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 cons tructed a plasmid with the firefly luciferase reporter gene linked to human IL-5 5' flanking sequence (nucleotides -507 to + 44).We have use d this plasmid to transfect the mouse EL4 T cell line, which can, unde r certain conditions, produce IL-5 transcripts. Phorbol 12-myristate 1 3-acetate, A23187 and N-6, 2'-O-dibutyryladenosine 3':5'-cyclic monoph osphate co-stimulation of EL4 cells transfected with the human IL-5/lu ciferase reporter gene construct resulted in maximal induction of the luciferase gene. Deletion analysis of the IL-5 promoter revealed the p resence of negative regulatory elements between nucleotides -404 and - 312 and two regions, located between nucleotides -312 and -227 and bet ween nucleotides -80 and -35, that are involved in the positive regula tion of the IL-5 promoter. Using electrophoretic mobility shift assays , we show that the positive element located between nucleotides -312 a nd -227 involves the binding of factors antigenically related to Oct1, Oct3A and Oct2B, to a perfect octamer motif located at position -244/ -237. Introduction of three point mutations in the octamer motif of th e IL-5/luciferase reporter gene plasmid, which results in the loss of competition for the factors binding to the IL-5 promoter sequence, red uced the production of luciferase from stimulated, transfected EL4 cel ls, by 90%. Octamer factors can also bind within the second positive r egulatory region.