Deciphering the regulatory code of a cell

描述（由申请人提供）：尽管在酿酒酵母中基因表达的调控已经得到了深入的研究，但关于这一过程的许多内容仍然未知。我们无法预测，而不是解释任何给定启动子序列的基因的表达模式，这就是例证。我们的长期目标是提供酿酒葡萄球菌基因调控网络的综合图谱，该图谱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。我们将结合现有的体外和体内方法来实现这一目标。我们将使用电泳凝胶迁移转移试验、酵母单杂交试验和一种新的方法来探测带有DMA寡核苷酸的蛋白质微阵列，确定100多个特异性未知的酵母转录因子的结合位点（目的1）。然后，我们将使用我们开发的SELEX方法的新实现开发酵母转录因子结合位点的综合权重矩阵（Aim 2）。这些结果将通过使用全基因组染色质免疫沉淀（ChlP-Chip）确定选定转录因子（Aim 3）的体内靶点来扩展。我们期望这些方法的结合将使我们能够确定几乎所有酵母转录因子的结合位点和靶基因。然后，我们将尝试通过确定转录因子结合位点如何促进基因表达来学习酵母启动子的结构原理。通过建立大量的潜在基因启动子文库，其中一组结合位点随机分布，我们可以确定结合位点的组合，从而确定特定的表达模式。这种方法最初将使用几个特征明确的结合位点进行开发和测试；我们期望它将为更全面的基因调控逻辑研究提供一个通用的工具。

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.