Pathogenic Alterations of the 3D Epigenetic Landscape in Dystrophin-Deficient Skeletal Muscles and Reversal by Dystrophin Re-Expression

项目摘要 缺乏肌营养不良蛋白（dys）和破坏dys相关蛋白复合物（DAPC）在膜 骨骼肌纤维被广泛认为是肌膜不稳定的主要原因， 杜氏肌营养不良症（DMD）患者的肌肉损失。除了结构功能外， dys参与调节其他下游细胞事件，包括控制细胞内的 基因组完整性和基因表达。确实，缺乏缺陷的肌肉表现出组蛋白的改变， 修饰，基因表达和非编码RNA，以及基因组不稳定性和核 异常基于染色体构象捕获（3C）的研究表明，基因组是折叠的， 转化为高级染色质相互作用。了解dys缺乏，失调 基因表达和改变的基因组拓扑结构对于全面了解DMD具有特殊意义 发病机制和评价旨在替代dys的治疗方法的有效益处 DMD男孩的表现。这项提案通过利用最先进的全基因组技术来解决这个问题。 方法（即启动子捕获-HiC，ChIP-seq，ATAC-seq和RNAseq）来检测微环境的扰动。 DMD肌肉的表观遗传景观和转录组，使用两个互补的实验模型- 基于患者iPSC的人DMD体内培养皿模型和mdx小鼠体内模型， 目标：目标1。鉴定调节基因表达的高级染色质相互作用的改变 在DMD肌肉中，我们将识别功能和结构之间的染色质相互作用的改变， 基因组元件，导致人类（hiPSC衍生的骨骼肌）中的致病基因表达， DMD的小鼠（mdx小鼠）模型。目标2.微-dys修复对逆转高- 顺序染色质相互作用调节DMD肌肉中的基因表达我们将评估 通过µ-dys恢复dys表达逆转（部分或完全）表观遗传和转录 目的1中鉴定的DMD肌肉的改变。目标3.生物信息学鉴定与分析 DMD肌肉的表观遗传和转录改变我们将进行一个综合的生物信息学 分析pcHi-C、RNA-seq、ATAC-seq和ChIP-seq数据以鉴定DMD相关致病性染色质 研究DMD MuSC和肌纤维中的DMD PCI相互作用及其对µ-dys表达的易感性。目标4。 收缩诱导的DMD致病基因位点的高级染色质相互作用的改变 我们将确定肌肉收缩对PCI的影响， 在dys缺乏和µ-dys恢复的情况下，致病基因位点的基因表达。 了解dys缺乏是否会导致致病性 DMD肌肉的转录输出，以及它们是否可以被µ-dys表达逆转，将不可避免地 扩展我们对DMD发病机制和基于µ-dys的基因疗法的治疗效果的知识。

PROJECT SUMMARY Lack of dystrophin (dys) and disruption of the dys-associated protein complex (DAPC) at the membrane of skeletal myofibers are widely recognized as the main cause of sarcolemma instability, leading to skeletal muscle loss in patients affected by Duchenne Muscular Dystrophy (DMD). In addition to its structural function, dys has been implicated in the regulation of additional downstream cellular events, including control of the genome integrity and gene expression. Indeed, dys-deficient muscles exhibit altered profiles of histone modifications, gene expression and non-coding RNA, as well as features of genomic instability and nuclear abnormalities. Chromosome conformation capture (3C)-based studies have revealed that the genome is folded into high-order chromatin interactions. Understanding the relationship between dys deficiency, dysregulated gene expression and altered genome topology is of special interest for the complete understanding of DMD pathogenesis and for the evaluation of the effective benefits of therapeutic approaches aimed at replacing dys expression in DMD boys. This proposal addresses this question by exploiting state-of-the-art genome-wide approaches (i.e. promoter capture-HiC, ChIP-seq, ATAC-seq and RNAseq) to detect perturbations of the epigenetic landscape and transcriptome in DMD muscles, using two complementary experimental models – patient iPSC-based in dish model of human DMD vivo and the mdx mouse model in vivo, by the following Aims: Aim 1. To identify alterations in high-order chromatin interactions that regulate gene expression in DMD muscles We will identify alterations of chromatin interactions between functional and structural genomic elements, leading to pathogenic gene expression in human (hiPSC-derived skeletal muscles) and mouse (mdx mice) models of DMD. Aim 2. Effect of µ-dys restoration on reversal of alterations in high- order chromatin interactions that regulate gene expression in DMD muscles We will evaluate whether restoration of dys expression by µ-dys reverses (partly or completely) the epigenetic and transcriptional alterations of DMD muscles identified in Aim 1. Aim 3. Bioinformatic identification and analysis of epigenetic and transcriptional alterations in DMD muscles We will perform an integrated bioinformatic analysis of pcHi-C, RNA-seq, ATAC-seq and ChIP-seq data to identify DMD-associated pathogenic chromatin interactions (DMD PCI) in DMD MuSCs and myofibers, and their susceptibility to µ-dys expression. Aim 4. Contraction-induced alterations in high-order chromatin interactions at pathogenic loci in DMD muscles and reversibility by dys restoration We will determine the effect of muscle contraction on PCI and gene expression at loci of pathogenic genes, within the context of dys deficiency and upon µ-dys recovery. Understanding whether dys deficiency causes epigenetic perturbations responsible for pathogenic transcriptional output of DMD muscles, and whether they could be reversed by µ-dys expression, will invariably extend our knowledge on DMD pathogenesis and on the therapeutic efficacy of µ-dys based gene therapies.