Genomic profiling of pathological R-loop formation in human diseases.

项目摘要/摘要 R-环是三链核酸结构，在转录过程中普遍形成 在前进的核糖核酸聚合酶后面的新生核糖核酸形成的双链DNA。来自我的最近的分析研究 研究小组已经证实，R环的形成很普遍，覆盖了高达5%的人类和老鼠 基因组。这使得R-环成为迄今为止最丰富的非B-DNA结构。在正常情况下，R- 环被认为是促进重要的核过程，如开放的染色质图案，高效 转录终止和DNA复制起点许可。在与以下疾病相关的病理情况下 然而，各种基因突变，功能失调的R环代谢被认为会导致DNA复制压力， 突变、DNA断裂和基因组不稳定。这些负面结果与 人类疾病的发病机制，包括神经发育/退行性疾病，如脆性X 综合征、肌萎缩侧索硬化症(ALS)和骨髓增生异常综合征(MDS)。 这项建议的主要目的是了解如何区分“好”和“坏”的R-环。主 推动这项工作的假设是，疾病状态下的R环功能障碍会导致R环分布的变化， 丰度、规模和/或周转率。该提案的总体目标是：1)开发新的 可以在单分子基础上精确测量R环足迹和R环周转率的技术 2)将这些方法应用于三种重要的人类疾病模型(ALS、MDS、Ewing 肉瘤)，这说明了疾病发病机制和R环功能障碍之间的可疑联系。这个 提出了以下目标。目的1：建立一种基于SMRT的单分子R环足迹方法。 目标2：在全球范围内衡量R循环周转率。目标3：在以下条件下衡量R环的形成和周转 与剪接功能障碍相关的病理状态。目标4：衡量EWSR1缺陷的影响 尤文肉瘤R环形成及转归的研究 这一提议将导致新的基因组学技术，以全面评估R环的形成和 单分子和全球层面的动力学。这些工具将使我们能够第一次识别 显著的分子特征，区分“正常”和“病态”R环，并提供新的见解 癌症和神经退行性疾病的分子机制。

PROJECT SUMMARY/ABSTRACT R-loops are three-stranded nucleic acid structures that universally form during transcription upon invasion of the duplex DNA by the nascent RNA behind the advancing RNA polymerase. Recent profiling studies from my group have established that R-loop formation is prevalent, covering up to 5% of the human and mouse genomes. This makes R-loops the most abundant non-B DNA structure to date. Under normal conditions, R- loops are thought to be facilitate important nuclear processes such as open chromatin patterning, efficient transcription termination, and DNA replication origin licensing. Under pathological conditions associated with various gene mutations, however, dysfunctional R-loop metabolism is thought to cause DNA replication stress, mutagenesis, DNA breakage, and genomic instability. These negative outcomes are relevant for the pathogenesis of human disorders, including neurodevelopmental / degenerative diseases such as Fragile X syndrome, Amyotrophic Lateral Sclerosis (ALS), and myelodisplastic syndromes (MDS). The main goal of this proposal is to understand what distinguishes “good” from “bad” R-loops. The main hypothesis driving the work is that R-loop dysfunction in disease states entails changes in R-loop distribution, abundance, size, and/or turnover rates. The overall objectives of the proposal are to: 1) develop new technologies that can accurately measure R-loop footprints on a single molecule basis, and R-loop turnover on a global scale; and 2) apply these methods to three important human disease models (ALS, MDS, Ewing sarcoma) that exemplify the suspected links between disease pathogenesis and R-loop dysfunction. The following Aims are proposed. Aim 1: Develop a single-molecule, SMRT-based, R-loop footprinting method. Aim 2: Measure R-loop turnover on a global scale. Aim 3: Measure R-loop formation and turnover under pathological conditions associated with splicing dysfunction. Aim 4: Measure the impact of EWSR1 deficiency on R-loop formation and turnover in Ewing sarcoma. This proposal will lead to new genomics technologies to comprehensively assess R-loop formation and dynamics at the single molecule and global levels. These tools will enable us to identify, for the first time, the salient molecular features that distinguish “normal” from “pathological” R-loops and provide novel insights into molecular mechanisms involved in cancer and neurodegenerative diseases.