4 P-LIKE ELEMENTS ARE REQUIRED FOR OPTIMAL TRANSCRIPTION OF THE MOUSEIL-4 GENE - INVOLVEMENT OF A DISTINCT SET OF NUCLEAR FACTOR OF ACTIVATED T-CELLS AND ACTIVATOR PROTEIN-1 FAMILY PROTEINS

We previously identified the P sequence as a critical regulatory eleme nt of the human IL-4 promoter. In the mouse IL-4 promoter, there are f ive elements homologous to the human P sequence designated conserved l ymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native prom oter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-repo rter constructs carrying mutated P-like elements showed that four P-li ke elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter, EMSA showed that both constitutive a nd inducible complexes bound to CLE0, P, P2 and P4, whereas only a con stitutive complex bound to P3, In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclea r factor of activated T cells (NFAT) family proteins as major componen ts, Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4, NFAT/AP-1 complex formed only with P and P2, Cross-competit ion assays among the P-like elements revealed element-specific and com mon complexes, Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of o ther elements did not, It would thus appear that components of each P- like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.