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

p53 动力学调控和功能的系统级分析

• 批准号: 9153885
• 负责人: Eric Batchelor
• 金额: $ 54.1万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153885
• 关键词: Affect; Apoptosis; Behavior; Biological; Cancerous; Cell Aging; Cell Cycle Arrest; Cell Death; Cells; Cellular Stress; Cellular biology; Characteristics; Complex; Computer Simulation; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Dose; Face; Fluorescence Microscopy; Frequencies; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Goals; Life; Malignant Neoplasms; Manuscripts; Measurement; Measures; Medicine; Methods; Mutate; Outcome; Output; Pathway interactions; Physiologic pulse; Property; Regulation; Reporter; Research; Series; Shapes; System; Techniques; Time; Translating; Validation; Work; base; chemical genetics; combat; extracellular; new technology; novel; novel strategies; response; synthetic biology; transcription factor; ultraviolet damage

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 FY2015: We have focused our efforts on two main sub-projects in FY2015. In sub-project 1, 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. In sub-project 2, we have made significant progress in the use of novel technologies to quantify expression of large sets of p53 regulated genes in single cells. We have submitted a manuscript describing the results of this project.

目的：在这个项目中，我们将结合计算和实验技术来表征健康细胞和癌细胞中 p53 动态响应重要的细胞应激。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。我们将利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通路行为。通过让我们研究在小规模相互作用水平上不明显的新兴特性，这种方法将为操纵电路功能提供新策略，以及对抗 p53 动态失调的癌症的新方法。材料和方法： 1. 使用合成生物学方法控制 p53 动态：我们将使用合成生物学方法扰乱 p53 动态的各种特征（例如，p53 脉冲幅度、持续时间和频率），并确定此类扰动对 p53 下游功能的影响。 2. 基于p53动力学识别靶基因表达模式：我们将在单细胞水平探讨p53动力学在调节100多个p53靶基因中的功能。将使用荧光转录报告基因的单细胞水平分析对重要靶基因进行更详细的研究验证。 2015 财年的进展： 2015 财年我们的工作重点是两个主要子项目。在子项目 1 中，我们开发了一种新的合成方法来控制 p53 定位，从而控制 p53 动力学的特定特征。使用该系统，我们观察到特定 p53 靶基因的差异调节。我们正在继续朝这个方向进行研究，扩大我们的分析范围，以包括 p53 调控的其他几个重要目标的测量。在子项目 2 中，我们在使用新技术量化单细胞中大量 p53 调控基因的表达方面取得了重大进展。我们已经提交了一份描述该项目结果的手稿。