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Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping


Yeast one-hybrid (Y1H) assays provide a gene-centered method for the identification of interactions between gene promoters and regulatory transcription factors (TFs). To date, Y1H assays have involved library screens that are relatively expensive and laborious. We present two Y1H strategies that allow immediate prey identification: matrix assays that use an array of 755 individual Caenorhabditis elegans TFs, and smart-pool assays that use TF multiplexing. Both strategies simplify the Y1H pipeline and reduce the cost of protein-DNA interaction identification. We used a Steiner triple system (STS) to create smart pools of 4–25 TFs. Notably, we uniplexed a small number of highly connected TFs to allow efficient assay deconvolution. Both strategies outperform library screens in terms of coverage, confidence and throughput. These versatile strategies can be adapted both to TFs in other systems and, likely, to other biomolecules and assays as well.

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Figure 1: Identified PDIs.
Figure 2: STS-based smart-pooling design.
Figure 3: Experimental validation of STS smart pools by Y1H assays using Pfat-5 as a DNA bait.
Figure 4: Comparison of different Y1H assays.


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We thank J. Dekker for critical reading of the manuscript; W. Harper (Harvard Medical School) for the Y187 yeast strain; N. Klitgord and M. Vidal (Dana-Farber Cancer Institute) for help with primer design; E. Méndez González (M-3 Informática, S.L.) for helpful suggestions regarding constraint programming and the sequencing staff at Agencourt Bioscience Corporation for technical assistance. This work was supported by BAEF fellowship for Biomedical and Biotechnology Research to V.V., and US National Institute of Diabetes and Digestive and Kidney Diseases grants (DK068429 and DK071713) to A.J.M.W.

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V.V., B.D., J.S.R.-H, H.E.A., C.A.G. and N.J.M. performed all experiments; B.D., M.I.B. and A.J.M.W. conceived the pooling strategy; M.I.B. created the STS design and deconvolution, and performed the bioinformatics analyses. M.R.B. provided TWINSCAN predictions. R.S. and L.D.-S. provided sequencing; V.V., J.S.R.-H. and A.J.M.W. wrote the manuscript.

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R.S. and L.D.-S. work for Agencourt bioscience.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 2–3, Supplementary Methods. (PDF 876 kb)

Supplementary Table 1

wTF2.1 and clone source and availability. (XLS 515 kb)

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Vermeirssen, V., Deplancke, B., Barrasa, M. et al. Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping. Nat Methods 4, 659–664 (2007).

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