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J Biol Chem, Vol. 274, Issue 42, 30066-30079, October 15, 1999

Sequences Required for Induction of Neurotensin Receptor Gene Expression during Neuronal Differentiation of N1E-115 Neuroblastoma Cells

Daniel Tavares, Keith Tully, and Paul R. Dobner

From the Department of Molecular Genetics and Microbiology,  Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01655

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me₂SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me₂SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me₂SO induction. The CACCC box is bound by at least one factor that is induced upon Me₂SO treatment of N1E-115 cells. The Me₂SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

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