ENKEPHALIN GENE-TRANSCRIPTION IN BOVINE CHROMAFFIN CELLS IS REGULATEDBY CALCIUM AND PROTEIN KINASE-A SIGNAL-TRANSDUCTION PATHWAYS - IDENTIFICATION OF DNASE I-HYPERSENSITIVE SITES

The bovine enkephalin gene is responsive to multiple signaling pathway s in primary chromaffin cell cultures. We examined the effects of acti vation of the calcium and protein kinase A pathways on accumulation of enkephalin peptide and mRNA, gene transcription, and chromatin struct ure in the 5' region of the gene. We show here that the increase of en kephalin mRNA and peptide after depolarization of chromaffin cells wit h KCl or activation of adenylate cyclase with forskolin is preceded by an increase in enkephalin gene transcription. Both enkephalin peptide and mRNA were reduced by co-treatment of KCl- or forskolin-stimulated cultures with phorbol esters. Three enhancer sequences that were prev iously shown to be responsive to calcium, protein kinase A, and phorbo l esters in the human gene in vitro were identified in the bovine enke phalin promoter, identifying a potential locus of control for these pa thways in vivo. DNase I hypersensitivity mapping identified two tissue -specific sites that are associated with enkephalin gene expression in adrenal medulla and chromaffin cells; site 1 is in the promoter, whic h contains the three enhancer elements, and site 2 is in the first int ron. These results suggest that regulation of the enkephalin gene in p rimary chromaffin cells by the calcium, protein kinase A, and protein kinase C signaling pathways occurs by modulation of transcription fact or activity at several discrete loci on the enkephalin gene.