Figure 6

The zinc finger domains and C-terminal region of the GATA4 protein are required for the transcriptional cooperation and direct interaction with WT1. A. The domain of the GATA4 protein involved in the transcriptional cooperation with WT1 was identified using the full-length GATA protein and four different GATA4 deletion mutants as indicated. HeLa cells were co-transfected with the -340 bp mouse Sry promoter (500 ng) along with an empty vector or expression vectors for the different GATA4 constructs in the absence (-) or presence (+) of a WT1(+KTS) expression vector (500 ng). All promoter activities are reported as fold activation over control ± S.E.M. The dotted line indicates the activation induced by WT1(+KTS) alone. *, Significantly different from the activation elicited by WT1 alone (P < 0.05). B. In vitro pull-down assays were performed using 500 ng of bacterially produced HIS-WT1(-KTS), HIS-WT1(+KTS) and HIS-LacZ fusion (-ve control) proteins and in vitro translated ³⁵S-labeled GATA4 proteins. After extensive washes, the bound proteins were separated on a 10% SDS-PAGE gel and visualized by autoradiography. Input corresponds to 10% of the total ³⁵S-GATA4 used in each assay. C. GATA4 interacts with both WT1(-KTS) and WT1(+KTS) isoforms. HeLa cells were transfected with expression vectors for either GATA4 alone or GATA4 in the presence of WT1 (+ or -KTS isoforms). A 100-μg aliquot of nuclear extract was then immunoprecipitated (IP) with an antibody for WT1. The precipitated material was then subjected to Western blot (WB) analysis for GATA4. The nuclear extracts used in the IPs were also directly immunoblotted (2.5 μg per lane) to control for the specificity of the GATA4 and WT1 antisera.