Abstract
Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage.
Highlights
The endothelium is a monolayer of cells which lines the blood vessel luminal side, in direct contact with the circulation
We identified several important regulatory regions and sites and showed that expression of VEstatin/egfl7 is directly regulated by the Erg and GATA-2 transcription factors and indirectly by Fli-1
In order to assess whether the VE-statin/egfl7 gene could be located in a larger cluster of endothelial-specific genes on chromosome 2, the expression levels of VE-statin/egfl7 and that of its flanking neighbors notch1 and agpat2 were measured
Summary
The endothelium is a monolayer of cells which lines the blood vessel luminal side, in direct contact with the circulation. Endothelial cells participate to or regulate thrombosis, leukocyte extravasation, vasodilatation, and angiogenesis. They express most several markers which define their identity. In addition to cis-acting regulation, epigenetic modifications directly participate to the endothelial-specific regulation of gene expression; eNOS is highly methylated in non-endothelial cells and is enriched in acetylated H3 and H4 histones in the regions surrounding the promoter and the transcription start site in endothelial cells [15,16]. Notch is enriched in acetylated histones along its regulatory regions in the first intron and third exon in endothelial cells [17] Other endothelial genes such as endothelial protein C receptor epcr [18] and von Willebrand factor vwf [19] are epigenetically regulated in endothelial cells
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