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Identification of a major positive regulatory element located 5' to the human zeta-globin gene.

Abstract
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.
AuthorsD E Sabath, K M Koehler, W Q Yang, K Patton, G Stamatoyannopoulos
JournalBlood (Blood) Vol. 85 Issue 9 Pg. 2587-97 (May 01 1995) ISSN: 0006-4971 [Print] United States
PMID7727787 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • DNA-Binding Proteins
  • Erythroid-Specific DNA-Binding Factors
  • GATA1 Transcription Factor
  • GATA1 protein, human
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Globins
  • Luciferases
Topics
  • Base Sequence
  • Binding Sites
  • DNA-Binding Proteins (metabolism)
  • Erythroid Precursor Cells (metabolism)
  • Erythroid-Specific DNA-Binding Factors
  • GATA1 Transcription Factor
  • Genes, Synthetic
  • Globins (biosynthesis, genetics)
  • Humans
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive (pathology)
  • Luciferases (biosynthesis, genetics)
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins (biosynthesis)
  • Regulatory Sequences, Nucleic Acid
  • Sequence Deletion
  • Transcription Factors (metabolism)
  • Transfection
  • Tumor Cells, Cultured

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