タイトル； Tip mark, Endothelial cell Specific Molecule (ESM) 1 gene regulation through the genome-wide FOXO1 enrichment study.
（熊本大学 生命資源研究・支援センター 分子血管制御分野）
Angiogenesis is the process of sprouting and extending new branches from existing vessels in response to pro-angiogenic signals. In the angiogenic environment, there are two types of endothelial cell (EC)s called “tip cells” which migrate by dynamic filopodia and make new sprouts at the leading front of growing vascular network, and “stalk cells” which stabilize new sprouts and form nascent vascular lumen following tip cells. The balanced tip/stalk selection is required for proper angiogenesis, and VEGF and Notch signaling pathways play crucial roles in this selection. Notch signaling directs ECs towards stalk cells, whereas VEGF stimulates tip cell formation. However, transcriptional program which determines tip cell identity under VEGF signaling remains unaddressed.
Forkhead box (FOX) O1 is a member of Forkhead-type transcription factor family which involved in various biological processes, including cell cycle regulation, apoptosis, and energy metabolism. Importantly, FoxO1 is implicated in angiogenesis. FoxO1-deficient mice die around embryonic day 11 due to EC abnormalities.
To delineate the biological roles of FOXO1 in ECs during angiogenesis, we performed ChIP-seq analysis using FOXO1 antibody after treatment with VEGF in human primary cultured endothelium. The FOXO1 peaks were co-enriched around the transcriptional active histone marks H3K4me3 and H3K27Ac, but not silencer mark H3K27me3, suggesting that the FOXO1 works as the permissive transactivator. Moreover, enrichment of FOXO1 was almost exclusively observed at the promotor region of Tip-marked genes, such as endothelial specific molecule (ESM)1, ANGPT2, CXCR4 and APLN. Especially, ESM1 gene has two significant FOXO-binding peaks within the proximal promoter and the 8.5 kb upstream region. To investigate whether the expression of ESM1 is actually regulated by FOXO1, we performed luciferase reporter assay using human ESM1 promotor and the upstream regions where FOXO1 bound. ESM1 promoter was transactivated by FOXO1 in ECs and its promoter activity was further increased by the upstream regions, indicating this region is the enhancer of ESM1. Finally, we carried out immunofluorescence staining of retina to confirm whether Foxo1 regulates Esm1 expression in physiological angiogenesis in vivo. Transactivated Esm1 was found in tip cells of the retinal vasculature, where Foxo1 is nuclear-translocated.
Taken together, these results suggest that FOXO1 is a key regulator for tip cells identify through regulating ESM1 expression. Further analysis of tip cell specified transcriptional program regulated by FOXO is require for answering the detailed molecular mechanism of angiogenesis.