This project focuses on the transcriptional control of the prodynorphin gene, which codes for the dynorphin family of opioid peptides. We have shown that during peripheral inflammation prodynorphin gene expression is greatly increased in spinal cord and, thus, may play a role in modulating pain. Fos, a transcription control protein, is also increased in our inflammation model. Fos binds to an AP-1-like DNA sequence located at -1546 from the transcription start site in the prodynorphin promoter. We call this sequence the DAP site for dynorphin AP-1-like site. A transient expression assay has been established to determine the functional efficacy of the DAP site in cell lines in vitro. Starting with approximately 2,000 bp of upstream sequence, several dynorphin promoter-CAT reporter plasmids have been constructed and analyzed by restriction digests or nucleotide sequencing. One series of clones contains the region surrounding the DAP site. We have found that this fragment confers high level constitutive expression when transiently transfected into HeLa cells or PC-12 cells. A 250 bp fragment adjacent to the DAP site was much less effective as an enhancer element. The entire 2,000 bp promoter fragment was also inefficient as an enhancer, suggesting that more proximal sequences have a modulatory role on expression driven by the DAP site. We have also obtained preliminary data on another region of the dynorphin promoter that showed strong complex formation with CNS nuclear protein extracts. This element, at -208, appears to bind a set of proteins distinct from the -1546 site and has homology to the Inr consensus YAYTCYYY. We have isolated a cDNA clone by southwestern screening of a rat brain cDNA library that binds to this element. This clone was sequenced and codes for a unique protein. Binding studies suggest that it forms a specific complex with the dynorphin Inr-like element. We are currently verifying our initial observations by producing the protein via recombinant methods and testing the purified protein for specific binding to the dynorphin Inr-like sequence.
