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 #
5R01HG006790-02
Application #
8463018
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
2013-03-01
Budget End
2014-02-28
Support Year
2
Fiscal Year
2013
Total Cost
$364,342
Indirect Cost
$124,643
Name
Washington University
Department
Genetics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wang, Jin-shan; Nymark, Soile; Frederiksen, Rikard et al. (2014) Chromophore supply rate-limits mammalian photoreceptor dark adaptation. J Neurosci 34:11212-21
Jiao, Yang; Lau, Timothy; Hatzikirou, Haralampos et al. (2014) Avian photoreceptor patterns represent a disordered hyperuniform solution to a multiscale packing problem. Phys Rev E Stat Nonlin Soft Matter Phys 89:022721