Fig. 3
HDAC3/SIRT1-induced deacetylation affect PLZF DNA binding and transcriptional activities. HDAC3 over-expression influence PLZF activities in vivo (a). Transcriptional repression by PLZF is blocked by HDAC3 (a.1). 293T cells were transfected with construct as indicated. Cells were lysated at 18 h post transfection and luciferase assays performed. Luciferase activity is expressed relative to the activity in the vector only sample (white bars). PLZF alone, light grey bars. HDAC3 alone, black bars. PLZF and HDAC3, dark grey bars, PLZF and HDAC3 with TSA treatment, striped bars and PLZF-Q mutant dotted bars. Each experiment was performed in triplicate and the data represents the average of at least three experiments. Errors bars standard error of mean. Lower panel control of PLZF expression using immunoblotting detection with a FLAG antibody (bottom panel α-FLAG). Chromatin immunoprecipitation of the PLZF target (a.2). Flagged PLZF transfected 293T cells were treated with either DMSO or 20 nM Trichostatin A, and used for chromatin immunoprecipitation with an antibody against either FLAG or a IgG antibody control. For each condition, the amount of the HoxB2 promoter DNA spanning a PLZF binding site bound by each antibody was amplified and quantified by real-time PCR. This was expressed to the signal obtained from the 5 % input chromatin samples. Transcriptional PLZF binding activity is regulated by PLZF acetylation (b). The activity of the chimeric protein 9znfPLZF-VP16 was tested in transient transfection experiments and compare to the Lex-VP16 chimeric protein. Where indicated HDAC1 or HDAC3 expression vectors were cotransfected with increasing amount amount (1, 50 ng; 2, 100 ng, 3, 150 ng). Each experiment was performed in triplicate and the data represents the average of at least three experiments. Errors bars standard error of mean. SIRT1 over-expression influence PLZF activities in vivo (c). Transcriptional repression by PLZF is blocked by HDAC3 (c.1). 293T cells were transfected with constructs as indicated. Cells were lysed at 18 h post transfection and luciferase assays performed. Luciferase activity is expressed relative to the activity in the vector only sample (white bars). PLZF alone, light grey bars. SIRT1 alone, black bars. PLZF and SIRT1, dark grey bars and PLZF and SIRT1 with nicotinamide treatment, striped bars. Luciferase activity is expressed relative to the activity in the vector only sample (white bars). Error bars standard error of the mean. 2.5 kb of the c-myc promoter, −1.8 kb to +0.7 kb relative to the P1 promoter, 5′ to luciferase. Lower panel control of PLZF expression using immunoblotting detection with a FLAG antibody (bottom panel α-FLAG). Chromatin immunoprecipitation of the PLZF target (c.2). Transfected cells were treated with either DMSO or 10 mM nicotinamide, and used for chromatin immunoprecipitation with an antibody against PLZF For each condition, the amount of c-myc promoter DNA spanning a PLZF binding site bound by each antibody was amplified and quantified by real-time PCR. This was expressed relative to the signal obtained from the 5 % input chromatin sample