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A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors

Nature Biotechnology volume 23, pages 988–994 (2005)Cite this article

Abstract

The DNA-binding specificities of transcription factors can be used to computationally predict cis-regulatory modules (CRMs) that regulate gene expression1. However, the absence of specificity data for the majority of transcription factors limits the widespread implementation of this approach. We have developed a bacterial one-hybrid system that provides a simple and rapid method to determine the DNA-binding specificity of a transcription factor. Using this technology, we successfully determined the DNA-binding specificity of seven previously characterized transcription factors and one novel transcription factor, the Drosophila melanogaster factor Odd-skipped. Regulatory targets of Odd-skipped were successfully predicted using this information, demonstrating that the data produced by the bacterial one-hybrid system are relevant to in vivo function.

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Figure 1: Overview of the bacterial one-hybrid system.
Figure 2: Binding site motifs for seven proteins determined using the B1H system.
Figure 3: Analysis of the DNA-binding specificity of Odd determined using the B1H system.
Figure 4: Altered gene expression after ectopic expression of D. melanogaster Odd.

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Acknowledgements

We would like to thank Keith Joung, Jessica Hurt, Carl Pabo and Hermann Bujard for precursor plasmids and strains. Henry Krause for providing the HSodd2 flies. Lucio Castilla, Sean Landrette, Marian Walhout, Marc Freeman, Tony Ip and the Drosophila Genomic Resource Center for various cDNAs. Nadine McGinnis and Robin Smith for technical support. Marian Walhout and Keith Joung for very helpful discussions. We thank the UCSC genome bioinformatics site and the Institute for Genomic Research, the Genome Sequencing Center at Washington University, Agencourt Bioscience Corporation and HGSC at Baylor College of Medicine for access to and analysis of unpublished Drosophila genome data. S.A.W. and X.M. were supported in part by the Concern Foundation and National Institutes of Health (NIH) grant 1R01GM068110; M.H.B. was supported in part by a Basil O'Connor Starter Research Award from the March of Dimes Birth Defects Foundation and by the American Cancer Society grant RSG-05-026-01-CCG. This work was supported by NIH grant 1R01GM068110 (S.A.W.). This work was supported by NIH grant 1R01GM068110 (S.A.W.) and ACS grant RSG-05-026-01-CCG (M.H.B.).

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Authors and Affiliations

  1. Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation St., Worcester, 01605, Massachusetts, USA

    Xiangdong Meng, Michael H Brodsky & Scot A Wolfe

  2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, 01605, Massachusetts, USA

    Scot A Wolfe

  3. Program in Molecular Medicine, University of Massachusetts Medical School, 364 Plantation St., Worcester, 01605, Massachusetts, USA

    Michael H Brodsky

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  1. Xiangdong Meng
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Correspondence to Scot A Wolfe.

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Supplementary information

Supplementary Fig. 1

Comparison of the number of self-activating clones in the original and purified library. (PDF 418 kb)

Supplementary Fig. 2

The Runt/Bgb heterodimer is required for reporter activation. (PDF 450 kb)

Supplementary Fig. 3

Growth rates of cells containing different Odd bait-prey combinations. (PDF 754 kb)

Supplementary Fig. 4

Schematic representation of the conserved Odd binding sites near hairy, paired, gooseberry and Goosecoid. (PDF 153 kb)

Supplementary Table 1

Details of the selections performed with each bait using the B1H system. (PDF 20 kb)

Supplementary Table 2

Unique insert sequences from the prey isolated using the B1H system with each bait. (PDF 61 kb)

Supplementary Table 3

Search results using a PWM for Odd built in Target Explorer. (PDF 82 kb)

Supplementary Methods (PDF 44 kb)

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Meng, X., Brodsky, M. & Wolfe, S. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nat Biotechnol 23, 988–994 (2005). https://doi.org/10.1038/nbt1120

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