Next-generation sequencing (NGS) technologies such as ChIP-seq are driving the discovery of putative cis-regulatory elements (CREs;i.e., enhancers/promoters) at an astonishing pace. Testing the regulatory potential of these predictions is a serious bottleneck in our efforts to understand the logic of gene regulation in the human genome. Currently there are no viable technologies for functionally testing the thousands of predictions that can be generated from even a single ChIP-seq experiment, let alone the millions of predictions being generated by ENCODE and other consortium-based efforts. Breaking the logjam of CRE predictions requires a new technology that enables massively parallel cis-regulatory analysis in mammalian cells. In this proposal, we introduce CRE-seq (Cis-Regulatory Element analysis by sequencing), a novel technique for assaying thousands of CREs in a single experiment in mammalian cells. Our strategy is to fuse libraries of CREs to barcoded reporter genes, transfect these libraries into cells and quantify their output by NGS. We will develop CRE-seq as an efficient, robust and highly parallel technology for assessing the cis-regulatory activity of thousands of ChIP-seq peaks in a single experiment. If successful, this strategy should make it possible to quantify the promoter activity of entire 'cis-regulomes'(i.e., the entire complement of cis-regulatory regions controlling gene expression in a given cell type). We will demonstrate the utility of this assay by studying cis-regulation in two different mammalian cell types, retinal photoreceptors (a differentiated neuronal cell type) and undifferentiated embryonic stem cells. CRE-seq promises to advance our understanding of human gene regulation and will serve as a novel source of personalized genomic information available for diagnosis and treatment of disease.

Public Health Relevance

New assays are needed to better understand the regions of the human genome that turn genes on and off. We propose to develop a novel technology for assaying thousands of these regions in a single experiment. Not only will this technique greatly accelerate the study of gene regulation, it will eventually provide insights into the influence of human genetic variation on disease.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG006790-03
Application #
8628861
Study Section
Special Emphasis Panel (ZHG1-HGR-M (J2))
Program Officer
Pazin, Michael J
Project Start
2012-04-24
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
3
Fiscal Year
2014
Total Cost
$373,880
Indirect Cost
$127,906
Name
Washington University
Department
Genetics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Sundaram, Vasavi; Choudhary, Mayank N K; Pehrsson, Erica et al. (2017) Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus. Nat Commun 8:14550
Shen, Susan Q; Myers, Connie A; Hughes, Andrew E O et al. (2016) Massively parallel cis-regulatory analysis in the mammalian central nervous system. Genome Res 26:238-55
White, Michael A; Kwasnieski, Jamie C; Myers, Connie A et al. (2016) A Simple Grammar Defines Activating and Repressing cis-Regulatory Elements in Photoreceptors. Cell Rep 17:1247-1254
Fiore, Chris; Cohen, Barak A (2016) Interactions between pluripotency factors specify cis-regulation in embryonic stem cells. Genome Res 26:778-86
Savic, Daniel; Roberts, Brian S; Carleton, Julia B et al. (2015) Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites. Genome Res 25:1791-800
White, Michael A (2015) Understanding how cis-regulatory function is encoded in DNA sequence using massively parallel reporter assays and designed sequences. Genomics 106:165-70
Xue, Yunlu; Shen, Susan Q; Jui, Jonathan et al. (2015) CRALBP supports the mammalian retinal visual cycle and cone vision. J Clin Invest 125:727-38
Xue, Yunlu; Shen, Susan Q; Corbo, Joseph C et al. (2015) Circadian and light-driven regulation of rod dark adaptation. Sci Rep 5:17616
Shen, Susan Q; Turro, Ernest; Corbo, Joseph C (2014) Hybrid mice reveal parent-of-origin and Cis- and trans-regulatory effects in the retina. PLoS One 9:e109382
Wang, Jin-shan; Nymark, Soile; Frederiksen, Rikard et al. (2014) Chromophore supply rate-limits mammalian photoreceptor dark adaptation. J Neurosci 34:11212-21

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