Fig. 9

A conserved enhancer upstream of HLX is regulated by ETS factors and is required for VEGF induction. (A) Transcription of HLX (as assessed by qRT-PCR of HLX pre-mRNA) reveals dynamic transcription, peaking at 15-30′ post-VEGF stimulation in HUVECs (n=3). (B) p300 is transiently recruited to a putative HLX enhancer element during VEGF stimulation, as assessed by ChIP assay (n=3). (C) p300 ChIP was performed in control and ERG knockdown cells. Shown are triplicate measures of a representative experiment of two. (D) Luciferase analysis of the HLX enhancer (HLX-3a), demonstrating that it is regulated by VEGF and ETS factors. ETS-binding sites were mutated in the HLX enhancer (HLX^ETSmut, see Materials and Methods). A representative experiment (of three) with triplicate determinations is shown. (E) Luciferase analysis of the HLX enhancer (HLX-3a), demonstrating that it is regulated by MAPK/ERK activity. A representative experiment (of two) with triplicate determinations is shown. (F) The human HLX enhancer (HLX-3b) is functional in ISVs in zebrafish during sprouting angiogenesis. Activity is lost when the ETS sites in the enhancer are mutated. Shown are representative images of embryos at 42 hpf. Quantification of enhancer activity is shown to the right. (G) CRISPR/Cas9-mediated deletion of a highly conserved enhancer upstream of HLX inhibits VEGF-mediated induction. Shown are a clonal scrambled-control line (Scr3) and three heterozygous deletion lines (ΔHLX15, ΔHLX17, ΔHLX21). Induction of DLL4 is included as a control. Knockdown of ERG affects the induction of HLX in the control line to a greater extent than in the deletion lines. n=2. (H) Schematic of the VEGF/MEK/ERK/ERG/p300 transcriptional pathway identified in this study.