The WT1 Wilms' tumor suppressor protein: Transcriptional activity and modulation of function by tumor-associated mutations.
Item
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Title
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The WT1 Wilms' tumor suppressor protein: Transcriptional activity and modulation of function by tumor-associated mutations.
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Identifier
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AAI9605651
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identifier
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9605651
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Creator
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Reddy, Josina Clare.
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Contributor
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Adviser: Jonathan D. Licht
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Date
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1995
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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The WT1 tumor suppressor gene is mutated in a small subset of patients with Wilms' tumor, a childhood kidney cancer. WT1 encodes four zinc finger DNA-binding transcription factors with both transcriptional activation and repression functions. In this thesis, I characterized the transcriptional effector functions of WT1 and studied the effect of tumor-associated WT1 mutations on WT1 protein function. WT1 activated a simple test promoter containing three Egr-1/WT1 sites upstream of the HSV-tk promoter in transient transfection experiments. The WT1(B) isoform was a slightly stronger transcriptional activator than WT1(A). WT1 also repressed transcription under certain circumstances. A GAL4-WT1 fusion protein repressed transcription through GAL4 binding sites, but failed to regulate transcription through WT1 binding sites, suggesting that the WT1 moiety might be in a non-native conformation. Native WT1 repressed the Egr-1 promoter when it was expressed from a CMV-based expression vector, while WT1 expressed from an RSV-based expression vector activated this same promoter. Co-transfection of this CMV expression vector greatly depressed the basal transcriptional level of the Egr-1 reporter construct, and in this depressed state, WT1 expressed from the RSV vector could repress the promoter. This suggests that the CMV and Egr-1 promoters compete with each other for transcription factors or cofactors which may modulate the transcriptional function of WT1. Expression of two genetically defined tumor-associated dominant negative WT1 alleles which yield WT1 proteins unable to bind DNA inhibited transcriptional activation by wild-type WT1. The WT1 protein self-associated and associated with mutant WT1 proteins in an in vitro biochemical assay. I therefore propose that these dominant negative WT1 proteins act by binding to wild-type WT1 and inhibiting its transactivation function. In contrast, a tumor-associated point mutation of WT1 yielded a protein which displayed DNA-binding, transactivation, transrepression, and self-association functions which were indistinguishable from the wild-type protein. This mutation may not be the primary etiologic event in this case of Wilms' tumor, and may be augmented by changes at a second Wilms' tumor locus.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
