Unexpected role for DUX transcription factors into the cytoplasm

Hundreds of double homeobox (DUX) genes map within dispersed 3.3-kb repeated elements and encode DNA-binding proteins. Among these we identified DUX4, a potent transcription factor that causes the FSHD muscular dystrophy. In the present study we identify skeletal muscle partners of either DUX4, DUX4c (an identical protein except for the end of the carboxyl-terminal domain) or DUX1 (limited to the double homeodomain). We first used the yeast two-hybrid system (Y2H) to screen an adult human skeletal muscle cDNA library with DUX1 or DUX4c that presented no or weak transcriptional activity in this system. We also expressed HaloTag-DUX4 or DUX4c fusion proteins in human muscle cells, co-purified them by affinity chromatography with their protein partners and identified these by mass spectrometry. We unexpectedly found desmin, an intermediate filament protein, other cytoskeletal proteins involved in actin-bundling or myofibrilar organization besides RNA-binding proteins involved in muscle-specific mRNA splicing and translation. Most of these partners play major roles in cytoskeletal organization during muscle differentiation. We confirmed DUX interaction with desmin as well as other partners involved in mRNA splicing by in vitro GST pull down, immunoprecipitation or by in situ proximal ligation assay in muscle cells. The functionality of these interactions was underscored by the observation of DUX4/DUX4c nucleo-cytoplasmic translocation upon myoblast fusion. Moreover, DUX4c and desmin immunostaining partially co-localized in transversal muscle sections of patients affected with Duchenne Muscular Dystrophy.
Our data suggest cytoplasmic functions for the numerous DUX gene products and identify novel toxicity pathways besides transcription activation for DUX4/DUX4c in FSHD muscles.