Analysis of genome wide transcriptional control in yeast

描述（由申请人提供）：酵母全基因组转录控制分析。该项目的长期目标是建立一个全面和预测的真核细胞功能转录调控网络模型。我们试图了解在应激条件下触发的全基因组转录变化，就个体转录调控因子的定量贡献而言，包括序列特异性DNA结合转录因子以及通过调节染色质结构影响基因表达的转录调控因子。我们将使用酵母作为模型系统，通过以下具体目标来解决这个领域的几个问题。首先，我们将全面确定每一个转录调节剂和染色质因子，可能调节酵母的应激反应。这将通过酵母株的表型生长分析来完成，其中转录因子功能通过缺失或过表达来调节。转录因子缺失和过表达菌株将在三种不同的应激条件下（热休克、营养饥饿和DNA损伤），通过在平板上和液体中培养来筛选生长缺陷。其次，我们将确定压力条件下关键监管机构的下游目标。我们将使用染色质免疫沉淀结合微阵列（ChIP-chip）技术，在应激反应中确定一组转录调节因子的直接结合靶标，这些转录调节因子被证明是必需的。我们还将在已知这些因子所必需的应激条件下，通过对被选中的转录因子删除的菌株进行基因表达谱分析，确定受这些因子积极和功能调节的基因。最后，我们将整合我们的实验基因组数据，建立一个预测和因果调控网络，以解释所有酵母基因在胁迫下的转录调控。我们将使用贝叶斯框架来建模和重建这个转录调控网络，它将最小限度地包含酵母中每个与应激相关的转录调控的目标，并理想地解释每个酵母基因在生理应激扰动下的调控。我们将使用外部实验数据和基因功能注释来测试我们网络的预测能力。网络中选定的预测调节关系将通过定向实验进行验证。

DESCRIPTION (provided by applicant): Analysis of genome-wide transcriptional control in yeast. The long term goal of this project is to develop a comprehensive and predictive model of a functional transcriptional regulatory network in a eukaryotic cell. We seek to understand genome-wide transcriptional changes that are triggered in response to stress conditions, in terms of the quantitative contributions of individual transcriptional regulators, including sequence-specific DNA binding transcription factors as well as transcriptional regulators that affect gene expression through modulating chromatin structure. We will use yeast as a model system to address several questions in this area through the following specific aims. First, we will comprehensively identify every transcriptional regulator and chromatin factor that potentially regulates stress responses in yeast. This will be accomplished through phenotypic growth assays of yeast strains in which transcription factor function is modulated by deletion or overexpression. Transcription factor deletion and overexpression strains will be screened for growth defects by spotting on plates and growth in liquid, under three different stress conditions - heat shock, nutrient starvation and DNA damage. Second, we will identify the downstream targets of key regulators under stress conditions. We will identify the direct binding targets of a prioritized set of transcription regulators during the stress responses that they are shown to be required for, using chromatin immunoprecipitation combined with microarrays (ChIP-chip). We will also identify the genes that are actively and functionally regulated by these factors, by carrying out gene expression profiling in strains deleted for the selected transcription factors, under the stress conditions that these factors are known to be required for. Finally, we will integrate our experimental genomic data to build a predictive and causal regulatory network to explain the transcriptional regulation of all yeast genes under stress. We will use a Bayesian framework to model and reconstruct this transcriptional regulatory network, which will minimally contain the targets of every stress-related transcriptional regulator in yeast, and ideally explain the regulation of every yeast gene under physiological stress perturbations. We will test the predictive ability of our network using both external experimental data and gene functional annotations. Selected predicted regulatory relationships in the network will be verified by directed experiments. PUBLIC HEALTH RELEVANCE: Analysis of genome-wide transcriptional control in yeast. This project will use the stress response in yeast as a model system to understand the global regulation of gene expression under physiological perturbation. Homologs of many of the transcriptional regulators active during this process in yeast, such as Heat Shock Factor and other factors that affect chromatin are directly implicated in cancer in mammalian cells. A global functional gene regulatory network such as we propose to construct will shed considerable light on the mechanism of global gene regulation in mammalian cells that is often impaired in disease.