Abstract 63: A Regulatory Enhancer Variant at the Hypertensive GRAF3 Locus Increases SMC-Selective Expression of GRAF3 by Promoting SRF Binding
We have recently shown that the Rho-specific GAP, GRAF3, is specifically expressed in smooth muscle cells (SMC) in mouse and humans and negatively regulates blood pressure by limiting Rho-dependent SMC contractility. Genome wide association studies identified a hypertension-associated locus within the first intron of the GRAF3 gene (currently defined by SNPs rs633185 and rs604723) and demonstrated that the minor allele was significantly correlated with decreased blood pressure in patients. The aims of the current study were to identify transcription mechanisms that regulate SMC-specific GRAF3 expression and to test the hypothesis that SNPs within the GRAF3 hypertension locus affect blood pressure by altering GRAF3 expression and RhoA activity. Using DNaseI Hypersensitivity of chromatin structure, ChIP-seq approaches for previously implicated transcription factors, and information from sequence/regulatory databases, we identified two regions within the human GRAF3 gene that exhibited strong SMC-selective transcription activity in cultured cells. Importantly, the enhancer at +52 Kb contains SNP rs604723 and mutation of the major C allele to the minor T allele significantly increased enhancer activity from 20- to 40-fold over promoterless. The DNA surrounding the minor T allele conforms to the consensus SRF binding element at 8 out of 10 residues. Gel shift and targeted ChIP assays revealed preferential binding of SRF to the minor allele sequence, and the SRF co-factor, myocardin, significantly transactivated the minor but not the major allele enhancer (p<0.05). Allele-specific quantitative RT PCR on RNA isolated from human aortic SMC heterozygous at rs604723 revealed T allele-specific mRNA expression. Finally, data within the Genotype-Tissue Expression (GTEx) database indicated that GRAF3 RNA levels in tibial artery samples were 3-fold higher in patients homozygous for the minor T allele compared to patients homozygous for the major C allele (p<1.5e-10). Taken together, our data indicate that the minor T allele at rs604723 upregulates GRAF3 transcription via an SRF-dependent mechanism and provides the first functional link between this SNP and blood pressure regulation.
Author Disclosures: K. Mangum: None. J. Taylor: None. C. Mack: None.
- © 2015 by American Heart Association, Inc.