DUX4-Induced Constitutive DNA Damage and Oxidative Stress Contribute to Aberrant Differentiation of Myoblasts from FSHD Patients
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Free Radical Biology and Medicine 99 (2016) 244–258 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Original article DUX4-induced constitutive DNA damage and oxidative stress contribute to aberrant differentiation of myoblasts from FSHD patients Petr Dmitriev a,b,1, Yara Bou Saada a,1, Carla Dib a, Eugénie Ansseau d, Ana Barat a, Aline Hamade e, Philippe Dessen c, Thomas Robert c, Vladimir Lazar c, Ruy A.N. Louzada g, Corinne Dupuy g, Vlada Zakharova f, Gilles Carnac b, Marc Lipinski a, Yegor S. Vassetzky a,f,n a UMR 8126, Univ. Paris-Sud, CNRS, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif, France b PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, F-34295 Montpellier cedex 5, France c Functional Genomics Unit, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif, France d Laboratory of Molecular Biology, University of Mons, 20 place du Parc, B700 Mons, Belgium e ER030-EDST, Department of Life and Earth Sciences, Faculty of Sciences II, Lebanese University, Lebanon f Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991 Moscow, Russia g UMR 8200, Univ., Paris-Sud, CNRS, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif, France article info abstract Article history: Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Received 31 March 2016 Western world, however, its etiology remains only partially understood. Here, we provide evidence of Received in revised form constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate 4 August 2016 oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant Accepted 8 August 2016 myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage ac- Available online 9 August 2016 cumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition re- Keywords: duced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the Facioscapulohumeral dystrophy culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment DUX4 during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in Oxidative stress Antioxidants myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts. & 2016 Elsevier B.V. All rights reserved. 1. Introduction and a coding region for the DUX4 gene. Several studies have documented a higher level of expression of DUX4 as well as several Facioscapulohumeral dystrophy (FSHD) is an autosomal domi- proximally located genes in FSHD cells and tissues (reviewed in nant disease manifesting mainly as wasting of specific groups of [2,3]). Transcriptomic and proteomic studies have revealed an facial and limb muscles. The prevalence of FSHD is estimated to be abnormal expression of a number of mRNA, microRNAs and pro- as high as 8 in 100,000 suggesting that it might be the most teins in in vitro cultured myoblasts isolated from FSHD patients as common muscular dystrophy in the Western world [1]. FSHD has compared to healthy controls [4–18]. been genetically linked to a shortened macrosatellite repeat array In vitro cultured FSHD myoblasts which suffer from a defect in D4Z4, specific polymorphisms and sequence variants on the long the myogenic differentiation program [5], demonstrate a low fu- arm of chromosome 4 (region 4q35) which together are thought to sion index and morphological abnormalities when differentiated create a context permissive for aberrant gene expression (re- into myotubes [18,19] and are sensitive to oxidative stress [19,20]. viewed in [2,3]). In agreement with these observations, it was demonstrated that Every D4Z4 repeat on chromosome 4q35 contains a promoter the expression of oxidative stress-related genes was altered in FSHD cells [7,8,11,12,20,21] and tissues [9,22] suggesting that FSHD cells might be subjected to an endogenous oxidative stress. n Corresponding author at: UMR 8200, Univ., Paris-Sud, CNRS, Institut de Can- Increasing evidence indicates that oxidative stress can reduce cérologie Gustave-Roussy, F-94805 Villejuif, France. fi E-mail address: [email protected] (Y.S. Vassetzky). the differentiation ef ciency of human myoblasts; persistent re- 1 These authors contributed equally to this work. active oxygen species (ROS) act as signaling molecules in several http://dx.doi.org/10.1016/j.freeradbiomed.2016.08.007 0891-5849/& 2016 Elsevier B.V. All rights reserved. P. Dmitriev et al. / Free Radical Biology and Medicine 99 (2016) 244–258 245 physiological processes in human skeletal muscle cells, they are and DNA in the cell. DNA damage and