Structural and Functional Studies of the Wilms' Tumour 1 Protein (WT1) in Interaction with Nucleic acids

Abstract: The WT1 protein, a product of the Wilms’ tumour 1 gene (WT1) is a zinc finger transcription factor implicated in a number of cellular processes particularly involved in the development of the urogenital system. Mutations in this gene have been implicated in abnormal development of the urogenital system resulting in syndromes such as the Denys-Drash, WAGR and Frasier and in the childhood kidney malignancy, Wilms’ tumour. Although WT1 was originally cloned as a tumour suppressor, it has most recently been described as an oncogene in some adult cancers. This dichotomous activity in cancer is cell and isoform specific though the exact mechanism responsible is yet unknown. In order to perform its regulatory activity WT1 interacts with DNA, RNA and proteins. The DNA and RNA binding activities of WT1 are restricted to the C-terminal zinc finger domain, which is made up of 4 C2H2 zinc fingers. Two of these isoforms are distinguished by the presence or absence of a 3 amino acid insert, Lysine-Threonine-Serine (KTS) in the linker between zinc-fingers 3 and 4. The –KTS and +KTS isoforms of WT1 differentially recognize DNA and RNA. Sequence examination of the zinc fingers of WT1 reveal significant differences between zinc finger 1 and the other 3 zinc fingers. Attempts at identifying DNA binding specificity for these zinc fingers have failed to successfully identify any specificity for zinc finger 1. We seek to elucidate the molecular mechanism that governs nucleic acid binding by WT1. Our biochemical and structural data enable the detailed description of the molecular interactions involved in the binding activity of the two major isoforms of WT1. These interactions reveal that the +KTS isoform binds DNA in a similar manner to the –KTS isoform. The +KTS isoform has until recently only been assigned a posttranscriptional role. These results provide the necessary detail to conclude that the +KTS isoform also possesses transcriptional capabilities similar to those of its counterpart, the -KTS isoform. We also show that the first zinc finger in this domain does not have DNA binding specificity and contributes very little to the overall affinity. The other 3 zinc fingers are specific DNA binding zinc fingers that define the transcriptional targets of WT1. Our studies with RNA reveal a synergistic effect between the KTS insert and the different zinc fingers but do not reveal any significant role for zinc finger 1. This study however suggests that the binding of WT1 to RNA depend strongly on the sequence and 3-dimensional structure of the RNA.