Deciphering the regulatory code of a cell

破译细胞的调控密码

• 批准号: 7619580
• 负责人: GARY D STORMO
• 金额: $ 42.94万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7619580
• 关键词: Algorithms; Animal Model; Binding; Binding Sites; Biological Assay; Cataloging; Catalogs; Cells; Code; Computing Methodologies; Conserved Sequence; Custom; DNA; DNA-Binding Proteins; Data; Development; Engineered Gene; Engineering; Frequencies; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genomics; Glucose; Goals; Hybrids; In Vitro; Knowledge; Learning; Libraries; Logic; Maps; Measurement; Metabolic Pathway; Methods; Modeling; Nature; Oligonucleotides; Pattern; Process; Promoter Regions; Protein Microchips; Regulator Genes; Research Personnel; Saccharomyces cerevisiae; Site; Specificity; Stem cells; Study models; Testing; Transcription factor genes; Weight; Work; Yeasts; cellular engineering; chromatin immunoprecipitation; combinatorial; design; gel mobility shift assay; genome-wide; in vivo; mathematical model; new technology; novel; predictive modeling; programs; promoter; research study; response; tool; transcription factor

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.

描述（由申请人提供）：尽管基因表达的调节已在酵母菌酿酒酵母中进行了深入研究，但关于这一过程的许多方面仍然未知。我们无法预测任何基因的启动子序列的表达模式，这是一个例证。我们的长期目标是提供酿酒酵母基因调节网络的综合图，该图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率转移测定法，一种酵母单杂交测定法以及一种用DMA寡核苷酸探测蛋白质微阵列的新方法。然后，我们将使用我们开发的SELEX方法的新实现来开发酵母转录因子的结合位点的全面重量矩阵（AIM 2）。这些结果将通过使用全基因组染色质免疫沉淀（CHLP-CHIP）确定所选转录因子的体内靶标（AIM 3）来扩展这些结果。我们预计这些方法的组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。通过创建大量的潜在基因启动子的文库，其中一组结合位点已被随机分布，我们可以确定确定特定表达模式的结合位点的组合。最初将使用一些表征良好的结合位点开发和测试这种方法。我们预计它将为基因调节逻辑的更全面研究提供一种通用工具。