The PU.I site is essential for the activity of the intronic enhancer. (A) Schematic representation of the mutant reporter constructs. Only the sequence around the tandem PU.I site is shown. The PU.I core binding sites are underlined. Mutations made are indicated in bold type. (B) EDN-CAT constructs containing the full promoter and intron with different mutations in the tandem PU.I site were transfected into HL60 and HL60 7.7 cells and treated with BA as described in Fig 2. Two days posttransfection, cells were harvested and assayed for CAT activity. Bars indicate the mean percentage of acetylation of at least four independent experiments. The standard deviation is indicated by error bars. The double mutant in the tandem PU.I site strongly reduces the activity of the EDN-CAT reporter construct.