The Transcriptional Effect of WT1 Is Modulated by Choice of Expression Vector

Josina C Reddy,Seiyu Hosono,Jonathan D Licht

doi:10.1074/jbc.270.50.29976

Josina C Reddy, Seiyu Hosono + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.50.29976

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Abstract

The WT1 Wilms' tumor suppressor gene encodes a zinc finger transcription factor which plays a critical role in renal and genitourinary development. The WT1 protein was reported to both activate and repress transcription. We found that the transcriptional effect of WT1 on the Egr1 promoter could be modulated by the use of expression vectors containing different promoters. WT1 activated the Egr1 promoter when expression of WT1 was driven by the Rous sarcoma virus promoter. In contrast, a cytomegalovirus (CMV) promoter-containing WT1 expression vector repressed the Egr1 promoter. However, WT1 activated transcription of a simple test promoter, EGR3tkCAT, regardless of the expression vector used. Co-transfection of the parental CMV-based vector strongly depressed the basal activity of the Egr1-CAT reporter, suggesting that the CMV promoter competes with the Egr1 promoter for transcription factors or co-factors which may be required for activation by WT1. In support of this hypothesis, WT1 was converted from an activator to a repressor by co-transfection of an excess of the parental CMV-based vector. These results provide an important caveat to the interpretation of co-transfection studies and confirm the bi-functional nature of the WT1 transcription factor.

Highlights

The Wilms’ tumor suppressor gene WT1 was isolated by positional cloning based on the presence of a constitutional deletion of chromosome 11p13 in patients with the WAGR syndrome (Wilms’ tumor, aniridia, genitourinary malformations, and mental retardation) [1, 2]
These results suggest that competition between the CMV and Egr1 promoters for transcription factors and/or cofactors results in changes in the basal activity of the Egr1 promoter and that only under conditions of low basal activity is the Egr1 promoter able to be repressed by WT1
To investigate the conditions that might affect the ability of WT1 to activate or repress transcription, we co-transfected both NIH 3T3 cells and CV1 cells with an Egr1 promoter-containing reporter gene and the RSV-WT1 expression vector

Summary

Introduction

The Wilms’ tumor suppressor gene WT1 was isolated by positional cloning based on the presence of a constitutional deletion of chromosome 11p13 in patients with the WAGR syndrome (Wilms’ tumor, aniridia, genitourinary malformations, and mental retardation) [1, 2]. We confirmed the importance of this promoter competition effect by demonstrating that WT1 expressed from the RSV vector was converted from an activator to a repressor of the Egr promoter by co-transfection of an excess of pCB6ϩ vector lacking a WT1 cDNA. Taken together, these results suggest that competition between the CMV and Egr promoters for transcription factors and/or cofactors results in changes in the basal activity of the Egr promoter and that only under conditions of low basal activity is the Egr promoter able to be repressed by WT1. Our results provide an important caveat regarding choice of expression vector in transfection experiments

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