title: The Role of D4Z4-Encoded Proteins in the Osteogenic Differentiation of Mesenchymal Stromal Cells Isolated from Bone Marrow.
abstract: Facioscapulohumeral muscular dystrophy (FSHD) is associated with an activation of the double homeobox 4 (DUX4) gene, which we previously identified within the D4Z4 repeated elements in the 4q35 subtelomeric region. The pathological DUX4 mRNA is derived from the most distal D4Z4 unit and extends unexpectedly within the flanking pLAM region, which provides an intron and polyadenylation signal. The conditions that are required to develop FSHD are a permissive allele providing the polyadenylation signal and hypomethylation of the D4Z4 repeat array compared with the healthy muscle. The DUX4 protein is a 52-kDa transcription factor that initiates a large gene deregulation cascade leading to muscle atrophy, inflammation, differentiation defects, and oxidative stress, which are the key features of FSHD. DUX4 is a retrogene that is normally expressed in germline cells and is submitted to repeat-induced silencing in adult tissues. Since DUX4 mRNAs have been detected in human embryonic and induced pluripotent stem cells, we investigated whether they could also be expressed in human mesenchymal stromal cells (hMSCs). We found that DUX4 mRNAs were induced during the differentiation of hMSCs into osteoblasts and that this process involved DUX4 and new longer protein forms (58 and 70 kDa). A DUX4 mRNA with a more distant 5' start site was characterized that presented a 60-codon reading frame extension and encoded the 58-kDa protein. Transfections of hMSCs with an antisense oligonucleotide targeting DUX4 mRNAs decreased both the 52- and 58-kDa protein levels and confirmed their identity. Gain- and loss-of-function experiments in hMSCs suggested these DUX4 proteins had opposite roles in osteogenic differentiation as evidenced by the alkaline phosphatase activity and calcium deposition. Differentiation was delayed by the 58-kDa DUX4 expression and it was increased by 52-kDa DUX4. These data indicate a role for DUX4 protein forms in the osteogenic differentiation of hMSCs.
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Laurence
authors.initials: L
authors.lastname: de la Kethulle de Ryhove
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Eugénie
authors.initials: E
authors.lastname: Ansseau
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Charlotte
authors.initials: C
authors.lastname: Nachtegael
authors.affiliation: 2 Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles , Brussels, Belgium .
authors.forename: Karlien
authors.initials: K
authors.lastname: Pieters
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Céline
authors.initials: C
authors.lastname: Vanderplanck
authors.affiliation: 3 Department of Embryology and Genetics, Vrije Universiteit Brussel , Jette, Belgium .
authors.forename: Mieke
authors.initials: M
authors.lastname: Geens
authors.affiliation: 3 Department of Embryology and Genetics, Vrije Universiteit Brussel , Jette, Belgium .
authors.forename: Karen
authors.initials: K
authors.lastname: Sermon
authors.affiliation: 4 Centre for Comparative Genomics, Murdoch University , Murdoch, and The University of Western Australia, Crawley, and Western Australian Neuroscience Institute, Nedlands, Western Australia, Australia .
authors.forename: Steve D
authors.initials: SD
authors.lastname: Wilton
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Frédérique
authors.initials: F
authors.lastname: Coppée
authors.affiliation: 2 Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles , Brussels, Belgium .
authors.forename: Laurence
authors.initials: L
authors.lastname: Lagneaux
authors.affiliation: 1 Laboratory of Molecular Biology, University of Mons , Mons, Belgium .
authors.forename: Alexandra
authors.initials: A
authors.lastname: Belayew
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: C121257:DUX4L1 protein, human; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D000469:Alkaline Phosphatase; D002118:Calcium