TRUNCATED WT1 MUTANTS ALTER THE SUBNUCLEAR LOCALIZATION OF THE WILD-TYPE PROTEIN

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, tha t is inactivated in a subset of Wilms tumors, Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observ ed dramatic differences in the subnuclear localization of the induced proteins, The WT1 isoform that binds with high affinity to a defined D NA target, WT1(-KTS), was diffusely localized throughout the nucleus, In contrast, expression of an alternative splicing variant with reduce d DNA binding affinity, WT1(+KTS), or WT1 mutants with a disrupted zin c-finger domain resulted in a speckled pattern of expression within th e nucleus. Although similar in appearance, the localization of WT1 var iants to subnuclear clusters was clearly distinct from that of the ess ential splicing factor SC35, suggesting that WT1 is not directly invol ved in pre-mRNA splicing, Localization to suhnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redis tribution of WT1(-KTS) from a diffuse to a speckled pattern, and the i nhibition of its transactivational activity. These observations sugges t that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments, Dominant-negative WT1 proteins physically associate wit h wild-type WT1 in vivo and may result in its sequestration within sub nuclear structures.