MAXIMAL ACTIVITY OF AN ERYTHROID-SPECIFIC ENHANCER REQUIRES THE PRESENCE OF SPECIFIC PROTEIN-BINDING SITES IN LINKED PROMOTERS

High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensit ivity site 2 (HS2) of the human beta-globin locus control region and t he globin gene promoters as a model to elucidate the mechanisms govern ing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroid-specific factors. We have compa red the enhancer activity of this core element with a synthetic enhanc er lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2( M)). In fetal/erythroid K562 cells, enhancement of a linked gamma-prom oter was significantly greater with wild-type HS2 than with HS2(M). In contrast, the increase in beta-promoter activity in these cells was e quivalent with either enhancer fragment. Truncation of the binding sit e for the fetal/erythroid-specific stage selector protein in the gamma -promoter abolished the additional enhancer activity of HS2. Similarly , insertion of the stage selector protein site into the beta-promoter boosted enhancer activity observed with HS2 but not HS2(M). In adult e rythroid MEL cells, enhancement of a linked beta-promoter was signific antly greater with HS2 than with HS2(M). This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the beta-promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/NF-EB motif of HS2 act in synergy.