GGAA binding proteins regulate βc chain promoter activity. (A) Cotransfection of Ets family members cEts-1, PU.1, and GABP enhances promoter activity in fibroblasts. COS-1 cells were transfected with 2 μg of promoter and 6 μg of Ets expression vector or pSG5 (negative control). Promoter activity of the constructs −2100/+600 and −226/+33 is enhanced 8 to 10 times, whereas construct −103/+33 is activated only 3 times, probably due to the high basal activity of −103/+33 in COS cells. (□) pSG5; (▨) cEts; (▪) PU.1; (▧) GABP+β. (B) Ets-binding sites −65 and −45 are crucial for promoter activity. Both binding sites were mutated in the −103/+33 construct and tested for promoter activity. Both single mutations and the double mutation show a clear decrease in promoter function in both U937 and HL-60 cells. Values are the mean of at least four independent experiments; the error bars indicate the standard deviation. LacZ determination was used to correct for transfection efficiency. (C) Point mutations in Ets-binding sites −65 and −45 prevent binding of several protein complexes. The −103/+33 fragments were used in a band shift assay, using 10 μg of nuclear extract HL-60 cells. When site −65 is mutated, complexes C2 and C3 are no longer observed (lanes 2 and 4), whereas binding of C1 is also decreased. Mutation of site −45 prevents complex C4 from binding (lanes 3 and 4). Similar results were obtained when using U937 cells (data not shown).