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

项目总结/摘要 R-环是三链核酸结构，其在转录过程中在侵入细胞后普遍形成。 双链体DNA被前进的RNA聚合酶后面的新生RNA所取代。我最近的侧写研究 研究小组已经确定R环形成是普遍的，覆盖了高达5%的人类和小鼠 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化，有效的 转录终止和DNA复制起点许可。在病理条件下， 然而，各种基因突变，功能失调的R环代谢被认为会导致DNA复制应激， 诱变、DNA断裂和基因组不稳定性。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染色体 综合征、肌萎缩性侧索硬化症（ALS）和骨髓增生综合征（MDS）。 这个建议的主要目标是了解什么区分“好”和“坏”的R-循环。主要 驱动这项工作的假设是疾病状态中的R环功能障碍引起R环分布的变化， 丰度、大小和/或周转率。该提案的总体目标是：1）开发新的 技术，可以准确地测量R环足迹的单分子的基础上，和R环周转 全球范围; 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），证实了疾病发病机制和R环功能障碍之间的可疑联系。的 提出了以下目标。目标1：开发一种基于SMRT的单分子R环足迹方法。 目标2：在全球范围内衡量R环周转率。目标3：测量R环的形成和周转， 与剪接功能障碍相关的病理状况。目的4：衡量EWSR 1缺陷的影响 对尤文肉瘤中R环形成和周转的影响。 这项提议将导致新的基因组学技术，以全面评估R环的形成， 单分子和全局水平的动力学。这些工具将使我们能够首次确定 突出的分子特征，区分“正常”与“病理性”R环，并提供新的见解， 参与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.