Fig. 4

Effect of PLZF deacetylation. PLZF occupancy of its endogenous DNA binding sites (a). Transfected 293T cells were used for chromatin immunoprecipitation with an antibody against PLZF or an antibody against histone H3 as positive control. For each condition, the amount of LINE-1, CRABPI and c-myc 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. PLZF localizes in specific subnuclear compartments in the presence or absence of HDAC3 and SIRT1 deacetylases (b). The nuclear localization pattern of PLZF (and indicated conditions) was analyzed in 293T cells transfected with PLZF alone (PLZF) or in the presence of HDAC3 (PLZF + HDAC3) and SIRT1 (PLZF + SIRT1), by indirect immunofluorescence and confocal microscopy, as reported previously [19], punctate nuclear distribution of wild-type PLZF was observed (PLZF). Only diffuse nuclear localization was observed when PLZF was co-expressed with HDAC3 (PLZF + HDAC3), whereas co-expression of PLZF and SIRT1 (PLZF + SIRT1) show both diffuse and punctate localization (at a lesser degree than PLZF alone). No immunofluorescence signal was observed when primary anti-PLZF monoclonal antibody was omitted from the experimental procedure