Figure 2

WT1–ZF protein specific binding to WT1 consensus motifs in WT1 promoter in vivo/in vitro. (a) Chromatin immunoprecipitation/PCR analysis for the cross-linked sheared chromatin from control MCF-7 cells (CON) and MCF-7 cells expressing HA-tagged zinc finger domain (HA/ZF-KTS, HA/ZF+KTS) of Wilms' tumor 1 suppressor gene zinc (indicated on right). Upper panel, schematic representation of PCR-amplified fragments P1 and P2 in endogenous human WT1 promoter. Input, mock control and immunoprecipitation with preimmune serum (PI), anti-WT1, anti-HA or anti-acetyl-histone H4 antibody are indicated. Left lane, 1 kb DNA ladder as marker. (b) Same chromatin immunoprecipitation/PCR analysis as (a) with aldehyde dehydrogenase 1 family member A2 (ALDH) promoter primers. (c) Coomassie blue-stained gel. The bacteria-expressed GST and GST/WT1–ZF fusion proteins (shown as GST, G/ZF-KTS and G/ZF+KTS) were purified, fractionated in a 10% SDS-PAGE and visualized by Coomassie blue stain. The molecular sizes of standard protein markers (M) are shown. (d) Competition electrophoretic mobility shift assays were performed using either GST only or GST/WT1–ZF fusion proteins (G/ZF-KTS, G/ZF+KTS) with the ³²P-labeled oligonucleotide of human WT1 proximal promoter in the absence (-) or presence of 20-fold or 100-fold molar excess of WT1 binding site wild-type or site mutant cold oligonucleotides (WT, Mut) as indicated. Migration of DNA-binding complexes induced by GST/ZF-KTS protein and free probe is shown by an arrow. The lane GST displayed no DNA-binding associated with purified GST protein. KTS, lysine–threonine–serine.