RUNX3-ERG Cooperation Orchestrates Endothelial Gene Regulation and Vascular Homeostasis

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.

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.

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.

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.