The function of the
zeta-globin promoter was studied using a series of
zeta-globin promoter deletion constructs to drive
luciferase expression in transiently transfected human
erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the
beta-globin locus control region and the
alpha-globin HS-40 enhancer. When transfected into K562 cells, which express
zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-
luciferase constructs and the alpha-
luciferase constructs when no enhancers or the
alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express
zeta-globin, the
zeta-globin promoters were at best 20% as active as the
alpha-globin promoters. When sequences from -417 to -207 5' to the
zeta-globin mRNA cap site were deleted, up to 95% of the
zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-
luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting
element, located between -417 and -207 bp 5' to the
zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This
element requires GATA-1 and additional unknown factors for maximal activity.