<i>SOX17</i> Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

Abstract

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) <i>SOX17</i> 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 <i>SOX17</i> cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of <i>SOX17</i>, which in turn reduces <i>SOX17</i> expression and contributes to disturbed endothelial cell function and PAH development. CRISPR manipulation and siRNA were used to modulate <i>SOX17</i> expression. Electromobility shift assays were used to confirm in silico<i>-</i>predicted TF differential binding to the <i>SOX17</i> variants. Functional assays in hPAECs were used to establish the biological consequences of <i>SOX17</i> loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed <i>SOX17</i> signaling. Mice with deletion of the <i>SOX17</i>-signal 1 enhancer region (<i>SOX17</i>-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. CRISPR inhibition of <i>SOX17</i>-signal 2 and deletion of <i>SOX17</i>-signal 1 specifically decreased <i>SOX17</i> expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both <i>SOX17</i> 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 <i>SOX17</i> silencing, including extracellular matrix regulation. <i>SOX17</i> 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. <i>SOX17</i> enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. Common PAH risk variants upstream of the <i>SOX17</i> promoter reduce endothelial <i>SOX17</i> 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.