CELLULAR TOXICITY OF DUX4 DEPENDS ON NUCLEAR LOCALIZATION

ARIAS CI; CONDE CB; ROSA AL

Pinamar, BsAs

Congreso; 41th Annual Meeting SAIB; 2005

SAIB, PABMB y SAN

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. We prepared anti-DUX4 antibodies that specifically recognize an endogenously expressed protein in the nuclei of the cultured human-muscle derived cells. 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. Amino acid residues at NLS-1 and NLS-2 were independently replaced by threonines and the subcellular distribution of the various DUX4 mutants was analyzed in transfected cells. DUX4 mutants that do not localize to the nuclei highlight residues that mediate the subcellular trafficking of DUX4. The typical DUX4-mediated cell death phenotype observed in transfection experiments was abolished in some DUX4 mutants. We propose that NLS-1 and NLS-2 are functional NLSs of DUX4 and that the nuclear entrance is required for  DUX4-mediated cell death.  Supported  by FONCYT (Argentina,) FSH Society (USA) and AFM (France).