Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

Abstract

The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

Structural Dynamics, Intrinsic Disorder, and Allostery in Nuclear Receptors as Transcription Factors

“…It was predicted that binding of DBDs to REs should cause acquisition of structure and function in the unstructured NTDs/AF1s of other SHRs (64,65) and that the structural changes should affect AF1 binding to and selection of CoFs. Consistent with this, it was shown subsequently that the REand AF1-dependent recruitment of TATA box-binding protein in vivo correlated with gene induction (66).…”

Binding of the N-terminal Region of Coactivator TIF2 to the Intrinsically Disordered AF1 Domain of the Glucocorticoid Receptor Is Accompanied by Conformational Reorganizations

“…and can induce genes and/or apoptosis in cells to nearly the same extent as steroid-bound holoGR (22). Our data indicate that GR500 alone significantly increased reporter activity compared with empty vector alone (Fig.…”

“…It has been proposed that such interactions are often accompanied by disorder-order transitions resulting in increased structure formation in ID regions (16,17). This hypothesis has been experimentally supported for GRs, where the ␣-helical content of the GR AF1 domain was found to increase upon binding the core of the TATA-binding protein (21)(22)(23). Similarly, the DNA binding of GRs lacking the LBD but retaining the N-terminal domain and the DNA-binding domain is able to alter the tertiary structure of the N-terminal domain of GR (24).…”

Regulation of the Structurally Dynamic N-terminal Domain of Progesterone Receptor by Protein-induced Folding

“…However, a core C-terminal domain of human TBP consisting of aa 159 -339 (TBP C ) was reported to interact with the NTDs of several SRs and to promote a more compact tertiary structure with increased ␣-helical content (14,(33)(34)(35)(36). In the case of glucocorticoid (GR) and mineralocorticoid receptors (MR), TBP C binding was also observed to be associated with an enhancement of NTD-dependent transcriptional activity (30,31,33). Coupled binding and folding events leading to disorderto-order transitions in the NTDs of the steroid receptors have been studied using isolated NTD peptides or a minimal AF1 subregion that may not represent the entire SR signaling spectrum.…”