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2005-10-25 - Colloque/Présentation - communication orale - Anglais - 1 page(s)

Laoudj-Chenivesse Dalila, Barro Marietta, Sauvage Sébastien, Tassin Alexandra , Ansseau Eugénie , Marcowycz Aline, Figlewicz Denise, Van Acker Anne-Marie, Leo Oberdan, Belayew Alexandra , Coppée Frédérique , "Overexpression of the double homeodomain protein DUX4c in FSHD muscle" in FSHD International Consortium Research Meeting, Salt Lake City, USA, 2005

  • 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) We have identified the putative double homeobox gene DUX4c in an isolated D4Z4 unit mapping 42 kb centromeric of the D4Z4 repeat array and encoding a 374-residue protein. DUX4c mRNA’s encompassing its open reading frame were detected when the natural gene was introduced by transfection into muscle cell cultures. 3’RACE experiments showed alternative splicing downstream of the STOP codon. A rabbit antiserum raised against a DUX4c-specific peptide detected a 47 kDa protein on a Western blot prepared with nuclear extracts of cells transfected with a DUX4c expression vector. In muscle cells transfected with the natural gene, the DUX4c protein was detected by immunoprecipitation of nuclear extracts with the rabbit antiserum, followed by a western blot with a very sensitive mouse monoclonal antibody raised against the carboxyl-terminal domain of DUX4 and cross reacting with DUX4c. This weak protein expression from the DUX4c natural gene was confirmed in the nucleus of a few transfected muscle cells by immunofluorescence. We have investigated the DUX4c expression by Western blot with this rabbit antiserum in muscle biopsies of patients with FSHD and controls, and in the primary myoblast cultures derived from them. DUX4c was detected in controls and at increased levels in all FSHD muscle biopsies analyzed. Interestingly, higher DUX4c expression was observed in samples with lower D4Z4 repeat copy number. Moreover, the strongest signal was found in a patient homozygous for the 4q35 deletion. In myoblast primary cultures (DMEM, 10% fetal calf serum and 1% Ultroser G), DUX4c was detected both in FSHD and control samples but when the culture medium was changed (SKGM and 10% fetal calf serum), it could not be detected in controls anymore. DUX4c expression was induced upon differentiation in both myoblast types with increased levels in FSHD samples. We then performed Western blots on biopsies of patients with Duchenne muscular dystrophy and found a higher DUX4c amounts as compared to controls. In myoblasts cultures, similar increases in DUX4c expression were detected during differentiation of Duchenne or control samples. Additional analyses will be needed to evaluate whether DUX4c expression might be linked in part to muscle regeneration. In another set of experiments, we transfected TE671 rhabdomyosarcoma cells grown in vitro with a pCINeo-DUX4c expression vector and observed induced Myf5 protein expression and DNA binding activity, but decreased MyoD and MEF2 activities. In conclusion, our data demonstrated that the DUX4c gene was functional, expressed in myoblasts, with higher levels in FSHD samples, and could affect some myogenic factor activities. DUX4c might not be involved in FSHD since a deletion extending from the D4Z4 repeat array to DUX4c was found in two affected families (Lemmers et al, 2003, Neurology 22, 178-83). Nevertheless, the second DUX4c allele is still present in these families and could be activated by transvection as recently reported for the FRG2 gene (Rijkers et al, 2004, J Med Genet. 41, 826- 36). We acknowledge funding by the MDA and the NIH (USA). Fellowships were from the FRIA (Belgium, to A.M.), the AFM (France, to S.S.) and the the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (France, to M.B.).