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2010-11-25 - Colloque/Présentation - poster - Anglais - 1 page(s)

Tassin Alexandra , Leroy Baptiste , Ansseau Eugénie , Pire Emilie, Faille Julie, Leclercq Thomas, Charron Sébastien, Laoudj-Chenivesse Dalila, Coppée Frédérique , Wattiez Ruddy , Belayew Alexandra , "Focus on nuclear proteins of FSHD primary myotubes by differential mass spectrometry." in VIIIè journées annuelles Société Française de Myologie, 26, 50, Nice, France, 2010

  • Codes CREF : Biologie moléculaire (DI3111), Acides nucléiques, synthèse des protéines (DI311A), Pathologies particulières (DI3370)
  • Unités de recherche UMONS : Protéomie et Microbiologie (S828), Biochimie métabolique et moléculaire (M122)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Our group has identified the DUX4 gene at the FSHD locus and shown its expression in affected but not control myoblasts (Gabriels et al., 1999; Kowaljow et al., 2007; Dixit et al., 2007). The DUX4 protein is a transcription factor that targets a number of genes, some of which encode other transcription factors among these, PITX1 (Dixit et al., 2007). Several studies investigated gene/protein expression in FSHD primary myoblasts or muscle biopsies. Since DUX4 is activated upon myoblast differentiation, we chose to compare the proteome of FSHD and control myotubes by gel free differential mass spectrometry analysis (LC-MS/MS) using an isotope coded protein labelling (ICPL). The genes coding differentially expressed proteins are putative DUX4/PITX1 targets. Their promoters were fused to the luciferase reporter to evaluate their response to DUX4/PITX1 in cotransfection experiments. 1/ With this approach we identified the GAL1 gene as a PITX1 target: its proximal promoter has a PITX1 binding site. GAL1 expression is induced in FSHD myotubes, or by forced DUX4 expression in control myoblasts and the protein is secreted. 2/ We wanted to focus on changes in the expression of transcription factors, splicing factors and other nuclear proteins. Because of the low level of these proteins as compared with the abundant cytosqueletal proteins in FSHD myotubes, we had to improve the method sensitivity. We have first adapted a nuclear extraction compatible with the LC-MS/MS method. Moreover, higher resolution LC separation and a modified version of ICPL was also tested (Leroy et al., 2010). Using this strategy, we have identified 649 proteins including 33% of predicted nuclear proteins involved in transcription regulation (FHL2, STAT1, SOX5), RNA maturation (splicing factors), DNA repair (KU86) or chromatin remodeling (SWI/SNF). Using this method, we also compared the nucleome of disorganized and atrophic myotubes as described by Barro et al., 2008. 3/ Although we could improve the sensitivity by this method, we have still not been able to detect DUX4 by mass spectrometry. Because of its central role in FSHD, we decided to use the Multiple Reaction Monitoring (MRM) approach to focus on a DUX4-specific signature. To determine DUX4-specific peptides, we analyzed by LC-MS/MS a DUX4-HaloTag protein produced in human muscle cell cultures and purified by affinity chromatography using a Halolink column (Promega). We detected four DUX4 peptides characterized by their m/z value and MS/MS spectra. All these peptides are in the NH2-terminal domain of DUX4, identical to DUX4c. No peptide of the DUX4-specific COOH-terminal domain was detected. Further experiments are in progress to achieve this aim.