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

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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.