Sequences Required for Neurotensin Receptor-1 Gene Expression in N1E-115 Neurosblastoma Cells: Critical Importance of a CACCC Element for Activation During DMSO-Induced Neuronal Differentiation: a Dissertation
Molecular Genetics and Microbiology
Department of Molecular Genetics and Microbiology
First Thesis Advisor
Dr. Paul R. Dobner
Gene Expression, Receptors, Neurotensin, Neuroblastoma
The promoter sequence of the mouse high affinity neurotensin receptor, Ntr-1, gene was cloned and characterized, sequences required for positive regulation in N1E-115 cells were localized, and at least two different peptides from these cells were shown to make specific contacts within the most potent positive regulatory element. A mouse neuroblastoma cell line, N1E-115, treated with 1.5% DMSO for 72 hours induces gene expression of both endogenous Ntr-l, and reporter constructs driven by the NTR-1 promoter, by 3 - 4 fold. The sequence ofthe NTR-1 promoter has no canonical TATA box, but is GC rich and contains consensus SP1, CACCC, CRE, and initiator elements. These elements are located within a 193 base positive regulatory region required for DMSO responsive activity and contains the transcriptional start site. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC rich palindrome are crucial cis-regulatory elements for DMSO induction. The SP1 element, an NGFI-A-related element, and the 5' end of the positive regulatory region are required for maintaining basal expression in N1E-115 cells. Cell type differences in the cis-regulatory elements that mediate both DMSO induction and maintenance of basal expression are observed. Characterization of proteins in N1E-115 cells that make specific contacts within the CACCC element identified at least two peptides with predicted sizes of 57 kd and 97 kd. Two dimensional UV crosslinking indicates that these proteins might contribute to inducible gel shift complexes that require the CACCC element. Several previously characterized CACCC binding proteins, belonging to the Kruppel-like family of transcription factors, were tested by supershift analysis for their ability to contribute to NTR-1 CACCC complexes. In fact, a protein closely related to SP1 does bind the CACCC element in N1E-115 cells, but of the other Kruppel-like protein tested, only BKLF contributes to a minor complex in N1E-115 cells. These results provide evidence for the complex regulation of Ntr-1 gene expression mediated by the cooperation of several cis-regulatory elements including a CACCC Kruppel-like binding element.
Tavares, DJ. Sequences Required for Neurotensin Receptor-1 Gene Expression in N1E-115 Neurosblastoma Cells: Critical Importance of a CACCC Element for Activation During DMSO-Induced Neuronal Differentiation: a Dissertation. (2000). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 40. DOI: 10.13028/mmfw-9q49. https://escholarship.umassmed.edu/gsbs_diss/40
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