Genomewide discovery & analysis of alternative promoters

全基因组发现

• 批准号: 7371108
• 负责人: RAMANA V DAVULURI
• 金额: $ 2.07万
• 依托单位: OHIO STATE UNIVERSITY
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371108
• 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

Promoters located at the 5'-ends of genes play a critical role in regulating transcriptional initiation. Emerging evidence suggests that a significant fraction of -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 ~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.

位于基因5末端的启动子在调节转录启动方面起着关键作用。新兴 有证据表明，很大一部分人类基因可能包含替代启动子， 在不同组织，细胞类型和/或 发展阶段。尽管可用于人类基因组序列的大量信息，但 仍然缺乏识别和表征基因基因座替代启动子的全面方法。 一种有效的方法是分析小鼠和人基因组中的直系同源序列。没有 对基因座的5'末端调节区域显示序列相似性的详细探讨 在人与啮齿动物之间。我们假设基因的基础（核）启动子区域是基因所需的 转录是人与小鼠之间的保守。计算方法将用于挖掘 人和小鼠基因组都识别功能性直系同源序列。派生的信息将 被添加到由美国开发的称为MPROMDB的哺乳动物启动子的原型数据库中。下一个， 这些计算得出的序列将经过实验验证。我们进一步假设 功能性基因组序列处于呈现开放染色质的正式环境中 配置，允许访问转录因子以驱动基因表达。而不是服用 分析基因表达的通常途径，我们将确定测试基因组的染色质状态 序列作为功能启动子的度量。染色质免疫沉淀（芯片）微阵列或 以前在我们实验室开发的所谓的CHLP-on-Chip分析将同时扩展到 评估250对人和小鼠直系同源的约1,500个推定的启动子的舒适素状态 基因。提出的计算，统计和高通量实验方法的组合 在此赠款中，应用将有助于更好地描述人类基因组中的基因调节区域，一个 未来基因组研究的巨大挑战。具体我们将：（1）开发用于 注释实验已知的替代启动子和第一个外显子。 （2）在片上进行CHLP和 荧光素酶测定以验证人和小鼠的计算注释的替代启动子序列 直系同源基因，（3）开发用于检测替代启动子和第一外显子的计算方法 在人类和小鼠基因组中。