A novel approach to improve comprehensive profiling of the epigenome and epitranscriptome

项目摘要 涉及基因组和转录组测序的生物医学研究的一个主要焦点是 了解基因在疾病中是如何调控的，也是如何失调的。表观基因组和表观基因组的综合分析 表位翻译组的改变是迫切需要的，以便有一个完整的细胞变化的分子图谱和 了解基因调控的分子机制。然而，标准的测序方法无法 捕获表观基因组的全部复杂性(DNA修饰和染色质可获得性)或 表位翻译组(RNA修饰)。当前基于Illumina的测序固有的问题包括： 引入了聚合酶链式反应偏向，阅读片段的长度较短，以及不能直接对RNA分子进行测序。至 为了满足这些需求，我们正在开发实验和计算方法，使我们能够(A) 在体内同时检测单个DNA长片段上的DNA修饰和DNA可及性 通过(B)直接分析分子序列，并将这些变化与表位转录组的影响相关联 来自单个RNA分子序列的全长替代RNA异构体、RNA编辑和RNA修饰。 我们的联合方法将依赖于牛津纳米孔长读技术，该技术能够区分 DNA和RNA中的修饰碱基，以及体内标记染色质可接近区域的方法。至 证明我们的方法的适用性和相关性，我们将在以下条件下进行这些实验 已知的影响染色质结构和表位转录组的生物条件。我们还计划(C) 关键染色质重塑被敲除后的表观基因组和表型转录的变化 基因和RNA结合蛋白，以测试这些调节因子是否以及在多大程度上影响表基因组和 墓志铭。我们的组合方法对修改和可访问的区域进行远程检测 DNA与检测特定异构体的RNA加工事件将提供急需的广泛 用于阐明涉及染色质的调控细胞信号反应的机制的实用工具 和转录组的改变。

Project Summary A major focus of biomedical research that involves sequencing of the genome and transcriptome is to understand how genes are regulated and also dysregulated in disease. Integrated analyses of epigenome and epitranscriptome changes are urgently needed to have a complete molecular profile of cellular changes and to understand the molecular mechanisms of gene regulation. Yet, standard sequencing methods are unable to capture the full complexity of the epigenome (DNA modifications and chromatin accessibility) or the epitranscriptome (RNA modification). Problems inherent to current Illumina-based sequencing include: introduction of PCR bias, short-length of the reads, and the inability to directly sequence RNA molecules. To address these needs, we are developing experimental and computational approaches that allow us to (a) simultaneously detect, in vivo, DNA modifications and DNA accessibility on long stretches of single DNA molecule sequences and correlate these changes with effects on the epitranscriptome by (b) directly profiling full-length alternative RNA isoforms, RNA edits, and RNA modifications from single RNA molecule sequences. Our combined approach will rely on Oxford Nanopore long-read technologies which is capable of distinguishing modified bases in DNA and RNA, and on in vivo methods of marking accessible regions of chromatin. To demonstrate the applicability and relevance of our methods, we will perform these experiments under biological conditions known to impact both chromatin structure and the epitranscriptome. We also plan to (c) profile epigenomic and epitranscriptomic changes in response to knockdown of key chromatin remodeling genes and RNA binding proteins to test if, and how broadly, these regulatory factors affect the epigenome and epitranscriptome. Our combined approach of long-range detection of modified and accessible regions of DNA with detection of isoform-specific RNA processing events will provide a much-needed broadly applicable tool to elucidate the mechanisms governing cell signaling responses involving chromatin and transcriptome alterations.