FIG. 3.

LXR represses basal transcription and interacts with corepressors. (a) CV-1 cells were transiently cotransfected with a luciferase reporter under the control of four copies of a GAL4 response element (4 × UAS) and either empty pCMX vector, a plasmid containing the GAL4 DNA binding domain (DBD) alone, or a construct with the GAL4 DBD fused to full-length LXRα or LXRβ. The cells were then treated with vehicle or 1 μM T1317 overnight. TK, thymidine kinase minimal promoter; Luc, luciferase. (b) LXRα and LXRβ interact with the nuclear receptor IDs of NCoR and SMRT. A mammalian two-hybrid system was established by transiently cotransfecting CV-1 cells with VP16 fusions of LXRα or LXRβ ligand binding domains together with GAL4 fusions of the receptor ID1 and ID2 of NCoR and SMRT and a luciferase reporter under the control of four copies of a GAL4 response element. The cells were treated with agonist overnight prior to being assayed for reporter activity. L.U., luciferase units. (c) Repression by LXR requires NCoR expression. MEFs isolated from NCoR^+/+ and NCoR^−/− embryos were transiently cotransfected with the GAL4 response element-luciferase reporter and the full-length LXRα- or LXRβ-GAL4 fusion constructs described for panel a. After an overnight incubation in the absence of ligand, the cells were lysed and analyzed for promoter activity. (d) The effect of a ligand was analyzed with MEFs transfected with the GAL4 one-hybrid system. NCoR^+/+ and NCoR^−/− MEFs were transfected with the same constructs as described for panel c, followed by addition of vehicle or T1317 (1 μM). (e) Overexpression of exogenous NCoR rescues repression of basal transcription in NCoR^−/− MEFs. The cells were cotransfected with the constructs described for panel c together with empty vector (pCMX) or full-length NCoR (pCMX NCoR). For panels a through e, all cells were cotransfected with a cytomegalovirus-β-Gal expression vector and luciferase values were normalized to those of β-Gal activity. In panel e, the luciferase activity for each control sample (cells transfected with GAL4-DBD) was assigned a value of 100%. The luciferase activities of cells transfected with GAL4-LXRs are represented as percentages of the respective control activity.