The proximal WT1 binding site in mouse Sry promoter is functional. A. A DNA-binding assay was performed with in vitro produced WT1 proteins and a 32P-labeled oligonucleotide probe corresponding to the consensus WT1 binding site of the human AMH promoter. WT1 binding (+ or - KTS isoforms) to the labeled probe was blocked by 10-fold excess of unlabeled probe (self) and unlabeled oligo corresponding to the proximal WT1 site (site 1) of the mouse Sry promoter. B. Western blot analysis of nuclear extracts (10 μg) from PGR 9E11 cells (a pig genital ridge cell line) using antisera against WT1 and GATA4. Nuclear extracts from HeLa cells overexpressing WT1(-KTS) and MA-10 Leydig cells were used as positive controls for WT1 and GATA4 expression, respectively. C. Transfection studies performed in homologous PGR cells confirm the importance of the proximal WT1 site for basal Sry promoter activity. PGR cells were transfected with the deleted or mutated mouse Sry promoter constructs (500 ng) as indicated. Results are shown as % activity relative to the intact -340 bp construct ± S.E.M. Like letters indicate no statistically significant difference between groups (P > 0.05). D. GATA4 is associated with the SRY promoter in PGR genital ridge cells. PGR cell lysates were prepared and interaction of GATA4 with the endogenous pig SRY promoter was studied by ChIP. An aliquot of chromatin preparation before immunoprecipitation (20% input) was used as positive control. Chromatin was precipitated with a GATA4 antiserum (αGATA4) or incubated with goat-IgG (IgG) which served as a negative control. A 320-bp DNA fragment spanning a portion of the SRY promoter containing the proximal WT1 binding site (ChIP targeting region 2) was amplified by PCR as indicated by the arrow. A more distal SRY promoter fragment lacking this WT1 site (ChIP targeting region 1) was not amplified.