Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

Abstract

Epigenetic processes involving long non-coding RNAs (lncRNAs) regulate endothelial genes expression. However, the underlying regulatory mechanisms causing endothelial dysfunction yet remain to be elucidated. Enhancer of Zeste Homologue 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischaemic angiogenesis. We used formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH) and identified new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 (ITGA4) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved EC migration and adhesion to fibronectin, in vitro. A-395 inhibitor re-directed MEG3-assisted chromatin remodelling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show context specific role for MEG3 in guiding EZH2 to repress ITGA4. Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.