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 prep rotachykinin-A promoter (rPPT) spanning - 865 to - 47. AP2 is a transc ription 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 c omplexes encompass a region which we had proposed might form a stem-lo op structure, allowing binding of the single stranded DNA binding prot ein to the DNA. Here using electrophoretic mobility shift analysis we demonstrate that the DNA region corresponding to the putative stem-loo p structure is bound by a variety of transcription factors, including in addition to AP2 the ubiquitous Spl. DNase 1 footprint analysis demo nstrates that binding to this domain by the proteins recognising the d ouble-stranded form of the cis acting element is mutually exclusive. A promoter fragment containing this domain demonstrated a DNase 1 footp rint over the 5' region of the stem-loop structure. Competition of the binding for this element by an oligonucleotide corresponding to the s tem-loop structure removed the 5' footprint and exposed a new footprin t over the 3' region of the stem-loop structure and extending for seve ral base pairs. This change in protection observed with DNase 1 digest ion also correlated with changes of the DNase 1 pattern at specific lo cations 3' of the proposed stem-loop structure. These changes correlat ed 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 mechanis ms.