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Fig. 4
p300 is dynamically recruited to the DLL4 enhancer and regulates sprouting angiogenesis. (A) Recruitment of p300 to the DLL4 enhancer located in intron 3 15-30′ after VEGF stimulation, as assessed by ChIP (n=5 for ERG and p300 ChIP, n=3 for H3K27ac ChIP). (B) p300 recruitment in response to VEGF stimulation requires ERG. Shown is a representative experiment of two with triplicate determinations. The extent of ERG knockdown as assessed by western blot is shown to the right. (C) Endogenous p300 and ERG physically interact by co-immunoprecipitation in HUVECs stimulated with VEGF. Shown is a representative experiment of three. (D) Exogenous Myc-p300 interacts with wild-type Flag-ERG in BAECs, but does not interact with phospho-mutant (S96A;S215A;S276A) Flag-ERG by co-immunoprecipitation. Shown is a representative experiment of three. (E) p300 activity is required for DLL4 mRNA induction in HUVECs in response to VEGF stimulation (n=5). c646 is a potent inhibitor of p300/CBP activity. (F) Inhibition of p300 activity suppresses ISV elongation in zebrafish. Quantification is shown to the right. Note: Quantification of the DMSO control is the same as that shown in Fig. 2B, as both inhibitors were used in the same experiment. Scale bars: 50 μm. NC, negative control (V5 antibody).