Systems-level analysis of the regulation and function of MYC

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MYC在各种细胞系统中的动态，并响应于一系列重要的生物刺激。我们将使用一系列定量方法（MYC蛋白表达的单细胞成像，MYC结合的全基因组分析，高通量转录组学分析等）来确定MYC调控和功能的系统水平特性。从这些方法中，我们的目标是开发新的策略，用于在疾病背景下操纵MYC活性，特别是MYC失调的癌症。材料和方法：为了测量MYC表达的动态，我们将主要使用活的单个细胞的长期延时荧光显微镜，其中MYC已被荧光标记。我们将使用化学和遗传扰动来改变MYC动力学，并确定对细胞功能的影响。我们将使用一套高通量测序技术来量化MYC结合和活性。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。2019财年的进展：我们专注于MYC抑制在细胞对DNA损伤的反应中的调节和功能。我们确定了肿瘤抑制基因p53是这种应激反应中MYC抑制的关键因素。我们鉴定了MYC基因远端的p53结合位点作为MYC抑制的关键位点，并发现p53是导致MYC抑制的MYC启动子处的DNA损伤依赖性染色质重排所需的。使用系统干扰MYC的表达，我们表明，正常的抑制MYC在DNA损伤反应是重要的抑制一般的转录，而在同一时间p53依赖性激活应激反应基因可以绕过一般的转录抑制。由于这种转录控制的“具有特异性激活的全局抑制”机制，维持细胞周期停滞并抑制程序性细胞死亡。我们现在正在确定MYC对其他刺激的动态反应，以及在健康和疾病状态下MYC的转录活性的动态影响。我们已经帮助启动了一项大规模的合作努力，以精确量化MYC在正常乳腺组织和B细胞中的转录组学、蛋白质组学和细胞表型水平上的功能。

PURPOSE: In this project, we will use a combination of computational and experimental techniques to characterize MYC dynamics in a variety of cell systems and in response to a range of important biological stimuli. We will use a range of quantitative approaches (single cell imaging of MYC protein expression, genome-wide analysis of MYC binding, high throughput transcriptomic analysis, etc) to identify systems-level properties of MYC regulation and function. From these approaches, we aim to develop novel strategies for manipulating MYC activity in disease contexts, especially cancers in which MYC is deregulated. MATERIALS AND METHODS: To measure the dynamics of MYC expression, we will primarily use long-term time-lapse fluorescence microscopy of living individual cells in which MYC has been fluorescently tagged. We will use chemical and genetic perturbations to alter MYC dynamics and determine the effects on cellular functions. We will use a suite of high-throughput sequencing technologies to quantify MYC binding and activity. Using computational modeling, we will integrate these data with measurements of cellular outcomes to predict pathway behavior in response to specific perturbations. PROGRESS IN FY2019: We focused on the regulation and function of MYC suppression during the cellular response to DNA damage. We identified the tumor suppressor p53 as a key factor in MYC repression in this stress response. We identified a p53 binding site distal to the MYC gene as a key site for MYC repression, and found that p53 is required for DNA-damage dependent chromatin rearrangements at the MYC promoter that lead to MYC repression. Using a system to perturb MYC expression, we showed that the normal repression of MYC during the DNA damage response is important for suppressing general transcription, while at the same time p53-dependent activation of stress response genes can bypass the general transcriptional inhibition. As a result of this "global inhibition with specific activation" mechanism of transcriptional control, cell cycle arrest is maintained and programmed cell death is suppressed. We are now determining the MYC dynamical response to other stimuli, and the impact of the dynamics on MYC's trascriptional activity in healthy and disease states. We have helped initiate a large-scale collaborative effort to precisely quantify MYC's function at the transcriptomic, proteomic, and cell phenotypic level in normal breast tissue and B cells.