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2004-10-26 - Colloque/Présentation - communication orale - Anglais - 1 page(s)

Ansseau Eugénie , Coppée Frédérique , D'Amico Eva, Marcowycz Aline, Mattéotti Christel, Sauvage Sébastien, Wiilocq Jonathan, Belayew Alexandra , "Functional study of the DUX4 and DUX4c genes" in FSHD International Consortium Research Meeting, Toronto, Canada, 2004

  • Codes CREF : Biologie moléculaire (DI3111), Pathologies particulières (DI3370)
  • Unités de recherche UMONS : Biologie moléculaire (M122)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) The FSHD candidate gene (DUX4) we propose maps within each of the D4Z4 units repeated in tandem at 4q35. We have shown that a double homeodomain protein with the apparent molecular weight (MW: 52 kDa) and charge (pI: 8.6) of DUX4 was expressed in FSHD but not control primary myoblasts. The DUX4 functional studies are done in parallel on the homologous DUX4c gene located in a single truncated D4Z4 element 42 kb centromeric of the D4Z4 array. A rabbit antiserum directed against a DUX4c-specific peptide detected a protein with the apparent DUX4c size (47 kDa) on a Western blot prepared with nuclear extracts of FSHD but not control myoblasts. The DUX4 and DUX4c proteins are 424- and 374-residue long, respectively, and present only 2 mismatches over their 342 amino-terminal residues. Their putative toxicity was evaluated in TE671 cells (human rhabdomyosarcoma) transfected with pCINeoDUX expression vectors. DUX4c induced cell proliferation and the Myf5 trans-factor that inhibits myoblast terminal differentiation. In contrast, DUX4 was toxic as evidenced by leakage of cytoplasmic lactate dehydrogenase leading to 50% cell death in 48h. DUX4 expression also activated the apoptosis effector caspases 3 and 7. In addition, DUX4 induced the muscle differentiation factors MyoD and MEF2, and led to premature fusion of the surviving cells. We used the yeast two hybrid system to identify DUX protein partners. DUX4 could not be used as a bait because it presented a very strong transcriptional activity. In contrast, DUX4c could be used to screen a cDNA library derived from human skeletal muscle, yielding 187 positive clones. Among the 150 ones analysed, two classes of putative DUX partners were identified: (i) cytoskeleton proteins: 105 clones encoded desmin, and 3 encoded alpha actinin 3 , (ii) transcription factors with a "zinc finger" domain either of the LIM (13 clones encoded FHL3 and 2 clones a LIM-like domain) or MYND type (2 different clones). A clone encoding importin/karyopherin 13 was also found: this protein mediates nuclear import of homeoproteins. A fraction of this cDNA library had previously been screened with DUX1 (a non-pathological homologue) and had yielded 35/42 positive clones encoding desmin. Reconstruction experiments in yeast had shown that the desmin interaction was mediated by the double homeodomain of either DUX1 or DUX4/4c. Confirmation of this interaction in vitro was obtained by GST pull down experiments and co-immunoprecipitations from extracts of cells transfected with a DUX expression vector. A DUX/desmin co-localisation was detected by immunofluorescence in these cells on narrow regions at the nuclear periphery. We further studied the DUX4 proximal promoter fused to a luciferase reporter gene in transient transfection experiments and evaluated several mutations in the YY1 and MyoD cis-elements. Binding of these proteins was analysed in electrophoretic mobility shift assays, and their impact on DUX4-LUC activity shown by co-transfection with the respective expression vectors. These experiments confirmed that YY1 had no impact on the DUX4 promoter activity in muscle cells, in contrast to MyoD that was a very potent inhibitor in TE671 cells. Since the DUX4 promoter was previously shown to be activated by Sp1, we expect DUX4 expression to occur in FSHD satellite cells or myoblasts, not in differentiated muscle fibers. We acknowledge funding by the ABMM (Belgium), AFM (France), MDA (USA), and the Frenzel (Germany) and Shaw-Fischer (USA) families. E.A., A.M., C.M. held FRIA fellowships (Belgium).