Abstract 2316: Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity.

Abstract

Abstract The p53, p63 and p73 family of sequence-specific transcription factors share similar biochemical properties and structural features resulting in the recognition of DNA with identical amino acids and similar binding affinity. However, loss of individual members results in very different phenotypes in mice as well as humans. We have addressed functional relationships between p53 family proteins using a combination of mutants, target response elements (REs) and sequence-specific transactivation assays based in yeast and human cells. p53 alleles exhibiting enhanced transcriptional activation and altered promoter selectivity were previously identified in the L1 and L3 loop of the DNA binding domain of the protein. Included was S121F that results in defective p21 induction and confers a strong apoptotic phenotype in mammalian cells. Guided by result with p53-S121F, -T123A and -S240N mutants, the corresponding p63 and p73 alleles were investigated for ability to drive transactivation at over fifty p53 REs in isogenic yeast reporter strains. The phenotypes observed with p53 alleles were reproduced with the p63- and p73- alleles. Focusing on p73-S139F (corresponding to p53-S121F) transactivation at many RE sequences, we identify and experimentally confirm an RE target sequence code that predicts enhanced or reduced transactivation potentials. The correlation is particularly strong for induction at p53 targets within promoters of genes associated with apoptosis. Furthermore, p73 S139F showed an enhanced DNA-binding cooperativity in vitro, and loop L1 shows conformational changes in the crystal structure of the protein-DNA complex. The altered function phenotype was confirmed not only in gene reporter assays but also for endogenous gene expression in the p53 null HCT116 human cell line, suggesting that target recognition can predict relative changes in transcription rates at different loci. Based on the correlation between RE code and associated gene functions, we propose the selection during evolution of an RE sequence code within target promoters that can affect intrinsic conformational flexibility / DNA binding affinity of p53 proteins thereby modulating in vivo selectivity, specifically favoring the activation of apoptotic target genes Citation Format: Yari Ciribilli, Paola Monti, Alessandra Bisio, H. T. Nguyen, A S. Ethayathulla, Micheal A. Resnick, Hector Viadiu, Gilberto Fronza, Alberto Inga. Mutations in the p53 gene family reveal conservation of structure-function in the L1 and L3 loops and a response element code for transcriptional specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2013-2316