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

描述（由申请人提供）：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cis调节模块（crm）的非编码序列片段调节（通常）相邻基因的表达。crm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的调控转录因子（TFs），其功能是激活或抑制靶基因；激活基因表达的CRMs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。在后生动物中鉴定组织/细胞类型特异性增强子仍然是一个重大挑战。此外，尽管最近技术取得了进步，但阻碍该领域快速发展的主要限速瓶颈是候选增强剂的实验测试仍然相当低通量。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代码。在本项目中，我们将重点研究发育中的果蝇胚胎中胚层作为模型系统。我们将识别顺式调控模块，并分析其组成的顺式调控代码，这些代码在体细胞中胚层（SM）创始细胞（fc）和融合能成肌细胞（fcm）以及心脏细胞（CCs）和心包细胞（PCs）中起作用。具体来说，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；测定果蝇胚胎中胚层中表达的约140个已知和预测的tf的DNA结合特异性；根据高分辨率TF-DNA结合特异性词典的高度组合输入，预测crm和推断顺式调控代码；并通过实验验证新发现的增强子和推断的顺式调控代码。重要的是，我们预计从这个项目中得到的技术、方法、工具和数据将普遍适用于其他系统和生物体。

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.