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动态。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动力学，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途径行为。通过允许我们研究在较小规模的相互作用水平上不明显的涌现特性，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动力学失调的癌症的新方法。材料与方法：根据p53刺激确定p53动力学的类别：我们将确定p53对广泛刺激的动力学反应，并根据p53动力学对应力进行分类。我们将使用长期延时荧光显微镜在单细胞中以高时间分辨率测量荧光标记p53的动态。2. 使用合成生物学方法来控制p53动力学：我们将使用合成生物学方法来干扰p53动力学的各种特征（例如，p53脉冲幅度，持续时间和频率），并确定这种扰动对p53下游功能的影响。3. 基于p53动力学的靶基因表达模式识别：我们将在单细胞水平上探讨p53动力学在100多个p53靶基因调控中的作用。通过对重要靶基因进行更详细的研究，将使用荧光转录报告器进行单细胞水平分析。2013财年进展：我们已经开始研究p53的广泛自然刺激，并确定了p53在应对新压力时的独特动态调控。我们目前正在描述这些新的反应，并确定产生它们的监管机制。我们还在开发通过使用化学和合成生物学扰动来操纵p53动力学的方法方面取得了进展。我们还在使用新技术来量化单个细胞中大量p53调控基因的表达方面取得了重大进展。

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. Determining classes of p53 dynamics based on p53 stimuli: We will determine the p53 dynamical response to a broad range of stimuli and classify stresses based on p53 dynamics. We will use long-term time-lapse fluorescence microscopy to measure the dynamics of fluorescently-tagged p53 at high temporal resolution in single cells. 2. 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. 3. 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 FY2013: We have begun looking at a broad range of natural stimuli of p53, and have identified unique dynamical regulation of p53 in response to novel stresses. We are currently characterizing these new responses and identifying the regulatory mechanisms that generate them. We have also made progress in developing methods to manipulate p53 dynamics through the use of chemical and synthetic biology perturbations. We have also made significant progress in the use of novel technologies to quantify expression of large sets of p53 regulated genes in single cells.