The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins

Abstract

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 Interactome of DUX4 Reveals Multiple Activation Pathways

“…One of the strongest interactions of the N-terminal is with the Complement component 1 Q subcomponent-binding protein (C1qBP), a highly acidic protein with multiple cellular localizations and functions 36 . Indeed, the DUX4-C1qBP interaction was previously reported in-vitro 15,26 and in-situ in human muscle sections 27 . In non-human myoblasts, C1qBP loss-of-function inhibits their proliferation and differentiation, leading to their death 37 .…”

“…They determined these interactions to be mediated by the N-terminal of DUX4, since they are also observed with shorter variants of the protein that lack the C-terminal. The interaction with C1qBP was also confirmed, by co-immunoprecipitation, at the endogenous level in FSHD muscle cells 15 , and recently by in situ proximity ligation assay on FSHD muscle sections 27 . Despite these efforts, our knowledge of the DUX4 interactome is still partial.…”

“…The molecular basis for this interaction may be the DUX4 KIX-binding domain in the activation region 33 , which corresponds to the endocytosis signal motif [ED]-X-X-X-L-[LI] that is recognized by AP-2 sigma 34 . We recently detected DUX4 both in nuclei and cytoplasm of myogenic progenitors in FSHD muscle sections 27 , thereby suggesting that cytoplasmic DUX4 may also directly interact with AP-2 in those cells.…”

The FSHD jigsaw: are we placing the tiles in the right position?

“…The hDUX4C protein seems to be associated with muscle regeneration. This implies that DUX4 and DUX4c in regenerating FSHD muscle cells might have antagonistic roles and that caution should be exerted with therapeutic agents aiming for DUX4 suppression as they might also repress the highly similar DUX4c and interfere with its physiological role [44,45].…”

Influence of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Possible Treatments

“…Another study demonstrated that the interaction between DUX4c and RBPs may play a role in muscle cell differentiation, repair, and mass maintenance particularly during the myoblast fusion stage. They also suggested that increased amounts of DUX4c or DUX4 proteins in FSHD muscle cells may obstruct C1QBP or other RBPs from performing their normal functions and impede the process of muscle regeneration, exacerbating the muscle pathology activated by DUX4 [123].…”