Mechanisms of Myotonic Dystrophy Type 2-causing CCTG DNA Repeat Instability

强直性肌营养不良 2 型导致 CCTG DNA 重复不稳定的机制

• 批准号: 9891078
• 负责人: Jane C Kim
• 金额: $ 9.71万
• 依托单位: CALIFORNIA STATE UNIVERSITY SAN MARCOS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-13 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891078
• 关键词: 5' Untranslated Regions; Affect; Affinity Chromatography; Alleles; Alternative Splicing; Animal Model; Area; Bacteria; Biological Models; Cell Culture System; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA replication fork; Disease; Drosophila genus; Equipment and supply inventories; Experimental Models; Frequencies; Genes; Genetic; Genetic Diseases; Genetic Screening; Genetic Transcription; Genome; Goals; Health; Heritability; Human; Incidence; Individual; Introns; Length; Maintenance; Mass Spectrum Analysis; Microsatellite Repeats; Modeling; Molecular; Molecular Biology; Muscle Weakness; Muscular Atrophy; Mutagenesis; Mutate; Myotonic Dystrophy; Myotonic dystrophy type 2; Neurodegenerative Disorders; Neuromuscular Diseases; Pathway interactions; Patients; Post-Translational Protein Processing; Preventive Intervention; Process; Proteins; RNA; RNA metabolism; Repetitive Sequence; Reporter; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Set protein; Structure; System; Tandem Repeat Sequences; Testing; Therapeutic; Therapeutic Intervention; Toxic effect; Trinucleotide Repeats; Work; Yeasts; base; chromatin modification; experimental study; innovation; intergenerational; member; novel; programs; repaired; transmission process

Project Summary Expansions of simple DNA repeats cause over 30 heritable neuromuscular and neurodegenerative disorders. For example, the two major types of myotonic dystrophy are repeat expansion disorders. DM1 is caused by long CTG trinucleotide repeats in the 5’ UTR of DMPK whereas DM2 is caused by long tetranucleotide CCTG repeats in an intron of ZNF9. Experimental systems from bacteria to Drosophila to human cells have been established to investigate the mechanisms by which CTG sequences expand, or increase in length. Such studies have implicated specific roles of DNA replication, DNA repair, and transcription in the expansion process. In contrast, CCTG repeats are more poorly understood, and it is unclear whether the same molecular mechanisms contributing to CTG repeat expansions also apply to CCTG repeats. This is a pressing question since individuals with DM2 have, on average, 5000 CCTG repeats in ZNF9. Moreover, the incidence of such long repeat lengths make delineating the mechanism of repeat contraction particularly attractive as a therapeutic prospect. Thus, the work proposed here seeks to establish budding yeast Saccharomyces cerevisiae as a robust experimental system to investigate CCTG repeat expansions and contractions. Specifically, the proposed research will investigate the genetic control of CCTG repeat instability and define the proteins and chromatin modifications associated with CCTG repeats. My overarching hypothesis is that the genes involved in CCTG repeat instability may partially overlap with those of CTG repeats, but there will be a unique set of players owing to the distinct effect of CCTG repeats on DNA secondary structure, chromatin state, and replication dynamics. Towards this end, I will determine the effect of CCTG repeat length, orientation, and transcription on expansions and contractions. I will identify genetic and molecular determinants of CCTG repeat instability using both candidate and unbiased genetic screening approaches. In particular, identifying genes involved in CCTG repeat contraction through an unbiased genetic screen will potentially uncover genes that have no previously known role in DNA repeat maintenance. Finally, I will define the protein and chromatin landscape at CCTG repeats and compare it to CTG repeats using the innovative CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach. Overall, the proposed research will have a major impact on our understanding of the molecular biology of CCTG repeats, which has significant implications for human health and disease. This work will identify potential targets for preventative and therapeutic interventions for DM2 and also serve as a model for understanding the replication and maintenance of microsatellite repeats responsible for other repeat expansion disorders.

项目概要 简单 DNA 重复序列的扩展会导致 30 多种遗传性神经肌肉和神经退行性疾病。 例如，强直性肌营养不良的两种主要类型是重复扩张障碍，DM1 是由长引起的。 CTG 三核苷酸重复出现在 DMPK 的 5’ UTR 中，而 DM2 是由长四核苷酸 CCTG 重复引起的 已经建立了从细菌到果蝇再到人类细胞的实验系统。 研究 CTG 序列扩展或长度增加的机制。 相比之下，DNA 复制、DNA 修复和转录的特定作用。 CCTG重复序列人们对它知之甚少，也不清楚是否具有相同的分子机制 对 CTG 重复扩展的贡献也适用于 CCTG 重复，这是一个紧迫的问题，因为个人。 DM2 在 ZNF9 中平均有 5000 个 CCTG 重复，而且如此长的重复长度的发生率。 因此，描述重复收缩的机制作为一种治疗前景特别有吸引力。 这里提出的工作旨在将芽殖酵母酿酒酵母建立为强大的实验 具体而言，本研究将研究 CCTG 重复扩张和收缩的系统。 研究 CCTG 重复不稳定性的遗传控制并定义蛋白质和染色质修饰 我的总体假设是与 CCTG 重复不稳定性相关的基因。 可能与 CTG 重复赛有部分重叠，但由于不同的特点，将会有一组独特的球员 CCTG 重复对 DNA 二级结构、染色质状态和复制动力学的影响。 最后，我将确定 CCTG 重复长度、方向和转录对扩展和转录的影响 我将使用这两种候选药物来确定 CCTG 重复不稳定性的遗传和分子决定因素。 以及公正的基因筛选方法，特别是鉴定参与 CCTG 重复收缩的基因。 通过公正的基因筛选将有可能发现DNA中以前未知的作用的基因 最后，我将定义 CCTG 重复的蛋白质和染色质景观，并将其与 CTG 重复使用基于 CRISPR 的创新染色质亲和纯化和质谱法 总体而言，拟议的研究将对我们的理解产生重大影响。 CCTG 重复的分子生物学，对人类健康和疾病具有重要意义。 这项工作将确定 DM2 预防和治疗干预措施的潜在目标，并作为 用于理解负责其他重复的微卫星重复的复制和维护的模型 扩张障碍。