Figure 3

ChIP verification of WT1 and SP1 binding to endogenous VEGF promoter and sequence conservation. Functional WT1 and SP1 TFBS in the VEGF promoter region were previously identified by EMSA and luciferase reporter assays [15]. (A) ChIP analysis of chromatin from WT1 transfected 293 kidney cells verified that these TFBS were functional. Lanes 1 and 7 show the 1 Kb ladder, lane 2 shows the No DNA PCR control, and lane 3 shows PCR amplified input DNA. Lanes 4, 5, and 6 show PCR amplified DNA immunoprecipitated by IgG (no antibody control), WT1 or SP1 antibodies, respectively. (B) ChIP analysis of chromatin from WT1 transfected LNCaP cells verified these TFBS were functional in prostate cancer cells as well. Lanes as described in section (A). (C) Predicted TFBS are based on human sequences and marked by boxes as described in Figure 1. These functional WT1 (human 1755–1771), EGR1 (human 1717–1733) and SP1 (human 1721–1735) sites were conserved between primates (human, chimpanzee, and macaque) and dogs, but not in rodents; and the SP1 site overlapped with the EGR1 site.