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: 8349510
• 负责人: Eric Batchelor
• 金额: $ 38.55万
• 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/8349510
• 关键词: Apoptosis; Apoptotic; Behavior; Biological; Cancerous; Cell Cycle Arrest; Cells; Cellular Stress; Cellular biology; Chemicals; Complex; Computer Simulation; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Dependence; Dose; Engineering; Equipment; Face; Feedback; Fluorescence Microscopy; Foundations; Gamma Rays; Gene Expression; Gene Targeting; Genes; Goals; Image; Individual; Interview; Kinetics; Laboratories; Life; Malignant Neoplasms; Measurement; Measures; Medicine; Microscope; Molecular; Molecular Profiling; Mutate; Oncogenes; Outcome; Output; Pathology; Pathway interactions; Pattern; Physiologic pulse; Positioning Attribute; Postdoctoral Fellow; Process; Property; Publishing; RNA Interference; Regulation; Reporter; Research; Resolution; Series; Shapes; Stimulus; Stress; System; Systems Biology; Techniques; Time; Translating; Ultraviolet Rays; Validation; Work; base; biological adaptation to stress; chemical genetics; combat; extracellular; fluorescence microscope; interest; novel strategies; research study; response; small molecule; 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 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).

目得：在这个项目中，我们将使用计算和实验技术的组合来表征健康和癌细胞中的p53动态响应重要的细胞应激。为了测量电路组件的动态，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涌现特性，这种方法将提供操纵电路功能的新策略，以及对抗p53动力学失调的癌症的新方法。材料与方法：1.根据p53刺激确定p53动力学的类别：虽然对p53对γ辐射的反应有很大的兴趣，但它只是可以激活p53的众多应激之一，包括其他形式的DNA损伤，核糖体应激和致癌基因。我们已经表明，p53显示出显着不同的动力学行为时，暴露于紫外线辐射的梯度脉冲。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对广泛刺激的动态反应，并根据p53动态对应激进行分类。我们将使用长时间的延时荧光显微镜来测量单细胞中荧光标记的p53在高时间分辨率下的动态。识别不同的动力学类别和每个类别中聚集的压力将为理解p53网络的先前未知的调节水平提供基础，并将为研究p53动力学的功能提供信息。 2.基于p53动力学识别靶基因表达模式：我们对p53动力学类别的发现表明，p53靶基因可能显示出影响细胞反应的复杂动力学表达模式。例如，当p53经历γ型动力学时，某些促凋亡基因可能以逐步方式表达，但当p53经历UV型动力学时，以快速饱和动力学表达。这可以转化为细胞凋亡的差异触发，这与不同的细胞命运响应于γ或UV辐射而发生的事实一致。由于p53调控超过100个基因，我们将开始采用qPCR方法来探测p53动力学的功能。将使用荧光转录报告基因进行单细胞水平分析，通过对重要靶基因进行更详细的研究进行验证。我们将分析基因表达谱的刺激内个别p53动态类和跨不同的类。我们预测，依赖于p53动态的表达模式的基因将集群的动态类。为了验证基因表达对p53动力学的依赖性，我们将比较压力反应曲线和我们扰动p53动力学的条件下的曲线。在初步研究中，我们已经使用小分子、反馈调节剂的RNAi和合成工程反馈来干扰对伽马或UV的动态响应。 2011财年的进展：NCI病理学实验室的系统生物学部分于2011年5月成立，由我（Eric Batchelor博士）雇用。自从我到达以来，我已经完成了物理实验室和办公空间的建设和装修的监督。我已经采购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一台倒置荧光显微镜，带有用于长期延时成像实验的环境室，目前仍在采购过程中。预计显微镜将于2012财年第一季度到货并安装。我目前正在审查系统生物学科的两名博士后研究员和一名全职生物学家的申请和面试候选人。在我到达《分子系统生物学》杂志后不久，一些研究发现（Batchelor E等人，单细胞中p53的刺激依赖动力学。摩尔7：488，2011）。