A STRUCTURE-FUNCTION ANALYSIS OF TRANSCRIPTIONAL REPRESSION MEDIATED BY THE WT1, WILMS-TUMOR SUPPRESSOR PROTEIN

The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-co ntaining transcription factor (WT1). WT1 binds to the consensus sequen ce (5'-GCGGGGGCG3') and represses transcription when bound to this sit e in vivo. The mechanism of repression is not yet defined. To investig ate the mechanisms of transcriptional repression and map the domains o f WT1 responsible, we constructed hybrid proteins between the yeast GA L4 1-147 DNA binding domain and WT1. Fusion of a 298 amino acid glutam ine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding dom ain created a potent transcriptional repressor. The use of N- and C-te rminal truncations of this segment demonstrated that as few as 96 amin o acids were required for active repression by GAL4-WT1 hybrid protein s in NIH3T3 fibroblasts. However, the truncated GAL4-WT1 fusion protei ns functioned poorly as repressors in embryonic kidney-derived 293 cel ls, suggesting cell type-specific requirements for transcriptional rep ression. Site-directed mutagenesis of the WT1 repression domain reveal ed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression fu nction. Single repressor binding sites placed upstream of the transcri ption start site conferred WT1-mediated repression to a heterologous p romoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression. Significant repression of tr anscription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transc ription initiation. We conclude that the transcriptional repression fu nction of WT1 is contained in the N-terminal, non-DNA binding domain o f the protein and that repression can be functionally transferred to a heterologous DNA binding domain.