Functional Interaction of the Glucocorticoid Receptor Activation Function 1 Domain with TATA‐Binding Protein

Abstract

The transcriptional mechanism of the glucocorticoid receptor (GR) is unclear. The N-terminal AF1 region of GR has been shown to be natively unstructured, but possesses much of the transcriptional potency. The canonical mechanism of GR is that it first binds to its DNA site and then collects a heterocomplex of proteins which modifies chromatin and other proteins that bridge to the basal transcription machinery, anchored by the TATA-binding protein (TBP) bound at its DNA site. However, recent work has shown that TBP interacts directly with the unstructured GR AF1 to induce folding. Here, we show that the TBP:GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by various methods, and confirm that the interaction functions in vivo. TBP binds the AF1C domain of GR with a 1:2 stoichiometry with a nanomolar Kd. In vivo fluorescence resonance energy transfer (FRET) experiments using fluorescently labeled TBP and GR variants, transiently transfected into CV-1 cells, show appropriate in vivo GR:TBP interactions. AF1-deletion variants showed FRET efficiencies similar to that of co-expressed CFP and YFP, indicating that the interaction is dependent on the AF1 domain. AF1-dependent functional in vivo GR:TBP interaction was shown by expressing increasing amounts of TBP which stimulated expression of transfected and endogenous GR-driven reporter.