Cellular Toxicity of DUX4 Depends on Nuclear Localization

CONDE CB; ARIAS CI; ROSA AL

Salt Lake City, USA

Congreso; Facioscapulohumeral Muscular Dystrophy International Research Consortium Research Workshop Meeting 2005; 2005

Facioscapulohumeral Muscular Dystrophy Society

DUX4 is a putative double-homeobox protein encoded at D4Z4, the 3.3-kb tandem repeat contracted on 4q35 in patients with FSHD. A nearly identical ORF (DUX4c) is located ~40 kb centromeric to D4Z4. Anti-DUX4 antibodies prepared in our laboratory specifically recognize a DUX4-related endogenously-expressed protein in cultured human adult and fetal rhabdomyosarcoma cells. The protein is not DUX4c as indicated by lack of immunostaining using a specific anti-DUX4c antibody (provided by A. Belayew, Belgium). The endogenously expressed DUX4-related protein, 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. PCRmediated mutagenesis was used to study the functionality of two putative nuclear localization signals (NLS-1 and NLS-2) present in DUX4. Selected amino acid residues at NLS-1 and NLS-2 were independently replaced by threonines and the sub-cellular distribution of the various DUX4 mutants was analyzed in transfected cells. DUX4 mutants that do not localize to the nuclei highlight amino acid residues that mediate sub-cellular trafficking of this protein. The typical DUX4-mediated cell death phenotype observed in transfection experiments was abolished in some of these DUX4 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 by The FSH Society (USA), FONCYT (Argentina) and AFM (France).