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）重组到表位标记40个转录因子进行染色质免疫沉淀（ChIP），然后测序以绘制全基因组范围的结合位点。ENCODE项目的一个主要障碍是针对每个待分析因子的芯片级抗体的可用性。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。由于内源性调控序列的存在，从BACs中表达表位标记的因子确保了这些因子在接近生理水平的表达，这些内源性调控序列驱动每个标记的因子从其原生的本地基因组背景中表达。我们建议使用这种方法分析转录因子的多样性，我们已经证明了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（Poser et al. 2008; Hua， Kittler和White 2009）。该项目的目标是将我们的方法与ENCODE项目相结合，对其进行更广泛的转录和染色质相关因子的测试，并将该方法扩展到ENCODE所需的生产水平。拟议的项目包括怀特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。