Tools for Single Molecule and Single Cell Epigenomic Analysis

哺乳动物的表观遗传特征包括组蛋白的共价修饰和胞嘧啶的甲基化。 它们的正确放置是许多生物过程的基础。分析表观基因组中的特征是 对了解人类生物学、诊断疾病、监测对表观基因组的反应很重要 修改药物，促进新药开发。这样的检测也将促进新出现的 基于胚胎和诱导的多能干细胞的治疗，这需要将细胞修改为 假定分化细胞处于表观遗传状态。用于组蛋白修饰的最先进的分析方法 染色质免疫沉淀(CHIP)，随后进行全基因组测序。甲基化的DNA可以是 通过免疫沉淀和测序，或通过亚硫酸氢盐测序进行鉴定。有两个 所有这些方法都存在根本限制。首先，他们一次只质疑一个表观遗传特征。 表观遗传特征出现在组合中，而这些组合而不是单个特征调节着 潜在的基因。除非可以同时检测和测量多个要素，否则不可能 可以肯定地知道，当组合在给定的基因上共存时。其次，化验使用的是细胞数量 并报告种群内的平均表观遗传状态，而不是表观遗传状态的实际分布 存在于组成种群的单个DNA分子上。第三，芯片经常使用大量的 染色质使得在稀有或不可能培养的细胞中检测多种表观遗传特征是不切实际的。在……里面 在这一应用中，我们寻求开发一种转化技术--单染色质分子分析 纳米流体学(SCAN)可以克服这些限制，并彻底改变表观基因组研究。在……里面 扫描，染色质分子被结合到识别不同表观遗传特征的荧光探针上，然后 通过纳米流体通道由电压驱动，其中单分子的荧光特性是 检测到。通过使用多个探测器，每个探测器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，从而精确计数多个表观遗传 同时提供多种功能。我们的第一代设备是在分析模式下运行的，简单地计算 功能。我们的第二代设备在准备模式下运行，使我们能够分类和分离 带有明确的表观遗传学特征的分子。在这项建议中，我们寻求进一步发展这一未来 代表观基因组学技术。首先，我们将对分析装置和分析物制备进行修改，以 将样品吞吐量提高两个数量级。第二，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，我们将使用我们的制备设备来分离染色质 定义表观遗传学特征，对DNA进行测序，并将我们的结果与当前CHIP-SEQ获得的数据进行比较 方法：研究方法。

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.