Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time FCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBε, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent-NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ε, Gro-β/γ and Naf-1 promoters directly bound NF-κ/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.