Comprehensive Identification of Worm and Fly Transcription Factor Binding Sites

我们的项目旨在完成果蝇和线虫的全套转录因子(TF)识别的调控元件的目录。在最初的modENCODE项目中，开发了一条实验管道，并将其应用于每个生物体中的大约100个TF，剩下大约600个TF用于研究每个苍蝇和蠕虫。为了实现这一规模扩大，该项目建立在集团在初始阶段取得的进展的基础上，还结合了生产管道，以提高效率并实现规模经济。对于这两种生物，总体策略是通过大插入克隆的重组工程与增强的绿色荧光蛋白(EGFP)序列融合来标记转录因子基因，并通过转基因将标记的基因引入基因组。使用高质量的抗绿色荧光蛋白抗体的ChLP-seq是在具有最大绿色荧光蛋白表达的发育阶段(S)进行的，通过现有的RNA-seq表达数据来增强。PeakSeq和其他软件对比对的序列读数进行分析，以确定候选结合位点和可能的靶基因。我们将优先考虑具有人类同源基因的函数，以最大限度地提高数据的更广泛效用。对于每个生物体中的40个TF，我们还将研究特定组织或细胞亚群的TF表达，以评估整个动物ChlP-seq分析的特异性和敏感性。我们还将在每个生物体中进行100个转录因子的RNAi，然后进行RNA-SEQ，以验证所谓的峰及其分配的目标基因。最后，我们将整合不同数据集的信息，以构建由TF结合位点数据暗示的调控网络。我们将与ENCODE关于人类转录因子的项目进行协调，我们的数据将提供关键的体内和发育调控信息，这些信息对于描述从根本上保守的以及人类特有的转录因子特性至关重要。

Our project seeks to complete the catalog of the regulatory elements recognized by the full set of transcription factors (TFs) in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. In the initial modENCODE project, an experimental pipeline was developed and applied to ~100 TFs in each organism, leaving approximately 600 TFs to study for each fly and worm. To achieve this scale-up, the project builds on the advances made by the groups in the initial phase and also combines the production pipelines to increase efficiency and to realize economies of scale. For both organisms, the overall strategy tags transcription factor genes by fusion with an enhanced Green Fluorescent Protein (eGFP) sequence through recombineering of large insert clones, and introducing the tagged genes into the genome by transgenesis. ChlP-seq using a high quality anti-GFP antibody is performed on the developmental stage(s) with maximal GFP expression, as augmented by available RNA-seq expression data. The aligned sequence reads are analyzed by PeakSeq and other software to identify candidate binding sites and likely target genes. We will prioritize TFs with human homologs to maximize the broader utility of the data. For 40 TFs in each organism we will also investigate TF expression of specific subsets of tissues or cells to estimate the specificity and sensitivity of whole animal ChlP-seq assays. We will also perform RNAi of 100 TFs in each organism, followed by RNA-seq, to validate called peaks and their assigned target genes. Finally, we will integrate the information for the different data sets to construct regulatory networks implied by the TF binding site data. We will coordinate with ENCODE projects on human TFs, and our data will provide key in vivo and developmental regulatory information that will be essential to delineate both fundamentally conserved as well as human-specific properties of TFs.