Studies of p53 -mediated mdm2 and gadd45 gene activation in nuclear chromatin.
Item
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Title
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Studies of p53 -mediated mdm2 and gadd45 gene activation in nuclear chromatin.
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Identifier
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AAI9959242
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identifier
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9959242
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Creator
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Xiao, Gu.
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Contributor
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Adviser: Jill Bargonetti
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Date
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2000
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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 tumor suppressor p53 is a transcription factor with the sequence-specific DNA binding activity. A variety of its target genes have been identified when p53 was overexpressed. However, none of the sequence of the binding site completely fits the consensus sequence, and whether p53 is able to bind to these binding sites in vivo still unknown. In addition, protein products of the p53-target genes have diverse functions. In this study, we have investigated p53-mediated mdm2 and gadd45 gene activation in two systems. One is that it utilizes murine cells containing a temperature sensitive p53-Val 135. At 32°C, p53-Val135 assumes a wild-type conformation, while at 37°C this p53 is conformationally mutant. The other system is by an inducible promoter that regulates p53 levels to analyze p53-mediated gene activation both in the presence and absence of DNA damage. Gadd45 protein induces cell growth arrest and protects genome integrity while Mdm2 protein promotes p53 degradation and blocks its transcriptional activity. Additionally, the transcriptional activity of p53 was considered to be part due to the increasing protein levels. Here, we provide evidence that (1) p53 binds specifically to the murine mdm2 P2 promoter in nuclear chromatin, and this binding correlates with mdm2 endogenous mRNA induction; (2) the mdm2 P2 promoter is maintained in a nucleosome free state and activation of the mdm2 gene does not require chromatin remodeling; (3) the p53 target genes p21, gadd45, and mdm2 are differentially activated in the presence and absence of DNA damage. Both induced p53 and DNA damage signaling are necessary to activate the gadd45 gene; (4) the p53-mediated gadd45 induction by camptothecin did not require a further increase in nuclear p53 levels, or DNA binding activity, or phosphorylation of p53 at serine 15 and acetylation of p53 at lysine 382; (5) constitutive DNase I hypersensitivity was detected by indirect Southern blot analysis at both the gadd45 promoter and its putative p53-RE, but after camptothecin treatment increased DNase I sensitivity was only detected at the gadd45 promoter region. We conclude that both mdm2 and gadd45 regulatory regions are preset for rapid activation by p53. Up-regulation of mdm2 is independent of change of chromatin structure. Induction of gadd45 gene expression by p53 and DNA damage signaling alters the configuration of chromatin at the gadd45 promoter region. Finally, the stability of p53 and the transcriptional activity of p53 are regulated at different levels.
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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.
