HLA CLASS-I HEAVY-CHAIN GENE PROMOTER ELEMENTS MEDIATING SYNERGY BETWEEN TUMOR-NECROSIS-FACTOR AND INTERFERONS

The cytokines tumor necrosis factor (TNF), beta interferon (IFN-beta), and IFN-gamma increase major histocompatibility complex class I molecule expression. A greater than additive (i.e., synergistic) induction of class I heavy-chain mRNA is observed in HeLa cells treated with TNF in combination with either type of IFN. To define the cis-acting elements mediating cytokine synergy, the promoter of a human major histocompatibility complex class I heavy-chain gene (HLA-B7) was placed in front of a reporter gene and transfected into HeLa cells. Deletion analysis mapped the elements required for synergy to a 40-bp region containing a kappa B-like element, which is necessary for the response to TNF, and an interferon consensus sequence (ICS), which is necessary for the responses to IFNs. When the orientation of these elements was reversed or their normal 20-bp spacing was reduced by 5 or 10 bp, i.e., one half or one full turn of the DNA helix, essentially equivalent responses were obtained, suggesting that these parameters are not critical. In electromobility shift assays, a p50-containing NF-kappa B nuclear factor from TNF-treated cells binds kappa B-containing probes, and ISGF-2 from IFN-gamma-treated cells binds ICS-containing probes. A probe containing both the kappa B and ICS elements (kappa B-ICS) forms a novel complex with nuclear factors isolated from cells treated with both TNF and IFN-gamma; this complex also forms when nuclear factors from individually cytokine-treated cells are mixed in vitro. The natural variant ICS found in HLA-A responds to IFN-gamma and can mediate synergy with TNF. However, the variant kappa B found in HLA-C does not respond to TNF, nor can it mediate synergy between TNF and IFN-gamma. These observations suggest that synergy between TNF and IFNs in the induction of HLa class I gene expression results from the sum of individual interactions of cytokine-activated enhancer-binding factors with the transcription initiation complex.