The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct

Abstract

The t(11; 22)(p13; q12) translocation associated with desmosplastic small round cell tumor results in a chimeric molecule fusing the amino terminal domain (NTD) of the EWS1 gene to three of the four carboxyterminal zinc ®ngers of the WT1 tumor suppressor gene. Since the DNA binding domains of WT1 and EWS/WT1 are structurally di erent, we have assessed the functional consequences of the EWS/WT1 fusion. We ®nd that the EWS/WT1 protein has a higher binding a nity for a given recognition target than the WT1 product. This is unlike other fusion products involving translocation of the NTD of EWS to DNA binding domains in which DNA binding speci®city and a nity is not changed. We demonstrate that EWS/WT1 is a nuclear protein and that the NTD of EWS contains (a) nuclear localization signal(s). We also ®nd that the integrity of a domain within the WT1 zinc ®ngers, responsible for mediating interaction between WT1 and the transcriptional repressor par-4, is disrupted in the EWS/WT1 fusion product. Deletion analysis of the NTD of EWS indicated that integrity of the entire domain was necessary to achieve full transactivation potential.

Analysis of WT1 target gene expression in stably transfected cell lines

“…Similar to the present study no eect of wild type WT1 could be detected on the expression of PDGFA and a number of other putative target genes. Most recently, Kim et al (1998) have shown that DNA binding by the EWS-WT1 fusion protein is much stronger compared to wild type WT1, providing additional evidence for distinct functions of these genes.…”

PDGF-A, PDGF-Rβ, TGFβ3 and bone morphogenic protein-4 in desmoplastic small round cell tumors with EWS-WT1 gene fusion product and their role in stromal desmoplasia: an immunohistochemical study

“…[9][10][11] Alternatively, the fusion protein may also ctivate target genes that are not regulated by wild-type WT1 due to changes in the DNA binding domain. 12 A previous in vitro study has shown that PDGF-A was upregulated by EWS-WT1 in desmoplastic small round cell tumor specimens as well as in an inducible cell line. 9 Expression of PDGF-A, a potent mitogen and growth factor for fibroblasts, has been speculated to play a role in the characteristic tumor-associated desmoplasia in desmoplastic small round cell tumor, the extent of which varies from case to case.…”

Rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax : Bcl-xL ratio through concurrent mechanisms dependent and independent of its mTOR inhibitory activity

“…However, EWS/WT1 downregulation did not correlate with rapamycin-induced apoptosis, suggesting that the known targets of this aberrant transcription factor are most likely not involved in the apoptotic process induced by rapamycin. In agreement with its distinct DNA-binding specificity relative to WT1 (Kim et al, 1998a), microarray analysis has shown that several apoptosis-related genes are regulated by EWS/ WT1 (ÀKTS) (Ito et al, 2003). Interestingly, the normal WT-1 protein is also expressed in DSRCT (Ladanyi and Gerald, 1994), and its activity is believed to be overcome by the stronger activity of EWS/WT1 (Scharnhorst et al, 2001).…”