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）：我们建议使用细菌性人造染色体（BAC）重新组合到表位标签每年40转录因子，用于染色质免疫沉淀（CHIP），然后进行测序以映射结合位点基因组宽。编码项目的一个主要障碍是为要分析的每个因素提供芯片级抗体。染色质相关蛋白的表位标记使用每个因子使用相同的表位特异性抗体提出了芯片的替代方法。从BAC中表达表位的标记因子可确保由于存在内源性调节序列而在近生物学水平上表达这些因子，这些序列的存在，这些序列从其本地局部基因组环境中驱动每个标记的因子。我们建议使用这种方法分析各种转录因子，我们已经为20多种核受体类蛋白，叉子域蛋白，JUN和FOS以及其他几种因素证明了这一点（Poser等，2008； Hua，Kittler和White 2009）。该项目的目的是将我们的方法与编码项目相结合，对其进行测试，以确定转录和染色质相关因素的多样性，并将其扩展到编码所需的生产水平。拟议的项目涉及白人和斯奈德实验室之间的正式合作，以及与其他资助的编码和人类表观基因组项目的集成。 公共卫生相关性：使用BAC重新组合方法，我们建议通过消除抗体生产和测试的辛苦步骤，从而系统地进行表位标签转录和CHIP-SEQ的染色质因子，以加快当前大规模映射项目的速度，例如DNA元素（编码）项目的诸如DNA元素（编码）项目的速度。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。该技术还将实现对固定抗体抗体生产的固定蛋白的芯片分析，因此使用传统因子特异性抗体芯片方法对哺乳动物细胞进行映射不切实际。