Identification of the promoter region of the P2RX4 gene

The human P2X(4) purinergic receptor is an ATP gated cation-selective channel, which can be upregulated following nerve injury or stimulation by various cytokines. However, the transcriptional control of this regulation is unknown. In this study, the transcription initiation site was estimated to be 72 bp upstream of ATG start codon by using a novel sequencing based primer extension method with 5'-FAM tagged primers. To delineate the promoter region of the P2RX4 gene which encodes the P2X(4) receptor, we constructed 8 fragments (size range 100-4500 bp) covering the 4.5 Kb upstream region of the P2RX4 gene. A dual-colour luciferase reporter vector system was used to measure the promoter activities in both transfected HEK-293 cells and COS-7 cells for each fragment extracted from 5 to 7 randomly picked colonies. The 62 bp sequence upstream of the initiation site showed promoter activity. A putative GATA-2 binding site (-29 to -20) within this region was required for high promoter activity and GATA-2 was found to be one of the transcriptional factors binding to P2RX4 promoter by both fluorescent super electrophoresis mobility shift assay and immunoprecipitation using streptavidin coated Dynabeads and biotin-labeled double-strand DNA probes. A single nucleotide polymorphism with minor allele frequency of 0.23 was found within the GATA-2 binding site of P2RX4 promoter region which significantly reduced gene transcription. In conclusion, our data has identified the first transcription factor involved in P2X receptor expression.