Blood pressure–associated polymorphism controls ARHGAP42 expression via serum response factor DNA binding
Xue Bai, … , Joan M. Taylor, Christopher P. Mack
Xue Bai, … , Joan M. Taylor, Christopher P. Mack
Published January 23, 2017
Citation Information: J Clin Invest. 2017;127(2):670-680. https://doi.org/10.1172/JCI88899.
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Research Article Genetics Vascular biology

Blood pressure–associated polymorphism controls ARHGAP42 expression via serum response factor DNA binding

Abstract

We recently demonstrated that selective expression of the Rho GTPase-activating protein ARHGAP42 in smooth muscle cells (SMCs) controls blood pressure by inhibiting RhoA-dependent contractility, providing a mechanism for the blood pressure–associated locus within the ARHGAP42 gene. The goals of the current study were to identify polymorphisms that affect ARHGAP42 expression and to better assess ARHGAP42’s role in the development of hypertension. Using DNase I hypersensitivity methods and ENCODE data, we have identified a regulatory element encompassing the ARHGAP42 SNP rs604723 that exhibits strong SMC-selective, allele-specific activity. Importantly, CRISPR/Cas9–mediated deletion of this element in cultured human SMCs markedly reduced endogenous ARHGAP42 expression. DNA binding and transcription assays demonstrated that the minor T allele variation at rs604723 increased the activity of this fragment by promoting serum response transcription factor binding to a cryptic cis-element. ARHGAP42 expression was increased by cell stretch and sphingosine 1-phosphate in a RhoA-dependent manner, and deletion of ARHGAP42 enhanced the progression of hypertension in mice treated with DOCA-salt. Our analysis of a well-characterized cohort of untreated borderline hypertensive patients suggested that ARHGAP42 genotype has important implications in regard to hypertension risk. Taken together, our data add insight into the genetic mechanisms that control blood pressure and provide a potential target for individualized antihypertensive therapies.

Authors

Xue Bai, Kevin D. Mangum, Rachel A. Dee, George A. Stouffer, Craig R. Lee, Akinyemi Oni-Orisan, Cam Patterson, Jonathan C. Schisler, Anthony J. Viera, Joan M. Taylor, Christopher P. Mack

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Figure 3

The minor T allele at rs604723 promotes SRF binding.

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The minor T allele at rs604723 promotes SRF binding. (A) Schematic of se...
(A) Schematic of sequence conservation at the center of the DHS2 region and of CArG homology at the rs604723 SNP. (B) Gel shift assays were performed by combining recombinant SRF (rSRF) with radiolabeled 100-bp oligonucleotide probes containing the major or minor alleles at rs633185 and rs604723. Representative image shown from n = 2. (C) Biotin-labeled 20-bp oligonucleotides containing the major C or minor T allele at rs604723 or a consensus CArG element were conjugated to streptavidin beads and incubated with HuAoSMC nuclear extracts. Washed immunoprecipitates were analyzed for the presence of SRF by Western blotting. Data are representative of 2 separate experiments. (D) ChIP assays were used to measure SRF binding to the DHS2 region in cultured HuAoSMCs and HuCoSMCs that are heterozygous (CT) and homozygous major (CC) at the rs604723 SNP, respectively. Data represent mean ± SEM of n = 4 experiments; *P < 0.05 vs. IgG in HuAoSMCs (Student’s t test). (E) SRF-ChIP immunoprecipitates from HuAoSMCs were subjected to a TaqMan-based assay that discriminates between the major and minor alleles at the rs604723 SNP. Data represent mean ± SEM of n = 4 experiments; *P < 0.01 vs. major allele (Student’s t test). (F) Increasing amounts of DNase I (0–1 μg) were added to permeabilized nuclei isolated from HuAoSMCs. Following genomic DNA isolation, allele-specific primers were used to amplify a 300-bp region containing the rs604723 SNP. Data represent mean ± SEM of n = 3 experiments; *P < 0.05 (Student’s t test).