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

项目总结 肌营养不良蛋白(dystrophin，dys)的缺失和dys相关蛋白复合体(dys-Associated Protein Complex，DAPC)的破坏 骨骼肌纤维被广泛认为是肌膜不稳定的主要原因，导致骨骼 杜氏肌营养不良症(DMD)患者的肌肉损失。除了其结构功能外， DYS参与了对其他下游细胞事件的调节，包括对 基因组完整性和基因表达。事实上，强直性肌无力的肌肉表现出组蛋白的改变。 修饰、基因表达和非编码RNA，以及基因组不稳定和核的特征 异常现象。基于染色体构象捕捉(3C)的研究揭示了基因组是折叠的 转化为高阶染色质相互作用。了解Dys缺乏、调节失调之间的关系 基因表达和基因组拓扑改变对于全面了解DMD具有特殊的意义 发病机制和评估旨在取代强直性脊柱炎的治疗方法的有效益处 在DMD男孩中的表现。这项建议通过利用全基因组最先进的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 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- 以患者IPSC为基础的DISH人DMD活体模型和MDX小鼠活体模型，方法如下 目的：目的1.确定调控基因表达的高阶染色质相互作用中的变化 在DMD肌肉中，我们将确定功能和结构之间染色质相互作用的变化 基因组元件，导致致病基因在人类(HiPSC来源的骨骼肌)和 小鼠(MDX小鼠)DMD模型。目的2.µ-dys修复体逆转高位牙周病患者牙周组织的改变 调控DMD肌肉基因表达的染色质相互作用我们将评估 用µ-dys恢复Dys的表达可部分或完全逆转表观遗传和转录 AIM中确定的DMD肌肉的变化1.目的3.生物信息学鉴定和分析 DMD肌肉的表观遗传和转录变化我们将进行一项综合生物信息学研究 分析PCI-C、RNA-SEQ、ATAC-SEQ和CHIP-SEQ数据以鉴定与DMD相关的致病染色质 DMD-MUSCs和肌纤维的相互作用(DMD-PCI)及其对µ-dys表达的敏感性。目标4. 收缩引起的DMD致病基因座高阶染色质相互作用的改变 肌肉与DYS修复的可逆性我们将确定肌肉收缩对经皮冠状动脉介入治疗和 在Dys缺乏症和恢复后致病基因座位上的基因表达。 了解DyS缺乏症是否导致表观遗传扰动 DMD肌肉的转录输出，以及它们是否可以被µ-dys表达逆转，总是 扩大我们对DMD发病机制和基于µ-dys的基因疗法的治疗效果的了解。

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.