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Background  We have previously demonstrated that double homeobox 4 centromeric ( DUX4C ) encoded for a functional DUX4c protein upregulated in dystrophic skeletal muscles. Based on gain- and loss-of-function studies we have proposed DUX4c involvement in muscle regeneration. Here, we provide further evidence for such a role in skeletal muscles from patients affected with facioscapulohumeral muscular dystrophy (FSHD). Methods DUX4c was studied at RNA and protein levels in FSHD muscle cell cultures and biopsies. Its protein partners were co-purified and identified by mass spectrometry. Endogenous DUX4c was detected in FSHD muscle sections with either its partners or regeneration markers using co-immunofluorescence or in situ proximity ligation assay. Results We identified new alternatively spliced DUX4C transcripts and confirmed DUX4c immunodetection in rare FSHD muscle cells in primary culture. DUX4c was detected in nuclei, cytoplasm or at cell–cell contacts between myocytes and interacted sporadically with specific RNA-binding proteins involved, a.o., in muscle differentiation, repair, and mass maintenance. In FSHD muscle sections, DUX4c was found in fibers with unusual shape or central/delocalized nuclei (a regeneration feature) staining for developmental myosin heavy chain, MYOD or presenting intense desmin labeling. Some couples of myocytes/fibers locally exhibited peripheral DUX4c-positive areas that were very close to each other, but in distinct cells. MYOD or intense desmin staining at these locations suggested an imminent muscle cell fusion. We further demonstrated DUX4c interaction with its major protein partner, C1qBP, inside myocytes/myofibers that presented features of regeneration. On adjacent muscle sections, we could unexpectedly detect DUX4 (the FSHD causal protein) and its interaction with C1qBP in fusing myocytes/fibers. Conclusions DUX4c upregulation in FSHD muscles suggests it contributes not only to the pathology but also, based on its protein partners and specific markers, to attempts at muscle regeneration. The presence of both DUX4 and DUX4c in regenerating FSHD muscle cells suggests DUX4 could compete with normal DUX4c functions, thus explaining why skeletal muscle is particularly sensitive to DUX4 toxicity. Caution should be exerted with therapeutic agents aiming for DUX4 suppression because they might also repress the highly similar DUX4c and interfere with its physiological role. Graphical Abstract

The ectopic expression of the DUX4 protein in muscle cells is the underlying cause of Facioscapulohumeral Muscular Dystrophy (FSHD). DUX4 is a potent transcription factor that activates a large number of genes in a dysregulated manner, but the direct protein interactions involved in this activation are only partially known. Here, we tailored an affinity purification and mass spectrometry (AP-MS) analysis to the unique features and functions of DUX4, to provide a more complete view of its interactome. We also obtained and verified all-atom models for two of the major interactions by employing cross-linking and mass spectrometry (CL-MS), computational modeling, and guided mutation studies. We find that DUX4 interacts strongly with two homologous transcription activators, PTOV1 and MED25, in addition to the previously characterized CBP/p300. The interaction with the PTOV1/MED25 domain involves the wrapping of the last thirty residues of the DUX4 activation region around the domain in a very extensive interface. Hence, DUX4 has the capacity to both open the chromatin and directly recruit the Mediator complex. DUX4 also binds to all members of the RFPL4 family, which are among the strongest genes it activates. These interactions are mediated through a hitherto unrecognized motif in the DUX4 disordered linker region. This feedback mechanism suggests that DUX4 may be inhibited by its own activation products, and explains its typical pulsed expression profile. We also found SIX1 and the AP-2 complex as strong DUX4 C-terminal interactors. A separate analysis of interactions involving the N-terminal of DUX4 revealed enrichment of proteins that are involved in DNA repair following double-strand breaks. Overall, these findings reveal new activation pathways for DUX4, which may be modulated in future strategies to control its toxicity. This study also showcases the synergy between CL-MS and deep-learning based modeling for the structural elucidation of challenging protein-protein interactions.