The

Flk-1 promoter contains 2 functional Ets sites.(A) Nucleotide sequence and putative Ets binding sites of theFlk-1 promoter spanning bp −640 to +16. Sequences matching putative Ets binding sites are underlined. The nucleotide sequence of the Flk-1 promoter spanning bp −640 to bp +299 is deposited in GenBank (accession number AF 153057). (B) 5′-Deletion analysis of the Flk-1 promoter. Luciferase reporter gene constructs were cotransfected with either a c-Ets1 expression vector (black bars) or pcDNA3 (gray bars) as control. Values that are significantly (P < .05, Student t test) below the corresponding values of the previous construct are marked with an asterisk. (C) Mutational analysis of the Ets sites E#3 and E#6. Luciferase reporter gene constructs containing the 939-bpFlk-1 promoter with the wild-type sequence or mutations in the Ets sites E#3 or E#6 (black cross) were cotransfected with either a c-Ets1 expression vector (black bars) or pcDNA3 (gray bars). The sequence 5′-CGGA-3′ of the Ets sites E#3 or E#6 (see Figure 6) was mutated to 5′-CccA-3′. The activation of both mutant promoters by c-Ets1 was significantly lower (P < .05; Studentt test) than the activation of the wild-type promoter by c-Ets1. The promoter activities of the pcDNA3 transfections in A and B were arbitrarily set at 1, and promoter activities were determined as described in “Materials and methods.” The relative induction levels shown in B and C are not directly comparable because the 5′-UTR is lacking in the constructs used for the analysis in B.