Diversity Within the Master Regulatory p53 Transcriptional Network: Impact of Sequence, Binding Motifs and Mutations Public Deposited

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  • March 20, 2019
Creator
  • Jordan, Jennifer
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • In response to cellular stress and DNA damage, the master regulatory gene p53 directly controls the differential expression of target genes within its extensive transcriptional network through promoter response elements (REs) to elicit many biological responses, including cell cycle arrest and apoptosis. Tetrameric p53 binds the consensus sequence RRRCWWGYYY (n= 0-13) RRRCWWGYYY, where R=purine, W=A/T and Y=pyrimidine. Phenotypic diversity within the p53 transcriptional network occurs as a result of a variety of factors including cell type, post-translational modifications, stress-inducing stimuli, and mutations in p53 that alter transactivation function through changes in the strength of gene activation or spectra of genes regulated. We have developed an isogenomic diploid yeast-based reporter system to evaluate the contribution of target binding sequence, organization and level of p53 on transactivation at target REs by wild-type and mutant p53. The chromosomal position for all of the human derived REs was identical and the number of p53 molecules/cell could be varied over a hundred-fold using an integrated rheostatable GAL1::p53 promoter that is sensitive to level of galactose in the medium. We confirm transactivation by WT p53 differs between REs where small differences in sequence can contribute significantly to levels of transactivation. Through deconstruction of the canonical RE and evaluating transactivation from various sequences and binding motifs, we have challenged the view of what constitutes a functional p53 target. We show small increases in distance between decamer half-sites greatly reduce p53 transactivation. Furthermore, we demonstrate that substantial sequence-dependent transactivation can occur from ½- and ¾-site REs. Importantly, the presence of these noncanonical REs greatly expands the p53 master regulatory network. In addition, we have determined the functional fingerprints of missense mutations to demonstrate that altered function p53 mutations can occur in breast cancers and the transcriptional effects are often subtle. Finally, we have identified super-trans sequences which enhance the efficiency of p53 transactivation by greatly lowering the number of molecules required. These sequences provide a useful tool for addressing wild-type and mutant p53 function. Overall, our findings demonstrate that RE sequence, organization, level of p53 and mutations can strongly impact the ability of the master regulator p53 to transactivate downstream targets, thus diversifying its transcriptional network.
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  • In Copyright
Advisor
  • Perou, Charles
  • Resnick, Michael
  • Sharpless, Norman
  • Kaufmann, William
  • Strahl, Brian
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2008
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