Direct detection of epigenetic modifications in their native chromatin contexts

描述（由申请人提供）：表观遗传修饰已被认为是基因表达水平改变、细胞状态和发育潜力规范以及癌症病因学的重要因素。特别是，组蛋白的翻译后修饰已被证明是表观遗传信息的关键载体。研究这些表观遗传修饰的传统技术，如染色质免疫沉淀（ChIP），通常集中在一次检查一个标记。然而，多种表观遗传信号可能共同作用，形成一个复杂的转录和细胞调控代码。质谱法通过直接观察蛋白质分子或蛋白质复合物上组蛋白修饰的组合而独特地适用于研究组蛋白修饰的组合。在本提案中，我们将采用ChIP中使用的一些策略来实现组蛋白表观遗传修饰的多重识别和量化。作为这些工作的一部分，我们还将通过直接探测回收的组蛋白修饰来评估ChIP常用组蛋白修饰抗体试剂的特异性。我们将通过开发一种新的基于杂交的选择性富集目标基因组区域的方法，将这些技术扩展到特定位点的研究。在这些研究中，使用交联（如ChIP）将维持细胞的天然染色质环境，以便发现组蛋白修饰的共发生。我们希望这些技术能够应用于对组蛋白修饰的详细研究，尤其是对那些多能性所需的组蛋白修饰。

DESCRIPTION (provided by applicant): Epigenetic modifications have been recognized as important factors in modification of gene expression levels, specification of cellular state and developmental potential, and etiology of cancer. In particular, post- translational modifications to histone proteins have been shown to be a key carrier of epigenetic information. Traditional techniques of studying these epigenetic modifications, such as chromatin immunoprecipitation (ChIP), have generally focused on examination of one mark at a time. However, it is likely that multiple epigenetic signals act together to form a complex code of transcriptional and cellular regulation. Mass spectrometry is uniquely suited to studying combinations of histone modifications by directly observing them together on protein molecules or in protein complexes. In this proposal, we will adopt some strategies used in ChIP to enable multiplex recognition and quantification of epigenetic modifications to histones. As part of these efforts, we will also assess the specificity of commonly used histone modification antibody reagents for ChIP by directly probing the modifications of the histones recovered. We will extend these techniques to the study of specific loci by developing a novel hybridization-based method of selective enrichment of targeted genomic regions. Throughout these studies, the use of cross-linking (as in ChIP) will maintain the native chromatin context of the cell so that co-occurrence of histone modifications can be discovered. We expect these technologies to be applicable to detailed studies of histone modifications present at developmentally important loci, especially those required for pluripotency. PUBLIC HEALTH RELEVANCE: The study of the transmission and regulation of epigenetic information is both the natural extension of and equally important to the Human Genome Project. Furthering our understanding in this area will help us learn about the developmental potential of stem cells and enable advances in regenerative medicine.