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The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that plays an important role in controlling urogenital development. Previously, WT1 has been shown to inhibit cell growth and to repress transcription initiated from the promoters of a number of growth-promoting genes. However, few physiological target genes that are transcriptionally activated by WT1 have been established. Using suppression subtractive hybridization polymerase chain reaction, we isolated a WT1 target gene that is up-regulated about 15-fold in cells expressing WT1. The gene was identified as retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46), a nuclear protein that interacts physically with Rb and is a component of the human mSin3 co-repressor complex. Cells transfected with RbAp46 cDNA formed fewer colonies than the control cells, and RbAp46 suppressed the growth rate (by about 2-fold) of transfected cells. In the developing kidney and gonad, RbAp46 exhibits an expression pattern similar to that of WT1. We conclude that RbAp46 has strong growth inhibition activity and may function as an important mediator of WT1's function.

The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that represses transcription of a number of growth-promoting genes and inhibits cell growth. The transcripts of wt1 undergo two alternative splicing events, giving rise to four isoforms of mRNA in constant ratios. The first alternative splice introduces an extra exon 5, which encodes 17 amino acid residues inserted between the transcription regulatory domain and the DNA binding domain of WT1. Previously, we demonstrated that the 17-amino acid domain functioned as a transcription repressor when it was fused with the DNA binding domain of WT1. We have now identified a point mutation within exon 5 of wt1 in a sporadic unilateral Wilms' tumor patient. The mutation changes the last of the 17 amino acids from asparagine to serine. The protein isoform of WT1 carrying this mutation exhibited a 2-3-fold lower transcription-repressing activity than wild-type WT1 in transient cotransfection assays. The mutation also decreased growth-inhibiting activity of WT1 in two osteosarcoma cell lines, U2OS and Saos-2. By diminishing transcription-repressing and growth-inhibiting activities of WT1, this naturally occurring mutation within exon 5 of wt1 may disturb the normal function of the protein and lead to the uncontrolled cell growth characteristic of Wilms' tumor.