Identifying transcription factor binding sites in the C. elegans genome

描述(由申请人提供)： 项目摘要我们的大约25,000个基因中的每一个在不同的组织或不同的条件下的差异表达对我们的正常发育和功能至关重要。事实上，转录因子(TF)或其结合的顺式调节基因组DNA元件(TF结合位点或TFBs)的突变导致差异基因表达的变化可导致多种人类疾病，包括几种先天性疾病和癌症。因此，为了充分了解正常发育和病理，并设计有效的治疗方法，了解哪种转铁蛋白调节哪种基因的表达，在什么地方和哪种(发育)条件下是至关重要的。此外，了解每个转铁蛋白结合的元件以及这些转铁蛋白在基因组中的位置也是至关重要的。这是基因组科学中的一个重大挑战，因为人们对大多数后生动物转录因子的靶标、结合部位、转录活性和生物学功能知之甚少。我们使用线虫线虫作为模型来应对这一挑战。我们的长期目标是全面定位和表征所有线虫TF和所有基因调控区之间的蛋白质-DNA相互作用，并确定所有负责的TFBs。目前，芯片是鉴定TF-DNA相互作用最常用的方法。虽然功能强大，但后生动物芯片仅限于少数广泛和高度表达的TF，并且有合适的抗体可用。为了能够以独立于条件的方式鉴定各种后生动物蛋白质-DNA相互作用，我们开发了一种高通量版本的酵母单杂交(Y1H)系统。我们的Y1H系统可以与我们创建的几个Gateway资源一起使用，包括由6,000个启动子组成的启动子组，以及所有940个预测蠕虫TF中约80%的ORF克隆。在这里，我们建议通过Y1H分析首先定位所有可用的基因启动子与TF之间的蛋白质-DNA相互作用，然后利用这些相互作用来计算描述TFBS，从而在所有线虫启动子中识别TFBS。项目叙述我们的每个基因在不同组织或不同条件下的表达对我们的正常发育和功能至关重要。事实上，转录因子或它们所结合的基因组DNA序列的突变可能会导致多种人类疾病，包括几种先天性疾病和癌症。我们将确定哪种转铁蛋白调节哪种基因的表达，以深入了解正常发育和疾病，并设计有效的治疗方法。由于这样的研究在人类或老鼠的基因组水平上是不可行的，我们选择了蠕虫(线虫)作为一个模型系统。

DESCRIPTION (provided by applicant): Project summary The differential expression of each of our ~25,000 genes in different tissues or under different conditions is critical for our proper development and function. Indeed, changes in differential gene expression caused by mutations in transcription factors (TFs) or the cis-regulatory genomic DNA elements they bind to (TF binding sites, or TFBSs) can result in a variety of human diseases, including several congenital disorders and cancer. In order to fully understand both normal development and pathologies, and to design effective therapeutics it is therefore critical to understand which TF regulates the expression of which gene, where and under which (developmental) conditions. In addition, it is essential to know the elements each TF binds to and where in the genome these TFBSs are located. This is a major challenge in genomic science as very little is known about the targets, binding sites, transcriptional activity and biological function for the majority of metazoan TFs. We use the nematode C. elegans as a model to address this challenge. Our long-term goal is to comprehensively map and characterize the protein-DNA interactions between all C. elegans TFs and all gene regulatory regions, and to identify all responsible TFBSs. Currently, ChIP is the most popular method to identify TF-DNA interactions. Although powerful, metazoan ChIP is limited to the few TFs that are widely and highly expressed, and for which suitable antibodies are available. To enable the identification of a wide variety of metazoan protein-DNA interactions in a condition-independent manner, we developed a high-throughput version of the yeast one-hybrid (Y1H) system. Our Y1H system can be used with several Gateway resources we created, including a promoterome that consists of 6,000 promoters, as well as ORF clones for ~80% of all 940 predicted worm TFs. Here, we propose to identify TFBSs throughout 30% of all C. elegans promoters by first mapping protein-DNA interactions between all available gene promoters and TFs by Y1H assays, and then to use these interactions to computationally delineate TFBSs. Project narrative The expression of each of our genes in different tissues or under different conditions is critical for our proper development and function. Indeed mutations in transcription factors or the genomic DNA sequences they bind to can result in a variety of human diseases, including several congenital disorders and cancer. We will identify which TF regulates the expression of which gene to gain insight into both normal development and disease, and to design effective therapeutics. Since such studies are not feasible at the genome scale in humans or mice, we have chosen the worm (C. elegans) as a model system.