FIG. 5.

PLZF lysine mutants assessed for transcriptional activity. Lysine (K) residues in the ninth zinc finger of PLZF were changed to arginines (R) by introducing single point mutation in the PLZF DNA coding sequence. A mutant lacking all the zinc finger 9 acetylation sites (K647/650/653-R) and each mutation separately (K647-R, K650-R or K653-R), were compared to wild-type PLZF (wild type) in its ability to repress transcription using cotransfection experiments in 293T cells. For each mutant, equal amounts of each expression vector and luciferase reporter with a minimal herpes simplex virus thymidine kinase promoter and two PLZF binding sites were cotransfected into 293T cells, and luciferase activity was determined 24 h later. The expression vector for the p300 HAT was cotransfected in each case to enhance PLZF-mediated repression of the luciferase reporter. Each lysine mutation occurring in zinc finger 9 has an impact on the repression activity of the PLZF protein. Levels of transiently expressed proteins were monitored by Western blot analysis using an anti-PLZF antibody (WBαPLZF).