A Comprehensive Catalog of Dnasel Hypersensitive Sites

该提案的总体目标是建立一个全面的，高质量的，高分辨率的人类和小鼠DNase 1超敏位点（DHS）的目录，跨越所有主要的组织谱系。在UW ENCODE中心先前成功的基础上，我们计划定位DNasel超敏位点，定义其中DNasel足迹的位置，并继续提供相关的协同注释，包括RNA-seq，组蛋白修饰和CTCF，以及DNA甲基化。我们生产工作的首要重点是数据质量。因此，样本将以流水线方式进行严格筛选，只有一组选择进入全基因组数据收集。为了确保尽可能广泛地覆盖独特和非独特的基因组区域，我们将采用比前一个项目期更高分辨率、更高覆盖率的测序策略，显著提高数据的信息含量。该提案整合了UW-FHCRC小鼠ENCODE中心，该中心将与人类项目密切合作，以在精心匹配的细胞和组织中生成调控DNA的比较目录，提供无与伦比的资源。由于DNase 1超敏位点是广谱人类顺式调节序列的通用标记，通过将DHS分类为主要功能类别（包括启动子、远端元件（增强子、LCR）和绝缘子），将大大增强目录的实用性。我们计划系统地将远端DHS与其同源启动子连接，并使用体细胞中核酸酶介导的远端DHS敲除对这些连接进行体内验证。

The overall aim of this proposal is to establish a comprehensive, high-quality, high-resolution catalogues of human and mouse DNasel hypersensitive sites (DHSs) spanning all major tissue lineages. Building on the prior success of the UW ENCODE center, we plan to localize DNasel hypersensitive sites, to define the locations of DNasel footprints therein, and to continue to provide relevant synergistic annotations including RNA-seq, histone modifications, and CTCF, as well as DNA methylation. The overriding focus of our production effort has been on data quality. Accordingly, samples will be rigorously screened in a pipeline fashion, with only a select set advancing to whole-genome data collection. To ensure the broadest possible coverage of both unique and non-unique genomic territories, we will employ a higher resolution, higher coverage sequencing strategy than the prior project period, significantly enhancing the information content of the data. This proposal integrates the UW-FHCRC Mouse ENCODE Center, which will be closely aligned with the human project to generate a comparative catalogue of regulatory DNA in carefully matched cells and tissues, providing an unparalleled resource. Since DNasel hypersensitive sites are generic markers of a broad spectrum of human cis-regulatory sequences, the utility of the catalogue will be greatly enhanced by the classification of DHSs into major functional categories including promoters, distal elements (enhancers, LCRs), and insulators. We plan to systematically connect distal DHSs with their cognate promoters and to perform in vivo validation of these connections using nuclease-mediated knockouts of distal DHSs in somatic cells.