Deciphering the regulatory code of a cell

说明（由申请人提供）：尽管在酵母S.然而，关于这一过程的许多信息仍然未知。我们无法预测，而不是解释，任何基因的启动子序列的表达模式就是一个例子。我们的长期目标是提供一个全面的S。酿酒酵母基因调控网络，可用于开发基因表达的预测模型。第一个任务是完成转录因子及其结合位点的目录。我们将结合使用现有的体外和体内方法来实现这一目标。我们将确定的100多个转录因子的酵母，其特异性仍然未知（目标1），使用电泳凝胶迁移率变动分析，酵母单杂交试验，和一种新的方法来探测蛋白质微阵列与DMA寡核苷酸的结合位点。然后，我们将开发全面的权重矩阵的结合位点的酵母转录因子（目标2）使用一种新的实现的SELEX方法，我们已经开发。这些结果将通过使用全基因组染色质免疫沉淀（ChIP-芯片）确定所选转录因子（Aim 3）的体内靶标来扩展。我们期望这些方法的结合将使我们能够确定几乎所有的酵母转录因子的结合位点和靶基因。然后，我们将试图通过确定转录因子结合位点如何促进基因表达来了解酵母启动子的结构原理。通过建立一个潜在基因启动子的大型文库，其中一组结合位点随机分布，我们可以确定决定特定表达模式的结合位点的组合。这种方法将最初开发和测试使用几个很好的特点结合位点，我们希望它将提供一个通用的工具，更全面的研究基因调控的逻辑。

DESCRIPTION (provided by applicant): Although the regulation of gene expression has been intensively studied in the yeast S. cerevisiae, much about this process remains unknown. This is exemplified by our inability to predict, as opposed to explain, the expression pattern of any gene given its promoter sequence. Our long-term goal is to provide a comprehensive map of the S. cerevisiae gene regulatory network that can be used to develop predictive models of gene expression. The first task is to complete the catalog of transcription factors and their binding sites. We will use a combination of existing in vitro and in vivo methods to accomplish that goal. We will identify the binding sites of the more than 100 transcription factors of yeast whose specificity remains unknown (Aim 1) using electrophoretic gel mobility shift assays, a yeast one-hybrid assay, and a novel method to probe protein microarrays with DMA oligonucleotides. We will then develop comprehensive weight matrices of the binding sites of yeast transcription factors (Aim 2) using a novel implementation of the SELEX method we have developed. These results will be extended by determining the in vivo targets of selected transcription factors (Aim 3) using genome-wide chromatin immunoprecipitation (ChlP-Chip). We expect that the combination of these approaches will enable us to determine the binding sites and target genes of nearly all transcription factors of yeast. We will then attempt to learn the architectural principles of yeast promoters by determining how transcription factor binding sites contribute to gene expression. By creating large libraries of potential gene promoters in which a set of binding sites have been randomly distributed, we can ascertain the combinations of binding sites that determine specific expression patterns. This approach will be initially developed and tested using a few well characterized binding sites; we expect it will provide a general tool for more comprehensive studies of the logic of gene regulation.