Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In nonaffected individuals, this locus compr ises 10-100 tandem copies of members of the 3.3 kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for whi ch no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5 kb EcoRI geno mic fragment comprising the only two 3.3 kb elements left in the affected D 4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the t wo 3.3 kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box . Transient expression of a luciferase reporter gene fused to 191 bp of thi s promoter, demonstrated strong activity in transfected human rhabdomyosarc oma TE671 cells that was affected by mutations in the TACAA or GC box. In a ddition, these 3.3 kb repeats include an open reading frame (ORF) starting 149 bp downstream from the TACAA box and encoding a 391 residue protein wit h two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two S-35 CYs/ S-35 Met labeled produc ts with apparent molecular weights of 38 and 75 kDa on SDS-PAGE, correspond ing to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3 kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein. (C) 1999 Elsevier Science B.V. All rights reserved.