Enhancer Scanning to Locate Regulatory Regions in Genomic Loci

2015-12-10 17:16:37

Nature; 10 December 2015: DOI:10.1038/nprot.2015.136

Melissa Buckley, Anxhela Gjyshi, Gustavo Mendoza-FandiƱo, Rebekah Baskin, Renato S Carvalho, Marcelo A Carvalho, Nicholas T Woods, Alvaro N A Monteiro


This protocol provides a rapid, streamlined and scalable strategy to systematically scan genomic regions for the presence of transcriptional regulatory regions that are active in a specific cell type. It creates genomic tiles spanning a region of interest that are subsequently cloned by recombination into a luciferase reporter vector containing the simian virus 40 promoter. Tiling clones are transfected into specific cell types to test for the presence of transcriptional regulatory regions. The protocol includes testing of different single-nucleotide polymorphism (SNP) alleles to determine their effect on regulatory activity. This procedure provides a systematic framework for identifying candidate functional SNPs within a locus during functional analysis of genome-wide association studies. This protocol adapts and combines previous well-established molecular biology methods to provide a streamlined strategy, based on automated primer design and recombinational cloning, allowing one to rapidly go from a genomic locus to a set of candidate functional SNPs in 8 weeks.


Genome-wide association studies (GWASs) have been extremely successful in identifying a large number of predisposition loci for a series of human diseases and conditions including cancer. These risk loci are physical locations that are defined by tagging SNPs associated with risk, but for which the mechanism underlying susceptibility is generally unknown. It is normally hypothesized that susceptibility is conferred by genetic variation, which is represented by the tagging SNP or by those in linkage disequilibrium (LD) with it, with a functional impact on coding or noncoding elements of the genome.

Typically, functional analysis starts by identifying a comprehensive set of candidate functional SNPs operating in the locus. It then proceeds by applying progressively stringent criteria to discard variants that do not contribute functionally and that are unlikely to be driving association with risk. The identification of functional SNPs allows the generation of several testable hypotheses of how different alleles might functionally contribute to cancer risk5. Functional SNPs might occur in coding regions, in which case the determination of the target gene is straightforward (i.e., the gene in which the coding SNP is located). Next, these coding SNPs are assessed for the impact of different alleles on protein stability and function. However, cancer susceptibility loci identified by GWASs are overwhelmingly located in noncoding regions.

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Key Words

Gene regulation | Genetic variation | Reporter genes | Transcriptional regulatory elements