Figure 4

The mouse Sry promoter requires an intact proximal WT1 binding site for full transcriptional synergism by GATA4 and WT1. A. Deletion and mutation analysis of mouse Sry 5' flanking sequences in HeLa cells. HeLa cells were co-transfected with the different mouse Sry promoters (500 ng) as indicated along with an empty vector (control) or expression vectors for GATA4 and/or WT1(+KTS). B. The mouse Sry promoter contains two low affinity GATA binding elements (named sites A and B) located between -340 and -70 bp. An EMSA was performed with recombinant GATA4 protein and a ³²P-labeled oligonucleotide probe corresponding to the consensus GATA element from the proximal murine Star promoter [70]. Competition with unlabeled probe (self) and oligonucleotides corresponding to GATA sites A and B of the mouse Sry promoter was used to assess the affinity of GATA4 binding to these sites. C. The low affinity GATA binding sites (A and B) of the proximal mouse Sry promoter are functional. The remaining GATA4/WT1 synergism observed on the -340 bp Sry construct harboring the WT1 site mutation (-340 bp WT1 mut.) is abolished when two different GATA4 DNA-binding mutants (C294A or ΔT279) are used. For the wild-type and mutated GATA4 constructs, a truncated GATA4 protein (aa 201–440; see diagram in Fig. 6A) was used. For all transfection experiments, promoter activities are reported as fold activation over control ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05).