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探测电路并确定重要的调节机制，如反馈。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的动态类型：虽然人们对p53对伽马辐射的反应非常感兴趣，但它只是众多可以激活p53的应激中的一种，包括其他形式的DNA损伤、核糖体应激和癌基因。我们已经证明，当暴露于紫外线辐射时，p53表现出明显不同的梯度脉冲动力学行为。我们的初步研究还发现，某些化学物质激活p53可以产生无阻尼脉冲或分级脉冲。我们将确定p53对各种刺激的动态反应，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。识别不同的动态类别和每个类别中聚集的应力将为理解p53网络以前未知的调节水平提供基础，并将为p53动态功能的研究提供信息。2. 基于p53动态识别靶基因表达模式：我们对p53动态分类的发现表明p53靶基因可能表现出影响细胞反应的复杂动态表达模式。例如，当p53经历γ型动力学时，某些促凋亡基因可能以阶梯方式表达，但当p53经历uv型动力学时，某些促凋亡基因以快速饱和动力学表达。这可以解释为细胞凋亡的不同触发，这与不同的细胞命运发生在对γ或紫外线辐射的反应中是一致的。由于p53调控超过100个基因，我们将首先采用qPCR方法来探索p53动态的功能。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。我们将分析单个p53动态类别和不同类别的刺激基因表达谱。我们预测依赖于p53动态的表达模式的基因将按动态类聚类。为了验证基因表达对p53动力学的依赖性，我们将比较应激反应谱和干扰p53动力学的条件下的谱。在初步研究中，我们使用了小分子、反馈调节器的RNAi和合成工程反馈来干扰对伽马或紫外线的动态响应。2011财年的进展：2011年5月，NCI病理实验室的系统生物学部门在我（Eric Batchelor博士）的雇佣下成立。自我来到这里以来，我已经完成了对物理实验室和办公空间的建设和装修的监督。我已经购置了进行实验所需的大部分科学设备。该项目最大和最重要的设备是用于长期延时成像实验的带环境室的倒置荧光显微镜，目前仍在采购过程中。预计显微镜的到达和安装将在2012财年第一季度进行。目前，我正在为系统生物学部门的两名博士后和一名全职生物学家进行申请和面试。在我进入分子系统生物学（Batchelor et al.，单细胞中p53的刺激依赖动力学）后不久，提出的项目的一些研究结果发表了。摩尔。系统。《生物》，2011年第7卷第488页)。

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 probe circuits and identify important regulatory mechanisms, such as feedbacks. 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: While there has been a great deal of interest in the p53 response to gamma radiation, it is only one of numerous stresses that can activate p53, including additional forms of DNA damage, ribosomal stress, and oncogenes. We have shown that p53 shows the strikingly distinct dynamical behavior of graded pulses when exposed to UV radiation. Our preliminary studies have also found that activation of p53 by certain chemicals can generate either undamped pulses or graded pulses. 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. Identifying the different dynamical classes and the stresses that cluster in each class will provide a foundation for understanding a previously unknown level of regulation of the p53 network, and will inform research into the function of p53 dynamics. 2. Identifying target gene expression patterns based on p53 dynamics: Our discovery of p53 dynamical classes suggests that p53 target genes may show complex dynamical expression patterns that impact cellular responses. For example, it may be that certain pro-apoptotic genes are expressed in a step-wise manner when p53 undergoes gamma-type dynamics, but are expressed with fast saturation kinetics when p53 undergoes UV-type dynamics. This could be translated into differential triggering of apoptosis, which is consistent with the fact that distinct cell fates occur in response to gamma or UV radiation. Since p53 regulates over 100 genes, we will begin by taking a qPCR approach to probe the function of p53 dynamics. Validation by more detailed studies of important target genes will be performed using single-cell level analysis with fluorescent transcriptional reporters. We will analyze gene expression profiles for stimuli within individual p53 dynamical classes and across distinct classes. We predict that genes with expression patterns that are dependent on p53 dynamics will cluster by the dynamical classes. To verify the dependence of gene expression on p53 dynamics, we will compare stress-response profiles to profiles for conditions in which we have perturbed p53 dynamics. In preliminary studies, we have used small molecules, RNAi of feedback regulators, and synthetic engineered feedbacks to perturb the dynamical response to gamma or UV. PROGRESS IN FY2011: The Systems Biology Section of the NCI Laboratory of Pathology was established in May 2011 with my (Dr. Eric Batchelor) hiring. Since my arrival, I have completed the overseeing of the construction and furnishing of the physical lab and office space. I have procured the majority of the scientific equipment required to perform the experiments. The largest and most important equipment for the project, an inverted fluorescence microscope with environmental chamber for the long-term time-lapse imaging experiments, is currently still in the procurement process. It is anticipated that the arrival and installation of the microscope will occur in the first quarter of FY2012. I am currently in the process of reviewing applications and interviewing candidates for the two postdoctoral fellows and one FTE Biologist position for the Systems Biology Section. Some of the research findings from which the proposed project developed were published shortly after my arrival in Molecular Systems Biology (Batchelor E et al., Stimulus-dependent dynamics of p53 in single cells. Mol. Syst. Biol. 7: 488, 2011).