Identifying transcription factor binding sites in the C. elegans genome

描述（由申请人提供）： 在不同的组织或不同的条件下，我们的约25，000个基因中的每一个的差异表达对于我们的正常发育和功能至关重要。事实上，由转录因子（TF）或其结合的顺式调节基因组DNA元件（TF结合位点或TFBS）突变引起的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。为了充分了解正常发育和病理，并设计有效的治疗方法，因此关键是要了解哪种TF调节哪种基因的表达，在哪里和在什么（发育）条件下。此外，了解每个TF结合的元件以及这些TFBS在基因组中的位置是至关重要的。这是基因组科学中的一个重大挑战，因为对大多数后生动物TF的靶点、结合位点、转录活性和生物学功能知之甚少。我们用线虫C. elegans作为一个模型来应对这一挑战。我们的长期目标是全面绘制和表征所有C.线虫TF和所有基因调控区，并确定所有负责TFBS。目前，ChIP是鉴定TF-DNA相互作用的最流行的方法。虽然功能强大，但后生动物ChIP仅限于广泛和高度表达的少数TF，并且可以获得合适的抗体。为了能够以不依赖于条件的方式鉴定多种后生动物蛋白质-DNA相互作用，我们开发了酵母单杂交（Y1 H）系统的高通量版本。我们的Y1 H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，我们建议在所有C的30%中识别TFBS。首先通过Y1 H分析绘制所有可用基因启动子和TF之间的蛋白质-DNA相互作用，然后使用这些相互作用来计算描绘TFBS。我们每个基因在不同组织或不同条件下的表达对于我们的正常发育和功能至关重要。事实上，转录因子或其结合的基因组DNA序列的突变可导致多种人类疾病，包括几种先天性疾病和癌症。我们将确定哪种TF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。由于这种研究在人类或小鼠的基因组规模上是不可行的，我们选择了蠕虫（C。elegans）作为模型系统。

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.