High-Throughput Identification of Tissue/Cell-Type-Specific Cis Regulatory Module

组织/细胞类型特异性顺式调控模块的高通量鉴定

• 批准号: 8004415
• 负责人: MARTHA L BULYK
• 金额: $ 57.44万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004415
• 关键词: Address; Adult; Applications Grants; Benchmarking; Binding Sites; Biological; Biological Assay; Biological Models; Biology; Boston; Cardiac; Cardiac Myoblasts; Cell fusion; Cells; Child; Code; Consultations; DNA Binding; DNA analysis; Data; Development; Dictionary; Drosophila genus; Educational workshop; Electronic Mail; Embryo; Enhancers; Extramural Activities; Funding; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Genomics; Goals; Grant; Guidelines; Heart; Heart Diseases; Human Resources; Indium; Laboratories; Leadership; Letters; Mesoderm; Mesoderm Cell; Muscle Development; Muscular Dystrophies; Mutation; Myoblasts; Myocardium; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; Natural regeneration; Organism; Participant; Pathway interactions; Pericardial body location; Persons; RNA Interference; Research Personnel; Resolution; Resources; Signal Transduction; Specificity; Stretching; Surveys; System; Techniques; Technology; Telephone; Testing; Time; Tissues; United States National Institutes of Health; Validation; Vertebrates; Visit; Work; base; cell type; combinatorial; congenital heart disorder; fly; gain of function; high throughput technology; in vivo; loss of function; member; mutant; new technology; novel; public health relevance; research study; technology development; tool; transcription factor

In metazoans, gene expression is regulated in a tissue/cell-type specific manner predominantly via stretches of noncoding sequence referred to as cis regulatory modules (CRMs) that regulate the expression of (typically) the adjacent gene(s). CRMs usually contain 1 or more DNA binding sites for 1 or more sequence-specific, regulatory transcription factors (TFs) that function to activate or repress the target gene(s); CRMs that activate gene expression are frequently referred to as "transcriptional enhancers", and have been the focus of many computational and experimental studies. Identification of tissue/cell-type-specific enhancers in metazoans remains a significant challenge. Moreover, despite recent technological advances, a major, rate-limiting bottleneck that is impeding rapid progress in the field is the still quite low-throughput experimental testing of candidate enhancers. The overarching goals of this project are to develop and apply novel 'wet-lab' technology for high-throughput experimental identification of tissue/cell-type-specific transcriptional enhancers. In this project we will focus on the developing embryonic mesoderm in Drosophila as a model system. We will identify cis regulatory modules and analyze their constituent cis regulatory codes that operate in somatic mesoderm (SM) founder cells (FCs) and fusion competent myoblasts (FCMs). Specifically, we will: develop and apply novel 'wet-lab' technology for high-throughput experimental identification of tissue/cell- type-specific transcriptional enhancers; determine the DNA binding specificities of ~75 known and predicted TFs expressed in the Drosophila embryonic mesoderm; predict CRMs and infer cis regulatory codes considering highly combinatorial input from large, high-resolution TF-DNA binding specificity dictionaries; and experimentally validate newly discovered enhancers. Importantly, we anticipate that the technologies, approaches, tools, and data resulting from this project will be generally applicable to other systems and organisms.

在后生动物中，基因表达以组织/细胞类型特异性方式调节 主要通过称为顺式调节的非编码序列延伸 调节（通常）相邻基因表达的模块（CRM）。客户关系管理系统 通常包含 1 个或多个 DNA 结合位点，用于 1 个或多个序列特异性、调节性 具有激活或抑制靶基因功能的转录因子 (TF)；客户关系管理系统 激活基因表达的物质通常被称为“转录增强子”， 并已成为许多计算和实验研究的焦点。鉴别 后生动物中组织/细胞类型特异性增强剂的研究仍然是一个重大挑战。 此外，尽管最近技术取得了进步，但一个主要的速率限制瓶颈是 阻碍该领域快速进展的是仍然相当低通量的实验测试 候选增强子。 该项目的总体目标是开发和应用新型“湿实验室”技术 用于组织/细胞类型特异性转录的高通量实验鉴定 增强剂。在这个项目中，我们将重点关注胚胎中胚层的发育 果蝇作为模型系统。我们将识别顺式监管模块并分析其 在体细胞中胚层 (SM) 创始细胞中运行的顺式组成调控代码 （FC）和具有融合能力的成肌细胞（FCM）。具体来说，我们将： 开发和应用 新颖的“湿实验室”技术，用于组织/细胞的高通量实验鉴定 类型特异性转录增强子；确定约 75 的 DNA 结合特异性 果蝇胚胎中胚层中表达的已知和预测的 TF；预测 CRM 和推断顺式监管代码考虑了来自大型、 高分辨率 TF-DNA 结合特异性字典；并通过实验验证 新发现的增强子。重要的是，我们预计这些技术， 该项目产生的方法、工具和数据将普遍适用于 其他系统和生物体。