DESCRIPTION (provided by applicant): Our proposed experiments will identify mechanisms that cause mis-expression of DUX4 and lead to pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). FSHD is the most prevalent myopathy afflicting both children and adults, but no therapy is known. However, FSHD research has now entered an important new stage as DUX4 has emerged from studies in multiple laboratories, including our own, as the consensus FSHD candidate gene. Current evidence supports a model for FSHD pathology in which stable expression of a polyadenylated DUX4 mRNA splicing variant, termed DUX4-fl (fl = full-length), leads to production of a pathogenic DUX4-FL protein. This model is compatible with our finding, based on analysis of a large new library of myogenic cells and biopsies, that DUX4-fl mRNA and DUX4-FL protein are expressed at a much higher level and in a much higher fraction of myonuclei in FSHD than in healthy control muscle cells. Thus, in FSHD patients, clinically apparent muscle weakness may develop as DUX4-FL-induced pathological changes accumulate over time, whereas in healthy control muscles, the extremely low level of DUX4- FL is apparently insufficient to induce overt pathology. Although the FSHD-associated genetic lesion does not obviously alter protein coding or mRNA sequences, there is strong evidence that aberrant epigenetic regulation underlies DUX4 mis-expression. Our preliminary studies, for example, showed that DUX4-fl expression levels in FSHD myogenic cells could indeed be altered by manipulations of epigenetic status, e.g., DNA methlyation. Based on these epigenetic studies and our complementary analyses of DUX4-FL expression, we propose and will test the hypothesis that epigenetic dysregulation leads to aberrantly increased expression of DUX4-fl in FSHD vs. unaffected myogenic cells and thus to pathology. In Specific Aim 1, we wil identify epigenetic and non-epigenetic mechanisms that regulate DUX4-fl mRNA expression levels in skeletal muscle cells. In Specific Aim 2, we will analyze DUX4-FL expression in biopsies and also use single cell analyses to determine how DUX4-FL expression alters the fate of FSHD vs. unaffected cels. In Specific Aim 3, we will identify gene networks that are differentially regulate by the DUX4 protein isoforms and determine if they are disrupted in DUX4-fl-expressing FSHD cells. The results of our studies, in which we will systematically investigate the expression, regulation, and function of DUX4 isoforms in FSHD and control muscle cells, will provide the vital information needed to guide development of FSHD therapies specifically targeted to DUX4.

PUBLIC HEALTH RELEVANCE: The goals of our experiments are to identify pathogenetic mechanisms and potential new therapeutic strategies for facioscapulohumeral muscular dystrophy (FHSD). FSHD is the most prevalent muscular dystrophy that afflicts children and adults. This disease is severely debilitating and treatments are lacking, so new therapies are critically needed.