Two N-Terminal Self-Association Domains Are Required for the Dominant Negative Transcriptional Activity of WT1 Denys-Drash Mutant Proteins

Abstract

Patients with Denys-Drash syndrome (DDS) have been shown to be constitutionally heterozygous for mutations of theWT1 gene. Almost all DDS mutations inactivate or remove the DNA-binding zinc finger region of WT1 and the resulting mutant proteins appear to act in a dominant negative manner. This may occur via WT1 self-association, which has been shown to involve the first 180 amino acids. By creating a series of N-terminal deletions, we have further investigated WT1 self-association using a yeast di-hybrid system and anin vitroprotein binding assay. Our results suggest that there are two distinct domains within the N-terminal region facilitating self-association, residing from amino acids 1-45 and 157-253. Co-transfection of WT1 with progressively shorter N-terminal constructs demonstrates that both of these sites are required for a dominant negative activity as assessed by activation of a reporter construct.