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 2

An enhancer within the ARHGAP42 first intron displays strong SMC-specific and allele-specific activity and is required for endogenous ARHGAP42 expression.

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An enhancer within the ARHGAP42 first intron displays strong SMC-specifi...
(A) Map of the chromatin determinations used to characterize potential regulatory elements near the ARHGAP42 BP-associated locus. The SNPs that define the BP-associated allele (r2 > 0.8) are shown at the top. (B) The indicated DNase-hypersensitive (DHS) regions were cloned into the pGL3 luciferase vector and transfected into primary human bronchial SMCs and mouse ECs. Luciferase activity in cell lysates was measured 2 days later and is expressed as fold over the promoterless pGL3 vector. Data represent mean ± SEM of n = 6 experiments; *P < 0.001 vs. in ECs (Student’s t test). (C) Site-directed mutagenesis was used to test the effects of the major/minor alleles on DHS1 and DHS2 enhancer activity. Data represent mean ± SEM of n = 6 experiments; *P < 0.01 vs. the major allele (Student’s t test). (D) Schematic of the 102-bp deletion (in red) generated by our CRISPR/Cas9–mediated gene editing protocol. (E) ARHGAP42 message was measured by semiquantitative RT-PCR in human bronchial SMC cultures transfected with expression plasmids encoding Cas9 and the guide RNAs shown in D (n = 5). The reduction in ARHGAP42 expression was normalized to the efficiency of DHS2 deletion, which ranged from 45% to 95%. Data represent mean ± SEM of n = 5 separate experiments; *P < 0.05 vs. cells transfected with empty guide RNA expression plasmid (Student’s t test).