Systems-level analysis of the regulation and function of c-Myc

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不同细胞系统中c-Myc对c-Myc激活信号的反应动力学。通过允许我们研究在较小规模相互作用水平上不明显的紧急性质，这种类型的方法将为操纵电路功能提供新的策略，以及抗击癌症的新方法，其中c-Myc动力学被解除调控。材料和方法：为了测量电路元件的动力学，我们将主要使用c-Myc已被荧光标记的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c-Myc的动力学，并确定对细胞功能的影响。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路径行为。2015财年的进展：在小鼠胚胎成纤维细胞中，编码绿色荧光蛋白GFP的基因已经插入到两个c-Myc等位基因的内源基因座中，我们发现了刺激后c-Myc激活的新的、复杂的动力学。我们已经根据负责产生响应的一般网络结构来描述这些动态。目前的工作集中在获得对这一调控的更详细的机制理解，以及识别观察到的c-Myc动力学的表型序列。我们还扩展了这项工作，以研究c-Myc网络和p53网络对各种刺激的共同调节。这项工作是与David Levens(LP/CCR)合作进行的。

PURPOSE: In this project, we will use a combination of computational and experimental techniques to characterize c-Myc dynamics in a variety of cell systems in response to c-Myc activating cues. 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 c-Myc dynamics are deregulated. MATERIALS AND METHODS: To measure the dynamics of circuit components, we will primarily use long-term time-lapse fluorescence microscopy of living individual cells in which c-Myc has been fluorescently tagged. We will use chemical and genetic perturbations to alter c-Myc dynamics and determine the effects on cellular functions. Using computational modeling, we will integrate these data with measurements of cellular outcomes to predict pathway behavior in response to specific perturbations. PROGRESS IN FY2015: Using mouse embryonic fibroblasts in which the gene encoding the green fluorescent protein GFP has been inserted into the endogenous loci of both c-Myc alleles, we have identified novel, complex dynamics of c-Myc activation following stimulation. We have characterized these dynamics in terms of general network structure responsible for generating the response. Current work is focused on obtaining a more detailed mechanistic understanding of this regulation, as well as identifying phenotypic comsequences of the observed c-Myc dynamics. We have also extended this work to look at co-regulation of the c-Myc network and the p53 network in response to various stimuli. This work is being performed in collaboration with David Levens (LP/CCR).