Sox transcription factor mediated transcriptional regulation of Robo4 expression and function

Abstract

Our laboratory has identified Roundabout4 (Robo4) as a bonafide angiogenic receptor. Robo4 knockdown (KD) endothelial cells show decreased migration and zebrafish (zf) embryos show intersomitic vessel (ISV) defects. We have reported that Robo4 affected cell migration results in imbalanced active Cdc42 and disrupted actin organization but precise molecules that interact with Robo4 remain unknown. To identify the proteins that bind and orchestrate Robo4 mediated angiogenesis, we performed Yeast 2 Hybrid (Y2H) screen with cytoplasmic zf Robo4 as bait and zf cDNA as prey. Y2H identified many SOX transcription factor (TF) proteins as potential binding partners. Based on their vascular specificity we focused on Sox18 and Sox7 and immune precipitation confirmed that Robo4 binds to both. In vivo, Robo4 KD in conjunction with Sox18 and Sox7 KD decreased Fli ISV expression, indicating disrupted angiogenesis. Bioinformatics on Robo4 promoter reveals Sox TF binding sites and signifies transcriptional regulation of Robo4 via Sox. Using transactivation assays we have identified that Sox18/7 temporally induce Robo4 promoter. Sox18/7 double KD decreases Robo4 transcription and expression in zf vasculature. In sum, our data indicate that Robo4 and Sox TF interact genetically and Sox TF mediated transcriptional and post‐translational Robo4 expression and function is critical in angiogenesis.Grant Funding Source: NIH