Identification of direct targets of plant transcription factors using the GR fusion technique.

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.