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: 8327886
• 负责人: KEVIN P. WHITE
• 金额: $ 89.24万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8327886
• 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）重组工程来标记40个转录因子的表位，用于染色质免疫沉淀（ChIP），然后测序以在全基因组范围内定位结合位点。ENCODE项目的一个主要障碍是要分析的每个因素的ChIP级抗体的可用性。染色质相关蛋白的表位标记为ChIP提供了一种替代方法，对每个因子使用相同的表位特异性抗体。从BAC表达表位标记的因子确保了因子以接近生理水平表达，这是由于存在内源性调节序列，所述内源性调节序列驱动每个标记的因子脱离其天然局部基因组环境。我们建议使用这种方法分析多种转录因子，我们已经证明了超过20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（Poser et al. 2008; Hua，Kittler and白色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。拟议的项目涉及白色和斯奈德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。 公共卫生关系：使用BAC重组工程方法，我们建议系统地为ChIP-seq标记表位标签转录和染色质相关因子，以通过消除抗体生产和测试的费力步骤来加速当前的大规模作图项目，例如DNA元件百科全书（ENCODE）项目。这里提出的技术有可能促进哺乳动物转录因子和其他染色质结合蛋白的结合位点的大规模鉴定。该技术还将使得能够对与ChIP级抗体生产无关的蛋白质进行ChIP分析，因此使用常规因子特异性抗体ChIP方法对哺乳动物细胞进行作图是不切实际的。