Alternative Promoter Usage in Specific Gene Expression

描述：（由申请人提供）转录调控在人类中极其重要且高度复杂。 在这个项目中，我们专注于组织特异性方式的替代第一外显子的转录调控。 第一，外显子在真核生物基因组中的特征通常很差。 此外，替代的第一外显子可以位于彼此远离的基因组位置，导致近端启动子的不同使用。 在文献中有许多关于负责组织特异性表达的广泛分离的替代启动子的实例。 这个建议的中心假设是，第一外显子的选择性剪接是指示替代近端启动子的使用，这将是一个重要的因素，在我们的功能性顺式元件簇，调节差异组织特异性基因表达的鉴定。 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康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。 我们将通过计算表征ENCODE项目选择的30 Mb人类序列中的所有基因，确定约100个具有第一外显子剪接的基因，研究主要组织类型中剪接变体的表达水平，并研究这些基因的启动子中的顺式元件，这些元件可以解释其转录调控。 该项目有四个具体目标：1）计算分析ENCODE序列，并选择约100个表现出第一外显子使用和组织特异性表达模式的基因：2）实验验证目标1中鉴定的所有基因的第一外显子使用，并测量其在10种主要组织类型中的表达水平; 3）计算预测基因的替代近端启动子中的顺式元件簇，并选择约40个最可信的预测，这些预测可以解释组织特异性表达;和4）在五个细胞系中使用转染测定实验性地测试~40个计算预测。

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.