Modulation of Nur77-DNA Interactions by the Glucocorticoid Receptor

Nuclear receptors (NRs) comprise a superfamily of (ligand-)regulated transcription factors that are pivotal in orchestrating gene networks essential for development, metabolism, and cellular homeostasis. Their activity is critical for normal physiology, and consequently, dysregulation of NR signaling is implicated in a wide array of human diseases. Within this superfamily, the orphan nuclear receptor Nur77 and the glucocorticoid receptor (GR) are key regulators that exhibit significant crosstalk, primarily antagonistic, which is crucial for modulating inflammatory and stress responses. Despite the recognized importance of their interplay, the precise molecular mechanisms by which GR modulates Nur77's engagement with DNA remain incompletely defined. This study elucidates the direct impact of GR and its ligand, dexamethasone (Dex), on the DNA binding dynamics of Nur77. Single-molecule DNA tightrope assays revealed that Nur77 employs a three-dimensional diffusion-based search mechanism on non-specific DNA, characterized by transient interactions with two distinct dissociation kinetic profiles. GR significantly stabilizes Nur77-DNA interactions, evidenced by a shift towards longer residence times, primarily achieved by slowing the dissociation of the more transiently interacting Nur77 population. Conversely, single-molecule analysis and biochemical assays demonstrated that Dex alone markedly reduces Nur77's overall DNA binding affinity kinetics and frequency in a sequence-dependent manner, to such an extent that accurate quantification was unfeasible. These findings delineate distinct modulatory effects of the GR protein and its ligand on Nur77-DNA interactions, providing crucial biophysical insights into their complex regulatory interplay and revealing a direct, GR-independent impact of dexamethasone on Nur77's DNA engagement.