Systems-level analysis of the regulation and function of p53 dynamics

目的：在这个项目中，我们将使用计算和实验技术相结合的方法来表征健康细胞和癌细胞对重要细胞应激反应的P53动力学。为了测量电路元件的动态，我们将使用活细胞的长期延时荧光显微镜。我们将使用化学和遗传扰动来改变P53的动态，并确定对P53靶基因表达和细胞命运的影响。使用计算模型，我们将把这些数据与细胞结果的测量相结合，以预测响应特定扰动的路径行为。通过允许我们研究在较小规模相互作用水平上不明显的紧急特性，这种类型的方法将为操纵电路功能提供新的策略，以及对抗P53动态失调的癌症的新方法。材料和方法：1.利用合成生物学方法控制P53的动力学：我们将利用合成生物学的方法来干扰P53的各种动力学特征(如P53的脉冲幅度、持续时间和频率)，并确定这种干扰对P53‘S下游功能的影响。2.基于P53动态识别靶基因表达模式：我们将在单细胞水平上探讨P53动态在调控100多个P53靶基因中的作用。通过对重要目标基因进行更详细的研究进行验证，将使用带有荧光转录报告的单细胞水平分析。2016财年进展：我们开发了一种新的合成方法来控制P53的定位，从而控制P53的动态特性。利用这个系统，我们观察到了特定的p53靶基因的差异调控。我们正朝着这个方向继续我们的研究，扩大我们的分析范围，以包括对P53调控的其他几个重要靶点的测量。我们还量化了大量p53调控基因在单细胞中的表达，发现了与mRNA稳定性和共调节子网络相关的新调控模式。这些研究的结果发表在去年的《细胞系统》杂志上。

PURPOSE: In this project, we will use a combination of computational and experimental techniques to characterize p53 dynamics in healthy and cancerous cells in response to important cellular stresses. To measure the dynamics of circuit components, we will use long-term time-lapse fluorescence microscopy of living cells. We will use chemical and genetic perturbations to alter p53 dynamics and determine the effect on p53 target gene expression and cell fate. Using computational modeling, we will integrate these data with measurements of cellular outcomes to predict pathway behavior in response to specific perturbations. By allowing us to study emergent properties that are not evident at the level of smaller-scale interactions, this type of approach will provide novel strategies for manipulating circuit functions, as well as new ways to combat cancers in which p53 dynamics are dysregulated. MATERIALS AND METHODS: 1. Using synthetic biology approaches to control p53 dynamics: We will use synthetic biology approaches to perturb various characteristics of p53 dynamics (for example, p53 pulse amplitude, duration, and frequency), and determine the effect that such perturbations have on p53's downstream functions. 2. Identifying target gene expression patterns based on p53 dynamics: We will probe the function of p53 dynamics in the regulation of the over 100 p53 target genes at the single cell level. Validation by more detailed studies of important target genes will be performed using single-cell level analysis with fluorescent transcriptional reporters. PROGRESS IN FY2016: We have developed a novel synthetic method to control p53 localization, and as a consequence specific features of p53 dynamics. Using this system, we have observed differential regulation of specific p53 target genes. We are continuing our study in this direction, expanding our analysis to include the measurement of several other important targets of p53 regulation. We have also quantified the expression of large sets of p53 regulated genes in single cells, uncovering novel modes of regulation related to mRNA stability and co-regulatory subnetworks. Results from these studies have been published in "Cell Systems" this past year.