CHARACTERIZATION OF PROMOTER ELEMENTS REQUIRED FOR CELL-SPECIFIC EXPRESSION OF THE NEUROTENSIN NEUROMEDIN-N GENE IN A HUMAN ENDOCRINE CELL-LINE

Expression of the gene encoding neurotensin/neuromedin N (NT/N) Is mos tly limited to the brain and specialized enteroendocrine cells (N cell s) of the distal small intestine. We have analyzed the NT/N DNA sequen ces upstream of the RNA start site that direct cell-specific expressio n using a novel human endocrine cell line, BON, that resembles intesti nal N cells in several important aspects, including NT/N precursor pro tein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection as says with plasmids with progressive 5' deletions of the rat NT/N promo ter identified the proximal 216 bp of 5' flanking sequences as essenti al for high-level constitutive NT/N expression in BON cells. In additi on, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. The se elements include a proximal cyclic AMP response element (CRE)/AP-1- like moth (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and A TF proteins, a near-consensus glucocorticoid response element, and a d istal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In additi on, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel f actors that act as positive regulators of NT/N expression. DNase I foo tprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specificall y bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP -1-like motif and other upstream control elements play an important ro le in the high-level constitutive expression of NT/N in the human endo crine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/ N expression and to better understand the mechanisms responsible for t he terminal differentiation of the N-cell lineage in the gut.