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

p53 对基因毒性应激的动态反应调节单细胞中的随机基因表达

• 批准号: 421980029
• 负责人: Professor Dr. Alexander Loewer
• 金额: --
• 依托单位: Arbeitsgruppe Systembiologie der Stressantwort
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/421980029?language=en
• 关键词: Regulation; stochastic; gene; expression; single

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

肿瘤抑制因子p53是保护人类细胞免受癌性转化的主要保障之一。它在基因毒性应激反应中被激活，并通过改变数百个靶基因的表达来控制细胞反应。定量荧光延时显微镜显示，不同形式的基因毒性应激诱导单个细胞中p53核积累的特定动态，从均匀脉冲到单调增加不等。进一步的研究提供了强有力的证据，表明p53动态对于确定哪些靶基因表达至关重要，因此，对于细胞是否修复损伤并继续增殖或诱导终端细胞命运（如衰老或凋亡）至关重要。然而，p53细胞核浓度随时间变化而改变基因表达和细胞命运的分子机制尚不清楚。由于基因表达是一个固有的随机过程，启动子会随着RNA产生的爆发而在非活跃期和活跃期之间切换，因此这些机制问题需要在单个细胞水平上用单分子分辨率来解决。在这里，我们建议确定靶基因启动子活性的哪些特征在基因毒性应激反应中由p53调节，以及p53如何随时间在单个细胞中动态调节启动子活性。我们将使用单分子荧光原位杂交（smFISH）和数学建模来量化随机基因表达的特性，如在响应各种形式的DNA损伤的特定时间点上的爆发大小和频率。为了测量活细胞中的启动子活性，我们将使用MS2系统和cas9介导的基因组编辑来标记选定靶基因的内源性rna。然后，我们将通过药理学或遗传扰动系统地改变p53动力学，并通过定量活细胞荧光显微镜在转录起始位点定量RNA产生来监测启动子活性的变化。通过表征p53结合和靶基因启动子的表观遗传修饰，并结合时间分辨测量和靶向扰动，我们将进一步了解p53如何在分子水平上调节启动子活性。综上所述，本研究将使我们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。它将为肿瘤发生过程中p53的功能如何改变提供更深入的见解，并可能为恢复癌细胞中肿瘤抑制因子的活性提供新的策略