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

在后生动物中，基因的表达主要通过被称为顺式调节模块(CRM)的非编码序列来调节组织/细胞类型的特定方式，这些非编码序列调节(通常)邻近基因(S)的表达。CRM通常包含一个或多个序列特异性的调节性转录因子(TF)的一个或多个DNA结合位点，其功能是激活或抑制靶基因(S)；激活基因表达的CRM通常被称为转录增强因子，一直是许多计算和实验研究的重点。在后生动物中识别特定于组织/细胞类型的增强子仍然是一个重大挑战。此外，尽管最近取得了技术进步，但阻碍该领域快速发展的一个主要的限速瓶颈是对候选增强剂的实验测试仍然相当低。 该项目的总体目标是开发和应用新的“湿实验室”技术，用于高通量组织/细胞类型特定转录增强子的高通量实验鉴定。在本项目中，我们将以果蝇发育中的胚胎中胚层为模型系统。我们将识别顺式调节模块，并分析其在体细胞中胚层(SM)、创始细胞(FCS)和融合能力成肌细胞(FCM)中起作用的顺式调节编码。具体地说，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；确定在果蝇胚胎中胚层表达的~75个已知和预测的TF的DNA结合特异性；考虑大型高分辨率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；以及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.