Figure 9: Wt1 transcriptionally activates Pecam-1 and c-kit.

Schematic representation of the putative Wt1-binding sites in the Pecam-1 (a) and c-kit (d) promoters. Positions of the cloned promoters relative to the transcription start site, positions and sequences of the putative Wt1-binding sites and positions of the oligonucleotides used for CHIP analyses are indicated. For promoter-deletion constructs, the indicated Wt1-binding sites were removed from the promoter reporter constructs. Luciferase activity of reporter constructs carrying mouse Pecam-1 promoter (b) or c-kit promoter (f) in the presence of Wt1(−KTS) or Wt1(+KTS) expression constructs. Transient transfections were performed using HEK293 cells (n=12 each). The promoterless luciferase expression construct (pGl3basic) served as a negative control. ΔWTB indicates reporter constructs with deletion of the predicted Wt1-binding sites. Chromatin immunoprecipitation (ChIP, n=4) was performed in M15 cells using either monoclonal or polyclonal antibodies against Wt1 or anti-acetyl-histone H3 antibody. Normal rabbit serum served as a negative control. Input DNA was used as a positive control for quantitative PCRs (graphs on the left) or semiquantitative PCRs (representative agarose gels on the right) for the Pecam-1 promoter (c) or c-kit promoter (g) and respective 3′UTR sequences. Confocal images of double-labelled tumour sections for Wt1 and c-kit (e) revealed partial overlapping expression of both molecules within tumours. Scale bars, 50 μm. Data are the mean±s.e.m. **P<0.01, ***P<0.001.