Genome-wide siRNA screen for mediators of NF-κB activation

Although canonical NF kappa B is frequently critical for cell proliferation, survival, or differentiation, NF kappa B hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NF kappa B pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NF kappa B activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NF kappa B-dependent, we identified 155 proteins significantly and substantially important for NF kappa B activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NF kappa B activation. Relevance to other canonical NF kappa B pathways was extended by finding that 118 of the 155 LMP1 NF-kappa B activation pathway components were similarly important for IL-1 beta-, and 79 for TNF alpha-mediated NF kappa B activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NF kappa B activation. Most novel pathway components functioned upstream of I kappa B kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NF kappa B, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNF alpha, and IL-1 beta NF kappa B-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NF kappa B activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1 beta-, or TNF alpha-mediated canonical NF kappa B activation.