Résumé :
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Facioscapulohumeral muscular dystrophy (FSHD) is linked to deletions within the D4Z4 repeat array in 4q35. We have identified the DUX4 double homeobox gene within each D4Z4 unit (1). It encodes a transcription factor that is expressed in FSHD but not in control primary myoblasts (2, 3). DUX4 targets a number of genes, some of which encode other transcription factors such as PITX1, thus leading to a large transcription deregulation cascade (3, 4). We determined that DUX4 stable mRNAs only derived from the most distal D4Z4 unit and extended within the flanking pLAM region that provided an intron and a polyadenylation signal (3). This signal is required to develop FSHD as recently shown by others (5).We compared the proteome of FSHD and control myotubes by gel free differential mass spectrometry using isotope coded protein labelling (ICPL). Among the differentially expressed proteins, we detected increased amounts of galectin-1 (GAL-1). Although intracellular GAL-1 was either induced or unchanged in Western blot depending on the FSHD myoblast line, its secretion was always increased in the FSHD culture media upon secretome analysis and ELISA. Increased extracellular GAL-1 was observed by immunohistochemistry on 2 FSHD muscle biopsies.The GAL-1 promoter contains a putative PITX1 binding site. It was fused to the luciferase reporter gene and evaluated in C2C12 cells co-transfected with a DUX4 or PITX1 expression vector. Both factors strongly induced luciferase activity and 2 mutation of the PITX1 binding site are currently being tested. We confirmed endogenous GAL-1 induction at the protein level by Western blot on immortalized control myoblasts transfected with DUX4 or PITX1 expression vectors. In conclusion our data suggested that GAL-1 was an indirect DUX4 transcriptional target. Because of its function in muscle differentiation and regeneration (6) and its induction upon muscle dystrophy or injury (7), GAL-1 could contribute to FSHD. Further studies will investigate whether PITX1 directly binds to the GAL-1 promoter (mutagenesis, EMSA and ChIP analyses) and quantify GAL-1 mRNA levels (qRT-PCR).References: (1) Gabriels et al., 1999. Gene 236:25-32.(2) Kowaljow et al., 2007. Neuromuscul Disord 17:611-623.(3) Dixit et al., 2007. Proc Natl Acad Sci U S A 104:18157-18162. (4) Bosnakovski et al., 2008. EMBO J 27:2766-2779.(5) Lemmers et al., 2010. Science 329:1650-3.(6) Georgiadis et al., 2007.236:1014-24.(7)
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