Regulation of stochastic gene expression in single cells by the dynamic p53 response to genotoxic stress

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The tumor suppressor p53 is one of the major safeguards protecting human cells against oncogenic transformation. It is activated in response to genotoxic stress and controls the cellular response by changing the expression of hundreds of target genes. Quantitative fluorescent time-lapse microscopy revealed that different forms of genotoxic stress induce specific dynamics of p53 nuclear accumulation in individual cells, ranging from uniform pulses to monotonic increases. Further studies provided strong evidence that p53 dynamics are critical for determining which target genes are expressed and, as a consequence, whether a cell repairs the damage and continues to proliferate or induces terminal cell fates such as senescence or apoptosis. However, little is known about the molecular mechanisms translating time dependent changes of p53’s nuclear concentration to altered gene expression and cell fate. As gene expression is an inherently stochastic process with promoters switching between inactive and active periods with bursts of RNA production, such mechanistic questions need to be addressed with single molecule resolution at the level of individual cells.Here, we propose to determine which features of target gene promoter activity are regulated by p53 during the response to genotoxic stress and how p53 dynamics regulate promoter activity over time in individual cells. We will use single-molecule fluorescence in-situ hybridization (smFISH) and mathematical modeling to quantify properties of stochastic gene expression such as burst sizes and frequencies at selected time points during the response to various forms of DNA damage. To measure promoter activity in living cells, we will use the MS2 system and Cas9-mediated genome editing to tag endogenous RNAs of selected target genes. We will then systematically alter p53 dynamics by pharmacological or genetic perturbation and monitor the resulting changes in promoter activity by quantifying RNA production at the transcriptional start site with quantitative live-cell fluorescence microscopy. By characterizing p53 binding and epigenetic modifications at target gene promoters and combining time-resolved measurements with targeted perturbations, we will gain further insights into how p53 regulates promoter activity at the molecular level. Taken together, the proposed research will allow us to comprehensively understand the information flow from a stress stimulus to p53 dynamics, target gene expression and cell fate. It will provide deeper insights into how p53’s function is altered during tumorigenesis and potentially enable new strategies for restoring activity of the tumor suppressor in cancer cells