Figure 4

ChIP verification of WT1 and SP1 binding to endogenous AR promoter and sequence analysis. Functional WT1 TFBS in AR promoter region were previously identified by EMSA and reporter assays [14, 23]. (A) ChIP analysis of chromatin from WT1 transfected LNCaP prostate cancer cells verified these TFBS were functional. Lane 1 shows 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) Predicted TFBS are based on human sequences and marked by boxes as described in Figure 1. Evidence for conservation of the functional WT1 (human 1434–1450) TFBS was limited by lack of sequence information available for chimpanzee (and lack of conservation with macaque). Surprisingly the TCC rich EGR1 site (human 1524–1537), previously shown to bind WT1 in vitro [14], also showed no evolutionary conservation.