Fig. 4. Requirement of an Mef2-binding site for activity in heart and smooth muscle of the distal myocardin enhancer. (A) Binding of Mef2c to the Mef2-binding site in the minimal enhancer element MyE8. A ³²P-labeled oligonucleotide containing the conserved Mef2 site and Mef2c translated in reticulocyte lysate was used for electrophoretic mobility shift assays. DNA binding was seen only in reactions containing lysates with Mef2c. The DNA-Mef2c complex was supershifted using a Mef2c-specific antibody and unlabeled wild-type (WT) oligonucleotide efficiently competed for DNA binding, whereas unlabeled mutant (Mut) oligonucleotide did not. (B) Mutation of the Mef2-binding site in MyE4 (3 kb) enhancer completely abolishes transgenic expression at E12.5 in heart and smooth muscle. (C) Myocardin activates its own enhancer via Mef2. COS cells (24-well plates, 5x10⁴ cells/well) were transfected with 100 ng of the indicated MyE8-luciferase reporters (wild-type and Mef2 mutant), 50 ng expression vectors encoding Mef2c and myocardin proteins and 30 ng of pCMV-lacZ. Activation of the MyE8 reporter by Mef2c was stimulated by the addition of cardiac isoform of myocardin and required the Mef2-binding site. The empty expression vector (pcDNA3.1) had no effect on the wild-type and mutated MyE8-luciferase reporter (not shown); therefore, the results are expressed as fold activation over empty pcDNA.