Epigenetic alterations caused by HTLV-1 Tax. (A) Construction of Tax-expressing lentiviral vector. Tax-IRES-Venus expression is controlled by EF1α promoter. (B) Left: graph showing proportion of Venus⁺ cells in PBMC cultures that were transduced with Tax WT, Tax M22 (NF-κB–deficient), Tax C29A (nuclear localization–deficient), F-Tax (CREB-deficient), or empty lentivirus. DZNep (500 nM) or GSK126 (1 μM) were cotreated for 21 days to inhibit endogenous EZH2. Right: representative flow cytometry showing expansion of Venus⁺ (Tax-expressing) population by Tax expression, which was inhibited by GSK126 or DZNep treatment. PBMCs from 1 of 4 healthy donors was reproducibly immortalized by Tax expression, which is consistent with a previous report.⁵¹  (C) Expression levels of EZH2 and miR-31 in Tax-dependent immortalized cells (Tax-cell, 5 weeks post lentivirus transduction) quantified by quantitative reverse-transcription PCR (n = 3, mean ± SD, P < .05). (D) Representative flow cytometry showing H3K27me3 level in mock (activated PBMCs) and Tax-cell (30 weeks post-transduction). Staining without primary antibody was used as a negative control. (E) Methylation intensity profiling of each probe in ATL cells, normal CD4⁺ T cells, and Tax-cell. Relative H3K27me3 levels of normalized each probe (P < .05) are shown in histogram. (F) Heat map and hierarchical clustering of H3K27me3 level at regions (P < .05) in ATL cells, Tax-cell, activated CD4⁺ T cells, and control resting CD4⁺ T cells. (G) Venn diagram showing overlap between regions with H3K27me3 gain (P < .05) in ATL cells and Tax-cell compared with normal CD4⁺ T cells. (H) Box pot showing relative H3K27me3 levels at the overlapped 82.9% regions defined in (G) in resting and activated CD4⁺ T cells, Tax-cell, and ATL cells (*P < 1E−10). (I) Composite H3K27 methylation profiles of 12 050 probes that showed H3K27me3 gain in ATL cells compared with normal CD4⁺ T cells. (J) P value showing results of gene ontology analysis of genes that were associated with H3K27me3 gain in ATL and Tax-cells defined in (G) and downregulated in acute-type ATL (expression profile FC < −1.5, P < .05) compared with normal CD4⁺ T cells.