Rewiring cell cycle-regulated transcription in response to stress

 描述(由申请人提供) 通过细胞分裂周期调节的进程对于维持细胞周期的稳定性是重要的 并防止失控的细胞分裂。细胞周期的各个步骤由一个潜在的转录程序来排序，该程序协调基因的表达与周期中需要其功能的时间。这种严格调控的基因表达模式在几乎所有的癌细胞中都被打乱，这突显了它的重要性。高通量的基因组方法已经确定了在不同的真核生物中控制细胞周期调节基因表达的转铁蛋白网络，最被理解的是酵母酿酒酵母。流行的模型认为，细胞周期每个阶段的关键转录因子激活下游转录因子的表达，以推动细胞周期向前发展。然而，该模型并不能准确预测当单个转录因子被灭活或删除时观察到的许多下游影响。为了充分理解细胞周期控制的这一基本机制，需要更多地了解网络中转铁蛋白之间的协调。在这里，我们将通过描述每个细胞周期Tf(及其下游效应器)对特定环境或遗传扰动的反应来解决这一悬而未决的问题。在第一个目标中，我们将阐明网络中每个TF的表达、调节和活性的动态变化，以响应我们已经发现的通过细胞周期蛋白依赖的激酶(CDK1)改变TF磷酸化的环境条件。在第二个目标中，我们将剖析网络中每个转铁蛋白的多位点磷酸化如何影响其功能的潜在机制。此外，我们将使用系统的饱和突变与本体竞争分析相结合的方法来阐明模型TF非结构区域内多个残基的磷酸化是如何读出的TF功能的变化。通过详细研究这个TF网络的多个调控参数，我们的工作将导致对这个网络的一个完整的、机械性的看法，并对我们对细胞周期控制的理解产生重大影响。

 DESCRIPTION (provided by applicant) Regulated progression through the cell division cycle is important to maintain the stability of the genome and to prevent uncontrolled cell division. The steps of the cell cycle are ordered by an underlying transcriptional program, which coordinates the expression of genes with the times in the cycle when their functions are needed. This tightly regulated pattern of gene expression is disrupted in nearly all cancer cells, underscoring its importance. High-throughput genomic approaches have identified TF networks that control cell cycle- regulated gene expression in diverse eukaryotes, the best understood being the yeast Saccharomyces cerevisiae. The prevailing model suggests that key TFs at each stage of the cell cycle activate expression of downstream TFs to drive the cell cycle forward. However, this model does not accurately predict many of the downstream effects that are observed when individual TFs are inactivated or deleted. A greater understanding of the coordination between TF proteins in the network is needed to fully understand this fundamental mechanism of cell-cycle control. Here we will address this outstanding issue by characterizing the response of each cell cycle TF (and their downstream effectors) to defined environmental or genetic perturbations. In the first aim, we will elucidate the dynamics of changes in the expression, regulation, and activity of each TF in the network in response to environmental conditions that we have found alter TF phosphorylation by cyclin- dependent kinase (Cdk1). In the second aim, we will dissect the mechanisms underlying how multisite phosphorylation of each TF in the network impacts their functions. In addition, we will use systematic, saturating mutagenesis in combination with bulk competition assays to elucidate how phosphorylation of multiple residues within an unstructured region of a model TF is read out into a change in TF function. By examining multiple regulatory parameters of this TF network in detail, our work will lead to an integrated, mechanistic view of this network, and have a significant impact on our understanding of cell-cycle control.