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Plant Functional Genomics pp 123–138Cite as

Identification of Direct Targets of Plant Transcription Factors Using the GR Fusion Technique

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1284))

Abstract

The glucocorticoid receptor-dependent activation of plant transcription factors has proven to be a powerful tool for the identification of their direct target genes. In the absence of the synthetic steroid hormone dexamethasone (dex), transcription factors fused to the hormone-binding domain of the glucocorticoid receptor (TF-GR) are held in an inactive state, due to their cytoplasmic localization. This requires physical interaction with the heat shock protein 90 (HSP90) complex. Hormone binding leads to disruption of the interaction between GR and HSP90 and allows TF-GR fusion proteins to enter the nucleus. Once inside the nucleus, they bind to specific DNA sequences and immediately activate or repress expression of their targets. This system is well suited for the identification of direct target genes of transcription factors in plants, as (A) there is little basal protein activity in the absence of dex, (B) steroid application leads to rapid transcription factor activation, (C) no side effects of dex treatment are observed on the physiology of the plant, and (D) secondary effects of transcription factor activity can be eliminated by simultaneous application of an inhibitor of protein biosynthesis, cycloheximide (cyc). In this chapter, we describe detailed protocols for the preparation of plant material, for dex and cyc treatment, for RNA extraction, and for the PCR-based or genome-wide identification of direct targets of transcription factors fused to GR.

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Acknowledgments

This work was supported by IOS grant 1257111 to D.W, JSPS postdoctoral fellowships for research abroad to N.Y., NIH Developmental Biology Training Grant T32-HD007516 and NIH Ruth L. Kirschstein NRSA F32 Fellowship GM106690-01 to C.M.W., and Science Foundation Ireland to F.W.

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Authors and Affiliations

  1. Department of Biology, University of Pennsylvania, 415 S. University Ave., Philadelphia, PA, 19104-6018, USA

    Nobutoshi Yamaguchi, Cara M. Winter & Doris Wagner

  2. Department of Biology, Duke University, 90338, Durham, NC, 27708, USA

    Cara M. Winter

  3. Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland

    Frank Wellmer

Authors
  1. Nobutoshi Yamaguchi
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  2. Cara M. Winter
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  3. Frank Wellmer
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  4. Doris Wagner
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Corresponding author

Correspondence to Doris Wagner .

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Editors and Affiliations

  1. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Jose M. Alonso

  2. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Anna N. Stepanova

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Yamaguchi, N., Winter, C.M., Wellmer, F., Wagner, D. (2015). Identification of Direct Targets of Plant Transcription Factors Using the GR Fusion Technique. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_6

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  • DOI: https://doi.org/10.1007/978-1-4939-2444-8_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2443-1

  • Online ISBN: 978-1-4939-2444-8

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