A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo

Abstract

Genome-wide association studies have underscored the importance of the clustered neuronal nicotinic acetylcholine receptor subunit genes with respect to nicotine dependence as well as lung cancer susceptibility. CHRNB4, which encodes the nicotinic acetylcholine receptor 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 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. Similarly, we observed drastically reduced reporter gene expression at postnatal day 30 when compared to wild type 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. KeywordsNicotinic Acetylcholine Receptors; CHRNB4; Sp1; CACCC; Transgenic Mice During the development of the mammalian nervous system, transcriptional regulatory cascades act to define the properties of individual nerve cells by controlling gene expression during neuronal differentiation (Sauka-Spengler and Bronner-Fraser, 2008). These developmental gene networks must be tightly controlled in order to ensure that each neuron is capable of performing the biological functions required for neuronal communication (Eisen, 1991, Francis and Landis, 1999, Groves and Bronner-Fraser, 1999 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 November 10. (Whitehouse et al., 1988, Steinlein et al., 1995, De Fusco et al., 2000, Perry et al., 2001, Isacson et al., 2002, Perl et al., 2003, Teaktong et al., 2003. Hence, further elucidating the molecular mechanisms that act to control expression of the nAChR subunit genes will contribute not only to our understanding of neuronal development but to a greater understanding of the pathophysiol...

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