PROTEIN-KINASE A-DEPENDENT DEREPRESSION OF THE HUMAN PRODYNORPHIN GENE VIA DIFFERENTIAL BINDING TO AN INTRAGENIC SILENCER ELEMENT

Induction of the prodynorphin gene has been implicated in medium and l ong-term adaptation during memory acquisition and pain. By 5' deletion mapping and site-directed mutagenesis of the human prodynorphin promo ter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma c ells are regulated by the GAGTCAAGG sequence centered at position + 40 in the 5' untranslated region of the gene (named the DRE, for downstr eam regulatory element). The DRE repressed basal transcription in an o rientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blottin g and UV cross-linking experiments with nuclear extracts from human ne uroblastoma cells or human brain revealed a protein complex of approxi mately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for a n increase in prodynorphin gene expression. Our results suggest that u nder basal conditions, expression of the prodynorphin gene is represse d by occupancy of the DRE site. Upon PKA stimulation, binding to the D RE is reduced and transcription increases. We propose a model for huma n prodynorphin activation through PKA-dependent derepression at the DR E site.