ERG is a regulator of dynamic and reversible endothelial plasticity

BACKGROUND: Endothelial cells (ECs) orchestrate vascular homeostasis and resilience but can undergo reprogramming into a mesenchymal-like phenotype through an endothelial-to-mesenchymal transition (EndMT). Crucially, EndMT is a linchpin underlying several cardiometabolic diseases, but is almost universally studied as an endpoint. The transcription factor ERG (ETS-related gene) is critical to the maintenance of EC identity and function, yet the dynamic transcriptional and functional consequences of ERG loss on EndMT programs, and whether this can be reversed, has not been explored. METHODS: We modeled both acute and chronic ERG loss in human aortic ECs using siRNA knockdown and CRISPR/Cas9-mediated ERG deletion. We profiled temporal changes in chromatin accessibility (ATAC-seq), transcriptomic responses (RNA-seq), and endothelial phenotypes, including migration and barrier integrity. The temporal kinetics of ERG loss and restoration was assessed by comparing stable ERG knockout to transient ERG knockdown and recovery over time. The implications to human disease were deciphered by examining ERG gene regulatory networks in human atherosclerosis and linkage with genetic variation associated with human cardiovascular disease. RESULTS: Analysis of gene regulatory networks revealed profound and dynamic rewiring of endothelial and mesenchymal transcriptional programs upon loss of ERG. While endothelial identity was rapidly lost by 24 h of ERG knockdown, acquisition of mesenchymal identity, barrier dysfunction, and enhanced cell migration required 72 h to manifest. Loss of ERG was accompanied by a rapid reduction in accessibility of ETS motifs and an extensive gain in open chromatin containing AP1 motifs. Disease-relevant endothelial dysfunction programs were associated with dynamically reorganized transcriptional networks. Importantly, restoration of ERG expression reversed EndMT gene regulatory networks and phenotypes. CONCLUSIONS: Overall, this study highlights the ETS factor, ERG, as an essential transcriptional safeguard of endothelial identity and function, and demonstrates that ERG loss initiates a progressive, yet reversible, EndMT program with EC identity loss preceding a gain of mesenchymal gene regulatory networks and phenotypes. This study establishes loss of ERG as an early initiating event in EndMT and suggests that ERG-targeted therapies may hold promise for promoting endothelial resilience.