A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo.
Genome-wide association studies have underscored the importance of the clustered neuronal nicotinic acetylcholine receptor (nAChR) subunit genes with respect to nicotine dependence as well as lung cancer susceptibility. CHRNB4, which encodes the nAChR β4 subunit, plays a major role in the molecular mechanisms that govern nicotine withdrawal. Thus, elucidating how expression of the β4 gene is regulated is critical for understanding the pathophysiology of nicotine addiction. We previously identified a CA box regulatory element, (5'-CCACCCCT-3') critical for β4 promoter activity in vitro. We further demonstrated that a 2.3-kb fragment of the β4 promoter region containing the 5'-CCACCCCT-3' regulatory element in the β4 gene promoter (CA box) is capable of directing cell-type specific expression of a reporter gene to a myriad of brain regions that endogenously express the β4 gene. To test the hypothesis that the CA box is critical for β4 promoter activity in vivo, transgenic animals expressing a mutant form of the β4 promoter were generated. Reporter gene expression was not detected in any tissue or cell type at embryonic day 18.5 (ED 18.5). Similarly, we observed drastically reduced reporter gene expression at postnatal day 30 (PD30) when compared to wild type (WT) transgenic animals. Finally, we demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the mutant β4 promoter. Taken together these results demonstrate that the CA box is critical for β4 promoter activity in vivo.
Scofield
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MD
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01604, USA.
Tapper
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Gardner
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D000818:Animals; D001921:Brain / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D002460:Cell Line; D018507:Gene Expression Regulation, Developmental; D007763:Lac Operon; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics; D050436:Regulatory Elements, Transcriptional
C486720:Chrnb4 protein, mouse; D009419:Nerve Tissue Proteins; D011978:Receptors, Nicotinic