An ENCODE ChIP-seq pipeline using endogenously tagged human DNA-associated proteins

项目摘要/摘要 几乎十分之一的人类基因编码与细胞核中的染色体相互作用的蛋白质。其中大多数都是 DNA相关蛋白(简称DAP)通过以下方式参与调节基因的表达 作为基本转录机制的一部分，作为调节时空的转录因子 转录水平，或作为染色质状态调节。这些蛋白质是生物学中的关键成分，因为 转录调控是生物体发育的基本生物学过程，在 确定分化过程中的细胞状态，并引导对内部和 外部环境。因此，全面和详细地评估DAP的分子作用，即， 它们在整个人类基因组中相互作用，是基础和临床的一个基本长期目标 研究。为响应RFA-HG-16-002，《扩展DNA元素百科全书(ENCODE)》 人和老鼠(UM1)“，这个应用程序建议使用最近建立的”准备好铲子“管道来 在人类细胞系中映射DAP，克服了传统芯片序列非常高的故障率，这是一种广泛的 所使用的方法需要针对每个因子的特定抗体。这种名为CETCH-SEQ的新方法， 包括在编码每种蛋白质的内源位置增加一个表位标签，并使用染色质 抗表位通用抗体免疫沉淀和高通量测序 (CHIP-SEQ)以确定全基因组范围内的DAP-DNA关联。这条生产流水线将应用于每一个 在一组人类细胞系中表达但尚未被ENCODE定位的1,244个DAP。 在为期四年的项目中，这条管道将用于在一个人类细胞系中测试这些因素中的每一个，以及 对于四个人类细胞系中的100个DAP，允许表征细胞类型的差异。该项目将 也标记和检测少数DAP的多个等位基因版本，在这些DAP中，致病的或潜在的 已经确定了致病突变。该项目将制作全基因组DAP地图并识别 数百个人类调节蛋白的基序，为下一阶段的 编码项目。所有数据，以及有用的基因编辑质粒和标记人类的材料 细胞系，将向研究界免费提供。

Project Summary/Abstract Almost a tenth of human genes code for proteins that interact with chromosomes in the nucleus. Most of these DNA-associated proteins (referred to as DAPs) are involved in regulating the expression of genes, by serving as part of the basic transcriptional machinery, as transcription factors that regulate the spatial and temporal levels of transcription, or as chromatin state regulators. These proteins are key components in biology, as transcriptional regulation underlies fundamental biological processes in organismal development, in determining cell states during differentiation, and in directing physiological responses to the internal and external environment. Thus, comprehensive and detailed assessment of the molecular actions of DAPs, that is, where they interact throughout the human genome, is a fundamental long-term goal of both basic and clinical research. In response to RFA-HG-16-002, “Expanding the Encyclopedia of DNA Elements (ENCODE) in the Human and Mouse (UM1)”, this application proposes to use a recently-established "shovel ready" pipeline for mapping DAPs in human cell lines that overcomes the very high failure rates of traditional ChIP-seq, a widely- used approach that requires specific antibodies for each factor. The new approach, called CETCh-seq, involves adding an epitope tag at the endogenous locus encoding each protein, and using chromatin immunoprecipitation with a universal antibody against the epitope followed by high-throughput sequencing (ChIP-seq) to identify DAP-DNA associations genome-wide. This production pipeline will be applied to each of 1,244 DAPs that are expressed in a set of human cell lines and have not yet been mapped by ENCODE. During the four-year project, this pipeline will be used to test each of these factors in one human cell line, and for 100 of the DAPs, in four human cell lines, allowing characterization of cell-type differences. The project will also tag and assay multiple allelic versions of a small number of DAPs in which pathogenic or potentially pathogenic mutations have been identified. The project will produce genome-wide DAP maps and identify motifs for hundreds of human regulatory proteins, providing an important component for the next phase of the ENCODE Project. All data, as well as useful materials in the form of gene editing plasmids and tagged human cell lines, will be made freely available to the research community.