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: 9247485
• 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/9247485
• 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.

项目总结/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。大多数这些 DNA相关蛋白（称为DAP）参与调节基因的表达， 作为基本转录机制的一部分，作为调节空间和时间的转录因子， 转录水平，或作为染色质状态调节剂。这些蛋白质是生物学中的关键成分， 转录调控是生物体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，并在指导生理反应的内部和 外部环境因此，全面而详细地评估DAPs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。 research.为了响应RFA-HG-16-002，“在DNA中扩展DNA元件百科全书（ENCODE）”， 人和老鼠（UM 1）"，该申请提出使用最近建立的“铲就绪”管道， 在人类细胞系中绘制DAP，克服了传统ChIP-seq的极高失败率，这是一种广泛的 所使用的方法需要针对每个因子的特异性抗体。这种新方法被称为CETCh-seq， 包括在编码每种蛋白质的内源基因座处添加表位标签，并使用染色质 用针对表位的通用抗体进行免疫沉淀，然后进行高通量测序 （ChIP-seq）以鉴定全基因组的DAP-DNA关联。该生产管道将应用于 1，244个DAP在一组人类细胞系中表达，尚未被ENCODE定位。 在为期四年的项目中，该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， 在四种人类细胞系中，对100种DAP进行了分析，从而可以表征细胞类型差异。该项目将 还标记和测定少数DAP的多个等位基因版本，其中致病性或潜在的 已经鉴定出致病突变。该项目将制作全基因组DAP图谱， 为数百种人类调节蛋白的基序，为下一阶段的研究提供了重要组成部分。 ENCODE项目。所有的数据，以及有用的材料，在基因编辑质粒的形式和标记的人类 细胞系，将免费提供给研究界。