title: Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.
abstract: Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Eugénie
authors.initials: E
authors.lastname: Ansseau
authors.affiliation: Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH, United States of America.
authors.forename: Jocelyn O
authors.initials: JO
authors.lastname: Eidahl
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Céline
authors.initials: C
authors.lastname: Lancelot
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Tassin
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Christel
authors.initials: C
authors.lastname: Matteotti
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Cassandre
authors.initials: C
authors.lastname: Yip
authors.affiliation: Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH, United States of America.
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.affiliation: Laboratory of Proteomic and Microbiology, Research Institute for Biosciences, University of Mons, Mons, Belgium.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Leroy
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Céline
authors.initials: C
authors.lastname: Hubeau
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Cécile
authors.initials: C
authors.lastname: Gerbaux
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Samuel
authors.initials: S
authors.lastname: Cloet
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Armelle
authors.initials: A
authors.lastname: Wauters
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Zorbo
authors.affiliation: Pediatric Department, CHRU Montpellier, Montpellier, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Meyer
authors.affiliation: I.R.I.B.H.M., Free University of Brussels, Brussels, Belgium.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pirson
authors.affiliation: Laboratory of Physiology and Experimental Medicine, INSERM U1046, Montpellier, France.
authors.forename: Dalila
authors.initials: D
authors.lastname: Laoudj-Chenivesse
authors.affiliation: Laboratory of Proteomic and Microbiology, Research Institute for Biosciences, University of Mons, Mons, Belgium.
authors.forename: Ruddy
authors.initials: R
authors.lastname: Wattiez
authors.affiliation: Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH, United States of America.|Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH, United States of America.
authors.forename: Scott Q
authors.initials: SQ
authors.lastname: Harper
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Belayew
authors.affiliation: Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Coppée
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D003893:Desmin / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D024510:Muscle Development; D032446:Myoblasts / Q000502:physiology*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: C121257:DUX4L1 protein, human; C581310:DUX4L9 protein, human; D003893:Desmin; D018398:Homeodomain Proteins; C527880:IPO13 protein, human; D028884:Karyopherins; D016601:RNA-Binding Proteins; D014157:Transcription Factors
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