The Orphan Receptor/Corepressor DAX‐1 Modulates Human CAR Transcriptional Activity

Abstract

The human constitutive androstane receptor (CAR) is a nuclear receptor (NR) primarily expressed in liver that regulates genes involved in xenobiotic metabolism, as well as lipid and energy homeostasis. Screening a panel of phage display peptides identified DAX‐1, a NR with corepressor properties, as a potential CAR coregulator. We next determined that DAX‐1 dose‐dependently inhibited CAR‐mediated transactivation. Directed mutation studies of DAX‐1 NR interaction domains showed that mutation of two N terminal domain ‘LXXM/LL’ motifs had no impact on DAX‐1 repression of CAR activity, whereas mutation of downstream ‘LXXLL’ or ‘PCFQVLP’ motifs restored CAR activity to 50% or 65% of control, respectively. Further, deletion of a transcription silencing domain (TSD) located in the DAX‐1 carboxy‐terminal region restored CAR transactivation to 90% of control levels. Two‐hybrid data demonstrated that full length DAX‐1 inhibited CAR‐SRC1 interaction by 50%, while the interaction was once again restored to 90% of control upon deletion of the TSD. Results of an alpha screen assay corroborated a direct interaction between CAR and DAX‐1 that was enhanced by CAR ligands. To examine ex vivo effects of DAX‐1, primary human hepatocytes were transfected with DAX‐1 and subsequent RT‐PCR analyses established that DAX‐1 inhibited CAR‐mediated induction of the CYP2B6 target gene by CITCO. In summary, these studies show that DAX‐1 is a functional corepressor of CAR transcriptional activity. Supported by NIH GM066411