Mechanisms of DUX4 mediated FSHD pathology

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SUMMARY Facioscapulohumeral Muscular Dystrophy (FSHD) is one of the most prevalent myopathies affecting males and females of all ages, and currently there is no cure or therapeutic intervention available. The long-term goal of this project is to determine the epigenetic mechanisms leading to pathogenic gene expression in FSHD, and identify regulatory components that are viable targets for therapeutic development. FSHD is a complex genetic and epigenetic disease caused by chromatin relaxation of the D4Z4 macrosatellite repeat array at chromosome 4q35, which leads to aberrant expression of the DUX4 gene from the distal-most repeat unit. The DUX4 protein, in turn, activates a host of genes normally expressed in early development, which cause pathology when mis-expressed in adult skeletal muscle. This established model of FSHD pathogenesis has stimulated the search for DUX4- based therapeutic targets, including approaches to block DUX4 expression. While normal mechanisms of D4Z4 repression have been well-characterized, very little is known about the factors and pathways responsible for facilitating aberrant activation of DUX4. Previously, we showed that the degree and stability of epigenetic dysregulation at the FSHD-associated 4q35 locus is the key determinant for DUX4 expression, clinical presentation, and severity of FSHD pathology. Thus, targeting epigenetic dysregulation in FSHD is a potentially powerful therapeutic avenue. Such an approach would be greatly aided by a better understanding of the factors facilitating DUX4 expression, each of which represents a potential target for therapeutic inhibition. In a candidate-based screen, we identified several epigenetic regulators with druggable domains that function as novel activators of DUX4. Here, we will characterize these epigenetic facilitators of DUX4 expression and validate their potential as therapeutic targets in FSHD. We will determine the global effects of reducing the expression of these factors on skeletal muscle health and development using primary patient cells and novel animal models. Finally, we will test the translational potential of inhibiting these DUX4 regulators in an FSHD human xenograft mouse model derived from FSHD patient myoblasts with their endogenous D4Z4 arrays. DUX4 regulators will be inhibited by CRISPR inhibition and assayed for specificity. Successful completion of this project will provide key insights into the mechanisms of epigenetic dysregulation in FSHD and validate the translational potential of novel therapeutic targets in vivo.