Sp1 and Sp3 physically interact and co-operate with GABP for the activation of the utrophin promoter

The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin which, in the absence of dystrophin, can functionally substitute it. Utrophin is transcribed by two independently regulated promoters about 50 kb apart. The upstream promoter is TATA-less and contains a functional GABP binding site which, in muscle, restricts the promoter activity to post-synaptic nuclei. Transient transfections analysis of mutant promoters in rhabdomyosarcoma cells showed that the upstream promoter contains three functional GC elements that are recognised by Spl and Sp3 factors in vitro. Co-transfections of the promoter with Spl, Sp3 and GABP factors in Drosophila SL2 Schneider cells, which lack of endogenous Sp factors, demonstrated that both Spl and Sp3 are positive regulators of the utrophin promoter and that they activate transcription synergistically with GABP. Consistent with this result, we observed physical interaction of both Sp factors with the GABP alpha subunit in vitro. Functional domain interaction analysis of Spl and Sp3 revealed that both factors interact with GABP alpha through their DNA binding zinc finger domain. The modulation and correct interaction between Spl, Sp3 and GABP in muscle cells may be critical for the regulation of the utrophin promoter, and provide new targets for therapies of Duchenne muscular dystrophy. (C) 2001 Academic Press.