SOX17 enhancer variants disrupt transcription factor binding and enhancer inactivity drives pulmonary hypertension

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• dc.contributor.author Walters, Rachel
• dc.contributor.author Vasilaki, Eleni
• dc.contributor.author Arman, Jurjan
• dc.contributor.author Chen, Chien-Nien
• dc.contributor.author Wu, Yukyee
• dc.contributor.author Liang, Olin D.
• dc.contributor.author Ashek, Ali
• dc.contributor.author Dubois, Olivier
• dc.contributor.author Zhao, Lin
• dc.contributor.author Sabrin, Farah
• dc.contributor.author Cebola, Inês
• dc.contributor.author Ferrer, Jorge
• dc.contributor.author Morrell, Nicholas W.
• dc.contributor.author Klinger, James R.
• dc.contributor.author Wilkins, Martin R.
• dc.contributor.author Zhao, Lan
• dc.contributor.author Rhodes, Christopher J.
• dc.date.accessioned 2023-09-13T07:12:35Z
• dc.date.available 2023-09-13T07:12:35Z
• dc.date.issued 2023
• dc.description.abstract Background: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. Methods: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. Results: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. Conclusions: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.
• dc.description.sponsorship This work was supported by the NIHR BioResource, which supports the UK National Cohort of Idiopathic and Heritable PAH; the British Heart Foundation (BHF PG/19/17/34275, SP/12/12/29836), and the UK Medical Research Council (MR/K020919/1). This work was supported in part by a British Heart Foundation Centre for Research Excellence award to Dr Wilkins (RE/18/4/34215). Dr Rhodes is supported by a BHF Intermediate Basic Science Research fellowship (FS/15/59/31839) and Academy of Medical Sciences Springboard fellowship (SBF004\1095). Dr Morrell is a BHF Professor and NIHR Senior Investigator. Dr Aman is supported by an European Respiratory Society/European Molecular Biology Organisation Longterm Research Fellowship and by an Nederlandse Organisatie voor Wetenschappelijk Onderzoek VENI grant (09150161910155). Dr Ferrer is supported by Medical Research Council (MR/L02036X/1), Wellcome Trust Senior Investigator Award (WT101033), and European Research Council Advanced Grant (789055) awards. Dr Cebola is recipient of a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (224662/Z/21/Z). This work was also supported in part by grants from the National Institutes of Health P20 GM119943 (Drs Liang and Klinger), by the American Heart Association, Transformational Project Award 18TPA34110329 (Dr Liang), and by the Brown Physicians, Inc, Academic Assessment Research Award (Drs Klinger and Liang).
• dc.format.mimetype application/pdf
• dc.identifier.citation Walters R, Vasilaki E, Aman J, Chen CN, Wu Y, Liang OD, Ashek A, Dubois O, Zhao L, Sabrin F, Cebola I, Ferrer J, Morrell NW, Klinger JR, Wilkins MR, Zhao L, Rhodes CJ. SOX17 enhancer variants disrupt transcription factor binding and enhancer inactivity drives pulmonary hypertension. Circulation. 2023;147(21):1606-21. DOI: 10.1161/CIRCULATIONAHA.122.061940
• dc.identifier.doi http://dx.doi.org/10.1161/CIRCULATIONAHA.122.061940
• dc.identifier.issn 0009-7322
• dc.identifier.uri http://hdl.handle.net/10230/57855
• dc.language.iso eng
• dc.publisher Wolters Kluwer (LWW)
• dc.relation.ispartof Circulation. 2023;147(21):1606-21
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/789055
• dc.rights © 2023 The Authors. Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword SOX17 protein
• dc.subject.keyword Human
• dc.subject.keyword Hypertension
• dc.subject.keyword Pulmonary
• dc.title SOX17 enhancer variants disrupt transcription factor binding and enhancer inactivity drives pulmonary hypertension
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion