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: 8151048
• 负责人: MARTHA L BULYK
• 金额: $ 56.95万
• 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/8151048
• 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; Myoblasts; 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; cardiogenesis; cell type; combinatorial; computer studies; congenital heart disorder; fly; gain of function mutation; high throughput technology; in vivo; loss of function; member; mutant; new technology; novel; public health relevance; research study; technology development; tool; transcription factor

DESCRIPTION (provided by applicant): 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' technologies for high-throughput experimental identification of tissue/cell-type-specific transcriptional enhancers, and to use them to decipher cis regulatory codes that control gene expression in a tissue- and cell-type-specific manner. 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), and in cardial cells (CCs) and pericardial cells (PCs). Specifically, we will: develop and apply novel 'wet-lab' technologies for high-throughput experimental identification of tissue/cell-type-specific transcriptional enhancers; determine the DNA binding specificities of ~140 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 and inferred cis regulatory codes. Importantly, we anticipate that the technologies, approaches, tools, and data resulting from this project will be generally applicable to other systems and organisms. PUBLIC HEALTH RELEVANCE: 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. The overarching goals of this renewal project are to develop and apply novel 'wet-lab' technologies for high-throughput experimental identification of tissue/cell-type- specific transcriptional enhancers, and to use them to decipher cis regulatory codes that control gene expression in a tissue- and cell-type specific manner. In this project we will focus on the developing embryonic mesoderm in Drosophila as a model system. There is remarkable conservation of all the major regulatory components, including both signals and TFs, governing heart and muscle development in vertebrates and Drosophila. A deeper understanding of these pathways and their integration is essential for developing rational approaches to congenital heart disease and muscular dystrophies in children and to cardiac regeneration for acquired heart disorders in adults. We anticipate that the technologies, approaches, tools, and data resulting from this project will be generally applicable to other systems and organisms.

描述（由申请人提供）：在后生动物中，基因表达主要通过称为顺式调控模块（CRM）的非编码序列片段以组织/细胞类型特异性方式进行调控，这些非编码序列片段（通常）调控相邻基因的表达。CRM通常含有一个或多个DNA结合位点，用于激活或抑制靶基因的一个或多个序列特异性调节转录因子（TF）;激活基因表达的CRM通常被称为“转录增强子”，并且已经成为许多计算和实验研究的焦点。鉴定后生动物中的组织/细胞类型特异性增强子仍然是一个重大挑战。此外，尽管最近的技术进步，一个主要的，限速瓶颈，阻碍了该领域的快速发展是候选增强子的仍然相当低的吞吐量实验测试。该项目的总体目标是开发和应用新的“湿实验室”技术，用于组织/细胞类型特异性转录增强子的高通量实验鉴定，并使用它们来破译以组织和细胞类型特异性方式控制基因表达的顺式调控密码。在这个项目中，我们将集中在果蝇胚胎中胚层发育作为一个模式系统。我们将确定顺式调控模块，并分析其组成顺式调控代码，在体细胞中胚层（SM）创始人细胞（FC）和融合能力成肌细胞（FCM），并在胰岛细胞（CC）和心包细胞（PC）。具体而言，我们将：开发和应用新的“湿实验室”技术，用于组织/细胞类型特异性转录增强子的高通量实验鉴定;确定果蝇胚胎中胚层中表达的约140种已知和预测的TF的DNA结合特异性;预测CRM并推断顺式调节代码，考虑来自大型高分辨率TF-DNA结合特异性字典的高度组合输入;并通过实验验证新发现的增强子和推断的顺式调节密码。重要的是，我们预计该项目产生的技术，方法，工具和数据将普遍适用于其他系统和生物体。 公共卫生关系：在后生动物中，基因表达以组织/细胞类型特异性方式主要通过称为顺式调控模块的非编码序列的延伸来调控。这个更新项目的总体目标是开发和应用新的“湿实验室”技术，用于组织/细胞类型特异性转录增强子的高通量实验鉴定，并使用它们来破译以组织和细胞类型特异性方式控制基因表达的顺式调控密码。在这个项目中，我们将集中在果蝇胚胎中胚层发育作为一个模式系统。在脊椎动物和果蝇中，控制心脏和肌肉发育的所有主要调控成分（包括信号和转铁蛋白）都有显著的保守性。更深入地了解这些途径及其整合对于制定合理的方法来治疗儿童先天性心脏病和肌营养不良症以及成人获得性心脏病的心脏再生至关重要。我们预计，该项目产生的技术，方法，工具和数据将普遍适用于其他系统和生物体。