Fig. 5. Identification of Foxo proteins as mediators of myocardin expression. (A) Results from transfection assays in the 96-well plate that identified Foxo4 as an activator of the distal myocardin enhancer MyE8. In each well, we transfected the MyE8-luciferase reporter into COS cells along with pools of 100 cDNA clones from a mouse E10.5 expression library. The MyE8-luciferase plasmid was specifically activated in well A10. Sibselection from this pool identified Foxo4 as the activating cDNA. (B) Transient transfections in COS cells show that Foxo4 activates the MyE8 reporter, which contains five predicted Foxo-binding sites, with the consensus sequence AAAC/TA (see Fig. 3C). Foxo4 was less effective in activating the MyE8 reporter in which all five Foxo-binding sites were mutated. (C) DNA binding of Foxo4 to the five predicted Foxo-binding sites (see Fig. 3C) in minimal enhancer element MyE8 is shown. Five ³²P-labeled oligonucleotides containing the predicted Foxo-binding sites and Foxo4 translated in reticulocyte lysate were used in electrophoretic mobility shift assays. The strongest binding was seen for Foxo sites 3 and 4, weaker binding for sites 2 and 5, whereas Foxo4 did not bind to the first site. The ³²P-DNA-Foxo4 complexes were supershifted using a Foxo4-specific antibody and unlabeled, wild-type oligonucleotides efficiently competed for DNA binding. (D) Requirement of Foxo sites for activity of the 3 kb myocardin enhancer in vivo. F0 transgenic embryos generated with construct MyE4, (see Fig. 3A) containing mutations of the five Foxo-binding sites abolishes transgenic expression at E10.5 in heart and smooth muscle.