FIG. 5.

Tethered HDAC4 represses transcription. (A) Schematic representation of the luciferase reporter Gal4-tk-Luc. Upstream from the tk core promoter (−152 to +50) are five copies of the Gal4-binding site. (B) Repression of Gal4-tk-Luc by HDAC4 and its mutants in NIH 3T3 cells. The mutants dm1 to -3 and dm1/H803A are depicted in Fig. 4A; dm4 and dm5 contain the N-terminal 208 and 114 residues of HDAC4, respectively. Mammalian constructs expressing HDAC4 and its mutants fused to the C terminus of Gal4(1-147) were transfected into NIH 3T3 cells with the reporter Gal4-tk-Luc. Luciferase (Luc) activities were normalized to the internal β-galactosidase control; the normalized luciferase activity from the transfection without any effector plasmid was arbitrarily set to 1.0. (C) Expression of Gal4-HDAC4 and its mutants. Extracts (10 μg/lane), prepared from 293T cells transfected with expression plasmids for indicated fusion proteins, were subjected to Western blotting analyses using an anti-Gal4 antibody (RK5C1; Santa Cruz Biotechnology). Molecular size markers are shown at the right. (D) Repression of reporters with different core promoters by Gal4-dm3 in 293T cells. The reporters possess indicated core promoters replacing the tk region of Gal4-tk-Luc (A); 100 and 300 ng of expression plasmids were used as indicated.