The goal of our project has been the elucidation of the molecular events involved in the regulation of the acute phase response in man. We have concentrated our attention on the prototype acute phase reactant, C-reactive protein (CRP), which is known to mediate innate immunity to infectious agents, i.e. parasites. Previously we found that Interleukin-6 (IL-6), was necessary and sufficient to initiate CRP transcription in a human hepatoma cell culture system. Currently, we have determined the cis-acting elements and the trans-acting factors responsible for the regulatory control of acute phase gene expression. We have isolated the upstream promoter region for the CRP gene and have shown that this region confers inducibility. We have identified both positive and negative regulatory elements, which include two distal enhancers and two proximal IL-6 responsive elements (IL-6REs) flanking a negative regulatory region. Using mobility shift, methylation interference, and immunodepletion assays, we have identified the binding sites and the presence of a number of trans-acting factors, including: NF-IL6a, HNF-1a, HNF-3 and several Octamer-like factors. Site-specific mutagenesis of the two IL-6REs as well as other surrounding elements indicated a synergistic effect of the two IL-6REs when bound to NFIL-6a. We have mutated these sites in the promoter region into Gal4 or GCN4 binding sites. By domain swapping the DNA binding region of NFIL-6a with that of Gal4 or GCN4, while maintaining the NFIL-6a activation domain, we have produced a chimeric NFIL-6a factor. When this factor is co-transfected into hepatoma cell with the expression plasmid containing a Gal4 or GCN4 site we were able to reproduce the observed synergistic activation by IL-6. These findings support the conclusion that the synergism is due to protein-protein interaction with the activation domain of NFIL-6a and not through the DNA binding or dimerization domains. Currently, we are examining the modification of the different forms of NFIL-6a following IL-6 signal transduction, and have found phosphorylation patterns unique to the inducible species.
