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

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterised by remodelling of the pulmonary arteries, increased vascular resistance and right heart failure. Genome-wide association studies (GWAS) of idiopathic/heritable PAH established novel genetic risk variants including conserved enhancers upstream of transcription factor (TF) SOX17 containing two independent signals. SOX17 is an important transcription factor 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 hypothesised 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 small interfering RNA were used to modulate SOX17 expression. Electromobility shift assays (EMSA) were used to confirm in-silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAEC were used to establish the biological consequences of SOX17 loss. In-silico analysis using the connectivity map (CMap) were used to predict compounds that rescue disturbed SOX17 signalling. 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. EMSA demonstrated differential binding of hPAEC nuclear proteins to the risk and non-risk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody EMSA. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways upon SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAEC resulted in increased apoptosis, proliferation, and disturbance of barrier function. Using CMap, 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.