Facioscapulohumeral muscular dystrophy (FSHD) is linked to deletions in 4q35 within the D4Z4 repeat array in which we identified the double homeobox 4 (DUX4) gene. We found stable DUX4 mRNAs only derived from the most distal D4Z4 unit and unexpectedly extended to the flanking pLAM region that provided an intron and a polyadenylation signal. DUX4 encodes a transcription factor expressed in FSHD but not control primary myoblasts or muscle biopsies. The DUX4 protein initiates a large transcription deregulation cascade leading to muscle atrophy and oxidative stress, which are FSHD key features.
We now show that transfection of myoblasts with a DUX4 expression vector leads to atrophic myotube formation associated with the induction of E3 ubiquitin ligases (MuRF1 and Atrogin1/MAFbx) typical of muscle atrophy. DUX4 induces expression of downstream targets deregulated in FSHD such as mu-crystallin and TP53. We developed specific siRNAs and antisense oligonucleotides (AOs) targeting the DUX4 mRNA. Addition of these antisense agents to primary FSHD myoblast cultures suppressed DUX4 protein expression and affected expression of the above-mentioned markers.
These results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.
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"This main form of FSHD, referred to as FSHD1, accounts for approximately 95% of cases [Richards et al., 2012]. Such a heterozygous D4Z4-array reduction is associated with chromatin relaxation on specific permissive haplotypes [Lemmers et al., 2002], which might result in stabilization of the DUX4 transcript , encoded by the retrogene located in D4Z4, and expression of this transcription factor [Gabriëls et al., 1999; Dixit et al., 2007; Lemmers et al., 2010; Spurlock et al., 2010; Vanderplanck et al., 2011]. A form of FSHD not linked to D4Z4 contraction accounts for 5% of patients (contraction-independent FSHD) [van Overveld et al., 2003]. "
[Show abstract][Hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is linked to copy number reduction (n<10) of the 4q D4Z4 subtelomeric array, in association with DUX4-permissive haplotypes. This main form is indicated as FSHD1. FSHD-like phenotypes may also appear in the absence of D4Z4 copy number reduction. Variants of the SMCHD1 gene have been reported to associate with D4Z4 hypomethylation in DUX4-compatible haplotypes, thus defining FSHD2. Recently, mice carrying a muscle-specific knock-out of the protocadherin gene Fat1 or its constitutive hypomorphic allele were shown to develop muscular and non-muscular defects mimicking human FSHD. Here we report FAT1 variants in a group of patients presenting with neuromuscular symptoms reminiscent of FSHD. The patients do not carry D4Z4 copy number reduction, 4q hypomethylation or SMCHD1 variants. However, abnormal splicing of the FAT1 transcript is predicted for all identified variants. To determine their pathogenicity, we elaborated a minigene approach coupled to an antisense oligonucleotide (AON) assay. In vitro, four out of five selected variants induced partial or complete alteration of splicing by creating new splice sites or modifying splicing regulators. AONs confirmed these effects. Altered transcripts may affect FAT1 protein interactions or stability. Altogether, our data suggest that defective FAT1 is associated with an FSHD-like phenotype.This article is protected by copyright. All rights reserved
"Both of these pathways are involved in skeletal muscle differentiation, sarcomeric protein degradation and apoptosis [10,11]. DUX4 may also activate ubiquitin-mediated protein degradation pathways including E3 ubiquitin ligases such as Atrogin-1 and MuRF1 [12,13]. These muscle specific ligase enzymes specifically target sarcomeric proteins and have been implicated in the atrophic phenotype in FSHD myotubes [13,14]. "
[Show abstract][Hide abstract] ABSTRACT: Although muscle weakness is a hallmark of facioscapulohumeral muscular dystrophy (FSHD), the molecular mechanisms that lead to weakness in FSHD remain largely unknown. Recent studies suggest aberrant expression of genes involved in skeletal muscle development and sarcomere contractility, and activation of pathways involved in sarcomeric protein degradation. This study will investigate the contribution of sarcomeric protein dysfunction to the pathogenesis of muscle weakness in FSHD.
Evaluation of sarcomeric function using skinned single muscle fiber contractile studies and protein analysis in muscle biopsies (quadriceps femoris and tibialis anterior) from patients with FSHD and age- and gender-matched healthy controls. Patients with other forms of muscular dystrophy and inflammatory myopathy will be included as disease controls to assess whether results are due to changes specific for FSHD, or a consequence of muscle disease in general. A total of 56 participants will be included. Extensive clinical parameters will be measured using MRI, quantitative muscle studies and physical activity assessments.
This study is the first to extensively investigate muscle fiber physiology in FSHD following an earlier pilot study suggesting sarcomeric dysfunction in FSHD. The results obtained in this study will increase the understanding of the pathophysiology of muscle weakness in FSHD, and possibly identify novel targets for therapeutic intervention.
"The DUX4 gene is turned off when cultured pluripotent cells are differentiating . Transgene expression of DUX4 in various cultured transfected cells leads to apoptosis  and its expression in myoblasts disrupts the normal myogenic regulatory pathway , alters normal myotube morphology ,  and increases stress susceptibility . Expression of DUX4 in mice muscles causes a TP53-dependent myopathy, which is dependent on the integrity of its homeodomains . "
[Show abstract][Hide abstract] ABSTRACT: DUX4 (Double Homeobox Protein 4) is a nuclear transcription factor encoded at each D4Z4 unit of a tandem-repeat array at human chromosome 4q35. DUX4 constitutes a major candidate pathogenic protein for facioscapulohumeral muscular dystrophy (FSHD), the third most common form of inherited myopathy. A low-level expression of DUX4 compromises cell differentiation in myoblasts and its overexpression induces apoptosis in cultured cells and living organisms. In this work we explore potential molecular determinants of DUX4 mediating nuclear import and cell toxicity. Deletion of the hypothetical monopartite nuclear localization sequences RRRR(23), RRKR(98) and RRAR(148) (i.e. NLS1, NLS2 and NLS3, respectively) only partially delocalizes DUX4 from the cell nuclei. Nuclear entrance guided by NLS1, NLS2 and NLS3 does not follow the classical nuclear import pathway mediated by α/β importins. NLS and homeodomain mutants from DUX4 are dramatically less cell-toxic than the wild type molecule, independently of their subcellular localization. A triple ΔNLS1-2-3 deletion mutant is still partially localized in the nuclei, indicating that additional sequences in DUX4 contribute to nuclear import. Deletion of ≥111 amino acids from the C-terminal of DUX4, on a ΔNLS1-2-3 background, almost completely re-localizes DUX4 to the cytoplasm, indicating that the C-ter tail contributes to subcellular trafficking of DUX4. Also, C-terminal deletion mutants of DUX4 on a NLS wild type background are less toxic than wild type DUX4. Results reported here indicate that DUX4 possesses redundant mechanisms to assure nuclear entrance and that its various transcription-factor associated domains play an essential role in cell toxicity.