Abstract P1108: Functional Genomics Analysis Of Sox17 Locus To Define The Pathogenic Role Of FUBP1 In Pulmonary Arterial Hypertension

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility. Genome-wide Association Studies (GWAS) have identified SOX17 as the most significant PAH-associated genomic locus. The allele of tag SNP in this locus is associated with 1.8-fold higher PAH risk. It has been challenging to define the mechanisms underlying the contribution of the PAH-associated functional SNPs (fSNPs) to pathogenesis of the disease. Methods: We developed a post-GWAS functional genomics strategy to define the causative fSNPs and identify the associated biological mechanisms. This analysis includes Reel-seq (Regulatory element-sequencing), an EMSA-based high-throughput technique to identify fSNPs in a synthetic DNA library containing PAH-associated SNPs; SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs; and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to show allele-imbalanced binding of these proteins to fSNPs. The regulation of risk gene expression by these fSNP-binding proteins and their pathogenicity were determined in human pulmonary arterial endothelial cells (PAECs) and confirmed in PAH animal models and patients. Results: By using high-throughput Reel-seq and subsequent validation with EMSA, intergenic SNP rs4738801 in SOX17 locus was identified as a fSNP from a library containing 254 PAH-associated haplotype SNPs. This fSNP resides in a remote upstream enhancer region of SOX17, an endothelial effector increasingly associated with PAH pathogenesis. Using SDCP-MS and AIDP-Wb, we found that the transcription factor FUBP1 binds to rs4738801 risk allele C with lower affinity than non-risk allele G, resulting in a decrease in SOX17 expression. FUBP1 and target gene SOX17 controlled PAH-associated pathophenotypes in PAECs, including proliferation, apoptosis, and angiogenesis. Downregulated by the major acquired PAH trigger hypoxia, FUBP1 and SOX17 were decreased in lungs and pulmonary ECs isolated from PAH patients and mouse models. A 3.77-fold enrichment of fSNP rs4738801 risk allele C was found in patients with PAH induced by hypoxia, but not in PAH associated with connective tissue disease or congenital heart disease. Conclusions: FUBP1 controls SOX17 expression via allele-specific binding to PAH-associated fSNP rs4738801. The reduced binding of FUBP1 to risk allele C defines the genomic architecture contributing to the SOX17-dependent genetic susceptibility of PAH. The downregulation of FUBP1-SOX17 by hypoxia results in endothelial dysfunction, contributing to the acquired pathogenesis of PAH. These findings identify a novel role of FUBP1 in the functional regulation of SOX17 locus and elucidating a pathogenic mechanism that combines the acquired PAH-triggering factors and altered genetic susceptibility.