Résumé :
|
Communication n° 452 The FSHD candidate gene (DUX4) we propose maps within each of the D4Z4 units repeated in tandem at 4q35. An homologous DUX4c gene is located in a single truncated D4Z4 element 42 kb centromeric of the D4Z4 array. The DUX4 and DUX4c proteins are 424- and 374-residue long, respectively, and present identical homeodomain region (342 residues). Both proteins were detected by Western blot in FSHD but not control myoblasts. We used the yeast two hybrid system to identify DUX4c protein partners. DUX4 could not be used as a bait because of its very strong transcriptional activity. By screening a cDNA library (2.8 x 105 clones) derived from human skeletal muscle, 187 positive clones were isolated. Three clones encoded importin/karyopherin 13 that mediates nuclear import of homeoproteins. In addition, two classes of putative DUX partners were identified: (i) cytoskeleton proteins like desmin and alpha actinin 3, (ii) zinc finger proteins with LIM or MYND type domains. Different proteins bearing these domains are implicated in muscle development and differentiation. DUX4 and DUX4c exhibit a PXLXP motif found to mediate interaction with the MYND domain (Ansieau and Leutz, 2002, J. Biol. Chem., 277, 4906-10). These putative DUX partners are being further evaluated. This cDNA library was previously screened with DUX1 (a non-pathological homologue) yielding 35/42 positive clones encoding desmin. Reconstruction experiments in yeast showed that the desmin interaction was mediated by the double homeodomain of either DUX1 or DUX4/DUX4c. Confirmation of this interaction in vitro was obtained by desmin-GST pull down and co-immunoprecipitations from extracts of cells transfected with a DUX expression vector. A DUX/desmin co-localisation was detected by immunofluorescence in these cells on narrow regions at the nuclear periphery.
|