Identification of combinatorial histone post-translational modifications within t

描述（由申请人提供）：表观遗传学是研究环境因素如何在没有DNA突变的情况下改变基因组的解释以产生表型差异。DNA和组蛋白尾部的修饰影响染色质的遗传状态，并与转录、复制、修复、重组和基因沉默有关。组蛋白/表观遗传密码假说认为，组蛋白翻译后修饰（PTMs）的组合性质在不同的生物学输出中发挥作用，如基因表达的调节。Tackett实验室的长期目标之一是开发鉴定染色体位置背景下组合组蛋白PTMs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），允许研究人员仅将单个组蛋白PTM基因组定位到已知的DNA序列。在这份NIH博士后奖学金提案中，我们概述了染色体特定区域分离技术的发展，鉴定纯化染色体区域内单个组蛋白分子上PTMs的组合，鉴定与该纯化染色体区域相关的非组蛋白以及控制非特异性富集蛋白。基因转录检测将用于功能性检查我们的发现。这套技术将被称为chapp - ms用于质谱法染色质亲和纯化。我们假设组合组蛋白ptm的特定模式提供了调节基因表达的关键调节蛋白的结合，并且识别这一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癌症等疾病的靶标。我们将使用酿酒酵母作为模型系统来鉴定特定基因位点、相关蛋白和相关基因表达的组蛋白ptm的组合模式。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的动态染色质解离。特定目标2：设计酿酒葡萄球菌菌株，用于分离特定染色体切片，该染色体切片将用于组合组蛋白ptm和相关蛋白的质谱鉴定。

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.