title: Maximal activity of an erythroid-specific enhancer requires the presence of specific protein binding sites in linked promoters.
abstract: 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 hypersensitivity site 2 (HS2) of the human beta-globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing 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 compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2(M)). In fetal/erythroid K562 cells, enhancement of a linked gamma-promoter was significantly greater with wild-type HS2 than with HS2(M). In contrast, the increase in beta-promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site 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 erythroid MEL cells, enhancement of a linked beta-promoter was significantly 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-E2 motif of HS2 act in synergy.
authors.affiliation: St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Amrolia
authors.forename: W
authors.initials: W
authors.lastname: Gabbard
authors.forename: J M
authors.initials: JM
authors.lastname: Cunningham
authors.forename: S M
authors.initials: SM
authors.lastname: Jane
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D005914:Globins / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D005914:Globins