Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.
Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene (Fbxo30) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.
Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans.
Pancreatic cancer is an aggressive malignancy resistant to most conventional and experimental therapies, including conditionally replicative adenoviruses (CRAds). The incorporation of immunostimulatory genes such as interleukin-12 (IL-12) in these viruses may overcome some of their limitations, but evaluation of such vectors requires suitable preclinical models. We describe a CRAd in which replication is dependent on hypoxia-inducible factor (HIF) activity and alterations of the pRB pathway in cancer cells. Transgenes (luciferase or IL-12) were incorporated into E3 region of the virus using a selective 6.7K/gp19K deletion. A novel permissive model of pancreatic cancer developed in immunocompetent Syrian hamsters was used for in vivo analysis. We show that, in contrast with nonreplicating adenoviruses (NR-Ad), active viral production and enhanced transgene expression took place in vivo. A single intratumor inoculation of the CRAd expressing IL-12 (Ad-DHscIL12) achieved a potent antitumor effect, whereas higher doses of replication-competent adenoviruses carrying luciferase did not. Compared to a standard NR-Ad expressing IL-12, Ad-DHscIL12 was less toxic in hamsters, with more selective tumor expression and shorter systemic exposure to the cytokine. We conclude that the expression of IL-12 in the context of a hypoxia-inducible oncolytic adenovirus is effective against pancreatic cancer in a relevant animal model.
Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
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