MULTIPLE PROTEIN COMPLEXES, INCLUDING AP2 AND SP1, INTERACT WITH A SPECIFIC SITE WITHIN THE RAT PREPROTACHYKININ-A PROMOTER

We demonstrate that there is a unique AP2 binding site in the rat preprotachykinin-A promoter (rPPT) spanning - 865 to - 47. AP2 is a transcription factor whose expression in sensory neurons has been correlated with rPPT expression in these cells. This binding site is adjacent to an element we previously identified as binding a single stranded DNA binding protein which was also present in sensory neurons. These two complexes encompass a region which we had proposed might form a stem-loop structure, allowing binding of the single stranded DNA binding protein to the DNA. Here using electrophoretic mobility shift analysis we demonstrate that the DNA region corresponding to the putative stem-loop structure is bound by a variety of transcription factors, including in addition to AP2 the ubiquitous Spl. DNase 1 footprint analysis demonstrates that binding to this domain by the proteins recognising the double-stranded form of the cis acting element is mutually exclusive. A promoter fragment containing this domain demonstrated a DNase 1 footprint over the 5' region of the stem-loop structure. Competition of the binding for this element by an oligonucleotide corresponding to the stem-loop structure removed the 5' footprint and exposed a new footprint over the 3' region of the stem-loop structure and extending for several base pairs. This change in protection observed with DNase 1 digestion also correlated with changes of the DNase 1 pattern at specific locations 3' of the proposed stem-loop structure. These changes correlated with two DNA sequences which were homologous to one another and to a region within the proposed stem-loop structure. Our results indicate that AP2 could regulate rPPT gene expression by a variety of mechanisms.