Molecular Pathphysiology of FSHD muscular dystrophy via genome-wide approaches

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DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder that is characterized by the progressive weakness and wasting of the muscles from face, upper-arm and shoulder girdle to lower limb. While there is consensus that FSHD is a disorder of transcription and gene regulation, the molecular pathways leading to muscular dystrophy and other unique clinical features of the disease are far from clear. Our preliminary study of whole genome profiles of 125 muscle biopsies representing 12 neuromuscular disorders showed that paired-like homeodomain transcription factor 1 (PITX1) gene was specifically up-regulated in FSHD patients. The significant PITX1 over-expression we observed in FSHD can not be due to inflammation, degeneration/ regeneration, or other "dystrophic" changes in muscle, as no other muscle disease (including juvenile dermatomyositis, Duchenne dystrophy, and others) showed up- regulation. Based on our extensive preliminary data both in vitro and in vivo, we present a model where over-expression of PITX1 in adult muscle invokes key muscle atrophy pathways, and, further, that PITX1 is regulated by DUX4 expression. The goal of this current proposal is to further develop our pathophysiological model to show direct relationships between 4q35 deletions, DUX4, and PITX1. The proposed research relies heavily on temporal series, conducted both in vivo and in vitro. Gene/gene interactions will also be determined. In aim 1, we propose to determine if Pitxl is a direct target of DUX4. We will determine whether a putative DUX4 binding site in the promoter region of Pitxl is functional, and whether it is specifically and directly regulated by DUX4. Additional DUX4 targets will be identified by temporal profiling. Interaction between DUX4 and potential target genes will be determined. In aim 2, we propose to generate and characterize a conditional muscle-specific Pitxl transgenic mouse model. The phenotype will be evaluated for changes in various clinical, functional, biochemical, molecular, and histological parameters. The phenotype of myoblasts, including appearance, proliferation, differentiation and susceptibility to oxidative stress, will also be evaluated. In addition, we will determine whether the disease phenotype is reversible. In aim 3, we will define molecular transcriptional pathways downstream of Pitxl expression using the Pitxl transgenic mouse. Temporal expression profiling will be performed to construct the pathways regulated by Pitxl. Interactions between Pitxl and potential regulatory targets of Pitxl will be further studied. Our preliminary data showed that disease-specific up-regulation of DUX4 and Pitxl and downstream changes of genes involved in muscle wasting might be involved in the pathophysiology of FSHD. The proposed research will identify key players in the pathological cascades of FSHD and define the interactions among them, which could potentially be used for developing treatments of the disease.