Transcriptional regulation of the gene encoding the Mouse PAC1 receptor (mAdcyap1r1) gene

Walid El Bestawy, E M Ellis, E M Lutz. SIPBS, University of Strathclyde, Glasgow, UK

The 38 amino acid pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor PAC1r are well conserved across vertebrate species. Both have been implicated in playing a role in neural growth, development and stress-protection as well as immunomodulation and anti-inflammatory effects. The 18 exon mouse PAC1r gene (mADCYAP1r1) has been characterised previously using transcription factor (TF) binding domain analysis, identifying a minimum promoter region -180bp from exon 1, containing GC boxes, CCAAT- and AP-2 binding sites[1]. Binding of the TF Zac1 has been investigated using Electrophoretic mobility shift assays (EMSA)[2] but no detailed analysis has been carried out of the region downstream or upstream of -2500bp from exon 1 and a full characterisation of the TF that govern expression of the receptor has not been performed.

The aim of this study was to characterise the promoter of the mouse Adcyap1r1 gene and to identify TF that bind and regulate expression. Since the mAdcyap1r1 gene is well conserved across vertebrate species, a cross-species DNA comparison was performed using rVista and Genomatrix software to detect evolutionary conserved regions. Mouse, rat, human and chimpanzee sequences were aligned, and several cross-species conserved regions (showing >75% conservation) that are rich in overlapping TF binding sites have been identified. These regions contain TF-binding domains such as those bound by NF-1 and Sp-1 (constitutive), AP-1 (developmental) and CREB (inducible). Three conserved regions have been identified upstream of the transcriptional start site (Tss) in exon 1(designated+1): -4540 to -4260, -3845 to -3710, -2829 to -2627bp. Two more conserved regions have been identified downstream of exon 1 within intron 1: +1739 to +1965 and +2710 to +2860bp. There is also a conserved region flanking exon 1. Based on this bioinformatic analysis, the minimum promoter region appears to start -115bp upstream of the Tss rather than -180bp.

To test the function of these putative regulatory regions with respect to TF binding and gene regulation, several DNA fragments spanning from -6000bp to exon 2 were amplified by PCR from a BAC clone that contains the entire mAdcyap1r1 gene and upstream region. PCR fragments were cloned into TOPO cloning vectors and subcloned into pGL-3 Basic Luciferase reporter vector. Restriction mapping and DNA sequencing was used to confirm clone identities and orientation.

In conclusion, we have identified putative regulatory regions in the mouse Adcyap1r1 gene, and have created valuable tools that will allow the function of these regions to be evaluated. Future work will involve transfecting constructs into mouse Neuro-2A neuroblastoma cells as a neuroendocrine model and using a variety of conditions to study the regulation of gene expression. EMSA assays will also be used to identify TF that bind to these regions.

References:

[1] H. Aino, H. Hashimoto, N. Ogawa, a Nishino, K. Yamamoto, H. Nogi, S. Nagata, and a Baba, “Structure of the gene encoding the mouse pituitary adenylate cyclase-activating polypeptide receptor.,” Gene, vol. 164, no. 2, pp. 301–4, Oct. 1995.

[2] N. Rodríguez-Henche, F. Jamen, C. Leroy, J. Bockaert, and P. Brabet, “Transcription of the mouse PAC1 receptor gene: cell-specific expression and regulation by Zac1.,” Biochimica et biophysica acta, vol. 1576, no. 1–2, pp. 157–62, Jun. 2002.