Deciphering the Histone Code by Mass Spectrometry and High Dimensional Statistics

项目描述（由申请人提供）：该项目旨在确定染色质上的翻译后修饰（PTMs）组合与基因表达之间的精确关系。众所周知，染色质上的PTMs在记忆形成和成瘾中起着重要作用。个体组蛋白可能携带PTMs的各种组合，组蛋白编码假说提出PTMs的组合可能对基因调控具有特异性作用。通过确定基因表达的哪些变化与哪些组合同时发生，将有可能更好地理解成瘾的分子机制，并提出改善它们的治疗方法。为此，将使用Top - Down质谱法同时测量酿酒酵母在干扰条件和菌株下的标记组合，并使用超高通量测序来测量基因表达。然后，基于正则化和变量选择的统计技术将用于选择基因组，其表达与特定标记子集相对丰度的变化最密切相关。ChIP-seq将用于确定携带这些特殊标记组合的核小体的位置和迁移率。将确定通过质谱测定的标记组合的相对丰度是否等于通过ChIP-seq测定的具有这些组合的核小体的相对丰度；这将决定携带这些标记的核小体是被束缚的还是自由的。为了支持这些目标，将开发计算技术，以实现高通量数据收集和统计分析。初步的工作表明，使用该模型系统开发的方法可以直接转移到人类细胞培养和原代组织样本中，为将这些研究结果应用于成瘾背后的染色质重塑机制的未来工作提供了直接途径。

DESCRIPTION (provided by applicant): The project aims to determine the precise relationship between combinations of post- translational modifications (PTMs) on chromatin and gene expression. It is known that PTMs on chromatin play a major role in memory formation and addiction. Individual histones may bear various combinations of PTMs, and the histone code hypothesis proposes that combinations of PTMs may have specific effects on gene regulation. By determining what changes in gene expression occur simultaneously with which combinations, it will be possible to better understand the molecular mechanisms underlying addiction, and suggest therapies to ameliorate them. To this end, Top Down mass spectrometry will be used to simultaneously measure combinations of marks in S. cerevisiae under conditions and strains that perturb them, and ultra-high-throughput sequencing will be used to measure gene expression. Statistical techniques based on regularization and variable selection will then be used to select groups of genes whose expression is most tightly correlated with changes in the relative abundance of particular subsets of marks. ChIP-seq will then be used to determine the location and mobility of nucleosomes bearing these particular combinations of marks. It will be determined if the relative abundances of mark combinations as measured by mass spectrometry are equal to the relative abundances of nucleosomes with those combinations as measured by ChIP-seq; this will determine whether the nucleosomes bearing these marks are bound or free. To support these aims, computational techniques will be developed to enable high-throughput data gathering and statistical analysis. Preliminary work suggests that methods developed using this model system are straightforwardly transferrable to work in human cell culture and primary tissue samples, providing a direct pathway for applying the results of these studies to future work on the chromatin remodeling mechanisms underlying addiction. PUBLIC HEALTH RELEVANCE: Addiction and memory formation are now known to involve changes in molecular marks on proteins called histones, which act as a scaffold for DNA. This project aims to determine the precise meaning of combinations of these marks, and their effects on gene regulation.