title: 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.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle 98195, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Sabath
authors.forename: K M
authors.initials: KM
authors.lastname: Koehler
authors.forename: W Q
authors.initials: WQ
authors.lastname: Yang
authors.forename: K
authors.initials: K
authors.lastname: Patton
authors.forename: G
authors.initials: G
authors.lastname: Stamatoyannopoulos
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005813:Genes, Synthetic; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000473:pathology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005914:Globins; D008156:Luciferases