Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome

  • Ghislain BretonEmail author
  • Steve A. Kay
  • José L. Pruneda-Paz
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


Genetic and molecular approaches revealed that the circadian clock network structure is comprised of several interlocked positive and negative transcriptional feedback loops. The network evolved to sense and integrate inputs from environmental cues to adjust daily rhythms in physiological processes. Compiling evidence indicates that part of this regulation happens at the transcriptional level through subtle adjustments in the expression of core clock genes. Thus, to better understand the network and identify the molecular mechanisms of clock input pathways, it is imperative to determine how core clock genes are regulated. For this purpose we developed reagents for an unbiased approach to identify transcription factors (TFs) interacting with the promoters of core clock genes. At the center of this approach lies the yeast one-hybrid (Y1H) assay in which a pool of proteins fused to the GAL4 transcriptional activation domain are tested for their ability to interact with a selected promoter fragment in yeast cells. Taking advantage of the fact that Arabidopsis TF genes are well annotated, we generated a comprehensive TF clone collection (TF ORFeome) and used it to replace the standard cDNA pool strategy traditionally used in Y1H screens. The use of this TF clone collection substantially accelerates the comprehensive discovery of promoter-specific DNA binding activities among all Arabidopsis TFs. Considering that this strategy can be extended to the study of the promoter interactome of any Arabidopsis gene, we developed a low throughput protocol that can be universally implemented to screen the ~2000 TF clone library.

Key words

Yeast one-hybrid Transcription factor ORFeome Circadian clock Cis-regulatory network Protein–DNA interaction β-galactosidase reporter Plant genomics 



We thank Katia Bonaldi and Dawn H. Nagel for critical reading of the manuscript. The research reported in this publication was supported by the National Institute of General Medical Sciences, NIH, under grants R01GM056006, R01GM067837, and RC2GM092412 to S. A. K. and R01GM056006 to J. L. P.-P. as a coinvestigator.


  1. 1.
    Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246CrossRefPubMedGoogle Scholar
  2. 2.
    Wang MM, Reed RR (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364:121–126CrossRefPubMedGoogle Scholar
  3. 3.
    Li JJ, Herskowitz I (1993) Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262:1870–1874CrossRefPubMedGoogle Scholar
  4. 4.
    Pruneda-Paz JL, Breton G, Nagel D et al (2014) A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Rep 8(2):622–632PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Deplancke B, Dupuy D, Vidal M et al (2004) A gateway-compatible yeast one-hybrid system. Genome Res 14:2093–2101PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Deplancke B, Vermeirssen V, Arda HE et al (2006) Gateway-compatible yeast one-hybrid screens. CSH Protoc. doi: 10.1101/pdb.prot4590 PubMedGoogle Scholar
  7. 7.
    Reece-Hoyes JS, Walhout AJ (2012) Gene-centered yeast one-hybrid assays. Methods Mol Biol 812:189–208PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Pruneda-Paz JL, Breton G, Para A, Kay SA (2009) A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science 323:1481–1485PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Hens K, Feuz JD, Isakova A et al (2011) Automated protein-DNA interaction screening of Drosophila regulatory elements. Nat Methods 8:1065–1070PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Gubelmann C, Waszak SM, Isakova A et al (2013) A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks. Mol Syst Biol 9:682PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Reece-Hoyes JS, Barutcu AR, McCord RP et al (2011) Yeast one-hybrid assays for gene-centered human gene regulatory network mapping. Nat Methods 8:1050–1052PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Walhout AJ (2011) What does biologically meaningful mean? A perspective on gene regulatory network validation. Genome Biol 12:109PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Li L, Ljung K, Breton G et al (2012) Linking photoreceptor excitation to changes in plant architecture. Genes Dev 26:785–790PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Niwa M, Daimon Y, Kurotani K et al (2013) BRANCHED1 interacts with FLOWERING LOCUS T to repress the floral transition of the axillary meristems in Arabidopsis. Plant Cell 25:1228–1242PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Ito S, Song YH, Josephson-Day AR et al (2012) FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis. Proc Natl Acad Sci U S A 109:3582–3587PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Chow BY, Sanchez SE, Breton G et al (2014) Transcriptional regulation of LUX by CBF1 mediates cold input to the circadian clock in Arabidopsis. Curr Biol 24(13):1518–1524PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Burke D, Dawson D Steams T (2000) Methods in yeast genetics: a Cold Spring Harbor Laboratory Course Manual. pp 205Google Scholar
  18. 18.
    Helfer A, Nusinow DA, Chow BY et al (2011) LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock. Curr Biol 21:126–133PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Li JF, Sheen J (2012) DNA purification from multiple sources in plant research with homemade silica resins. Methods Mol Biol 862:53–59CrossRefPubMedGoogle Scholar
  20. 20.
    Lezin G, Kosaka Y, Yost HJ (2011) A one-step miniprep for the isolation of plasmid DNA and lambda phage particles. PLoS One 6:e23457PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Marra MA, Kucaba TA, Hillier LW (1999) High-throughput plasmid DNA purification for 3 cents per sample. Nucleic Acids Res 27:e37PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ghislain Breton
    • 1
    Email author
  • Steve A. Kay
    • 2
  • José L. Pruneda-Paz
    • 3
    • 4
  1. 1.Department of Integrative Biology and PharmacologyUniversity of Texas Health Science Center at HoustonHoustonUSA
  2. 2.Molecular and Computational Biology SectionUniversity of Southern CaliforniaLos AngelesUSA
  3. 3.Division of Biological SciencesUniversity of California San DiegoLa JollaUSA
  4. 4.Center for ChronobiologyUniversity of California San DiegoLa JollaUSA

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