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A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo - PubMed

  • ️Fri Jan 01 2010

A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo

M D Scofield et al. Neuroscience. 2010.

Abstract

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.

Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

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Figures

Fig. 1
Fig. 1

WT and mutant CA box β4 promoter transgenic constructs. (A) WT and mutant β4 promoter/lacZ transgene architecture. The β4/α3/α5 gene cluster is depicted as boxes with arrows depicting the direction of transcription. Below the clustered nAChR subunit genes is a schematic of the linearized construct used to generate the transgenic animals. MAR, matrix attachment region; NLS, nuclear localization sequence. Shown below this schematic are the nucleotide sequences of the WT and mutant CA boxes. Mutations made to the CA box in the mutant transgenic construct are shown in grey with asterisks over the mutated nucleotides. (B) β4 promoter/lacZ transcriptional activity in vitro. DNA from either the WT (black bars) or mutant (white bars) transgenic constructs was transfected into Neuro-2a (left) or SN17 cells (right) along with a luciferase construct in which the SV40 promoter drives expression of the firefly luciferase gene. β-Gal activity was normalized to luciferase activity in order to correct for differences in transfection efficiencies. The data shown here are an average of 3 independent experiments, error bars represent standard deviations of the means. Student t test indicated that CA box mutation significantly decreased the β-gal activity of the mutant transgenic construct in both cell lines, p<0.05.

Fig. 2
Fig. 2

nAChR β4 subunit promoter activity in ED18.5 transgenic mice. Sagittal sections of WT transgenic (A - D) and mutant CA box transgenic (E - H) ED18.5 mouse embryos are shown. These sections were simultaneously stained for β-Gal activity and then counter-stained with neutral red. (A and E) lower lumbar region of the spinal cord; (B and F), intestine; (C and G) cortex; (D and H) lower lumbar dorsal root ganglion (DRG). Arrows in panels A, C and D indicate β-gal-expressing cells.

Fig. 3
Fig. 3

nAChR β4 subunit promoter activity of in the CNS of PD30 transgenic mice. Coronal sections of WT transgenic (A - D) and mutant CA box transgenic (E - H) PD30 mouse brains are shown. These sections were simultaneously stained for β-Gal activity, and then counter-stained with neutral red. (A and E) piriform cortex (Pir); (B and F) medial habenula (Mhb); (C and G) subiculum (S); (D and H) hippocampus, corpus ammon layer 1 (CA1), dentate gyrus (DG); Arrows in panels A and B indicate β-Gal-expressing cells.

Fig. 4
Fig. 4

nAChR β4 subunit promoter activity of in the DRG of PD30 transgenic mice and in the Trigeminal ganglion of ED18.5 transgenic mice. Sections of WT transgenic line 54 (A) and mutant CA box transgenic line 28 (B) PD30 DRG are shown as well as WT transgenic line 54 (C) and mutant CA box transgenic line 28 (d) ED18.5 trigeminal ganglion. These sections were simultaneously stained for β-Gal activity and then counter-stained with neutral red. Arrows in panels A and C indicate β-Gal-expressing cells.

Fig. 5
Fig. 5

CA box mutation results in decreased association of Sp1 with the β4 promoter. ChIP experiments were performed using brain tissue from mutant β4 promoter/lacZ transgenic line 28. Transgenic brain ChIP-derived DNA was used as template for PCR designed to amplify either the WT endogenous mouse β4 promoter (black bars), or the CA box mutant transgenic rat β4 promoter (white bars). (H4) anti-acetylated histone protein H4; (IgG) normal rabbit IgG; (Sp1) Transcription factor Sp1; (No Ab) Mock IP. The data shown here are an average of 4 independent experiments, expressed as a percentage of a 2% un-precipitated input sample. One-way ANOVA statistical analysis performed on the WT β4 promoter values (black bars) indicated that ChIP values differed significantly from the total mean (ANOVA F3, 7 = 148.4, p<0.001). Tukey’s multiple comparison post-test resulted in a significant difference between the Sp1 ChIPs and the WT IgG and mock ChIP controls, p <0.01. One-way ANOVA statistical analysis performed on the transgenic mutant β4 promoter values (white bars) indicated that ChIP values differed significantly from the total mean (ANOVA F3, 7 = 94.62, p<0.001). Tukey’s multiple comparison post-test resulted in no significant difference between Sp1 ChIPs and the IgG and mock ChIP controls at the mutant β4 promoter. Error bars represent standard deviation of the means.

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