Comprehensive Identification of Worm and Fly Transcription Factor Binding Sites

我们的项目旨在完成由果蝇和线虫中的全套转录因子（TF）识别的调控元件的目录。在最初的modENCODE项目中，开发了一个实验管道，并将其应用于每种生物体中的约100个TF，为每种苍蝇和蠕虫留下约600个TF进行研究。为了实现这一规模扩大，该项目以各集团在初始阶段取得的进展为基础，并结合生产管道，以提高效率并实现规模经济。对于这两种生物体，总体策略通过与增强的绿色荧光蛋白（eGFP）序列融合来标记转录因子基因，所述融合通过大插入克隆的重组工程进行，并通过转基因将标记的基因引入基因组中。使用高质量抗GFP抗体的ChIP-seq在具有最大GFP表达的发育阶段进行，如通过可用的RNA-seq表达数据增强的。通过PeakSeq和其他软件分析比对的序列读段以鉴定候选结合位点和可能的靶基因。我们将优先考虑与人类同源的TF，以最大限度地扩大数据的效用。对于每种生物体中的40个TF，我们还将研究组织或细胞的特定子集的TF表达，以估计整个动物ChIP-seq测定的特异性和灵敏度。我们还将在每个生物体中进行100个TF的RNAi，然后进行RNA-seq，以验证所谓的峰及其指定的靶基因。最后，我们将整合不同数据集的信息，构建TF结合位点数据所隐含的调控网络。我们将与ENCODE项目对人类转铁蛋白进行协调，我们的数据将提供关键的体内和发育调控信息，这些信息对于描述转铁蛋白的基本保守特性和人类特异性特性至关重要。

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.