Characterization of the human interleukin-5 gene promoter: involvement of octamer binding sites in the gene promoter activity.
To investigate the human interleukin (IL)-5 gene promoter, we have constructed a plasmid with the firefly luciferase reporter gene linked to human IL-5 5' flanking sequence (nucleotides -507 to +44). We have used this plasmid to transfect the mouse EL4 T cell line, which can, under certain conditions, produce IL-5 transcripts. Phorbol 12-myristate 13-acetate, A23187 and N6, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate co-stimulation of EL4 cells transfected with the human IL-5/luciferase reporter gene construct resulted in maximal induction of the luciferase gene. Deletion analysis of the IL-5 promoter revealed the presence of negative regulatory elements between nucleotides -404 and -312 and two regions, located between nucleotides -312 and -227 and between nucleotides -80 and -35, that are involved in the positive regulation of the IL-5 promoter. Using electrophoretic mobility shift assays, we show that the positive element located between nucleotides -312 and -227 involves the binding of factors antigenically related to Oct1, Oct2A and Oct2B, to a perfect octamer motif located at position -244/-237. Introduction of three point mutations in the octamer motif of the IL-5/luciferase reporter gene plasmid, which results in the loss of competition for the factors binding to the IL-5 promoter sequence, reduced the production of luciferase from stimulated, transfected EL4 cells, by 90%. Octamer factors can also bind within the second positive regulatory region.
Gruart-Gouilleux
V
V
Sandoz Pharma Ltd., Basel, Switzerland.
Engels
P
P
Sullivan
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D016384:Consensus Sequence; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D017930:Genes, Reporter; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D013601:T-Lymphocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013945:Thymoma / Q000473:pathology; D013953:Thymus Neoplasms / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015848:Interleukin-5; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000001:Calcimycin; D003994:Bucladesine; D013755:Tetradecanoylphorbol Acetate