CHARACTERIZATION OF CONSTITUTIVE AND INDUCIBLE TRANSCRIPTION FACTORS BINDING TO THE P2 NF-AT SITE IN THE HUMAN INTERLEUKIN-4 PROMOTER

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secret ed by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites-the P2 site-located between -173 and -150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer protein s were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze th e P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, J un, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T -cells form two P2-binding complexes-a PMA/ionomycin-inducible and a c onstitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c , Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of th e two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU dom ains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at -173 to -150 of the IL-4 promoter is a binding site fo r NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with N F-ATp/c at this site. (C) 1997 Elsevier Science B.V.