Super-enhancer-driven core transcription factor FOXP1 delays endothelial cell senescence via phase separation-mediated SESN3 activation

<b>Rationale:</b> Endothelial cell senescence leads to endothelial dysfunction, thereby promoting the progression of atherosclerosis. Super-enhancers are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in human diseases. However, the underlying mechanisms by which super-enhancers regulate endothelial cell senescence remain unclear. This study reveals the effect of liquid-liquid phase separation (LLPS) mediated by super-enhancer-driven core transcription factor FOXP1 on endothelial cell senescence. <b>Methods:</b> The landscape of super-enhancers, chromatin accessibility, and transcriptome profiling were characterized during endothelial cell senescence by conducting CUT&Tag-seq with antibodies against H3K27ac, H3K4me1, and H3K4me3, along with assays for ATAC-seq and RNA-seq. The Coltron algorithm was used to identify core transcription factors in the process of endothelial cell senescence. Fluorescence recovery after photobleaching (FRAP), dCas9-KRAB CRISPRi, and the Optodroplet assay were utilized to confirm the phase separation properties of FOXP1. Functional experiments were employed to elucidate the effect of FOXP1 on endothelial cell senescence through LLPS. <b>Results:</b> Senescent endothelial cells undergo significant changes in their epigenome. FOXP1 is identified as a core transcription factor, driven by super-enhancers, which delays endothelial cell senescence and inhibits atherosclerosis. Moreover, FOXP1 undergoes LLPS, which the 19 phase-forming amino acids within the intrinsically disordered region of FOXP1 are capable of maintaining its ability to delay endothelial cell senescence. Mechanistically, FOXP1 activates the target gene SESN3 and inhibits the mTORC1 signaling pathway through phase separation, a key event in delaying endothelial cell senescence. The clinical evidences support the potential role of FOXP1 and SESN3 as protective factors against atherosclerosis. <b>Conclusion:</b> FOXP1 undergoes phase separation at its super-enhancer, recruiting transcription coactivators to form condensates. These condensates, in turn, facilitate binding with the SESN3 promoter and inhibit the mTORC1 signaling pathway, thereby delaying endothelial cell senescence.