Wilms Tumor suppressor
protein (WT1) is a
transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different
cancer forms. The
protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual
transcription factor - an unconventional
amino acid composition for zinc finger 1, and the insertion of a tri-
peptide KTS in some of the splice
isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall
DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal
DNA-recognition domain' for WT1, however the formation of a stable
protein-
DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected.