Analysis of genome wide transcriptional control in yeast

描述(申请人提供)：酵母全基因组转录控制的分析。这个项目的长期目标是在真核细胞中开发一个全面的和可预测的功能转录调控网络模型。我们试图从单个转录调控因子的数量贡献方面了解在胁迫条件下触发的全基因组转录变化，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一领域的几个问题。首先，我们将全面鉴定在酵母中可能调节应激反应的每一个转录调节因子和染色质因子。这将通过酵母菌株的表型生长分析来实现，在这种分析中，转录因子的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胁迫条件下-热休克、营养饥饿和DNA损伤-通过在平板上斑点生长和在液体中生长来筛选生长缺陷。第二，确定重点监管机构在压力条件下的下游目标。我们将使用染色质免疫沉淀和微阵列(芯片-芯片)相结合的方法，在应激反应期间识别一组优先考虑的转录调节因子的直接结合靶点。我们还将通过在选定转录因子缺失的菌株中进行基因表达谱分析，在已知这些因子所需的应激条件下，识别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来构建一个预测性和因果调控网络来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模拟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胁迫相关的转录调控因子的靶标，并理想地解释每个酵母基因在生理胁迫扰动下的调控。我们将使用外部实验数据和基因功能注释来测试我们的网络的预测能力。网络中选定的预测调控关系将通过定向实验进行验证。 公共卫生相关性：酵母全基因组转录控制的分析。本项目将以酵母菌的应激反应为模型系统，了解生理扰动下基因表达的整体调控。酵母中活跃在这一过程中的许多转录调节因子的同源基因，如热休克因子和其他影响染色质的因素，直接与哺乳动物细胞的癌症有关。我们建议构建的全球功能基因调控网络将对经常在疾病中受损的哺乳动物细胞的全球基因调控机制提供相当大的帮助。

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.