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

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节模块（CRM）的非编码序列的延伸来调节，所述顺式调节模块（CRM）调节（通常）相邻基因的表达。CRM通常含有1个或多个DNA结合位点，用于1个或多个序列特异性调节转录因子（TF），其功能是激活或抑制靶基因;激活基因表达的CRM通常被称为转录增强子，并且一直是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型的湿实验室技术，用于组织/细胞类型特异性转录增强子的高通量实验鉴定。在本项目中，我们将重点研究果蝇发育中的胚胎中胚层作为模型系统。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）。具体而言，我们将：开发和应用新技术湿实验室组织/细胞类型特异性转录增强子的高通量实验鉴定技术;确定果蝇胚胎中胚层中表达的约75种已知和预测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;并实验验证新发现的增强子。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。

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.