The single nucleotide polymorphism rs12041948 influences CDX4-Mediated RGS4 gene expression

The SNPs in the 5' regulatory region of the human RGS4 gene were reportedly associated with schizophrenia risk.

To elucidate the impact of the rs12041948 on CDX4 binding and the subsequent regulation of RGS4 gene expression, the dual-luciferase reporter assays, EMSAs, qPCR and western blotting were performed.

Haplotypes carrying the rs12041948 T allele exhibited significantly higher relative luciferase activity when compared with those carrying the C allele (P < 0.0001, 95% CI = 0.0133 to 0.0249). EMSAs indicated that recombinant CDX4 protein bound to DNA at region 1 (-1073 to - 1065, allele-specific for rs12041948) and region 2 (-307 to - 299) relative to TSS + 1. The pEGFP-N1-CDX4 vector co‑transfection significantly enhanced relative luciferase activity of all four recombinant vectors compared with pEGFP-N1-basic vector, with GTC haplotype vector showing the greatest increase, followed by GCC haplotype vector, mutant vector 1, and mutant vector 2 in descending order (GTC haplotype vector: adj. P = 0.0001, 95% CI [0.0173 to 0.0812]; GCC haplotype vector: adj. P = 0.0023, 95% CI [0.0092 to 0.0731]; mutant vector 1: adj. P = 0.0057, 95% CI [0.0067 to 0.0706]; mutant vector 2: adj. P = 0.0203, 95% CI [0.0029 to 0.0668]). Endogenous RGS4 mRNA and protein expression levels were also significantly increased following pEGFP-N1-CDX4 transfection when compared with pEGFP-N1-basic transfection (mRNA: adj. P < 0.0001, 95% CI = 1.137 to 1.542; protein: (adj. P = 0.0162, 95% CI = 0.2440 to 1.913).

CDX4 upregulated RGS4 expression, and this effect was allele-specific for rs12041948.