FSHD / OPMD / EDMD / DMI: P.362Regulation of facioscapulohumeral muscular dystrophy candidate protein DUX4

Abstract

The DUX4 gene associated with Facioscapulohumeral muscular dystrophy (FSHD) encodes a toxic transcription factor. Mechanisms that may confer toxic properties to the DUX4 protein are unknown. Characterizing biochemical properties of the DUX4 protein, including addressing whether DUX4 is post-translationally modified, will provide fundamental information required to design FSHD therapies aimed at inhibiting DUX4 protein. We have characterized the following biochemical properties; (1) DUX4 post-translational modifications: We performed mass spectrometry to identify DUX4 post-translational modifications (PTMs) followed by mutagenesis to determine the impact of these modified residues on DUX4 protein stability, toxicity, function, subcellular localization and biomarker expression. We have found a single methylated residue and phosphorylated residues within or near the double homeodomains alter DUX4′s function and toxicity without altering DUX4 protein stability or nuclear localization; (2) DUX4 modifying enzymes: To identify proteins that may interact with DUX4 transiently or indirectly (e.g. transcriptional complex) we performed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins. Additionally, we have quantified the activity of 245 human kinases on DUX4 in vitro. Our screen identified 92 kinases that significantly phosphorylate DUX4 protein and 13 exhibit very high activity. We are overexpressing these kinases in myoblasts and determining their impact on DUX4 toxicity. Additionally, we identified a class of modifying enzyme inhibitors that reduce DUX4 toxicity in human myoblasts and are continuing to screen commercially available inhibitors targeting DUX4 modifying enzymes. Our goal has been to investigate modifying enzyme contribution to DUX4 toxicity in FSHD muscle. Importantly, we report for the first time a pattern of PTMs that regulate DUX4 protein function, as well as a modifying enzyme class that when inhibited prevents DUX4-induced toxicity.