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开发一个全面的和预测性的真核细胞功能性转录调控网络模型。我们试图了解全基因组的转录变化，在响应压力条件下触发，在定量方面的贡献，个别转录调节因子，包括序列特异性DNA结合转录因子以及转录调节因子，通过调节染色质结构影响基因表达。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一领域的几个问题。首先，我们将全面确定每一个转录调节因子和染色质因子，可能调节酵母的应激反应。这将通过酵母菌株的表型生长测定来实现，其中转录因子功能通过缺失或过表达来调节。将通过在平板上点样并在液体中生长，在三种不同的胁迫条件下-热休克、营养饥饿和DNA损伤-筛选转录因子缺失和过表达菌株的生长缺陷。其次，我们将识别压力条件下关键监管者的下游目标。我们将确定一组优先的转录调节因子的直接结合目标，在应激反应，他们被证明是必要的，使用染色质免疫沉淀结合微阵列（ChIP芯片）。我们还将通过在已知需要这些因子的应激条件下，在缺失所选转录因子的菌株中进行基因表达谱分析，来鉴定受这些因子积极和功能性调节的基因。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，以解释压力下所有酵母基因的转录调控。我们将使用贝叶斯框架来建模和重建这个转录调控网络，它将最低限度地包含酵母中每个与压力相关的转录调控因子的靶点，并理想地解释生理压力扰动下每个酵母基因的调控。我们将使用外部实验数据和基因功能注释来测试我们的网络的预测能力。选择预测的调控关系的网络将被验证的定向实验。 公共卫生相关性：酵母全基因组转录控制分析。本计画将以酵母的胁迫反应为模式系统，了解生理扰动下基因表现的整体调控。在酵母中，在此过程中活跃的许多转录调节因子的同源物，如热休克因子和影响染色质的其他因子，直接涉及哺乳动物细胞中的癌症。一个全球性的功能基因调控网络，如我们建议构建将揭示相当多的哺乳动物细胞中的全球基因调控机制，往往是受损的疾病。

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.