Fig. 5.

PXR directly interacts with the DBD of FoxA2 to inhibit its binding to the responsible elements in vitro and in vivo. A. Mammalian two-hybrid assay was performed by co-transfecting pG5-Luc reporter plasmid with various combinations of pBIND, pACT, pBIND/mPXR and pACT/FoxA2 into HepG2 cells. At 24 h after transfection, cells were treated with DMSO or PCN for an additional 24 h. Relative luciferase activities were calculated by taking the activity obtained from the GAL4DBD- and VP16AD-transfected cells in the presence of DMSO as one. Bars indicate mean ± standard deviations. B. Schematic structure of FoxA2 and mPXR (Upper panel). In vitro translated ³⁵S-labeled mPXR was incubated with bacterially expressed GST-FoxA2 and its deletion mutants (middle panel). In vitro translated ³⁵S-labeled mPXR or the deletion mutants was incubated with bacterially expressed GST-FoxA2 (lower panel). GST-pull down assay was carried out as in EXPERIMENTL PROCEDURES. Bound proteins were detected by autoradiography. C. PXR LBD represses the FoxA2-mediated activation of mouse Cpt1a -2.4k reporter. pGL3/CPT1a -2.4k reporter plasmid and pcDNA3.1/FoxA2 were co-transfected with and without pcDNA3.1/mPXR-LBD-V5-His or pcDNA3.1/mPXR-DBD-V5-His into HepG2 cells. After 24 h, DMSO or PCN was added to the cells cultured for additional 24 h. Relative luciferase activities were calculated by taking the activity of the DMSO-treated cells transfected with pGL3/CPT1a -2.4k reporter plasmid alone as one. D. In vitro-translated FoxA2, mPXR and hRXR were incubated with ³²P-labeled double-stranded FoxA2 binding site of IGFBP-1 promoter. Competitor stands for fifty-fold excess cold probe co-incuabation. Shifted bands were separated by electrophoresis on a 4% polyacrilamide gel. Bands were detected by autoradiography. E. Activated PXR inhibits FoxA2 binding to Cpt1a and Hmgcs2 promoter in ChIP assay. Chromatin fragments were immunoprecipitated with anti-FoxA2 antibody from liver nuclear extracts. Precipitated Cpt1a and Hmgcs2 promoter DNA was semi-quantified by PCR as in EXPERIMENTAL PROCEDURES. D and P stands for DMSO and PCN treated mouse liver, respectively.