Suzanne van der Veen1, Miranda van Triest1, Eric Kalkhoven1, Saskia van Mil1
(1) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (2) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (3) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (4) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (5) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (6) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (7) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands. (8) Center for Molecular Medicine, University Medical Center Utrecht and Utrecht University, Utrecht, The Netherlands.
The Farnesoid X Receptor (FXR) is a master regulator of metabolism. In the liver, FXR is expressed as two isoforms: FXRα1 and FXRα2, which differ only in a four amino acid (MYTG) sequence downstream of the DNA binding domain (DBD), present in FXRα1, but absent in FXRα2. While both isoforms bind the canonical IR-1 (inverted repeat-1) DNA motif, FXRα2 additionally binds ER-2 (everted repeat-2) DNA motifs, thereby activating additional transcriptional programs involved in liver metabolism. We set out to investigate how these four amino acids influence FXR differential activation from IR-1 and ER-2 motifs.
To explore how the MYTG affects FXR activity, we performed mutagenesis to alter the MYTG region in FXRα1. Using alanine scanning, where each amino acid in the sequence was replaced with alanine individually or simultaneously, we assessed transcriptional activation from IR-1 or ER-2 motifs using luciferase reporter assays. Remarkably, all mutants retained the ability to activate transcription exclusively from the IR-1 motif, but did not gain ER-2 transcriptional activity. Further analysis by sequentially deleting amino acids from the MYTG sequence confirmed that neither the identity nor the number of residues is essential for IR-1 binding and activation, whereas only the complete deletion of the 4 residues results in gaining ER-2 transcriptional activity.
Structural insights from AlphaFold3 modeling revealed a distinct difference between the isoforms: the hinge region of FXRα2 adopts a unique angle relative to the ligand binding domain (LBD), compared to FXRα1. Interestingly, all mutants clustered structurally with FXRα1, congruent with their restricted transactivation capabilities.
In conclusion:
Neither the identity, nor the number of amino acids of MYTG in FXRα1 affects IR-1-mediated transactivation. However, the hinge’s structural orientation relative to the LBD appears critical for the ability of FXRα2 to transactivate from ER-2 DNA motifs.