Thorsten Kessler1, Jana Wobst2, Bernhard Wolf2, Juliane Eckhold2, Baiba Vilne2, Ronja Hollstein2, Simon von Ameln2, Tan An Dang2, Hendrik B Sager2, Philipp Moritz Rumpf2, Redouane Aherrahrou2, Adnan Kastrati2, Johan L M Björkegren2, Jeanette Erdmann2, Aldons J Lusis2, Mete Civelek2, Frank J Kaiser2, Heribert Schunkert1. 1. From Deutsches Herzzentrum München, Klinik für Herz-und Kreislauferkrankungen, Technische Universität München, Munich, Germany (T.K., J.W., B.W., B.V., S.V.A., T.A.D., H.B.S., P.M.R., A.K., H.S.); Sektion für Funktionelle Genetik am Institut für Humangenetik, Universität zu Lübeck, Germany (J. Eckhold, R.H., F.J.K.); Center for Public Health Genomics, Department of Biomedical Engineering, University of Virginia, Charlottesville (R.A., M.C.); DZHK e.V. (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany (A.K., H.S.); Department of Genetics and Genomic Sciences, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY (J.L.M.B.); Department of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia (J.L.M.B.); Institut für Integrative und Experimentelle Genomik and Universitäres Herzzentrum Lübeck, Universität zu Lübeck, Germany (J. Erdmann); DZHK e.V. (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Lübeck, Germany (J. Erdmann, F.J.K.); and Departments of Human Genetics and Medicine, David Geffen School of Medicine, University of California, Los Angeles (A.J.L., M.C.). thorsten.kessler@tum.de schunkert@dhm.mhn.de. 2. From Deutsches Herzzentrum München, Klinik für Herz-und Kreislauferkrankungen, Technische Universität München, Munich, Germany (T.K., J.W., B.W., B.V., S.V.A., T.A.D., H.B.S., P.M.R., A.K., H.S.); Sektion für Funktionelle Genetik am Institut für Humangenetik, Universität zu Lübeck, Germany (J. Eckhold, R.H., F.J.K.); Center for Public Health Genomics, Department of Biomedical Engineering, University of Virginia, Charlottesville (R.A., M.C.); DZHK e.V. (German Center for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany (A.K., H.S.); Department of Genetics and Genomic Sciences, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY (J.L.M.B.); Department of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia (J.L.M.B.); Institut für Integrative und Experimentelle Genomik and Universitäres Herzzentrum Lübeck, Universität zu Lübeck, Germany (J. Erdmann); DZHK e.V. (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Lübeck, Germany (J. Erdmann, F.J.K.); and Departments of Human Genetics and Medicine, David Geffen School of Medicine, University of California, Los Angeles (A.J.L., M.C.).
Abstract
BACKGROUND: A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. METHODS: Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses. RESULTS: Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. CONCLUSIONS: Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.
BACKGROUND: A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. METHODS: Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses. RESULTS: Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxidedonorsodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3expression correlated with less atherosclerosis in the aorta. CONCLUSIONS:Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.
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