Genomewide discovery & analysis of alternative promoters

全基因组发现

• 批准号: 7678211
• 负责人: RAMANA V DAVULURI
• 金额: $ 28.89万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-27 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678211
• 关键词: Antibodies; Applications Grants; Automobile Driving; Biological Assay; Chromatin; Code; Computing Methodologies; DNA; Data; Databases; Development; Environment; Euchromatin; Exons; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Heterochromatin; Histones; Human; Human Genome; Laboratories; Length; Luciferases; Lysine; Maps; Measures; Mining; Mus; Nucleic Acid Regulatory Sequences; Orthologous Gene; Pattern; Play; Polymerase Chain Reaction; Promoter Regions; Proteins; Research; Research Personnel; Rodent; Role; Route; Sampling; Specific qualifier value; Staging; Testing; Tissue Sample; Tissues; Transcription Initiation; Transcription Initiation Site; cell type; chromatin immunoprecipitation; computerized tools; data mining; functional genomics; genome sequencing; improved; mammalian genome; mouse genome; novel; programs; promoter; prototype; tool; transcription factor

DESCRIPTION (provided by applicant): Promoters located at the 5'-ends of genes play a critical role in regulating transcriptional initiation. Emerging evidence suggests that a significant fraction of approximately 30,000 human genes likely contain alternative promoters, which produce more elaborate regulation of gene expression in different tissues, cell-types and/or developmental stages. Despite vast information available for the human genome sequences, a comprehensive approach for identifying and characterizing alternative promoters of gene loci is still lacking. One effective approach is to analyze orthologous sequences in both mouse and human genomes. It has not been thoroughly explored as to what extent the 5'-end regulatory regions of a locus show sequence similarity between human and rodents. We hypothesize that the basal (core) promoter regions necessary for gene transcription are conserved between human and mouse. Computational approaches will be used to mine both human and mouse genomes to identify functional orthologous sequences. The derived information will be added into a prototype database for mammalian promoters, called MPromDb, developed by us. Next, these computationally derived sequences will be experimentally verified. We further hypothesize that a functional genomic sequence is in a euchromatic environment that presents an open chromatin configuration, allowing for the access of transcription factors for driving gene expression. Instead of taking the usual route to analyze gene expression, we will determine the chromatin status of a test genomic sequence as a measure for a functional promoter. The chromatin immunoprecipitation (ChIP) microarray, or the so-called ChlP-on-chip assay, previously developed in our laboratory will be extended to simultaneously assess the euchromatin status of approximately 1,500 putative promoters of 250 pairs of human and mouse orthologous genes. The combination of computational, statistical and highthroughput experimental approaches proposed in this grant application will help better characterize the gene regulatory regions in the human genome, one of the grand challenges of future genome research. Specifically we will: (1) Develop computational tools for annotating experimentally known alternative promoters and first exons. (2) Conduct ChlP-on-chip and luciferase assays to verify computationally annotated alternative promoter sequences of human and mouse orthologous genes, and (3) Develop computational methods to detect alternative promoters and first exons in the human and mouse genomes.

描述(申请人提供)：位于基因5‘端的启动子在调节转录起始中起着关键作用。新出现的证据表明，在大约30,000个人类基因中，有相当一部分可能含有替代启动子，这些启动子对不同组织、细胞类型和/或发育阶段的基因表达产生更精细的调节。尽管人类基因组序列提供了大量的信息，但仍缺乏一种全面的方法来识别和表征基因座的替代启动子。一种有效的方法是分析老鼠和人类基因组中的同源序列。一个基因座的5‘端调控区在多大程度上显示了人类和啮齿动物之间的序列相似性，目前还没有深入的研究。我们假设基因转录所需的基本(核心)启动子区域在人和鼠之间是保守的。计算方法将被用来挖掘人类和小鼠的基因组，以确定功能上的同源序列。得到的信息将被添加到我们开发的哺乳动物启动子原型数据库MPromDb中。接下来，将对这些通过计算得出的序列进行实验验证。我们进一步假设，功能基因组序列处于一个等染色质环境中，该环境呈现开放的染色质配置，允许转录因子访问以驱动基因表达。我们将不采用通常的方法来分析基因表达，而是确定测试基因组序列的染色质状态，作为功能启动子的衡量标准。染色质免疫沉淀(CHIP)微阵列，或所谓的ChlP-on-ChIP分析，将扩展到同时评估250对人和小鼠的250对同源基因中大约1500个假定的启动子的常染色质状态。在这项拨款申请中提出的计算、统计和高通量实验方法的组合将有助于更好地描述人类基因组中的基因调节区，这是未来基因组研究的重大挑战之一。具体来说，我们将：(1)开发计算工具，用于注释实验中已知的替代启动子和第一外显子。(2)进行ChlP-on-Chip和荧光素酶分析，以验证计算注释的人类和小鼠同源基因的替代启动子序列，以及(3)开发计算方法来检测人类和小鼠基因组中的替代启动子和第一外显子。