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

描述(申请人提供)：我们建议使用细菌人工染色体(BAC)重组工程，每年40个转录因子的表位标签(TAG)进行染色质免疫沉淀(ChIP)，然后进行测序，以绘制全基因组的结合部位图。ENCODE项目的一个主要障碍是每个待分析因素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中表达表位标记的因子确保了因子在接近生理水平的表达，因为存在内源调控序列，将每个标记的因子从其本地基因组环境中驱动出来。我们建议用这种方法分析各种转录因子，我们已经对20多种核受体类蛋白质、叉头区蛋白、Jun和Fos以及其他几种类型的因子(Poser等人)进行了分析。2008年；华为、基特勒和怀特2009年)。该项目的目标是将我们的方法与ENCODE项目相结合，测试其转录和染色质相关因子的更广泛多样性，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和人类表观基因组计划的整合。 公共卫生相关性：使用BAC重组工程方法，我们建议为CHIP-SEQ系统地表位标签转录和染色质相关因子，以通过消除繁琐的抗体生产和检测步骤来加快当前的大规模测绘项目，如DNA元素百科全书(ENCODE)项目。这项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。这项技术还将使芯片分析蛋白质成为可能，这些蛋白质是芯片级抗体生产的顽固性，因此使用针对哺乳动物细胞的传统因子特异性抗体芯片方法绘制图谱是不切实际的。

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.