Abstract
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.
Highlights
In these experiments, when double homeobox 4 (DUX4) was found in at least one myotube nucleus, the consecutive myonuclei were often DUX4 positive. These intensity gradients suggested that the DUX4 mRNA transcribed in one myonucleus was translated in the adjacent cytoplasm domain into proteins that could be imported in several neighbouring nuclei, a diffusion mechanism previously described for other muscle proteins [25,26,27,28, 30]
Several groups besides ours have demonstrated the presence of polyadenylated DUX4-fl mRNA in Facioscapulohumeral muscular dystrophy (FSHD) muscle cells [9,10,11,12]
In myoblasts grown 4 days in a differentiation medium we detected an increase in DUX4 protein by Western blot analysis (Fig. 5C), which correlated with a higher number of DUX4-positive nuclei (1/200; Fig. 2A and B)
Summary
Facioscapulohumeral muscular dystrophy (FSHD1A: OMIM #158900) is one of the most common hereditary muscle disorders, affecting seven individuals in 100,000 (http://www.orpha.net), and is associated with contractions of the D4Z4 repeat array in the 4q35 subtelo-. The encoded protein is expressed in healthy muscle cells and induced in FSHD [13] Because it activates myoblast proliferation and inhibits their differentiation, DUX4c might be involved in muscle regeneration, and changes in its expression could contribute to the FSHD pathology [13,14,15]. Our discovery of the functional DUX4 and DUX4c genes in repeated DNA elements has contributed to the obsolescence of the ‘junk DNA’ concept [16]
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