ORF48-01 - Mapping the Functional Landscape of the Farnesoid X Receptor N-Terminal Domain

The farnesoid X receptor (FXR) is a ligand-activated nuclear receptor expressed primarily in the liver, intestine, kidney, and adipose tissue, where it functions as an endocrine regulator by governing bile acid, carbohydrate, and lipid homeostasis. Structurally, FXR exhibits the canonical nuclear receptor architecture, comprising an intrinsically disordered N-terminal domain (NTD), a highly conserved DNA-binding domain (DBD), a flexible hinge region, and a C-terminal ligand-binding domain (LBD). Even though the DBD and LBD have been extensively characterized and there are ongoing studies on the hinge region, the role of the NTD remains largely unexplored. This study examines the functional role of the FXR-NTD in transcriptional regulation, coactivator recruitment, and interdomain interactions, and we hypothesize that the FXR-NTD harbors activation function regions essential for full receptor activity. To evaluate this hypothesis, constructs containing complete or partial deletions of the NTD, as well as specific motif-targeted mutations, were generated. Transcriptional activity and coactivator binding were assessed using luciferase reporter assays, while interdomain interactions were examined using mammalian two-hybrid assays. Deletion of the FXR-NTD significantly reduced transcriptional activity compared with wild-type FXR. Furthermore, deletion of selected regions within the FXR-NTD also resulted in diminished transcriptional activity, indicating that these regions are essential for NTD-mediated FXR transcriptional function. We identified novel motifs within these regions that play a pivotal role in FXR-dependent transcription by facilitating coregulator recruitment and interdomain communication. Collectively, these findings provide new insight into the contribution of the FXR-NTD to ligand-dependent and ligand-independent transcriptional regulation and highlight specific motifs and regions that are critical for its activity.