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Deciphering the regulatory code of a cell

破译细胞的调控密码

基本信息

批准号：
8000359
负责人：
GARY D STORMO
金额：
$ 11.88万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-08 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8000359
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

项目摘要

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)：尽管在酿酒酵母中基因表达的调控已经被深入研究，但关于这一过程的许多方面仍不清楚。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一个全面的酿酒酵母基因调控网络图，可以用来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。我们将使用现有的体外和体内方法相结合来实现这一目标。我们将使用凝胶迁移率变化分析、酵母单杂交分析和一种新的利用DNA寡核苷酸探测蛋白质芯片的新方法来鉴定100多个特异性未知的酵母转录因子的结合位点(目标1)。然后，我们将使用我们开发的SELEX方法的一种新实现方式来开发酵母转录因子结合位点的综合权重矩阵(目标2)。这些结果将通过使用全基因组染色质免疫沉淀(ChlP-Chip)来确定选定的转录因子(AIM 3)的体内靶标来扩展。我们希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部位和目标基因。然后，我们将尝试通过确定转录因子结合位点如何影响基因表达来学习酵母启动子的结构原理。通过创建大量潜在的基因启动子文库，其中一组结合位点已经随机分布，我们可以确定决定特定表达模式的结合位点的组合。这种方法最初将使用几个特征良好的结合位点进行开发和测试；我们预计它将为更全面地研究基因调控的逻辑提供一个通用工具。