Runx1 and C/EBPβ transcription factors directly up-regulate P2X3 gene transcription

Recent evidence indicates that transcription factor Runx1 modulates the expression of several phenotypic markers in dorsal root ganglia (DRGs) neurons, including the pain-related P2X3 receptor. In several cell lineages C/EBP transcription factors interact with the Runx factor family members to jointly bind and activate transcription of target genes. Here, we examine whether these two transcription factors directly regulate P2X3 gene expression. Through in silico analyses of the first 2kb of the P2X3 gene promoter we identified putative consensus-binding sites for both Runx1 and C/EBPβ transcription factors. Transient over-expression in PC12 cells of either Runx1 or C/EBPβ increases P2X3 gene promoter activity and co-expression of both factors results in an additive stimulatory effect on the promoter function. Accordingly, chromatin immunoprecipitation assays demonstrate that both Runx1 and C/EBPβ bind to the P2X3 promoter in PC12 cells expressing this gene. Site-directed mutagenesis of the proximal Runx1 and C/EBPβ consensus elements in the P2X3 promoter decrease Runx1- and C/EBPβ-mediated transcriptional activity. Moreover, C/EBPβ-mediated enhancement of the P2X3 promoter requires a functional Runx1 binding site. Altogether our results support a functional and coordinated role for Runx1 and C/EBPβ transcription factors during activation of P2X3 gene transcription.