Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

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BackgroundFacioscapulohumeral 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.Methodology/Principal FindingsWe 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.Conclusions/SignificanceThese results constitute a proof of concept for the development of therapeutic approaches for FSHD targeting DUX4 expression.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most frequent hereditary muscle disorders. It is linked to contractions of the D4Z4 repeat array in 4q35. We have characterized the double homeobox 4 (DUX4) gene in D4Z4 and its mRNA transcribed from the distal D4Z4 unit to a polyadenylation signal in the flanking pLAM region. It encodes a transcription factor expressed in FSHD but not healthy muscle cells which initiates a gene deregulation cascade causing differentiation defects, muscle atrophy and oxidative stress. PITX1 was the first identified DUX4 target and encodes a transcription factor involved in muscle atrophy. DUX4 was found expressed in only 1/1000 FSHD myoblasts. We have now shown it was induced upon differentiation and detected in about 1/200 myotube nuclei. The DUX4 and PITX1 proteins presented staining gradients in consecutive myonuclei which suggested a diffusion as known for other muscle nuclear proteins. Both protein half-lifes were regulated by the ubiquitin-proteasome pathway. In addition, we could immunodetect the DUX4 protein in FSHD muscle extracts. As a model, we propose the DUX4 gene is stochastically activated in a small number of FSHD myonuclei. The resulting mRNAs are translated in the cytoplasm around an activated nucleus and the DUX4 proteins diffuse to adjacent nuclei where they activate target genes such as PITX1. The PITX1 protein can further diffuse to additional myonuclei and expand the transcriptional deregulation cascade initiated by DUX4. Together the diffusion and the deregulation cascade would explain how a rare protein could cause the muscle defects observed in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contractions of the D4Z4 repeat array in 4q35. We have previously identified a double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription factor expressed in FSHD but not control myoblasts. DUX4 and its target genes contribute to the global dysregulation of gene expression observed in FSHD. We have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was detected in primary myoblast extracts by Western blot with a specific antiserum, and was induced upon differentiation. The protein was increased about 2-fold in FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c over-expression induced the MYF5 myogenic regulator and its DNA-binding activity. DUX4c might stabilize the MYF5 protein as we detected their interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in myoblast accumulation during mouse muscle regeneration DUX4c over-expression activated proliferation of human primary myoblasts and inhibited their differentiation. Altogether, these results suggested that DUX4c could be involved in muscle regeneration and that changes in its expression could contribute to the FSHD pathology.