Transcriptional regulation of the human chromogranin A gene by its 5' distal regulatory element: novel effects of orientation, structure, flanking sequences, and position on expression.

In previous studies of human chromogranin A (hCgA) gene expression, we had identified a 27 bp distal regulatory element (DRE) located between -576 and -550 bp that interacts with a CRE-containing promoter to enhance gene transcription specifically in neuroendocrine (NE) cells. To characterize the DRE we have now mutated its various parts and assessed the effects on protein binding by electrophoretic mobility shift assays (EMSAs) and hCgA transcription within BEN cells. We found that the sequence TGACTAA, an AP-1 binding site that we refer to as DRE-AP-1, was necessary but not sufficient to produce the DRE's enhancer effect. Moreover, while AP-1 (Jun/Fos) bound this site, binding was not correlated with transcriptional effects. Protein binding by the DRE-AP-1 could be attenuated by mutations of its flanking sequences, and transcriptional enhancement by the DRE was dependent on its orientation and spatial relationship to the hCgA proximal promoter. Mutation of the DRE-AP-1 to a consensus AP-1 did not produce greater transcriptional activity, even though it increased binding of nuclear factors. Co-transfection with c-jun and/or c-fos expression plasmids showed that the DRE was unresponsive to the over-expressed AP-1 proteins. Co-transfection with wild-type DRE oligonucleotides competitively inhibited DRE-mediated transcription, while co-transfection with mutant DRE oligonucleotides had a lesser effect. Our studies indicate that transcriptional enhancement of hCgA by the DRE is dependent on a unique NE-specific DRE-binding factor, which we refer to as DBF, that specifically and directionally binds the DRE to assemble and synergize a functional transcription complex.