Genomewide discovery & analysis of alternative promoters

描述（由申请人提供）：位于基因5'端的启动子在调控转录起始中起关键作用。新出现的证据表明，大约30,000个人类基因中的很大一部分可能包含替代启动子，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复杂的调节。尽管有大量关于人类基因组序列的信息，但一种鉴定和表征基因位点替代启动子的综合方法仍然缺乏。一种有效的方法是同时分析小鼠和人类基因组中的同源序列。人类和啮齿动物基因座的5′端调控区在多大程度上显示序列相似性，目前还没有得到充分的研究。我们假设基因转录所需的基础（核心）启动子区域在人和小鼠之间是保守的。计算方法将用于挖掘人类和小鼠基因组，以鉴定功能同源序列。所得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，这些计算得到的序列将被实验验证。我们进一步假设，功能性基因组序列处于染色质开放的常染色质环境中，允许转录因子进入驱动基因表达。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，作为功能性启动子的测量。先前在我们实验室开发的染色质免疫沉淀（ChIP）微阵列，或所谓的chlp芯片检测，将扩展到同时评估250对人类和小鼠同源基因的大约1500个假定启动子的常染色质状态。本基金申请中提出的计算、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控区域，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)进行芯片上的chlp和荧光素酶检测，验证计算注释的人类和小鼠同源基因的替代启动子序列；(3)开发计算方法，检测人类和小鼠基因组中的替代启动子和第一外显子。

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.