FIG. 2.

DN2 as a specific dominant-negative repressor of LXR transactivation. (A and B) An oxysterol-responsive LXRE-LUC reporter construct was cotransfected into HeLa cells, along with lacZ expression vector (100 ng) and expression vectors for ASC-2, DN2 (i.e., the ASC-2 residues 1431 to 1511), DN2/m, TRAP220, and SRC-1, as indicated. The solid and shaded bars indicate the absence and presence of 10 μM 22(R)-hydroxycholesterol, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the lacZ expression. Similar results were obtained with CV-1 cells (data not shown). The error bars indicate standard deviations. (C) ASC-2 recruitment to the LXRE region of SREBP-1c. 293T cells were cotransfected with expression vectors for DN2 (50 and 100 ng) and DN2/m (50 and 100 ng) in the presence of 5 μM T0901317, as indicated. Chromatin from these cells was isolated and immunoprecipitated (IP) by the indicated antibodies. The endogenous SREBP-1c-LXRE region present in the immunoprecipitated samples was amplified by PCR, and the input PCR (10%) is shown for loading controls. Similar results were also obtained in the absence of ligand (data not shown).