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The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
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 carboxy-terminal zinc fingers of the WT1 tumor suppressor gene. Since the DNA binding domains of WT1 and EWS/WT1 are structurally different, we have assessed the functional consequences of the EWS/WT1 fusion. We find that the EWS/WT1 protein has a higher binding affinity 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 specificity and affinity 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 find that the integrity of a domain within the WT1 zinc fingers, 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.
Insights
The EWS/WT1 fusion protein, resulting from a specific cancer translocation, exhibits increased DNA binding affinity compared to WT1. This altered binding and disrupted interaction with par-4 contribute to its oncogenic potential in desmoplastic small round cell tumors.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Desmoplastic small round cell tumor (DSRCT) is characterized by the t(11;22)(p13;q12) translocation.
- This translocation creates a fusion gene, EWS/WT1, combining the EWS1 amino-terminal domain (NTD) with WT1 tumor suppressor gene zinc fingers.
Purpose of the Study:
- To investigate the functional consequences of the EWS/WT1 fusion protein.
- To compare the DNA binding properties of WT1 and EWS/WT1.
- To elucidate the role of the EWS NTD in the fusion protein's function.
Main Methods:
- Assessed DNA binding affinity of WT1 and EWS/WT1.
- Determined the subcellular localization of EWS/WT1.
- Utilized deletion analysis of the EWS NTD.
- Investigated interactions with the transcriptional repressor par-4.
Main Results:
- EWS/WT1 demonstrates higher binding affinity for target DNA than WT1.
- EWS/WT1 is a nuclear protein, with the EWS NTD containing nuclear localization signals.
- The EWS/WT1 fusion disrupts the WT1 zinc finger domain interaction with par-4.
- Full transactivation potential of EWS/WT1 requires the integrity of the entire EWS NTD.
Conclusions:
- The EWS/WT1 fusion protein possesses altered DNA binding characteristics and subcellular localization compared to WT1.
- Disruption of WT1-par-4 interaction and enhanced DNA binding contribute to the oncogenic activity of EWS/WT1.
- The EWS NTD plays a critical role in mediating the functional properties of the EWS/WT1 fusion protein.