genetic instability are notably known to affect their morphology and function (for review known to be involved in the etiology of many human diseases see [23,24]); both increased mitochondrial production of ROS and including cancer, neurodegenerative disorders, and aging (for re- NF-kB signaling linked to increased production of ROS can lead to view see [28,29]) and were also described in Duchenne (DMD) and muscle atrophy [25,26]. Limb-girdle Muscular (LGMD) dystrophies, but not in FSHD [30] Several previous observations suggest that increased DUX4 (for review see [31]). expression might provoke oxidative stress. DUX4, when over- Here we describe a novel phenotypic feature of in vitro cultured expressed in mouse immortalized myoblasts C2C12 or human FSHD myoblasts: a high level of DNA damage lesions, increased rhabdomyosarcoma cell line RD, altered the expression of genes production of ROS and upregulation of many genes related to DNA controlling redox balance and genes involved in the Nrf2-medi- damage repair. Furthermore, we demonstrated for the first time ated oxidative stress response pathway [21,27]. Furthermore, in- that DUX4 overexpression in human immortalized myoblasts re- ducible DUX4 overexpression in C2C12 cells rendered them more sults in a higher level of ROS in these cells. We also demonstrated sensitive to paraquat, a pro-oxidant compound [27]. However, the increased production of ROS in cells overexpressing DUX4 has that DNA damage and increased production of ROS were closely never been demonstrated directly. implicated in FSHD pathophysiology and that treatment of FSHD Oxidative stress is known to induce toxic effects through pro- myoblasts with an antioxidant during myogenic differentiation duction of peroxides and free radicals that damage proteins, lipids, significantly improved the phenotype of resulting FSHD myotubes. Fig. 1. Supervised clustering of gene expression profiles of in vitro cultured primary myoblasts (A) isolated from five healthy individuals (Norm-2,3,4,6) and six FSHD patients (FSHD-A,B,C,D,E,F) and myotubes derived from these myoblasts (B). 246 P. Dmitriev et al. / Free Radical Biology and Medicine 99 (2016) 244–258 2. Results category, Cell motility and migration, while no significant functional category could be attributed to the genes downregulated in FSHD 2.1. Transcriptome profiling of FSHD myoblasts myotubes (Table 3 and Supplementary Table S5). Several transcriptome analyses of FSHD muscles have been 2.2. DNA damage in FSHD myoblasts carried out so far, but many of these studies were done either on biopsies or on non-purified myoblasts containing contaminating In order to substantiate the transcriptomic data, we tested the fibroblasts and adipocytes [7,10,12]. To gain insight into mechan- phosphorylation of histone H2A variant H2AX at Ser-139 (γH2AX), isms of FSHD, we have analyzed transcriptional profiles in purified a marker for DNA damage that correlated with the presence of þ CD56 myoblasts and myotubes derived from either normal DNA double-stranded breaks (DSB). Using immunoblotting we subjects or FSHD patients as described elsewhere [19]. Agilent found that the overall level of γH2AX in the protein extracts of Human Genome 8 Â 44K microarrays were used to identify dif- FSHD myoblasts was significantly higher as compared to normal ferentially expressed genes as described in Materials and Methods. myoblasts (Fig. 2A and B). Immunofluorescent microscopy re- Microarray analysis of FSHD myoblasts revealed 312 up- and 88 vealed a higher percentage of nuclei with γH2AX foci in FSHD downregulated protein-coding genes (Fig. 1A), while only 71 and myoblasts than in normal cells (Fig. 2C and D). The observation 38 protein-coding genes were respectively up- and downregulated that a large number of DNA Damage Response (DDR)-related genes in FSHD myotubes (Fig. 1B) as compared to myoblasts and myo- were upregulated in FSHD myoblasts and were accompanied by a tubes isolated from healthy subjects (Table 1, Supplementary significantly higher level of γH2AX foci as well as a higher level of Tables S1, S3–6). the overall γH2AX prompted us to test the integrity of DNA in The functional annotation of 312 genes upregulated in FSHD these cells. We quantified the number of apurinic/apyrimidinic myoblasts resulted in 8 superclusters, each integrating several si- (AP)-sites using ELISA. Single-stranded breaks (SSB) and DSB milar Gene Ontology (GO) functional categories (Supplementary breaks were quantified using single-cell gel electrophoresis (comet Table S2); the three