8th international congress , Myology: Estrogens interfere with DUX4 nuclear import

Introduction Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder characterized by skeletal muscle weakness and wasting, involving mainly the muscles of the upper part of the body. The major molecular event that drives FSHD is the epigenetic de-repression of the subtelomeric D4Z4 macrosatellite repeat array at chromosome 4q35, which leads to aberrant expression of the transcription factor Double Homeobox 4 (DUX4) and cytotoxicity in skeletal muscle cells (Lemmers et al., 2010). Currently, there is no cure for FSHD. DUX4 pathogenic activity is developed through its transcriptional function and is associated with its nuclear localization. The structure of the DUX4 protein is characterized by three monopartite nuclear localization signals (NLS) (Corona et al., 2013), whose function has not yet been investigated in human myoblasts. A recent paper published by our group demonstrated that in vitro muscle differentiation of myoblasts from FSHD patients is improved by estrogens treatment. Estrogen through the estrogen receptor β (ERβ) reduce DUX4 transcriptional activity and interfere with its recruitment in the nucleus (Teveroni et al., 2017). Results To elucidate the mechanism by which estrogens antagonize DUX4 activity, we analyzed the interactome of DUX4 in immortalized myoblasts differentiated for three days in presence or absence of 17β-estradiol (E2). The interactome analysis revealed that DUX4 binds some proteins of the Nuclear Pore Complex (NPC), the largest protein complex in eukaryotic cells that mediates macromolecular transport between nucleus and cytoplasm. Estrogen treatment decreases such binding without altering NPC protein levels. Immunofluorescence experiments confirmed increased co-localization of DUX4 with these proteins during myoblast differentiation. Overexpression of these proteins increases DUX4 transcriptional activity, supporting the relevance of such interaction for the development of DUX4 activity. Finally, a loss of function mutant in one of the three NLS shows decreased transcriptional function compared to wild-type DUX4. Conclusion Overall, these data suggest that the nuclear import of DUX4 is an active process and that estrogens can impair it. This research also introduces the possibility of developing therapeutic molecules targeting DUX4 nuclear transport, as a potential treatment for FSHD.