Figure 1.

Pharmacological activation of FXR increases its occupancy at FGF15 signaling pathway genes and increases their expression. A, FGF15/19-signaling components in hepatocytes. B and C, C57BL/6 mice were treated with GW4064 or vehicle for 1 hour, and liver re-ChIP assays (B) were performed to examine occupancy of FXR, RXRα, and RNA polymerase II at FXR-bound hepatic chromatin at the indicated genes, and (C) levels of the mRNA of the indicated genes were determined by q-RT-PCR; SEM, n = 3 mice; *, P < .05; **, P < .01; ***, P < .005; NS, not significant. D–F, βKL is a direct transcriptional target of FXR. D, A 700-bp fragment of βKL containing the WT or mutated IR2 motif or deletion mutations was cloned into pGL3-SV40-Luc as described in Materials and Methods. E, Luciferase reporter assays. Hepa1c1c7 cells were transfected with reporters and expression plasmids (0, 50, 100, and 200 ng) as indicated and treated with GW4064 (100nM) for 4 hours. Luciferase activity normalized to β-galactosidase activity was determined; SEM, n = 9. F, Gel mobility shift assays were performed using partially purified flag-FXR and flag-RXR, with a ³²P-labeled oligonucleotide containing the βKL IR2 motif as probe. Antibody to FXR or the Gal4 DBD (G4), or competitor oligonucleotides containing the WT or mutated IR2 motif of βKL gene or the IR1 motif of the Shp promoter region were added.