Cellular toxicity of DUX4, a protein encoded at the fascioscapulohumeral muscular dystrophy locus FSHD-1A, depends on nuclear localization

ARIAS CI; CONDE CB; ROSA AL

Iguassu Falls, Brasil

Congreso; 10th International Congress of the World Muscle Society; 2005

World Muscle Society

Fascioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited myopathy after Duchenne muscular dystrophy and myotonic dystrophy. A 3.3-kb tandem repeat on chromosome 4q35 (D4Z4) is contracted in patients with FSHD. D4Z4 contains an ORF encoding a putative double homeobox protein called DUX4. A nearly identical ORF (DUX4c) is located ~40 kb centromeric to D4Z4. We prepared anti-DUX4 antibodies that specifically recognize an endogenously expressed protein in cultured human adult and fetal rhabdomyosarcoma cells. This protein is not DUX4c as indicated by lack of immunostaining using a specific anti-DUX4c antibody (provided by A. Belayew, Belgium). Endogenously expressed DUX4, like DUX4 expressed in cell transfection experiments, is a ~46 kDa protein that localizes to the cell nuclei. Transient expression of DUX4 in cultured cells leads to cell death, and no stable transfectants constitutively expressing DUX4 could be isolated. The functionality of two putative nuclear localization signals (NLS-1 and NLS-2) present in DUX4 was studied using PCR-mediated mutagenesis. Arginine residues at NLS-1 and NLS-2 were independently replaced by threonines and the subcellular distribution of the various DUX4 mutants (DUX4RnT) was analyzed in transfected cells. DUX4RnT mutants that do not localize to the nuclei highlight arginine residues that mediate subcellular trafficking of DUX4. The typical DUX4-mediated cell death phenotype observed in transfection experiments was abolished in these DUX4RnT mutants. We propose that NLS-1 and NLS-2 are functional DUX4 NLSs and that nuclear entrance is required for DUX4-mediated cell death.  Supported in part by the FSH Society (USA), FONCYT (Argentina) and AFM (France).