Center for Functional Validation and Evaluation of ENCODE Enhancer Regions

项目摘要 每个Encode功能表征中心的目的是“开发和应用可泛化的 表征ENCODE项目中确定的候选功能元件在特定情况下作用的方法 生物学背景“。我们建议的中心将专注于候选增强子元件的表征。我们 将开发、改进和应用增强剂功能分析的实验方法。我们将使用两个非常 选择了不同的生物学模型，因为它们具有很高的潜力，可以作为研究的通用样本 在(I)疾病的遗传风险因素和(Ii)涉及癌症的体细胞突变的背景下的增强剂。 我们还将发展和完善我们在ENCODE细胞系中的实验方法，我们将保留25%的 我们为测试候选基因组元素所做的努力将在所有ENCODE中共同研究 功能表征中心。使用STARR-SEQ及其变体，我们将测试以下各项的充分性 候选增强子元件来调节基因表达。使用CRISPR-Cas9方法，我们将编辑 人类基因组，在其内源背景下测试候选增强子元件的必要性。我们会 利用这些方法检测遗传性DNA变异对增强子功能的影响 冠心病(CAD)，并检测获得性体细胞DNA突变对增强子的影响 在胰腺癌(胰腺导管腺癌-PDAC)模型中的作用。虽然我们的方法 必须需要生物信息学组件来利用ENCODE和其他现有数据集，以便 定义最佳候选增强子元件，以便在我们将测试的特定生物模型中进行测试，我们的 中心将专注于增强剂的实验表征，测试不同的组合 为系统化和精确化创建可扩展和可推广的协议的方法 增强子功能的表征。

Project Summary The purpose of each ENCODE Functional Characterization Center is “to develop and apply generalizable approaches to characterize the role of candidate functional elements identified the ENCODE project in specific biological contexts”. Our proposed Center will focus on characterization of candidate enhancer elements. We will develop, refine and apply experimental methods for functional assays of enhancers. We will use two very different biological models chosen for their high potential to act as generalizable exemplars for the study of enhancers in the context of (i) inherited risk factors for disease, and (ii) somatic mutations involved in cancers. We will also develop and refine our experimental methods in ENCODE cell lines, and we will reserve 25% of our efforts for testing candidate genomic elements that will be studied in common across all of the ENCODE Functional Characterization Centers. Using STARR-seq, and variations thereof, we will test for sufficiency of candidate enhancer elements to modulate gene expression. Using CRISPR-Cas9 methods we will edit the human genome, testing for necessity of candidate enhancer elements in their endogenous context. We will utilize these methods to examine the effects of inherited DNA variation on enhancer function in models of coronary artery disease (CAD), and to examine the effects of acquired somatic DNA mutations on enhancer function in models of pancreatic cancer (Pancreatic Ductal Adenocarcinoma – PDAC). While our approach necessarily requires a bioinformatics component to utilize ENCODE and other existing data sets in order to define the best candidate enhancer elements for testing in the specific biological models we will assay, our Center will be focused on experimental characterization of enhancers, testing different combinations of approaches in order to create extensible and generalizable protocols for systematic and accurate characterization of enhancer function.