Epigenetic regulation of Anoctaminl in pulmonary arterial hypertension

INTRODUCTION/HYPOTHESIS: Pulmonary arterial hypertension (PAH) is associated with substantial pulmonary vascular remodeling wherein endothelial cells (ECs) within the lumen hyper-proliferate leading to occlusive plexiform lesions. We previously reported that Anoctamin-1 (ANO1) expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the mechanism of ANO1 expression regulation in PAH remains unclear. DNA methylation has been recognized as an important mechanism in gene regulation. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically mediated via DNA methylation. METHODS/RESULTS: DNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next-Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5'-upstream, 5'-UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. To determine whether decreased DNA methylation changes human ANO1 gene expression, we then exposed normal human pulmonary artery ECs to RG108, which is a DNA methyltransferase inhibitor that broadly reduces DNA methylation. Our data show that cells treated with RG108 for 72 hours have a significant 2-fold increase in ANO-1 mRNA expression in comparison to control cells, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. We are developing CRISPR-based approaches to target these identified CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene. CONCLUSIONS: Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH. © FASEB.