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2006-10-09 - Colloque/Présentation - communication orale - Anglais - 1 page(s)

Ansseau Eugénie , Tassin Alexandra , Sauvage Sébastien, Laoudj-Chenivesse Dalila, Barro Marietta, Figlewicz Denise, Coppée Frédérique , Belayew Alexandra , "DUX4 gene expression in FSHD primary myoblasts" in FSHD International Consortium Research Meeting, New Orleans, USA, 2006

  • 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) Several years ago we have identified the DUX4 double homeobox gene within each unit of the D4Z4 repeat array that is contracted in FSHD. Demonstration of DUX4 expression in patient muscle cells proved technically very challenging because of its low level, its toxicity, and its homology to hundreds of DUX genes unlinked to FSHD. We could demonstrate expression of the homologous non-toxic DUX4c protein encoded by an isolated D4Z4 element 42 kb centromeric of the repeat array: it was induced by differentiation in control and FSHD myoblasts and was expressed at higher levels in biopsies of patients with low D4Z4 copy numbers. For DUX4 mRNA studies we used retro-transcription (RT) and amplification by PCR (see below). We cloned all the products we obtained in the pCR4 plasmid and sequenced individual clones to confirm they were identical to DUX4. We first set up optimal conditions on total RNA’s extracted from mouse C1C12 cells transfected with a pGEM plasmid containing either a single D4Z4 element, or a 13.5-kb EcoRI genomic fragment from the contracted 4q35 allele of a patient with only two D4Z4 units left. The extracted total RNA was digested with desoxyribonuclease I to eliminate putative DNA contamination. The same experimental conditions were then applied to human primary myoblasts. By 5’ RACE we found one major transcription initiation site mapping 48 bp downstream from a TACAA box, as well in transfected cells as in FSHD and control myoblasts. We performed RT with a DUX4-specific primer followed by amplification with DUX4-specific primers flanking its open reading frame. We could detect by agarose gel electrophoresis the expected 1.4-kb RT-PCR product as well in transfected cells as in primary myoblasts: the signal was much stronger in differentiated versus proliferating FSHD myoblasts but hardly detectable in control myoblasts. No product different from DUX4 was found upon sequencing, and no product was obtained upon omission of the RT step. For DUX4 protein detection, we have raised a monoclonal antibody against its carboxyl-terminal domain (297 residues) outside of the homeodomains that specifically detects the DUX4 (52 kDa) and homologous DUX4c (47 kDa) proteins on Western blots performed with extracts of cells transfected with pCI-neo-DUX expression vectors. The Western blot sensitivity was recently increased about 20-fold by use of a new peroxydase substrate (Pierce) and allowed detection of DUX4 in 6 additional FSHD myoblast lines. In conclusion these data underscore our hypothesis that the DUX4 gene is expressed in patients with FSHD.