Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Molecular Genetics and Microbiology
Medical Subject Headings
Activating Transcription Factor 1; Activating Transcription Factor 2; Adenocarcinoma; Animals; Base Sequence; Binding, Competitive; Cyclic AMP Response Element-Binding Protein; DNA Mutational Analysis; DNA-Binding Proteins; *Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neurotensin; Pancreatic Neoplasms; Peptide Fragments; Promoter Regions (Genetics); Protein Binding; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Sequence Deletion; Tissue Distribution; Transcription Factors; Tumor Cells, Cultured
Life Sciences | Medicine and Health Sciences
Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically 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 role in the high-level constitutive expression of NT/N in the human endocrine 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 the terminal differentiation of the N-cell lineage in the gut.
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Citation: Mol Cell Biol. 1995 Jul;15(7):3870-81.
Molecular and cellular biology
Evers, B. Mark; Wang, Xiaofu; Zhou, Zhichao; Townsend, Courtney M.; McNeil, Gerard P.; and Dobner, Paul R., "Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line" (1995). GSBS Student Publications. 270.