A Comprehensive Catalog of Dnasel Hypersensitive Sites

本提案的总体目标是建立一个全面、高质量、高分辨率的人类和小鼠DNasel超敏感位点（DHSs）目录，涵盖所有主要组织谱系。在UW ENCODE中心先前成功的基础上，我们计划定位DNasel超敏感位点，确定其中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，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，只有精选的一组才能进行全基因组数据收集。为了确保尽可能广泛地覆盖独特和非独特的基因组区域，我们将采用比先前项目期间更高分辨率、更高覆盖率的测序策略，显著增强数据的信息内容。该提案整合了UW-FHCRC小鼠ENCODE中心，该中心将与人类项目紧密结合，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DNasel超敏感位点是广泛的人类顺式调控序列的通用标记，通过将dhs分类为主要的功能类别，包括启动子、远端元件（增强子、lcr）和绝缘子，目录的实用性将大大增强。我们计划系统地将远端DHSs与其同源启动子连接起来，并在体细胞中使用核酸酶介导的远端DHSs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.