Tools for Single Molecule and Single Cell Epigenomic Analysis

单分子和单细胞表观基因组分析工具

• 批准号: 8683212
• 负责人: HAROLD G CRAIGHEAD
• 金额: $ 56.18万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683212
• 关键词: Address; Agreement; Antibodies; Basic Science; Binding; Biological Assay; Biological Process; Cell Separation; Cells; Chemicals; Chromatin; Clinical assessments; Complex; Computer-Assisted Image Analysis; Cultured Cells; Cytosine; DNA; DNA Sequence; DNA-Binding Proteins; Data; Data Collection; Data Set; Development; Devices; Embryo; Engineering; Epigenetic Process; Fluorescent Antibody Technique; Fluorescent Probes; Generations; Genes; Genetic Materials; Genome; Goals; Health; Histones; Human Biology; Image; Immunoprecipitation; Individual; Mammals; Measures; Medicine; Methods; Methylation; Microscope; Modification; Monitor; Patients; Pharmaceutical Preparations; Population; Preparation; Process; Property; Publishing; Reporting; Research; Sampling; Sorting - Cell Movement; Source; Surface; Technology; Testing; Therapeutic; Time; base; bisulfite; chromatin immunoprecipitation; cost; disease diagnosis; drug development; epigenome; epigenomics; fluorescence activated cell sorter device; fluorophore; genome-wide; histone modification; improved; induced pluripotent stem cell; information gathering; nanofluidic; next generation; operation; research study; response; single molecule; tool; trait; voltage

Epigenetic features in mammals include covalent modifications to histones, and methylation of cytosines. Their proper placement is fundamental to many biological processes. Assaying features in the epigenome is important for understanding human biology, diagnosing disease, monitoring responses to epigenome modifying drugs and facilitating development of new medicines. Such assays will also facilitate emerging therapeutics based on embryonic and induced pluripotent stem cells, which require modifying the cells to assume epigenetic states of differentiated cells. State-of-the-art assays for histone modifications use chromatin immunoprecipitation (ChIP), followed by genome wide sequencing. Methylated DNA can be identified by immunoprecipitation followed by sequencing, or by bisulfite sequencing. There are two fundamental limitations with all these approaches. First, they query only one epigenetic feature at a time. Epigenetic features arise in combinations, and those combinations rather than individual features regulate the underlying genes. Unless multiple features can be detected and measured simultaneously, it is not possible to know, with certainty, when combinations coexist on a given gene. Second, assays use populations of cells and report the average epigenetic states within the population, not the actual distribution of epigenetic states present on individual DNA molecules comprising the population. Third, ChIP often uses abundant amounts of chromatin making it impractical to assay multiple epigenetic features in rare or impossible to culture cells. In this application, we seek to develop a transforming technology, Single Chromatin molecule Analysis in Nanofluidics (SCAN) that can overcome each of these limitations and revolutionize epigenomic studies. In SCAN, chromatin molecules are bound to fluorescent probes recognizing distinct epigenetic features, then driven by voltage through nanofluidic channels where the fluorescent properties of single molecules are detected. By using multiple probes, each recognizing different features and carrying distinct fluorophores, we can directly detect their binding to individual molecules, allowing precise enumeration of multiple epigenetic features simultaneously. Our first-generation devices were operated in an analytical mode, simply counting features. Our second-generation devices were operated in a preparative mode, allowing us to sort and isolate molecules carrying defined epigenetic features. In this proposal, we seek to further develop this next generation epigenomics technology. First, we will modify the analytical device and analyte preparation to increase sample throughput by two orders of magnitude. Second, we will use the new analytical device to address selected questions in epigenomics. Third, we will use our preparative device to isolate chromatin with defined epigenetic features, sequence the DNA and compare our results to data obtained by current ChIP-seq methods.

哺乳动物的表观遗传特征包括对组蛋白的共价修饰和胞嘧啶的甲基化。 它们的适当放置是许多生物过程的基础。表观基因组中的分析特征是 对于了解人类生物学，诊断疾病，监测对表观基因组的反应很重要 修改药物并促进新药物的开发。这样的测定也将有助于新兴 基于胚胎和诱导多能干细胞的治疗剂，这些干细胞需要将细胞修改为 假设分化细胞的表观遗传态。用于组蛋白修饰的最新测定法 染色质免疫沉淀（CHIP），然后进行基因组广泛的测序。甲基化的DNA可以是 通过免疫沉淀，然后进行测序或硫酸盐测序。有两个 所有这些方法的基本限制。首先，他们一次只查询一个表观遗传特征。 表观遗传特征在组合中出现，这些组合而不是单个特征调节 基础基因。除非可以同时检测和测量多个功能，否则不可能 可以肯定地知道，当组合在给定基因上共存时。其次，测定使用细胞种群 并报告人口中平均表观遗传状态，而不是表观遗传状态的实际分布 存在于包括种群的单个DNA分子上。第三，芯片通常使用大量 染色质使得在稀有或不可能培养细胞中测定多个表观遗传特征是不切实际的。在 在此应用中，我们试图开发一种转化的技术 可以克服这些局限性并彻底改变表观基因组学研究的纳米流体（扫描）。在 扫描，染色质分子与荧光探针结合，以识别不同的表观遗传特征，然后 通过电压通过纳米流体通道驱动，单分子的荧光特性为 检测到。通过使用多个探针，每个探针都识别不同的特征并携带不同的荧光团，我们 可以直接检测它们与单个分子的结合，从而精确列举多个表观遗传学 同时功能。我们的第一代设备以分析模式运行，仅计数 特征。我们的第二代设备以准备模式运行，使我们能够对 携带定义表观遗传特征的分子。在此提案中，我们寻求进一步发展下一个 一代表观基因组技术。首先，我们将将分析设备和分析物的准备修改为 将样品吞吐量增加两个数量级。其次，我们将使用新的分析设备来 解决表观基因组学中选定的问题。第三，我们将使用制备装置将染色质与 定义的表观遗传特征，序列DNA并将我们的结果与当前ChIP-Seq获得的数据进行比较 方法。