Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric region. This deletion induces epigenetic modifications that affect the expression of several genes located in the vicinity. In each D4Z4 element, we identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription factor that plays a major role in the development of FSHD through the initiation of a large gene dysregulation cascade that causes myogenic differentiation defects, atrophy and reduced response to oxidative stress. Because miRNAs variably affect mRNA expression, proteomic approaches are required to define the dysregulated pathways in FSHD. In this study, we optimized a differential isotope protein labeling (ICPL) method combined with shotgun proteomic analysis using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56+ FSHD myoblasts were found to fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better understand the FSHD myogenic defect, our improved proteomic procedure was used to compare predominantly atrophic or disorganized myotubes to the same matching healthy control. FSHD atrophic myotubes presented decreased structural and contractile muscle components. This phenotype suggests the occurrence of atrophy-associated proteolysis that likely results from the DUX4-mediated gene dysregulation cascade. The skeletal muscle myosin isoforms were decreased while non-muscle myosin complexes were more abundant. In FSHD disorganized myotubes, myosin isoforms were not reduced, and increased proteins were mostly involved in microtubule network organization and myofibrillar remodeling. A common feature of both FSHD myotube phenotypes was the disturbance of several caveolar proteins, such as PTRF and MURC. Taken together, our data suggest changes in trafficking and in the membrane microdomains of FSHD myotubes. Finally, the adjustment of a nuclear fractionation compatible with mass spectrometry allowed us to highlight alterations of proteins involved in mRNA processing and stability.
Highlights
Facioscapulohumeral muscular dystrophy (FSHD1A: OMIM #158900) is one of the most frequently occurring hereditary muscle disorders
The myoblasts were propagated without dexamethasone and differentiation was induced upon confluence by decreasing the concentration of fetal bovine serum to 2% for 4 days
Protein quantification was based on the ICPL methodology, which consisted of labeling protein or peptide free amino groups (N-terminal and lysines) with amine-specific reagents containing different stable isotopes
Summary
Facioscapulohumeral muscular dystrophy (FSHD1A: OMIM #158900) is one of the most frequently occurring hereditary muscle disorders. A nuclear matrix attachment site (S/MAR) was found upstream of the repeated elements, which suggests that D4Z4 and the upstream genes reside in two chromatin loops. This site is weakened in FSHD myoblasts, enabling D4Z4 and the upstream genes to locate into a single loop and allowing cis-regulation [5]. A recent study indicated that Polycomb group proteins induced chromatin repression on large D4Z4 arrays in healthy cells, whereas a long non-coding RNA expressed from the contracted locus recruited the Trithorax group protein Ash1L and promoted histone H3 lysine 36 dimethylation and chromatin opening [6]. Several FSHD candidate genes located centromeric of D4Z4 have been proposed, including ANT1 (adenine nucleotide translocator 1 gene) [7], FRG1 (FSHD-related gene 1) [8], FRG2 [9] and DUX4c (double homeobox 4 centromeric) [10]
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