A role for Octamer binding protein motifs in the regulation of the proximal preprotachykinin-A promoter

We have recently developed cell line models that express the endogenous rat preprotachykinin-A (rPPT) gene and support reporter gene expression direct ed by the rPPT promoter. These are the neuronal derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. Reporter gen e activity in these cell lines is similar to that observed in primary cultu res of rat dorsal root ganglion neurons. Analysis of reporter gene expressi on supported by various fragments of the rPPT promoter demonstrated that al though -865 to +92 supported expression, addition of fragments between +92 and +500 led to repression of expression. Two previously defined octamer bi nding motifs, present both 5' and 3' of the major transcriptional start sit e, have been postulated to be potential enhancers of rPPT activity and we h ave now used these cell lines to determine the role of these regions in the rPPT promoter. Site specific mutagenesis of these elements has shown not o nly that both sites are potential enhancers of gene expression but also tha t the 3' element binds multiple factors, of which at least one has represso r function. Binding of this repressor protein over or adjacent to the 3' oc tamer binding protein site leads to the observed repression of promoter act ivity in the -865 to +500 construct relative to the to -865 to +92 the frag ment. (C) 2000 Harcourt Publishers Ltd.