Enhancing ENCODE Through a Transcription Factor Tagging Approach to ChIP-seq

通过 ChIP-seq 的转录因子标记方法增强 ENCODE

• 批准号: 7853780
• 负责人: KEVIN P. WHITE
• 金额: $ 90万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7853780
• 关键词: Affinity Chromatography; Antibodies; Antibody Formation; Bacterial Artificial Chromosomes; Binding Proteins; Binding Sites; C-terminal; Cell Line; Cells; Chicago; Chromatin; Chronic Myeloid Leukemia; Collaborations; DNA; Data; Elements; Endogenous Factors; Ensure; Epitopes; Funding; Genetic Transcription; Genomics; Goals; HeLa S3; Hela Cells; Human; Human Genome; Institutes; K-562; K562 Cells; Lead; Mammalian Cell; Maps; Methods; N-terminal; Nuclear Receptors; Peptide Hydrolases; Peptides; Physiological; Production; Protein Analysis; Proteins; Site; Speed; Systems Biology; TEV protease; Technology; Tertiary Protein Structure; Testing; Transfection; Validation; Work; base; chromatin immunoprecipitation; enhanced green fluorescent protein; genome-wide; public health relevance; stable cell line; success; transcription factor

DESCRIPTION (provided by applicant): We propose to use bacterial artificial chromosome (BAC) recombineering to epitope tag 40 transcription factors per year for chromatin immunoprecipitation (ChIP) followed by sequencing to map binding sites genome wide. A major hurdle for the ENCODE project is the availability of ChIP-grade antibodies for each factor to be analyzed. Epitope tagging of chromatin-associated proteins presents an alternative approach for ChIP, using the same epitope-specific antibody for each factor. Expressing epitope tagged factors from BACs ensures that the factors are expressed at near-physiological levels due to the presence of endogenous regulatory sequences that drive each tagged factor from its native local genomic context. We propose to analyze a diversity of transcription factors using this method, which we have already demonstrated for more than 20 nuclear receptor class proteins, a forkhead domain protein, Jun and Fos, and several other types of factors (Poser et al. 2008; Hua, Kittler and White 2009). The goal of this project is to integrate our approach with the ENCODE project, testing it for a wider diversity of transcription and chromatin-associated factors and scaling the approach to production levels necessary for ENCODE. The proposed project involves a formal collaboration between the White and Snyder labs, as well as integration with other funded ENCODE and human epigenome projects. PUBLIC HEALTH RELEVANCE: Using a BAC recombineering approach, we propose to systematically epitope tag transcription and chromatin associated factors for ChIP-seq to speed current large-scale mapping projects such as the Encyclopedia of DNA Elements (ENCODE) project by eliminating the laborious step of antibody production and testing. The technology presented here has the potential to facilitate the large-scale identification of the binding sites of mammalian transcription factors and other chromatin-binding proteins. This technology will also enable the ChIP analysis of proteins that are recalcitrant to ChIP grade antibody production and thus impractical to map using the conventional factor-specific antibody ChIP approach for mammalian cells.

描述（由申请人提供）：我们建议每年使用细菌人工染色体 (BAC) 重组技术对 40 个转录因子进行表位标记，进行染色质免疫沉淀 (ChIP)，然后进行测序以绘制全基因组结合位点图谱。 ENCODE 项目的一个主要障碍是每个待分析因素的 ChIP 级抗体的可用性。染色质相关蛋白的表位标记为 ChIP 提供了另一种方法，对每个因子使用相同的表位特异性抗体。从 BAC 中表达表位标记因子可确保这些因子以接近生理水平的水平表达，因为内源性调控序列的存在可驱动每个标记因子脱离其天然的局部基因组环境。我们建议使用这种方法分析多种转录因子，我们已经针对 20 多种核受体类蛋白、叉头结构域蛋白、Jun 和 Fos 以及其他几种类型的因子进行了证明（Poser 等人，2008 年；Hua ，基特勒和怀特 2009）。该项目的目标是将我们的方法与 ENCODE 项目相结合，测试其更广泛的转录和染色质相关因子的多样性，并将该方法扩展到 ENCODE 所需的生产水平。拟议的项目涉及 White 和 Snyder 实验室之间的正式合作，以及与其他资助的 ENCODE 和人类表观基因组项目的整合。 公共健康相关性：使用 BAC 重组工程方法，我们建议系统地进行 ChIP-seq 表位标签转录和染色质相关因子，以加快当前的大规模作图项目，例如 DNA 元素百科全书 (ENCODE) 项目，消除繁琐的步骤抗体生产和测试。这里介绍的技术有可能促进大规模鉴定哺乳动物转录因子和其他染色质结合蛋白的结合位点。该技术还可以对那些难以生产 ChIP 级抗体的蛋白质进行 ChIP 分析，因此使用传统的因子特异性抗体 ChIP 方法对哺乳动物细胞进行绘图是不切实际的。