title: Conserved high-affinity NF-kappa B binding site in the interferon regulatory factor-1 promoter is not occupied by NF-kappa B in vivo and is transcriptionally inactive.
abstract: The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast 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 not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.
authors.affiliation: Institut für Biochemie der Ludwig-Maximilians-Universität, Munich, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Rein
authors.forename: R
authors.initials: R
authors.lastname: Schreck
authors.forename: W
authors.initials: W
authors.lastname: Willenbrink
authors.forename: W J
authors.initials: WJ
authors.lastname: Neubert
authors.forename: H
authors.initials: H
authors.lastname: Zorbas
authors.forename: P A
authors.initials: PA
authors.lastname: Bäuerle
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D050856:Interferon Regulatory Factor-1; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D015335:Molecular Probes; D016328:NF-kappa B; D010750:Phosphoproteins; D014157:Transcription Factors; D013755:Tetradecanoylphorbol Acetate