Search

Background: Facioscapulohumeral muscular dystrophy (FSHD) is associated with DNA hypomethylation at the 4q35 D4Z4 repeat array. Both the causal gene DUX4 and its homolog DUX4c are induced. DUX4c is immunodetected in every myonucleus of proliferative cells, while DUX4 is present in only 1/1000 of myonuclei where it initiates a gene deregulation cascade. FSHD primary myoblasts differentiate into either atrophic or disorganized myotubes. DUX4 expression induces atrophic myotubes and associated FSHD markers. Although DUX4 silencing normalizes the FSHD atrophic myotube phenotype, this is not the case for the disorganized phenotype. DUX4c overexpression increases the proliferation rate of human TE671 rhabdomyosarcoma cells and inhibits their differentiation, suggesting a normal role during muscle differentiation. Methods: By gain- and loss-of-function experiments in primary human muscle cells, we studied the DUX4c impact on proliferation, differentiation, myotube morphology, and FSHD markers. Results: In primary myoblasts, DUX4c overexpression increased the staining intensity of KI67 (a proliferation marker) in adjacent cells and delayed differentiation. In differentiating cells, DUX4c overexpression led to the expression of some FSHD markers including β-catenin and to the formation of disorganized myotubes presenting large clusters of nuclei and cytoskeletal defects. These were more severe when DUX4c was expressed before the cytoskeleton reorganized and myofibrils assembled. In addition, endogenous DUX4c was detected at a higher level in FSHD myotubes presenting abnormal clusters of nuclei and cytoskeletal disorganization. We found that the disorganized FSHD myotube phenotype could be rescued by silencing of DUX4c, not DUX4. Conclusion: Excess DUX4c could disturb cytoskeletal organization and nuclear distribution in FSHD myotubes. We suggest that DUX4c up-regulation could contribute to DUX4 toxicity in the muscle fibers by favoring the clustering of myonuclei and therefore facilitating DUX4 diffusion among them. Defining DUX4c functions in the healthy skeletal muscle should help to design new targeted FSHD therapy by DUX4 or DUX4c inhibition without suppressing DUX4c normal function. [ABSTRACT FROM AUTHOR]

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 m RNA 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 m RNAs 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. [ABSTRACT FROM AUTHOR]