Differential Effects of TNF-α and IFN-γ on Gene Transcription Mediated by NF-κB-Stat1 Interactions

Abstract

Regulation of gene transcription by the cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) involves complex interactions between NF-κB and Stat families of transcription factors. The purpose of this study was to identify the spatial promoter requirements that govern cytokine synergy for gene transcription regulated by NF-κB and Stat factors. Using a set of transcription reporter-luciferase constructs, we show that the relative orientation of juxtaposed NF-κB-Stat (SIE) cis-elements determines the ability of TNF-α and IFN-γ to induce gene transcription. Further, NF-κB and Stat1 proteins directly regulate transcription by interacting cooperatively on NF-κB-SIE DNA binding in response to TNF-α plus IFN-γ. Coimmunoprecipitation provides evidence for a direct NF-κB/Stat1 protein-protein interaction. In contrast, IFN-γ inhibits TNF-α-induced transcription of an NF-κB reporter gene in a Stat1-dependent mechanism in 2fTGH fibroblasts. Similarly, Stat1 is inhibitory to NF-κB overexpression-induced transcription. IFN-γ and Stat1-dependent inhibition of NF-κB transcription occurs independent of TNF-α-induced NF-κB DNA binding. Interestingly, IFN-γ pretreatment of 2fTGH fibroblasts potentiates TNF-α induction of Stat1 DNA binding. Further, ChIP analysis was applied to detect cytokine-induced in vivo binding and transcriptional regulation of the human inducible nitric oxide synthase (iNOS) gene by NF-κB and Stat1. These data demonstrate complex transcriptional regulatory mechanisms elicited by TNF-α and IFN-γ and have potentially important implications for other genes differentially controlled by cytokines.