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

抑癌基因P53是保护人类细胞免受致癌转化的主要保护措施之一。它在应对遗传毒性应激时被激活，并通过改变数百个靶基因的表达来控制细胞反应。定量荧光时间推移显微镜显示，不同形式的遗传毒性应激诱导单个细胞中P53核积累的特定动力学，从均匀的脉冲到单调的增加。进一步的研究提供了强有力的证据表明，P53的动态对于决定哪些靶基因的表达至关重要，因此，细胞是修复损伤并继续增殖，还是诱导终末细胞命运，如衰老或凋亡。然而，将P53‘S核浓度的时间依赖性变化转化为基因表达和细胞命运改变的分子机制尚不清楚。由于基因表达是一个内在的随机过程，启动子随着RNA的产生而在非活跃期和活跃期之间切换，这类机制问题需要在单个细胞水平上用单分子分辨来解决。在这里，我们建议确定在对遗传毒性胁迫的反应中，靶基因启动子活性的哪些特征受到p53的调控，以及随着时间的推移，p53动态如何调节单个细胞中的启动子活性。我们将使用单分子荧光原位杂交(SmFISH)和数学模型来量化随机基因表达的特性，如在对各种形式的DNA损伤的反应中，选定时间点的猝发大小和频率。为了测量活细胞中启动子的活性，我们将使用MS2系统和Cas9介导的基因组编辑来标记选定目标基因的内源RNA。然后，我们将通过药理学或遗传扰动系统地改变P53的动力学，并通过用定量活细胞荧光显微镜定量转录起始点的RNA生产来监测由此导致的启动子活性的变化。通过表征p53与靶基因启动子的结合和表观遗传修饰，并将时间分辨测量与靶向扰动相结合，我们将在分子水平上进一步了解p53如何调控启动子活性。综上所述，这项拟议的研究将使我们能够全面了解从应激刺激到p53动态、靶基因表达和细胞命运的信息流。这将为深入了解P53‘S在肿瘤发生过程中的功能变化提供更深入的见解，并有可能为恢复肿瘤细胞中肿瘤抑制因子的活性提供新的策略

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