IDENTIFICATION OF A TPA-RESPONSIVE ELEMENT MEDIATING PREFERENTIAL TRANSACTIVATION OF THE GALANIN GENE PROMOTER IN CHROMAFFIN CELLS

The gene encoding the neuropeptide galanin is upregulated by second me ssenger signal transduction pathways in bovine chromaffin cells. To id entify its transcriptional regulatory elements, 5'-flanking sequences of the galanin gene were transiently transfected into primary cultures of bovine chromaffin cells within reporter gene constructs. Multiple regions of the galanin 5' flank seem to be necessary for basal activit y. The most promoter-proximal of these regions contains a sequence (TG ACG) -66 to -62 nucleotides upstream from the transcriptional start si te which mediates stimulation by 12-O-tetradecanoylphorbol- 13 acetate (TPA), as demonstrated by site-directed mutagenesis and cis-activatio n experiments. This cis-regulatory element mediates preferential TPA s timulation of transcription from the galanin promoter in chromaffin ce lls compared with bovine endothelial or HeLa cells, DNA-protein bindin g assays indicate that an oligonucleotide that includes the galanin TP A-responsive element (GTRE) binds specifically to proteins from nuclea r extracts of chromaffin cells. TPA treatment persistently increases t his binding activity in chromaffin but not in endothelial cells. Mutat ion of the galanin promoter within the -66 to -62 region renders it un responsive to transcriptional stimulation by TPA, and a correspondingl y mutated oligonucleotide fails to bind chromaffin cell nuclear protei ns in a gel-shift assay. Chromaffin cell nuclear extracts also contain proteins that bind consensus TPA-responsive (TRE) and cyclic AMP-resp onsive (CRE) elements. GTRE, TRE, and CRE oligonucleotides all compete more efficiently for protein binding to their labeled congeners than for protein binding to either of the other labeled oligonucleotides, s uggesting that the GTRE, TRE, and CRE oligo nucleotides each bind uniq ue as well as common proteins, likely to be members of the Jun/Fos and cAMP-responsive element-binding protein/activating transcription fact ors (CREB/ATF) families of transcription factors, in chromaffin cells.