Figure 3

The RND1 locus contains an active endothelial specific enhancer with Notch binding sites. (A) RND1 is induced by EGTA at similar rates and magnitude as well-characterized HES and HEY Notch targets. (B) ENCODE database view of the RND1 locus shows a DNAse hypersensitivity peak at the promoter region in all cell types (blue bar) and a second peak upstream that appears only in endothelial cell types (yellow bar). The endothelial-specific peak shows histone methylation patterns consistent with active enhancers (insert box). Gray bars mark CTCF-binding insulator regions. (C) Enhancer region sequence in the RND1 locus with two Notch consensus sequence (green highlight). Also highlighted is the primer sequence for ChIP PCR (orange highlight). (D) ChIP assay of the putative endothelial-specific RND1 enhancer with antibodies against the intracellular domain of Notch1 (N1ICD). N1ICD binds to the RND1 enhancer after EGTA induction (left) or DLL4 overexpression, but binding is blocked with CpE treatment, indicating that active Notch signaling is required. (E) HUVECs transfected with siCon or siRND1 or infected with Control or Notch1IC lentiviral constructs were serum-starved for 3 h, treated with 100 ng/ml EGF, and analyzed for Ras activity by G-LISA after 0, 5, and 10 min. (F) HUVECs as in (E) were treated with 50 nM thrombin and analyzed for RhoA activity by G-LISA after 0, 2, and 5 min.