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Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping


A major challenge in systems biology is to understand the gene regulatory networks that drive development, physiology and pathology. Interactions between transcription factors and regulatory genomic regions provide the first level of gene control. Gateway-compatible yeast one-hybrid (Y1H) assays present a convenient method to identify and characterize the repertoire of transcription factors that can bind a DNA sequence of interest. To delineate genome-scale regulatory networks, however, large sets of DNA fragments need to be processed at high throughput and high coverage. Here we present enhanced Y1H (eY1H) assays that use a robotic mating platform with a set of improved Y1H reagents and automated readout quantification. We demonstrate that eY1H assays provide excellent coverage and identify interacting transcription factors for multiple DNA fragments in a short time. eY1H assays will be an important tool for mapping gene regulatory networks in Caenorhabditis elegans and other model organisms as well as in humans.

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Figure 1: eY1H assays.
Figure 2: Sampling sensitivity and reproducibility of eY1H assays.
Figure 3: eY1H assays with 50 previously published C. elegans gene promoters as DNA baits.
Figure 4: Automated quantification of eY1H assays using SpotOn.


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We thank members of the Walhout laboratory for discussions and critical reading of the manuscript, S. Lee for preparing media, K. Salehi-Ashtiani (Center for Cancer Systems Biology) for transcription factor Entry clones, J. Boeke for advice on the creation of Y1H-aS2, and C. Boone for general advice on the robotic pipeline and the use of different yeast strains. This work was supported by US National Institutes of Health (NIH) grant GM082971 to A.J.M.W. Research in the Dekker laboratory is supported by NIH grant HG003143 and a W.M. Keck Foundation Distinguished Young scholar award. C.L.M. and C.P. are supported by NIH grant HG005084 and US National Science Foundation grant DBI 0953881.

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Authors and Affiliations



J.S.R.-H. and A.J.M.W. conceived the project; A.K. and S.S. performed the eY1H assays; A.D. created the tools for automated eY1H assay quantification in collaboration with B.L., C.P., J.D., J.S.R.-H. and C.L.M.; S.K. cloned additional transcription factor–encoding ORFs; J.S.R.-H. and A.J.M.W. wrote the paper.

Corresponding authors

Correspondence to John S Reece-Hoyes or Albertha J M Walhout.

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The authors declare no competing financial interests.

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Supplementary Figs. 1–6, Supplementary Tables 1–9 (PDF 92727 kb)

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Reece-Hoyes, J., Diallo, A., Lajoie, B. et al. Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping. Nat Methods 8, 1059–1064 (2011).

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