Alternative Promoter Usage in Tissue-specific Gene Expr*

描述：(申请人提供)转录调控在人类中是极其重要和高度复杂的。在这个项目中，我们专注于以组织特异性的方式对备选第一外显子的转录调控。首先，在真核生物基因组中，外显子的特征常常很差。此外，可选择的第一外显子可以位于彼此相隔很远的基因组位置，导致近端启动子的不同使用。文献中有许多广泛分离的替代启动子负责组织特异性表达的例子。这一建议的中心假设是，第一外显子的选择性剪接指示了选择性近端启动子的使用，这将是我们识别调节组织特异性差异基因表达的功能顺式元件簇的重要因素。 该项目将翁的实验室的计算专业知识与波士顿大学的丁和坎托最近开发的极其敏感和定量的基因表达技术紧密结合起来。我们将对ENCODE项目选择的人类30Mb序列中的所有基因进行计算表征，确定约100个具有选择性第一外显子剪接的基因，研究剪接变体在主要组织类型中的表达水平，并研究这些基因启动子中可能解释其转录调控的顺式元件。 该项目有四个具体目标：1)通过计算分析ENCODE序列，并选择同时显示可供选择的第一外显子和组织特定表达模式的约100个基因；2)通过实验验证Aim 1中确定的所有基因的第一外显子使用，并测量其在十种主要组织类型中的表达水平；3)通过计算预测基因的可选近端启动子中的顺式元件簇，并选择约40个可解释组织特定表达的最可信的预测；以及4)使用在五个细胞系中的转染试验对~40个计算预测进行实验测试。

DESCRIPTION: (provided by applicant) Transcriptional regulation is extremely important and highly complex in humans. In this project, we focus on the transcriptional regulation of alternative first exons in a tissue specific manner. First exons are often poorly characterized in eukaryotic genomes. Furthermore, alternative first exons can reside in genomic locations that are far apart from each other, leading to distinct usage of proximal promoters. There are numerous examples in the literature on widely separated alternative promoters that are responsible for tissue specific expression. The central hypothesis of this proposal is that alternative splicing of first exons is indicative of alternative proximal promoter usage, which will be an important factor in our identification of functional cis-element clusters that regulate differential tissue-specific gene expressions. The project tightly couples the computational expertise in Weng's laboratory with the extremely sensitive and quantitative gene expression technique recently developed by Ding and Cantor at Boston University. We will computationally characterize all genes in the 30 Mb of human sequence selected by the ENCODE project, identify ~ 100 genes with alternative first exon splicing, study the expression levels of splicing variants in major tissue types, and investigate the cis-elements in the promoters of these genes that could account for their transcriptional regulation. The project has four specific aims: 1) Computationally analyze the ENCODE sequences and select ~100 genes that exhibit both alternative first exon usage and tissue specific expression patterns; 2) Experimentally validate first exon usage for all genes identified in Aim 1 and measure their expression levels in ten major tissue types; 3) Computationally predict cis-element clusters in the alternative proximal promoters of the genes and select ~40 most confident predictions that may account for tissue-specific expressions; and 4) Experimentally test the ~40 computational predictions using transfection assays in five cell lines.