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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.