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

项目摘要 简单DNA重复序列的扩张会导致30多种可遗传的神经肌肉和神经退行性疾病。为 例如，强直性肌营养不良的两种主要类型是重复扩张性障碍。DM1是由龙引起的 DMPK的5‘非编码区存在CTG三核苷酸重复序列，而DM2是由长四核苷酸CCTG重复序列引起的 在ZNF9的一个内含子中。从细菌到果蝇再到人类细胞的实验系统已经建立起来 研究CTG序列扩展或长度增加的机制。这类研究已经 涉及DNA复制、DNA修复和转录在扩增过程中的特定作用。相比之下， 对CCTG重复序列的了解更少，目前还不清楚是否存在相同的分子机制 有助于CTG重复扩增的CCTG重复也适用于CCTG重复。这是一个紧迫的问题，因为个人 对于DM2，在ZNF9中平均有5000个CCTG重复。此外，如此长的重复长度的发生率 使描绘重复收缩的机制作为一种治疗前景特别吸引人。因此， 这里提出的工作旨在建立萌芽酵母酿酒酵母作为一个强大的实验 研究CCTG重复扩张和收缩的系统。具体而言，拟议的研究将 研究CCTG重复序列不稳定性的遗传控制，确定蛋白质和染色质修饰 与CCTG重复相关联。我的主要假设是，参与CCTG的基因重复不稳定 可能与CTG重复部分重叠，但由于不同的球员将有一组独特的球员 CCTG重复序列对DNA二级结构、染色质状态和复制动力学的影响。朝向这个方向 最后，我将确定CCTG重复序列的长度、方向和转录对扩增和 宫缩。我将使用两个候选者来确定CCTG重复不稳定的遗传和分子决定因素 和无偏见的基因筛查方法。特别是，识别与CCTG重复收缩有关的基因 通过无偏见的基因筛查，可能会发现以前在DNA中没有已知作用的基因 重复维护。最后，我将在CCTG Repeats上定义蛋白质和染色质景观，并将其与 CTG重复使用创新的基于CRISPR的染色质亲和纯化的质谱分析 (CRISPR-CHAP-MS)方法。总体而言，拟议的研究将对我们对 CCTG的分子生物学重复，对人类健康和疾病具有重要意义。这 这项工作将确定DM2预防和治疗干预的潜在目标，并作为 理解负责其他重复的微卫星重复的复制和维持的模型 扩张性疾病。

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.