Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene

Xiaofu Wang,Pat Gulhati,Jing Li,Paul R Dobner,Heidi Weiss,Courtney M Townsend,B Mark Evers

doi:10.1074/jbc.m110.145664

Abstract

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut.

Highlights

Health Grants R37 AG10855 and R01 DK48489. 1 To whom correspondence should be addressed: Markey Cancer Center, abundance in the jejenum and ileum of the gastrointestinal (GI) tract, is released by intraluminal fats [1,2,3]
5Ј-Deletion and Mutational Analysis Identifies Multiple Sites in the Proximal human NT/N (hNT/N) Promoter That Are Required for Constitutive NT/neuromedin N (NT/N) Expression—We had shown previously that the proximal 216 bp of the rat NT/N gene is sufficient for near maximal expression in the BON human endocrine cell line, which constitutively expresses NT/N and possesses many characteristics noted in enteroendocrine cells of the intestine [24]
The findings in this study identify crucial cis-regulatory elements of the hNT/N gene required for cell-specific transcriptional activity in the novel human endocrine BON cell line

Summary

Introduction

Health Grants R37 AG10855 and R01 DK48489. 1 To whom correspondence should be addressed: Markey Cancer Center, abundance in the jejenum and ileum of the gastrointestinal (GI) tract, is released by intraluminal fats [1,2,3]. Performed using the hNT/N promoter (Ϫ175 to ϩ26) linked to the luciferase reporter gene, and the resulting plasmids were transiently transfected into BON cells (Fig. 4D).

Results

Conclusion