Differential transcriptional activation in vitro by NF-κB/Rel proteins

Abstract

Distinct NF-κB subunit combinations contribute to the specificity of NF- κB-mediated transcriptional activation and to the induction of multiple cytokine genes including interferon-β (IFN-β). To evaluate the regulatory influence of different homo- and heterodimers, NF-κB subunits were analyzed for transcriptional activity in vitro using test templates containing two types of NF-κB recognition elements (the human immunodeficiency virus type 1 enhancer and the IFN-β-positive regulatory domain II (PRDII) as well as IFN- β PRDIII-PRDI-PRDII linked to the -56 minimal promoter of rabbit β-globin. Recombinant NF-κB subunits (p50, p65, c-Rel, p52, and IκBα) and interferon regulatory factor 1 were produced from either Escherichia coli or baculovirus expression systems. Transcriptional analysis in vitro demonstrated that 1) various dimeric complexes of NF-κB differentially stimulated transcription through the human immunodeficiency virus enhancer or PRDII up to 20-fold; 2) recombinant IκBα specifically inhibited NF-κB-dependent transcription in vitro; and 3) different NF-κB complexes and interferon regulatory factor 1 cooperated to stimulate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains of the IFN-β promoter. These results demonstrate the role of NF-κB protein dimerization in differential transcriptional activation in vitro and emphasize the role of cooperativity between transcription factor families as an additional regulatory level to maintain transcriptional specificity.