriching proteins. Gene transcription assays will be used to functionally examine our findings. This suite of techniques will be referred to as ChAP-MS for chromatin affinity purification with mass spectrometry. We hypothesize that specific patterns of combinatorial histone PTMs provide for binding of key regulatory proteins that modulate gene expression, and that development of technologies to identify this will allow epigenetists to identify targets for diseases like cancer that alter normal gene expression. We will use Saccharomyces cerevisiae as a model system to identify the combinatorial pattern of histone PTMs at a specific gene locus, associated proteins, and associated gene expression. The following aims will be pursued: Specific Aim 1: Optimize chemical cross-linking technology to prevent dynamic chromatin dissociation during isolation and purification of a specific chromosomal region. Specific Aim 2: Engineer a S. cerevisiae strain for the isolation of a particular chromosomal section that will be subjected to mass spectrometric identification of combinatorial histone PTMs and associated proteins. PUBLIC HEALTH RELEVANCE: Histone post-translational modifications (PTMs) activate or repress transcription leading to disease and cancer. The identification of combinatorial histone PTMs will allow epigenetists to identify targets for diseases like cancer that alter normal gene expression.