Biological interaction between EZH2 and HTLV-1 Tax. (A) EZH2 promoter activity in the presence or absence of HTLV-1 genes. 293FT cells were transfected with plasmid vectors carrying EZH2 promoter reporter and indicated HTLV-1 cDNAs. The luciferase activities were quantified at 48 hours post-transfection (n = 3, mean ± SD, P < .05). (B) Tax-dependent EZH2 gene activation analyzed by luciferase reporter assay. Signaling inhibitors (DHEMQ for NF-κB inhibition; cyclosporin A [CsA] for NAFT inhibition; U0126 for ERK1/2 inhibition) were cotreated. A Tax M22 was used as a NF-κB–deficient mutant (n = 3, mean ± SD, P < .01). (C-D) Interaction between Tax and PRC2. 293T cells were transfected with plasmids for expression of FLAG-Tax (C), or FLAG-EZH2 and untagged Tax (D), respectively. Nuclear cell extracts from each sample were immunoprecipitated with an anti-FLAG antibody. Coprecipitated proteins were prepared and analyzed by western blotting with indicated antibodies. H.C., IgG heavy chain. (E) Pull-down assay with GST-EZH2. Expression vectors for deletion mutant series of GST-tagged EZH2 were established (top). 293T cells were transfected with the plasmids encoding GST-EZH2 series and untagged Tax, and then subjected in GST pull-down assay. Purified GST-EZH2 series and copurified PRC2 components were detected by western blotting with indicated antibodies (bottom). (F) Co-immunoprecipitation (Co-IP) between endogenous Tax and EZH2 in HTLV-1–infected HUT102 cells. The same amounts of anti-EZH2 antibody and non-specific IgG were used in the immunoprecipitation, followed by detection with indicated antibodies. (G) Confocal microscopy data showing subcellular localizations of endogenous EZH2 and Tax in HUT102. The scale bar indicates 10 μm. Arrowheads indicate colocalized spots. (H) Confocal microscopy data showing subcellular localizations of endogenous EZH2 and ectopically expressed viral proteins that were detected by anti-EZH2 and anti-FLAG antibodies, respectively, in HeLa cells. The scale bars indicate 10 μm. Arrowheads indicate colocalized spots. (I) Venn diagram showing the overlap between genes (Coc analysis FC > 0 vs control IgG) associated with EZH2 occupancy, H3K27me3 accumulation, and Tax binding in HTLV-1–infected MT-2 cells. (J) Composite enrichment profiles of Tax, EZH2, and H3K27me3 relative to the closest TSS at Tax-bound genes (solid line, 5377 genes, Coc FC > 0 vs control IgG) and Tax-unbound genes (dashed line, 14 937 genes, Coc FC < 0 vs control IgG) in MT-2 cells.