Regulation of Mouse Neuropeptide Y Y1 Receptor Gene Transcription: A Potential Role for Nuclear Factor-κB/Rel Proteins
- 1Institute of Pharmacology and Experimental Therapeutics, Medical School, University of Torino, 10125 Torino, Italy (R.M., A.O., S.R.G., C.E.), and 2Division of Pharmacology, Department of Biomedical Sciences and Biotechnology, University of Brescia, 25124 Brescia, Italy (M.G.)
Abstract
We previously isolated a 1.3-kb genomic fragment in the 5′-flanking region of the murine neuropeptide Y (NPY) Y1 receptor gene, which is able to drive the expression of LacZ reporter gene in neuronal cells. We determined the ability of deletion mutants of this region to modulate transcription of the heterologous luciferase gene in the Y1 receptor-expressing neuroblastoma/glioma NG108-15 cells and the Y1 receptor-deficient 293 cells. Results suggest the presence of a cell type-specific core promoter (−399 to −218 from the initiator ATG) and, upstream, of two positive and two negative regulatory elements. Sequence analysis of the Y1 receptor promoter identified two decameric sequences corresponding to consensus binding sites for nuclear factor-κB/Rel proteins. Gel shift analysis indicated that a 29-bp oligonucleotide comprising the two putative κB sites, which we refer to as Y1-κB sequence, specifically binds κB-related complexes in nuclear extracts from rat brain areas, NG108-15 cells, and the murine T cell clone A.E7. In nuclear extracts from A.E7 and NG108-15 cells, the Y1-κB sequence specifically binds an additional complex whose molecular nature remains to be elucidated. Through transient transfection studies, we also demonstrated that the Y1-κB sequence acts as an enhancer element, inferring its potential role in regulation of the Y1 receptor gene expression.
Footnotes
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Send reprint requests to: Carola Eva, Ph.D., Instituto di Farmacologia e Terapia Sperimentale, Via Pietro Giuria, 13, 10125 Torino, Italy. E-mail: eva{at}medfarm.unito.it
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↵1 The corrected DNA sequence is in the EMBL/GenBank database (accession No. Z18281).
- Abbreviations:
- NPY
- neuropeptide Y
- Y1R-LUC
- Y1 receptor/luciferase fusion gene
- κB-Y1-LUC
- Y1-κB/Y1receptor/luciferase fusion gene
- Y1-κB
- wild-type κB site(s) from the Y1 receptor gene
- mY1-κB
- mutated κB site(s) from the Y1 receptor gene
- DTT
- dithiothreitol, HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- IL
- interleukin
- Ig-κB
- κB sequence from the immunoglobulin κ light chain enhancer region
- AP-1
- activator protein-1
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- Received May 30, 1996.
- Accepted October 14, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
