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。 它们的正确放置对许多生物过程至关重要。分析表观基因组的特征是 对于理解人类生物学、诊断疾病、监测对表观基因组的反应 改进药物和促进新药开发。这种分析也将促进新兴的 基于胚胎干细胞和诱导多能干细胞的治疗，其需要修饰细胞以 呈现分化细胞的表观遗传状态。用于组蛋白修饰的最先进测定法 染色质免疫沉淀（ChIP），然后进行全基因组测序。甲基化DNA可以是 通过免疫沉淀随后测序或通过亚硫酸氢盐测序鉴定。有两 所有这些方法的基本局限性。首先，他们一次只查询一个表观遗传特征。 表观遗传特征出现在组合中，这些组合而不是个体特征调节了基因的表达。 潜在基因除非可以同时检测和测量多个特征，否则不可能 确切地知道特定基因上的组合何时共存。第二，测定使用细胞群 并报告人口中的平均表观遗传状态，而不是表观遗传状态的实际分布 存在于构成群体的单个DNA分子上。第三，ChIP经常使用大量的 染色质，使得在罕见或不可能培养的细胞中测定多种表观遗传特征不切实际。在 本申请中，我们寻求开发一种转化技术，单染色质分子分析， 纳米流体（SCAN）可以克服这些限制，并彻底改变表观基因组研究。在 扫描，染色质分子结合到识别不同表观遗传特征的荧光探针上， 由电压驱动通过纳米流体通道，其中单分子的荧光特性 检测到通过使用多个探针，每个探针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，允许精确计数多种表观遗传 特色同时我们的第一代设备以分析模式运行， 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 携带明确的表观遗传特征的分子。在这一建议中，我们寻求进一步发展这一未来 一代表观基因组学技术。首先，我们将修改分析设备和分析物制备， 将样品处理量提高两个数量级。其次，我们将使用新的分析设备， 解决表观基因组学中的一些问题。第三，我们将使用我们的制备设备分离染色质， 定义的表观遗传特征，对DNA进行测序，并将我们的结果与当前ChIP-seq 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.