Fig. 4

ZNF326 up-regulates HDAC7 expression at the transcriptional level. a-c After transfection of ZNF326 in HEK293 cells, the level of β-catenin nuclear import was assessed by cytosolic fractionation assay (a) and immunofluorescence assay (b), β-Tublin and LaminB1 were the loading controls for cytoplasm and nucleus, respectively. Chromatin was immunoprecipitated with the Myc-tag antibody or control IgG after transfection of Myc-ZNF326 plasmid in U87, followed by qPCR using primer pairs spanning the human HDAC7 promotor. Results are presented as percentage immunoprecipitated over input and are representative of three independent experiments (c). e-f Information about luciferase reporter plasmids of HDAC7 promoter region plasmid (d) and the domain structure of ZNF326 (f). ZNF326 could promote the wild-type luciferase reporter activity of HDAC7 promoter, and had no effect on the mutants. HEK293 cells were co-transfected with ZNF326 and HDAC7 promotor-luciferase reporters. Renilla luciferase served as a control for signal normalisation (e). g Transcriptional activation domain (TAD) and zinc-finger structure are essential for ZNF326 to promote HDAC7 transcription. HDAC7 transcriptional activity was measured by luciferase report assay after transfection of HDAC7 promotor-luciferase reporter plasmid and ZNF326 wild-type, as well as mutants in HEK293 cells. Data from a representative experiment are plotted as the mean of three replicates plus the standard deviation. h-k In the HEK293 cell line, HDAC7 mRNA and protein levels were detected by qPCR (h) and immunoblotting (j) after transfection of ZNF326 wild-type and mutants. After transfection of siRNA-ZNF326 in U87 cells, the mRNA and protein levels of HDAC7 were significantly downregulated (i, k). GAPDH served as a loading control. Columns: mean numbers. Bars: S.D. (*: P < 0.05; **: P < 0.01; ***: P < 0.001)