Analysis of the AP-1 sites in the IL-2 promoter.
We have investigated the role of the two AP-1 sites, located at approximately -150 and -180 bp relative to the transcription start site, in induction of the IL-2 promoter through the TCR/CD3 complex. We show that only the proximal (-150 bp) AP-1 site is functional in vitro, as judged by its ability to bind nuclear proteins from T cells stimulated with Ag or anti-CD3 epsilon. The inducible nuclear proteins binding to this site have the characteristics of AP-1, as judged by their kinetics of induction, the ability to compete and be competed efficiently by a metallothionein AP-1 site oligonucleotide, and their reaction with antibodies to Fos and Jun proteins. Mutations in the proximal AP-1 site greatly diminish or abrogate induction of the IL-2 promoter, indicating that the site is also functional in vivo. Although the distal (-180 bp) AP-1 site is incapable of direct binding to nuclear proteins from activated T cells, a mutation in this site diminishes IL-2 promoter induction, suggesting that this site may also be functional in vivo. Cotransfection of a 5' IL-2-chloramphenicol acetyltransferase plasmid with c-Fos and/or c-Jun enhances the induction of IL-2-chloramphenicol acetyltransferase activity, confirming that the IL-2 promoter contains a functional AP-1 site. Both AP-1 sites may be targets for c-Fos action, as inferred from the results of experiments in which c-Fos was cotransfected with internal deletion mutants of the IL-2 promoter lacking either AP-1 site. Northern analysis indicates that mRNAs for at least six members of the Fos/Jun family (c-fos, fosB, fra-1, c-jun, junB, and junD) are expressed in activated Ar-5 cells; thus the AP-1 sites of the IL-2 promoter may bind different dimeric Fos/Jun complexes at different times after T cell activation, perhaps mediating both positive and negative regulation of the IL-2 promoter.
Jain
J
J
Division of Tumour Virology, Dana-Farber Cancer Institute, Boston, MA 02115.
Valge-Archer
V E
VE
Rao
A
A
D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D016762:Genes, fos; D016758:Genes, jun; D007376:Interleukin-2 / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D007376:Interleukin-2; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger