Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

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) 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 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of , which in turn reduces expression and contributes to disturbed endothelial cell function and PAH development.

Methods: CRISPR manipulation and siRNA were used to modulate expression. Electromobility shift assays were used to confirm in silicopredicted TF differential binding to the variants. Functional assays in hPAECs were used to establish the biological consequences of loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed signaling. Mice with deletion of the -signal 1 enhancer region (-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia.

Results: CRISPR inhibition of -signal 2 and deletion of -signal 1 specifically decreased expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both 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 silencing, including extracellular matrix regulation. 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. 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 promoter reduce endothelial 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.