RUNX3-ERG Cooperation Orchestrates Endothelial Gene Regulation and Vascular Homeostasis

BACKGROUND: Transcriptional regulation is fundamental to vascular homeostasis, ensuring vessel stability and function. Disruption of this regulation underlies pathological angiogenesis in cancer and vascular malformations. While RUNX3 (Runt-related transcription factor 3), a Runt-domain transcription factor, is established as a tumor suppressor and regulator of lymphocyte development, its role in endothelial cells has remained unknown. METHODS: To define the vascular function of RUNX3, we generated endothelial-specific RUNX3 knockout mice and analyzed postnatal retinal angiogenesis and oxygen-induced retinopathy. Mechanistic insights were obtained through ATAC-sequencing data analysis, coimmunoprecipitation, proximity ligation assay, electrophoretic mobility shift assay, and ChIP-qPCR. RESULTS: Loss of endothelial RUNX3 induced hypersprouting angiogenesis with excessive tip cell formation and reduced DLL4 (delta-like ligand 4) expression. RUNX3 deficiency disrupted vascular integrity by lowering VE-cadherin (vascular endothelial cadherin) and claudin-5 levels, while increasing ICAM1 (intercellular adhesion molecule 1) expression, retinal hemorrhage, and leukocyte infiltration. In the oxygen-induced retinopathy model, endothelial RUNX3 deletion aggravated pathological neovascularization. Mechanistically, RUNX3 cooperated with ERG (ETS-related gene) at ETS:RUNX motifs to regulate genes controlling sprouting ( DLL4 ), junctional stability ( CDH5 , CLDN5 ), and leukocyte adhesion ( ICAM1 ). RUNX3 formed complexes with ERG, p300, and HDAC3 (histone deacetylase 3), and proximity ligation assays showed that these interactions were enriched in the nucleus upon VEGF-A (vascular endothelial growth factor-A) stimulation. ChIP-qPCR further demonstrated that RUNX3 was required for the recruitment of these coregulators to target loci. Through differential coregulator engagement, RUNX3 functioned bidirectionally, acting as a transcriptional activator of CDH5 , CLDN5 , and DLL4 while serving as a repressor of ICAM1 . Consistently, single-cell transcriptome analysis of human arteriovenous malformations revealed dysregulated RUNX3 and target gene expression, underscoring translational relevance. CONCLUSIONS: This study provides the first evidence that RUNX3 is a pivotal transcriptional coordinator in endothelial cells. By integrating ERG and chromatin regulators at ETS:RUNX motifs, RUNX3 safeguards the balance between angiogenic activation and vascular stability. These findings establish RUNX3 as a gatekeeper of endothelial transcriptional identity and highlight its potential as a therapeutic target in pathological angiogenesis.