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Enhancer scanning to locate regulatory regions in genomic loci

Abstract

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.

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Figure 1
Figure 2: Strategies for analyzing regions containing candidate functional SNPs.
Figure 3: Vector maps.
Figure 4: Guide for transfections in a 96-well format.
Figure 5: Typical anticipated results.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) Genetic Association and Mechanisms in Oncology (GAME-ON) through the National Cancer Institute (NCI) U19 award (no. CA148112), the Ovarian Cancer Research Foundation (no. 258807), the Phi Beta Psi Foundation and in part by the Molecular Genomics Core Facilities at the Moffitt Cancer Center through its NCI Cancer Center Support Grant (grant no. P30-CA76292). M.B. is an ARCS (Achievement Rewards for College Scientists) fellow and a recipient of the Ruth L. Kirschstein National Research Service Award (no. F31 CA165528). R.B. is a trainee on an NIH R25T award (no. CA147832). R.S.C. is supported by a fellowship from the Brazilian National Council for Scientific and Technological Development (CNPq). We thank A. Valle, P. Cilas Jr., B. Reid and X. Li for technical assistance.

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Authors and Affiliations

Authors

Contributions

M.B., A.G., R.S.C., N.T.W. and A.N.A.M. conceived the project and designed the experiments. M.B., G.M.-F., A.G., R.B. and R.S.C. performed the experiments. M.B., G.M.-F., A.G., R.B., R.S.C., M.A.C., N.T.W. and A.N.A.M. performed the analysis and contributed to the discussion and overall data interpretation. M.B., G.M.-F., A.G. and A.N.A.M. wrote the paper. All authors provided intellectual input and approved the final version of the manuscript.

Corresponding author

Correspondence to Alvaro N A Monteiro.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Steps to generate a BED file containing SNP coordinates from LDlink or SNAP Proxy. Contains step-by-step examples of data generated from LDlink or SNA Proxy and formatted for uploading onto the UCSC Human Genome Browser. (XLSX 718 kb)

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Buckley, M., Gjyshi, A., Mendoza-Fandiño, G. et al. Enhancer scanning to locate regulatory regions in genomic loci. Nat Protoc 11, 46–60 (2016). https://doi.org/10.1038/nprot.2015.136

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