Biphasic myopathic phenotype of mouse DUX, an ORF conserved FSHD-related repeats

Brunel University, United Kingdom
PLoS ONE (Impact Factor: 3.23). 09/2009; 4(9):e7003. DOI: 10.1371/journal.pone.0007003
Source: PubMed


Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX), embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models highlight the myopathic nature of sequences within the FSHD-related repeat array.

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Available from: Zhaohui Xu, Oct 06, 2015
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    • "The D4Z4 transcript encodes the DUX4 protein, a double-homeodomain presumptive transcription factor [12] which kills myoblasts and other cells when expressed at high levels [13,14]. DUX4 and homologues of DUX4 also have effects when expressed in myoblasts at low, non-toxic levels: MyoD expression is lost and cells lose the ability to differentiate into myotubes [13,15,16]. Because of these pathological activities, DUX4 is the key molecular target in the development of a pharmacological treatment for FSHD. "
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    ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein. The DUX4 protein is therefore the primary molecular target for therapeutic intervention. We have developed a high-throughput screen based on the toxicity of DUX4 when overexpressed in C2C12 myoblasts, and identified inhibitors of DUX4-induced toxicity from within a diverse set of 44,000 small, drug-like molecules. A total of 1,280 hits were then subjected to secondary screening for activity against DUX4 expressed by 3T3 fibroblasts, for absence of activity against the tet-on system used to conditionally express DUX4, and for potential effects on cellular proliferation rate. This allowed us to define a panel of 52 compounds to use as probes to identify essential pathways of DUX4 activity. We tested these compounds for their ability to protect wild-type cells from other types of cell death-inducing insults. Remarkably, we found that 60% of the DUX4 toxicity inhibitors that we identified also protected cells from tert-butyl hydrogen peroxide, an oxidative stress-inducing compound. Compounds did not protect against death induced by caspase activation, DNA damage, protein misfolding, or ER stress. Encouragingly, many of these compounds are also protective against DUX4 expression in human cells. These data suggest that oxidative stress is a dominant pathway through which DUX4-provoked toxicity is mediated in this system, and we speculate that enhancing the oxidative stress response pathway might be clinically beneficial in FSHD.
    Skeletal Muscle 02/2014; 4(1):4. DOI:10.1186/2044-5040-4-4
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    • "In the same study, it was proposed that the DUX4 gene in the most distal D4Z4 element was the only copy that could be transcribed into a polyadenylated mRNA. The polyadenylation will stabilize the transcript to produce a functional DUX4 protein which has been shown to be toxic to muscle cells (Kowaljow et al., 2007; Bosnakovski et al., 2009). This hypothesis was supported by the recent genetic study which showed that stable DUX4 mRNA is essential to FSHD pathogenesis (Lemmers et al., 2010). "
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    ABSTRACT: Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.
    Biology Open 07/2012; 1(7):629-639. DOI:10.1242/bio.20121305 · 2.42 Impact Factor
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    • "When human DUX homeodomains (DUX4, DUXA and DUXB) are compared with sDUX and human PRD class homeodomains, sDUX and all DUX proteins cluster together, further supporting their close relationship (Figure 8). Pax3 and Pax7 have been proposed to represent potential competitors for the same DNA binding sites as DUX, a hypothesis based on their similar homeodomain sequences[20,27]. However, as our tree demonstrates, Pax3/7 are not the only candidates that could be considered in this context, and indeed there are other PRD class homeodomains (e.g. "
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    ABSTRACT: DUX4 is causally involved in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). It has previously been proposed to have arisen by retrotransposition of DUXC, one of four known intron-containing DUX genes. Here, we investigate the evolutionary history of this multi-member double-homeobox gene family in eutherian mammals. Our analysis of the DUX family shows the distribution of different homologues across the mammalian class, including events of secondary loss. Phylogenetic comparison, analysis of gene structures and information from syntenic regions confirm the paralogous relationship of Duxbl and DUXB and characterize their relationship with DUXA and DUXC. We further identify Duxbl pseudogene orthologues in primates. A survey of non-mammalian genomes identified a single-homeobox gene (sDUX) as a likely representative homologue of the mammalian DUX ancestor before the homeobox duplication. Based on the gene structure maps, we suggest a possible mechanism for the generation of the DUX gene structure. Our study underlines how secondary loss of orthologues can obscure the true ancestry of individual gene family members. Their relationships should be considered when interpreting the relevance of functional data from DUX4 homologues such as Dux and Duxbl to FSHD.
    BMC Evolutionary Biology 11/2010; 10(1):364. DOI:10.1186/1471-2148-10-364 · 3.37 Impact Factor
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