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

目得：在这个项目中，我们将使用计算和实验技术相结合，以确定在重要的细胞应激压力的调节p53动力学的作用。为了测量下游途径的动力学，我们将利用下面描述的一套补充方法来测定癌性和非转化细胞系统中的p53活性。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。使用计算建模，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。通过允许我们研究在小规模相互作用水平上不明显的新兴特性，这种方法将为操纵生物电路功能提供新的策略，并产生新的方法来对抗p53动力学失调的癌症。材料与方法：1.使用光遗传学和小分子扰动方法来控制p53动力学：我们将使用光遗传学和小分子扰动方法来改变p53动力学的特征（例如，p53脉冲幅度，持续时间或频率），并确定这种扰动对p53转录活性和细胞命运的影响。2.基于p53动力学识别p53靶表达模式：我们将在单细胞和细胞群体水平上探索p53动力学在调节超过100个p53靶中的功能。将使用荧光报告分子进行单细胞水平分析，以及在关键时间点对固定细胞进行免疫荧光成像和流式细胞术，通过对重要靶标进行更详细的研究进行验证。3.我们正在开发方法来创建具有由内源性p53振荡器驱动的新靶点的所需表达模式的合成系统。使用这个系统，我们正在恢复癌细胞中的细胞死亡途径，这些细胞被突变以进行适当的凋亡调节。4.突变p53和患者样本中的p53动力学：我们正在确定常见的p53突变如何改变p53动力学，以及如何影响下游细胞命运调控。我们正在使用癌细胞系，并计划使用患者来源的肿瘤样本。2019财年的进展：1.我们已经开发了一种新的合成方法来控制p53的本地化，并因此p53动力学的特定功能。使用这个系统，我们观察到特定的p53靶基因的差异调节。我们已经表征了p53动力学对几个重要基因调控特征的影响，包括基因表达中的噪声。一份描述我们结果的手稿已经提交。2.我们扩展了去年发表在“细胞系统”上的先前工作的结果，从转录水平上的p53靶点分析转移到p53靶点的蛋白表达动态分析。从这些研究中，我们已经确定并表征了p53调控的几种细胞命运途径的关键介质的表达动态。我们的研究结果发表在《细胞生物学杂志》上。3.我们开发了通过调节被p53振荡激活的合成靶点来调节不同表达模式和细胞命运结果的系统。我们的研究结果已发表在Scientific Reports上。4.我们已经开发了含有p53突变的细胞系，并正在分析它们的动力学及其对转录组的影响。

PURPOSE: In this project, we will use a combination of computational and experimental techniques to identify the role of p53 dynamics in the regulation of important cellular stress stresses. To measure the dynamics of downstream pathways, we will make use of a suite of complementary approaches described below to assay p53 activity in both cancerous and non-transformed cellular systems. 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 biological circuit functions, as well as generate new ways to combat cancers in which p53 dynamics are dysregulated. MATERIALS AND METHODS: 1. Using optogenetic and small molecule perturbation approaches to control p53 dynamics: We will use optogenetic and small molecule perturbation approaches to alter characteristics of p53 dynamics (for example, p53 pulse amplitude, duration, or frequency), and determine the effect that such perturbations have on p53's transcriptional activity and cell fate. 2. Identifying p53 target expression patterns based on p53 dynamics: We will probe the function of p53 dynamics in the regulation of the over 100 p53 targets at the level of single cells and in cell populations. Validation by more detailed studies of important targets will be performed using single-cell level analysis with fluorescent reporters, as well as immunofluorescent imaging and flow cytometry of fixed cells at key time points. 3. We are generating methods to create synthetic systems with desired expression patterns of novel targets driven by the endogenous p53 oscillator. Using this system, we are restoring cell death pathways in cancer cells that are mutated for proper apoptotic regulation. 4. p53 dyanmics for mutated p53 and in patient samples: WE are determining how common p53 mutations alter p53 dynamics, and how that impacts downstream cell fate regulation. We are using cancer cell lines and plan to use patient-derived tumor samples. PROGRESS IN FY2019: 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 observed differential regulation of specific p53 target genes. We have characterizing the impact of p53 dynamics on several important gene regulatory features, including the noise in gene expression. A manuscript describing our results has been submitted. 2. We extended the results from our previous work published in "Cell Systems" last year, moving from the analysis of p53 targets at the transcript level to analysis of protein expression dynamics of p53 targets. From these studies, we have identified and characterized expression dynamics for key mediators of several cell fate pathways regulated by p53. Our results have been published in the Journal of Cell Biology. 3. We developed systems to regulate distinct expression patterns and cell fate outcomes through modulation of synthetic targets engineered to be activated by p53 oscillations. Our results have been published in Scientific Reports. 4. We have developed cell lines harboring p53 mutations and are analyzing their dynamics and their effect on the transcriptome.