Genomewide discovery & analysis of alternative promoters

全基因组发现

• 批准号: 7033451
• 负责人: RAMANA V DAVULURI
• 金额: $ 32.5万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033451
• 关键词: chromatin; clinical research; genes; genome; human

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（由申请人提供）：位于基因5'末端的启动子在调节转录启动中起着至关重要的作用。新兴的证据表明，大约30,000个人类基因可能包含替代启动子的大量部分，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致的调节。尽管可用于人类基因组序列的大量信息，但仍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。尚未彻底探索该基因座的5'末端调节区域显示人与啮齿动物之间的序列相似性的程度。我们假设基因转录所需的基础（核）启动子区域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以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。接下来，将对这些计算得出的序列进行实验验证。我们进一步假设功能性基因组序列是在呈现开放式染色质构型的整体环境中，可以访问转录因子以驱动基因表达。我们将确定测试基因组序列的染色质状态，而不是采取通常的途径来分析基因表达，作为功能启动子的度量。以前在我们的实验室中开发的染色质免疫沉淀（CHIP）微阵列或所谓的CHLP-on-ChIP测定法，将扩展以同时评估大约1,500个人类和小鼠矫正基因的250对推测启动子的舒适素状态。在本赠款应用中提出的计算，统计和高通量实验方法的结合将有助于更好地表征人类基因组中的基因调节区域，这是未来基因组研究的巨大挑战之一。具体而言，我们将：（1）开发用于注释实验已知的替代启动子和第一外显子的计算工具。 （2）进行CHLP片和荧光素酶测定法以验证人和小鼠直系同源基因的计算注释的替代启动子序列，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。