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

使用内源标记的人类 DNA 相关蛋白的 ENCODE ChIP-seq 流程

• 批准号: 9420660
• 负责人: Eric Mendenhall
• 金额: $ 195.94万
• 依托单位: HUDSON-ALPHA INSTITUTE FOR BIOTECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9420660
• 关键词: Alleles; Antibodies; Basic Science; Binding; Biological Assay; Biological Process; Biology; Cell Line; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosomes; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; DNA; DNA Binding; Data; Data Coordinating Center; Data Set; Development; Disease; Elements; Encyclopedia of DNA Elements; Environment; Epitopes; Failure; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Genomics; Goals; HepG2; High-Throughput Nucleotide Sequencing; Human; Human Biology; Human Cell Line; Human Genome; Human Genome Project; Individual; K-562; Light; Location; Maps; Measurement; Molecular; Mus; Mutation; Neurons; Pathogenicity; Pathologic; Pathology; Phase; Physiological; Plasmids; Positioning Attribute; Production; Property; Protein Isoforms; Protein Microchips; Proteins; Protocols documentation; Quality Control; Recording of previous events; Regulator Genes; Regulatory Element; Research; Technology; Testing; The Cancer Genome Atlas; Transcriptional Regulation; Validation; Variant; cell type; chromatin immunoprecipitation; data sharing; flexibility; genetic makeup; genetic regulatory protein; genome editing; genome wide association study; genome-wide; human DNA; human disease; improved; induced pluripotent stem cell; novel strategies; repository; response; success; transcription factor

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.

项目摘要/摘要 几乎十分之一的人基因代码与核中染色体相互作用的蛋白质代码。其中大多数 通过服务，与DNA相关蛋白（称为DAPS）参与调节基因的表达 作为基本转录机械的一部分，作为调节空间和临时性的转录因子 转录水平，或作为染色质状态调节剂。这些蛋白质是生物学的关键组成部分， 转录调节是有机发育中基本生物学过程的基础， 在分化过程中确定细胞状态，并指导对内部和内部和 外部环境。这是对DAP的分子作用的全面和详细评估，也就是说 他们在整个人类基因组中相互作用的地方是基本和临床的基本长期目标 研究。为了响应RFA-HG-16-002，“扩展了DNA元素的百科全书（encode） 人类和鼠标（UM1）”，该申请建议使用最近建立的“铲子就绪”管道 在人类细胞系中映射DAP，从而克服了传统芯片seq的极高失败率， 使用的方法需要针对每个因素的特定抗体。新方法，称为cetch-seq， 涉及在编码每种蛋白质的内源基因座上添加情节标签，并使用染色质 具有针对表位的通用抗体的免疫沉淀，然后进行高通量测序 （chip-seq）识别全基因组DAP-DNA关联。该生产管道将应用于每个 1,244个DAP在一组人类细胞系中表达，并且尚未被编码映射。 在四年项目中，该管道将用于在一个人类细胞系中测试这些因素，并且 对于100个DAP，在四个人类细胞系中，可以表征细胞类型的差异。该项目将 还标记并测定多种少量DAP的私人版本，其中病原或潜在 已经确定了致病性突变。该项目将产生全基因组DAP地图并识别 数百种人类调节蛋白的基序，为下一阶段的下一阶段提供了重要组成部分 编码项目。所有数据以及以基因编辑质粒形式的有用材料并标记为人类 细胞系将免费提供给研究社区。