Genomewide discovery & analysis of alternative promoters

位于基因5 '端的启动子在调节转录起始中起关键作用。新兴 有证据表明约30，000个人类基因中的很大一部分可能含有替代启动子， 其对不同组织、细胞类型和/或细胞中的基因表达产生更精细的调节 发育阶段尽管人类基因组序列的信息量很大， 仍然缺乏鉴定和表征基因座的替代启动子的综合方法。 一种有效的方法是分析小鼠和人类基因组中的正向同源序列。它没有 基因座的5 '端调控区在多大程度上显示出序列相似性， 人类和啮齿动物之间的差异我们假设基因启动子的基础（核心）区域， 转录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性正向序列。衍生信息将 将其添加到我们开发的哺乳动物启动子原型数据库MPromDb中。下一个是 这些计算得到的序列将被实验验证。我们进一步假设， 功能基因组序列处于常染色质环境中， 这种构型允许转录因子进入以驱动基因表达。而不是采取 通常的途径来分析基因表达，我们将确定测试基因组的染色质状态 序列作为功能性启动子的量度。染色质免疫沉淀（ChIP）微阵列，或 我们实验室先前开发的所谓ChIP芯片检测将被扩展到同时 评估250对人类和小鼠直向染色体的约1，500个推定启动子的常染色质状态 基因.结合计算，统计和高通量的实验方法提出了 在这项拨款申请将有助于更好地描述人类基因组中的基因调控区域， 未来基因组研究的巨大挑战。具体来说，我们将：（1）开发计算工具， 注释实验已知的替代启动子和第一外显子。(2)进行ChIP芯片， 荧光素酶测定以验证计算注释的人和小鼠的替代启动子序列 直系同源基因，和（3）开发计算方法来检测替代启动子和第一外显子 在人类和老鼠的基因组中。

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.