Thesis
Transcriptional regulation of the mouse PAC1 receptor gene
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2013
- Thesis identifier
- T13646
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The G-protein coupled receptor PAC₁ has been implicated in playing a role in neural growth, development and stress-protection, cognition, homeostasis, immunomodulation and anti-inflammatory effects. Previously, an investigation of PAC₁ gene (mADCYAP1r1) revealed a minimum promoter region -113bp to +206 relative to the transcriptional start site. Binding of the Zac1 transcription factor has been investigated but no detailed analysis has been carried out of the region downstream or upstream of -2598bp from exon 1 and a full characterisation of the Transcription Factors (TF) that govern expression of the receptor has not been performed. The aim of this study was to characterise the promoter of the mAdcyap1r1 gene and to identify transcription factor binding sites, explore their role in controlling expression. Since the mAdcyap1r1 gene is well conserved across vertebrate species, a cross-species DNA comparison was performed to detect evolutionary conserved regions. Mouse, rat, human and chimpanzee sequences were aligned, and several cross-species conserved regions rich in transcription factor binding sites have been identified and reported. 3 upstream regions AdU1, AdU2 and AdU3; 2 downstream AdD1 and AdD2; and a new minimum promoter region Ad1 -80 and including most of exon 1 to +353. 279 transcription factor binding sites were found in these regions. Luciferase reporter gene assays of these regions was performed in Neuro-2a cell lines, α-T3 neuroendocrine models and Cos-7 cells as a negative control that don't express PAC1. Results indicated a functional new basal promoter region, Ad1, that was expressed in Neuro-2a. Upstream and downstream regions showed Neuro-2a specificity but were 20 fold lower than expression from Ad1. Hydrogen peroxide treatment gave increased expression via the AdU1, Ad1 and AdD2 regions mainly in Neuro-2a cells. Electrophoretic Mobility Shift Assays indicated binding of nuclear extracts to most promoter regions. Promoter Expression data gave functional insight to the bioinformatic analysis, and transcription factor binding sites indicate further roles to be investigated in future studies.
- Resource Type
- DOI
- Date Created
- 2013
- Former identifier
- 1004657
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