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

Regulation of gene transcription by the cytokines tumor necrosis factor- alpha (TNF- alpha ) and interferon- gamma (IFN- gamma ) involves complex interactions between NF- Kappa B 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- Kappa B and Stat factors. Using a set of transcription reporter-luciferase constructs, we show that the relative orientation of juxtaposed NF- Kappa B-Stat (SIE) cis-elements determines the ability of TNF- alpha and IFN- gamma to induce gene transcription. Further, NF- Kappa B and Stat1 proteins directly regulate transcription by interacting cooperatively on NF- Kappa B-SIE DNA binding in response to TNF- alpha plus IFN- gamma . Coimmunoprecipitation provides evidence for a direct NF- Kappa B/Stat1 protein-protein interaction. In contrast, IFN- gamma inhibits TNF- alpha -induced transcription of an NF- Kappa B reporter gene in a Stat1-dependent mechanism in 2fTGH fibroblasts. Similarly, Stat1 is inhibitory to NF- Kappa B overexpression-induced transcription. IFN- gamma and Stat1-dependent inhibition of NF- Kappa B transcription occurs independent of TNF- alpha -induced NF- Kappa B DNA binding. Interestingly, IFN- gamma pretreatment of 2fTGH fibroblasts potentiates TNF- alpha 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- Kappa B and Stat1. These data demonstrate complex transcriptional regulatory mechanisms elicited by TNF- alpha and IFN- gamma and have potentially important implications for other genes differentially controlled by cytokines.