Characterization of an E-box-dependent cis element in the smooth muscle alpha-actin promoter

Identification of the regulators of smooth muscle specific gene expression is critical for understanding smooth muscle cell (SMC) differentiation and- the alterations in SMC phenotype seen in vascular diseases. Previous studie s have identified that a 2-bp mutation in a conserved cia-acting element (T GTTTATC) in the promoter of the chicken smooth muscle (SM) alpha-actin gene abolished nuclear factor binding and decreased transcriptional activity of a 271-bp SM alpha-actin promoter fragment when transfected into rat aortic SMC. However, the promoter region containing this conserved sequence has n egative cis regulatory activity when studied in homologous systems. The goa l of the present studies was to further characterize the transcriptional ac tivity of the rat SM alpha-actin promoter region between -224 and -236 that is conserved across mammals. DNAse I analysis and electrophoretic mobility shift assays demonstrated that SMC nuclear proteins bound an extended sequ ence (TGTTTATCCCCATAA). Transient transfection experiments of wild-type and mutant rat SM alpha-actin promoter-luciferase constructs into rat aortic S MC revealed that promoter activity was enhanced by mutations of specific nu cleotides in the TGTTTATCCCCA region. Interestingly, the TGTTTATCCCCA eleme nt in the rat SM alpha-actin promoter is centered between 2 canonical E-box es. Mutations of the flanking E-boxes abolished the enhancement in promoter activity seen with mutation of the TGTTTATCCCCA element alone, Thus studie s provide evidence for a regulatory cassette in the rat SM alpha-actin prom oter that regulates gene expression via combinatorial interactions between 2 E-boxes and a newly described TGTTTATCCCCA element.