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Identification of a major positive regulatory element located 5' to the
human zeta-globin gene
DE Sabath, KM Koehler, WQ Yang, K Patton and G Stamatoyannopoulos
Department of Laboratory Medicine, University of Washington, Seattle 98195,
USA.
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.
Volume 85,
Issue 9,
pp. 2587-2597,
05/01/1995
Copyright © 1995 by The American Society of Hematology
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