IDENTIFICATION OF 2 NOVEL REGULATORY ELEMENTS WITHIN THE 5'-UNTRANSLATED REGION OF THE HUMAN (A)GAMMA-GLOBIN GENE

Interaction between the stage selector element (SSE) in the proximal g amma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin pr omoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma- gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell li ne, K562, we have shown that the core enhancer of hypersensitivity sit e 2 can preferentially interact with the proximal gamma-promoter in th e absence of the SSE, completely silencing a linked beta-promoter. Mut ation of a 20-base pair sequence of the gamma-gene 5'-untranslated reg ion (UTR) led to derepression of beta-promoter activity. A marked acti vation of gamma-promoter activity was also observed with this mutation , suggesting the presence of a repressor. Fine mutagenesis dissected t hese activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15 . Electromobility shift assays utilizing this sequence demonstrated bi nding of a fetal and erythroid-specific protein. The repressor activit y of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid trans cription factor GATA-1. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcript ional and developmental regulation.