Elucidating Mechanisms Mediating Genome Recognition and Cell Fate Determination by p53
The tumor suppressor p53 is the central integrator of stress response pathways in human cells. In unstressed cells, p53 is kept at low levels by proteasome-mediated degradation. Once activated, stabilized p53 triggers the appropriate cell fate by selectively activating one of a diverse set of gene expression programs (e.g., cell cycle arrest, apoptosis, DNA repair). A central event in that process is the binding of tetrameric p53 to 20-bp response elements (REs) in the human genome at the consensus sequence (RRRCWWGYYYN0-13RRRCWWGYYY, R=A or G, W=A or T, Y=C or T, N=any base). The unusually degenerate nature of the p53 RE provides the potential mechanism for selective regulation of distinct downstream programs. The main aim of this study was to decipher the RE code and to elucidate DNA-binding mechanisms that allow for differential recognition of genomic targets by p53. In general, REs of pro-survival genes are recognized with higher affinity, while those connected to cell death are recognized with lower affinity, and this quantitative differentiation has been proposed as the basis of p53 target gene selectivity and cell fate decisions. Here we identify distinct molecular mechanisms that mediate p53 binding to high- and low-affinity REs. These include a predominantly non-polar binding mechanism, at higher affinity REs linked to pro-survival genes, (e.g., CDKN1A) and a predominantly polar binding mechanism, at lower affinity REs linked to pro-apoptotic targets (e.g., BAX). We further show that the key determinants of p53 DNA-binding mechanisms and p53 RE code are embedded in the DNA shape. Specifically, we demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. We confirmed these findings by genome editing of the pro-apoptotic BAX RE, which reprogrammed the stress-responsive induction pattern of the BAX gene, and resulted in an in vivo cell fate transformation. These data identify novel mechanisms mediating p53 dose-dependent DNA-binding selectivity and suggest diverse biophysical DNA-binding modes as an additional regulatory mechanism used by transcription factors to trigger distinct downstream transcriptomes and cell fates.
Farkas, Marina, "Elucidating Mechanisms Mediating Genome Recognition and Cell Fate Determination by p53" (2021). ETD Collection for Thomas Jefferson University. AAI28646345.