Fig. S4

Identification and analysis of the Ephb4-2 and Ephb4-10 enhancers. a. The 5’ upstream region of the human EPHB4 gene as seen on UCSC Browser (http://genome.ucsc.edu). Histone marks (light blue) and DNase I hypersensitivity (HS, peaks shown as a heat map, different cell type names written on left with endothelial lines denoted in red) are concentrated around the two putative enhancers, marked as -2 and -10 and by red horizontal lines. Conservation between the human sequence and mouse, manatee, chicken, xenopus and zebrafish is indicated by black vertical lines, demonstrating that neither enhancer is conserved beyond manatee. b. Probability weight matrices from JASPAR showing the consensus binding motifs for ERG, ETV2 and SMAD (referred to in text as the SBE). Red boxes denote the core base pairs used for analysis of Ephb4-2 and Ephb4-10 sequences. Dotted line denotes additional base pairs considered to decide which EBE most adhered to consensus. c-d. ClustalW alignment of the Ephb4-2 (c) and Ephb4-10 (d) mouse and human enhancer sequence annotated with conserved ETS binding elements (EBE, yellow) and SMAD binding elements (SBE, blue). Numbers refer to mutations analysed in Figure 5 and Supplementary Fig. 5. Bold EBE refer to those that adhere to a more stringent consensus binding motif denoted by red dashed lines in b. Base-pairs mutated in mutCONT transgene are denoted in orange text. e. IGV browser view of 5’ region of EPHB4 gene and upstream sequence incorporating ERG ChIP-seq data from Fish et al10. Both Ephb4 putative enhancers (red horizontal bars) have significant ERG binding peaks in HUVECs (yellow peaks). f. Table summarizing Ephb4-2:GFP and Ephb4-10:GFP transgene activity as detected by GFP expression in 48hpf transient transgenic zebrafish. g-j. Relating to Fig. 4c, expression in the transgenic zebrafish line tg(Ephb4-2:GFP; kdrl:HRAS-mCherry) as shown by representative embryos at 20hpf (g), 24hpf (h), 36hpf (i) and 96hpf (j). a=dorsal aorta, v=dorsal/ventral veins.