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 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.
Evers
B M
BM
Department of Surgery, The University of Texas Medical Branch, Galveston 77555, USA.
Wang
X
X
Zhou
Z
Z
Townsend
C M
CM
McNeil
G P
GP
Dobner
P R
PR
D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000099169:Fos-Related Antigen 1
D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009496:Neurotensin; D010446:Peptide Fragments; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D000099169:Fos-Related Antigen 1; C042090:neuromedin N