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

人类疾病中病理性 R 环形成的基因组分析。

• 批准号: 9357618
• 负责人: Frederic Louis Chedin
• 金额: $ 31.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9357618
• 关键词: Acute Myelocytic Leukemia; Amyotrophic Lateral Sclerosis; Automobile Driving; Binding; Bone neoplasms; Cells; Childhood; Chromatin; Chromatin Loop; DNA; DNA Damage; DNA Footprint; DNA Structure; DNA biosynthesis; DNA replication origin; DNA sequencing; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Degenerative Disorder; Disease; Disease model; EWS-FLI1 fusion protein; EWSR1 gene; Embryo; Escherichia coli; Event; Ewings sarcoma; Fibroblasts; Fragile X Syndrome; Functional disorder; Gene Expression; Gene Mutation; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; High-Throughput Nucleotide Sequencing; Human; Human Genome; Hybrids; Immunoprecipitation; Knockout Mice; Licensing; Life; Link; Malignant Neoplasms; Maps; Measures; Metabolism; Methods; Molecular; Mus; Mutagenesis; Mutate; Mutation; Nature; Neurodegenerative Disorders; Neurologic; Neurons; Nuclear; Oncogenic; Outcome; Pathogenesis; Pathologic; Pattern; Pharmaceutical Preparations; Physiological; Play; Population; Process; RNA; RNA Splicing; Residual state; Resolution; Role; Single-Stranded DNA; Site; Spliceosomes; Stress; Structure; Syndrome; Technology; Testing; Time; Work; actionable mutation; base; bisulfite; genetic technology; genomic profiles; helicase; human disease; insight; knockout animal; mammalian genome; mouse genome; new technology; novel; nucleic acid structure; single molecule; tool; transcription termination

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.

项目摘要/摘要 R环是在入侵后在转录过程中普遍形成的三链核酸结构 前进的RNA聚合酶后面的新生RNA的双链DNA。我最近的研究研究 小组已经确定R-loop形成很普遍，覆盖了多达5％的人和小鼠 基因组。这使得R-loop成为迄今为止最丰富的非B DNA结构。在正常情况下，r- 循环被认为是快乐的重要核过程，例如开放染色质图案，高效 转录终止和DNA复制起源许可。在与之相关的病理状况下 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 人类疾病的发病机理，包括神经发育 /退化性疾病，例如脆弱x 综合征，肌萎缩性侧索硬化症（ALS）和骨髓增生综合征（MDS）。 该提案的主要目标是了解什么区别“好”与“坏” R环。主 推动工作的假设是疾病状态中的R环功能障碍需要R-loop分布的变化， 抽象，大小和/或周转率。该提案的总体目标是：1）开发新的 可以单个分子准确测量R-loop足迹的技术，然后在R-loop周转 全球规模； 2）将这些方法应用于三种重要的人类疾病模型（ALS，MDS，EWING） 肉瘤示例疾病发病机理与R环功能障碍之间的可疑联系。 提出了以下目标。 AIM 1：开发一种基于SMRT的单分子，R-loop足迹方法。 AIM 2：在全球范围内测量R-loop周转。目标3：测量R环的形成和周转 目标4：衡量EWSR1缺乏症的影响 在Ewing肉瘤中的R环形成和营业额上。 该建议将导致新的基因组技术，以全面评估R环的形成和 单分子和全球水平的动力学。这些工具将使我们首次确定 显着的分子特征，将“正常”与“病理” R环区分开，并提供新颖的见解 涉及癌症和神经退行性疾病的分子机制。