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节和失调的。表观基因组和 迫切需要表位转录组的变化，以获得细胞变化的完整分子谱， 了解基因调控的分子机制。然而，标准测序方法无法 捕获表观基因组的全部复杂性（DNA修饰和染色质可及性）或 epitranscriptome（RNA修饰）。当前基于Illumina的测序固有的问题包括： PCR偏倚的引入、读段的短长度以及不能直接测序RNA分子。到 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DNA修饰和DNA可及性 分子序列，并通过（B）直接分析将这些变化与对表位转录组的影响相关联 全长替代RNA同种型、RNA编辑和来自单个RNA分子序列的RNA修饰。 我们的组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，以及标记染色质可接近区域的体内方法。到 为了证明我们的方法的适用性和相关性，我们将在 已知影响染色质结构和表转录组的生物条件。我们还计划（c） 响应于关键染色质重塑的敲低的表观基因组学和表转录组学变化谱 基因和RNA结合蛋白，以测试这些调控因子是否以及在多大程度上影响表观基因组， epitranscriptome。我们的组合方法，远程检测的修改和可访问的区域， 检测同种型特异性RNA加工事件的DNA将提供急需的广泛的 一种适用于阐明涉及染色质的细胞信号转导反应机制的工具 和转录组改变。

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.