AN UPSTREAM STIMULATORY FACTOR (USF) BINDING MOTIF IS CRITICAL FOR RAT PREPROTACHYKININ-A PROMOTER ACTIVITY IN PC12 CELLS

We demonstrate the presence of a functional E box motif in the proxima l rat preprotachykinin-A (rPPT) promoter. This element (spanning nucle otides -67 to -47) exhibits the sequence 5'-CACGTG-3' which is recogni zed and bound by the basic helix-loop-helix family of regulatory prote ins. We also show that at least one of the factors bound to this rPPT promoter element in both HeLa and PC12 nuclear extract is the ubiquito usly expressed transcription factor, the upstream stimulatory factor ( USF). Mutation of this element by insertion of a 10 bp linker into the E box motif, in an rPPT promoter fragment spanning -865 to +92, destr oys the ability of this promoter fragment to support reporter gene exp ression in a PC12 cell model of rPPT promoter activity. The data indic ate that this rPPT E box element is likely to function as an important cis-regulatory domain in the rPPT promoter.