In Vivo Characterization of Major ENCODE-Predicted Classes of Noncoding Elements

主要编码预测非编码元素类别的体内表征

• 批准号: 10241190
• 负责人: Len Alexander Pennacchio
• 金额: $ 135.88万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241190
• 关键词: Alleles; Biochemical; Biological; Biological Assay; Biology; Boundary Elements; CRISPR/Cas technology; Categories; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Complex; DNA; DNA Methylation; Data; Data Set; Development; Disease; Elements; Embryonic Development; Encyclopedias; Engineering; Enhancers; Excision; Gene Expression; Gene Transfer Techniques; Genes; Genetic Enhancer Element; Genetic Transcription; Genomics; Goals; Gold; Health; Human; Human Genome; Human body; In Vitro; Individual; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Lead; Link; Methods; Modeling; Molecular; Molecular Profiling; Mus; National Human Genome Research Institute; Organ; Organism; Phase; Phenotype; Process; Property; Regulatory Element; Reporter; Research Personnel; Resources; Scheme; Technology; Testing; Time; Tissues; Transgenic Mice; Transgenic Organisms; Untranslated RNA; Variant; Work; base; epigenomics; experimental study; genome editing; genome-wide; genomic data; human disease; in vivo; insight; mouse genome; programs; protective allele; risk variant; synergism; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY We propose to establish a Center for In Vivo Characterization of ENCODE Elements (CIViC) as part of ENCODE Phase 4. Understanding the function of the 98% of the human genome that is noncoding remains one of the most pressing challenges in genomics. The ENCODE Program has enabled major progress toward obtaining genome-wide molecular signatures associated with the human and mouse genome. During ENCODE3 our group contributed to the mapping of enhancer-associated marks, DNA methylation, and transcriptomes from multiple mouse tissues across closely spaced time points of embryogenesis, resulting in >750 datasets defining the in vivo epigenomic landscape during mammalian development. Our group has also characterized over 3,000 candidate enhancer sequences in transgenic mouse assays, including more than 400 through our participation in ENCODE2 and ENCODE3. Despite this progress, enhancers are only one of many noncoding molecular functions that have been inferred from ENCODE data. Other major proposed categories of noncoding sequences identified through ENCODE and other publicly available data sets include DNA elements with predicted functions, such as “super-enhancers” (very large enhancers with possibly distinct functions) or chromatin domain boundary elements. They also include sequence classes of unknown function primarily defined by specific assays, such as differentially methylated regions (DMRs). The functional impact of these different classes of noncoding sequences on organismal biology and human health remains minimally explored, representing a major limitation of the ENCODE encyclopedia. The Center for In Vivo Characterization of ENCODE Elements will use CRISPR/Cas9 genome editing to systematically explore the biological significance of several classes of noncoding function based on ENCODE3 data. Leveraging the streamlined set of mouse engineering tools available in our laboratory, we will: 1. Perform integrative analysis of ENCODE3 and complementary data sets to identify and prioritize representative sequences from 3 different classes of noncoding elements (enhancers and super-enhancers, boundary elements, DMRs); 2. Systematically delete a total of 48 representative sequences in mice and perform RNA-seq and gross organismal phenotyping to understand the in vivo consequences of these deletions; 3. Continue to make transgenic enhancer characterization capabilities available to ENCODE investigators to validate and calibrate enhancer prediction methods. We will also use transgenics and CRISPR knock-in editing to test human disease-associated alleles of ENCODE-predicted enhancer elements. All efforts will be closely coordinated with other ENCODE4 functional characterization groups to focus on common sets of elements to be characterized using the full ENCODE-wide arsenal of in vitro and in vivo characterization methods. Our results will provide an understanding of the in vivo significance of different classes of noncoding elements and thereby substantially increase the value of the ENCODE encyclopedia.

项目概要 我们建议建立一个编码元件体内表征中心（CIViC），作为 ENCODE 阶段 4：了解 98% 的人类基因组非编码部分的功能 基因组学中最紧迫的挑战之一。 ENCODE 计划在以下方面取得了重大进展： 获得与人类和小鼠基因组相关的全基因组分子特征。期间 ENCODE3 我们的小组对增强子相关标记、DNA 甲基化和 来自胚胎发生的紧密间隔时间点的多个小鼠组织的转录组，从而产生 > 750 个数据集定义了哺乳动物发育过程中的体内表观基因组景观。我们组还有 在转基因小鼠试验中表征了 3,000 多个候选增强子序列，其中包括 400 多个 通过我们参与 ENCODE2 和 ENCODE3。尽管取得了这些进展，增强剂只是众多增强剂之一 从 ENCODE 数据推断出的非编码分子功能。其他主要提议类别 通过 ENCODE 和其他公开可用的数据集识别的非编码序列包括 DNA 具有预测功能的元素，例如“超级增强子”（非常大的增强子，可能具有不同的功能） 函数）或染色质域边界元素。它们还包括未知功能的序列类 主要通过特定测定来定义，例如差异甲基化区域（DMR）。功能影响 这些不同类别的非编码序列对生物体生物学和人类健康的影响仍然很小 探索，代表了 ENCODE 百科全书的一个主要局限性。体内中心 ENCODE Elements 的表征将使用 CRISPR/Cas9 基因组编辑来系统地探索 基于 ENCODE3 数据的几类非编码函数的生物学意义。利用 我们实验室提供简化的鼠标工程工具集，我们将： 1. 执行综合分析 ENCODE3 和补充数据集来识别和优先考虑来自 3 个不同的代表性序列 非编码元素类别（增强子和超级增强子、边界元素、DMR）； 2. 系统删除小鼠体内共计48条代表性序列，并进行RNA-seq和gross 有机体表型分析，以了解这些缺失的体内后果； 3.继续制作 转基因增强子表征功能可供 ENCODE 研究人员验证和校准 增强子预测方法。我们还将使用转基因技术和CRISPR敲入编辑来测试人类 ENCODE 预测的增强子元件的疾病相关等位基因。所有努力都将密切协调 与其他 ENCODE4 功能特征组一起关注常见的元素集 使用完整的 ENCODE 范围的体外和体内表征方法进行表征。我们的成果 将提供对不同类别非编码元件的体内重要性的理解，从而 大幅提升ENCODE百科全书的价值。