Identification of DRIP205 as a coactivator for the Farnesoid X receptor.
Farnesoid X receptor (FXR) is a bile acid sensor that regulates the expression of a number of genes the products of which control bile acid and cholesterol homeostasis; however, the role of DRIP205 in FXR-mediated gene regulation remains unexplored. In this study we demonstrate that DRIP205 binds FXR in a ligand-dependent manner in vitro and in vivo. Glutathione S-transferase pull-down assays showed that DRIP205 binds FXR in response to bile acid ligands in a dose-dependent fashion and that the potency of this interaction is associated with the ability of the ligand to activate FXR. In addition, the FXR-DRIP205 interaction required the presence of an intact LXXLL nuclear receptor box 1 (N-terminal) motif of DRIP205. In gel shift assays FXR was also able to recruit DRIP205 in the context of a DNA-bound FXR/RXR (retinoid X receptor) heterodimer. In transient transfection assays, DRIP205 efficiently enhanced a bile acid-activated FXRE-driven reporter gene in a dose-dependent manner in cells overexpressing FXR/RXR, demonstrating that DRIP205 enhances FXR-mediated transactivation. By contrast, an FXRW469A mutant in the activation function 2 domain that does not bind to DRIP205 was unable to activate ligand-stimulated FXR transcription, indicating that DRIP205 is recruited to activation function 2 of FXR. Requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was evaluated using an RXR heterodimerization-deficient FXR mutant (FXRL433R). FXRL433R was not able to bind to DRIP205 and failed to enhance an FXRE-driven reporter gene. In addition, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression, indicating that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulated the induction of endogenous FXR target gene mRNA expression by ligand. Together, these results demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the importance of DRIP205 in modulating the bile acid response of FXR target genes.
Pineda Torra
Inés
I
Department of Microbiology and Urology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
Freedman
Leonard P
LP
Garabedian
Michael J
MJ
D020816:Amino Acid Motifs; D001647:Bile Acids and Salts / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D056913:Mediator Complex Subunit 1; D011485:Protein Binding; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D000099150:Receptor, Farnesoid X-Activated
D001647:Bile Acids and Salts; D004247:DNA; D004268:DNA-Binding Proteins; D005982:Glutathione Transferase; D008024:Ligands; D056913:Mediator Complex Subunit 1; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated; C538855:MED1 protein, human