CONSERVED HIGH-AFFINITY NF-KAPPA-B BINDING-SITE IN THE INTERFERON REGULATORY FACTOR-I PROMOTER IS NOT OCCUPIED BY NF-KAPPA-B IN-VIVO AND ISTRANSCRIPTIONALLY INACTIVE

The promoter of the interferon regulatory factor-1 (IRF-1) gene contai ns at position -47 to -38 an evolutionary conserved binding sequence f or the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enh ancer. In this study, we show by in vitro assays using purified NF-kap pa B that the kappa B motif in the IRF-1 promoter binds the factor spe cifically and with high affinity comparable to various other cis-actin g kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol a cetyl transferase (CAT) reported gene in response to stimulation of L9 29 fibroblasts with various NF-kappa B inducers, such as tumor necrosi s factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). M utation of the binding site completely abolished transcriptional induc ibility of the heterologous promoter. Surprisingly, the same IRF-1 kap pa B motif in context of the homologous IRF-1 promoter was transcripti onally inactive in CAT assays. The very weak induction of the IRF-1 pr omoter in response to TNF treatment or infection of fibroblasts with N ewcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. An alysis of the occupational state of the chromosomal IRF-1 kappa B Site by in vivo footprinting revealed that no footprint was induced over t he kappa B motif in the IRF-1 promoter after PMA treatment of L929 fib roblast cells, despite the simultaneous induction of IRF-1 mRNA and NF -kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may n ot participate in transcriptional regulation tinder conditions activat ing NF-kappa B DNA binding and gene expression. (C) 1995 Wiley-Liss, I nc.