98% of the mammalian genome is non-coding, a significant fraction of which has cis- regulatory function. If we are ever to interpret the effects of the vast number of non-coding polymorphisms in the human population, we must gain a more complete understanding of the cis-regulatory grammar of gene expression. Fueled by genomic-scale technologies, the identification of potential cis-regulatory elements (CREs) is proceeding rapidly. Unfortunately, the rate of discovery of putative CREs far outpaces our capacity to functionally analyze them in vivo. Clearly, there is an urgent need for new high-throughput technologies to elucidate the functional architecture of CREs in mammalian cells. Reporter gene analysis is the standard assay for dissecting the function of mammalian CREs, but the time- and labor-intensive nature of the assay makes it impractical for testing large numbers of CREs. We propose to harness the power of Next Generation Sequencing (NGS) to create a system for the multiplexed analysis of reporter genes in mammalian cells. Our strategy is to fuse libraries of CREs to barcoded reporter genes and quantify their output by NGS. We will demonstrate the utility of this assay by studying cis-regulation in a mammalian neuronal cell type, the retinal photoreceptor cell.
In Aim 1 we will dissect, at single nucleotide resolution, the photoreceptor- specific Rhodopsin (Rho) promoter. More than 15,000 mutant promoters will be assayed in a single, multiplexed assay in living retinas, an experiment that is not possible with any existing methodologies. The data from this experiment will allow us, for the first time, to quantify the relationship between evolutionary sequence conservation and cis-regulatory activity at nucleotide resolution.
In Aim 2 we will deploy the assay to test 1,000 Chip-seq "peaks" that are enriched for binding to the key photoreceptor transcription factor, Crx. In a single, multiplexed experiment we will test all 1,000 genomic sequences for their ability to drive photoreceptor- specific expression in vivo. In addition, in Aim 2 we will assay combinatorial libraries of synthetic promoters composed of binding sites for Crx and two other transcription factors, Nrl and Nr2e3, known to play a key role in controlling photoreceptor-specific gene expression. The output of all these experiments will be analyzed using a formal thermodynamic model of combinatorial cis-regulation. Our intention is to demonstrate, in a relatively short time frame, that it is possible to unravel the cis-regulatory grammar of an important mammalian cell type in vivo. This study will serve as a proof-of-concept that CRE analysis can be made as high- throughput as CRE discovery.

Public Health Relevance

Differences in the DNA sequence of the human genome account for variation between individuals in their susceptibilities to many diseases. Many of these genetic differences occur in regions of the genome containing stretches of DNA that control which genes are turned on and off. The goal of the proposed research is to develop a new methodology for determining the function of these control regions so that we can better predict the effects of human genetic variation on disease susceptibility.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HG006346-02
Application #
8309063
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Pazin, Michael J
Project Start
2011-08-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2012
Total Cost
$228,000
Indirect Cost
$78,000
Name
Washington University
Department
Genetics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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
Wang, Jin-shan; Nymark, Soile; Frederiksen, Rikard et al. (2014) Chromophore supply rate-limits mammalian photoreceptor dark adaptation. J Neurosci 34:11212-21
White, Michael A; Myers, Connie A; Corbo, Joseph C et al. (2013) Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks. Proc Natl Acad Sci U S A 110:11952-7