GSBS Dissertations and Theses

Approval Date

9-8-2010

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Subjects

Receptors, Nicotinic; Nerve Tissue Proteins; Gene Regulatory Networks; Signal Transduction; Dissertations, UMMS

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.

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Copyright is held by the author, with all rights reserved.

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