Fig. 4

ETV2 activates FLK1 expression through AP-1 binding sites in Flk1 enhancer region. a Schematic diagram of Flk1-promoter-enhancer (p/e)-luciferase plasmid. Three potential AP-1 binding elements were marked as diamonds. b, c Luciferase-based promoter assay. b pGL3-Flk1 p/e was transiently co-transfected with pCMV-Etv2, pMCL-MAP2K1-8E, pCMV-c-Jun ND (dominant negative form of c-Jun) into HEK/293T cells. n = 3, **p < 0.01, ***p < 0.001. c pGL3-luciferase construct carrying wild type (Wt), putative AP-1 binding site mutants (Mt #1, Mt #2, Mt #3) or enhancer deletion mutant (del) of Flk1 p/e was co-transfected with pCMV-c-Jun and c-Fos into HEK/293T cells. Firefly luciferase activity was normalized by Renilla luciferase activity. n = 3, ***p < 0.001. d A working model for ETV2-miR126/MAPK in regulating FLK1 expression. In addition to the direct binding of ETV2 on the ETS binding sites in FLK1 gene, ETV2 activates miR-126, which can target SPRED1, thereby activating the MAPK pathway. JUN/FOS, a downstream signaling complex of the MAPK pathway, subsequently activates gene transcription of FLK1 via the AP1 binding sites