Fig. S3

Fig. S3

Figure S3 (Related to Figure 4): The Dll4 F2 enhancer contains multiple ETS binding sites. (A) ClustalW analysis of the 802-bp Dll4 F2 evolutionarily conserved region (ECR), comparing mouse, human, and opossum sequences. The region contains 9 perfectly conserved ETS consensus (GGA(A/T)) sequences (red boxes). Murine F2 contains a total of 14 ETS sites, with five more non-conserved sites (blue boxes). The minimal 36-bp enhancer (F2-6) is highlighted (yellow box). Asterisks denote conserved nucleotides across all three species. (B) Recombinant ETS1 DNA-binding domain (DBD) was used in EMSA with radiolabelled probe encompassing a confirmed ETS-binding site from the Mef2c F7 enhancer. Lane 1 contains reticulocyte lysate lacking ETS1 DBD. ETS1 DBD bound the bona fide ETS-binding site in the control probe (Mef2c F7) (lane 2) and this specific binding was competed away by unlabeled control probe (lane 3), but binding was not competed by a control probe with a mutated ETS site (lane 4). Unlabeled probes corresponding to each of the potential ETS sites in the Dll4 F2 enhancer were used as competitors for ETS1 DBD binding to the control probe (lanes 5-18). The Dll4 F2 sites competed for ETS DBD binding to varying degrees. Probes encompassing ETSB, ETS-D, ETS-H, ETS-I, ETS-J, and ETS-L competed most efficiently (lanes 8, 10, 13, 14, 15, 17, respectively). In addition to the GGA(A/T) core, ETS-B and ETS-H possessed (or overlapped with) canonical ETS sites (cETS). cETS-1/ETS-B was a consistent competitor (lanes 5 and 8), and directly bound the ETS1-DBD (panel C), but cETS-2 (lane 6) and ETS-H (lane 13) did not compete consistently, and they did not bind the ETS1-DBD directly (data not shown). (C) EMSA showing binding of ETS1 DNA binding domain (DBD) to the ETS-B site of F2-6. Binding was competed by an excess of unlabeled wild-type but not ETS-B mutant probe. (D) EMSA was performed using F2 probe and recombinant ETV2 protein. Wild-type F2 sequence effectively competed for ETV2 binding, but this competition was lost when the ETS-B site was mutated.