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.

Public Health Relevance

Most of the human genome is comprised of noncoding sequence, which contains millions of regulatory DNA elements that orchestrate the complex activities of individual genes as the human body develops, functions, and reacts to disease processes. While the ENCODE project has made incredible progress towards mapping these sequences and defining different classes based on their biochemical properties, their general function in the context of a living organism is poorly understood. This project will use targeted removal of individual regulatory sequences from the mouse genome as a model to understand the importance of noncoding sequences defined by the ENCODE project for the development and survival of mammalian organisms including humans.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project with Complex Structure Cooperative Agreement (UM1)
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Special Emphasis Panel (ZHG1-HGR-L (O1))
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Feingold, Elise A
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Lawrence Berkeley National Laboratory
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United States
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Dickel, Diane E; Ypsilanti, Athena R; Pla, Ramón et al. (2018) Ultraconserved Enhancers Are Required for Normal Development. Cell 172:491-499.e15
Stender, Stefan; Smagris, Eriks; Lauridsen, Bo K et al. (2018) Relationship between genetic variation at PPP1R3B and levels of liver glycogen and triglyceride. Hepatology 67:2182-2195