CCR4 is transcriptionally activated by IRF4 and NF-κB in ATL cells. (A) Among 399 genes selected in Figure 4A, 30 were found to be associated with super-enhancers in more than 5 of 11 ATL samples, including 10 primary ATL cases (ATL1-10) and TL-Om1. The heat map on the left shows the differential mRNA expression of these 30 genes upon knockdown of IRF4 and p65 in TL-Om1 cells. Super-enhancer status of these gene loci in T-ALL cells (KOPT-K1 and Jurkat cells), normal T-cells (thymus, Th1, Th2, and Th17) and ATL cells (TL-Om1 and 10 primary ATL samples) is shown on the right. Red, super-enhancer. (B) The ChIP-seq gene tracks represent the binding of IRF4 and p65, as well as the presence of H3K27ac marks near the CCR4 gene locus in various cell samples. See Figure 4D legend for details. The red lines indicate super-enhancer positions. (C-D) CCR4 mRNA expression in 2 ATL/HTLV-1-transformed T-cell lines (TL-Om1 and MT-2) was measured by qRT-PCR, using 2 different primer sets (P1 and P2) on day 3 after lentiviral transduction with various shRNAs. Expression was normalized to that of the internal control (GAPDH) and os presented as fold-changes compared with the sh-GFP control: as mean of SD of duplicates. *P < .05; **P < .01; ***P < .001 by a 2-sample, 2-tailed t-test compared with the sh-GFP control. (E) dCas9-KRAB-expressing TL-Om1 cells were transduced with 2 independent doxycycline (Dox)-inducible sgRNAs (sgRNA-1 and sgRNA-2) targeting the IRF4-NFκB/p65-bound region marked by the red arrowhead in Figure 5B. The expression levels of CCR4 were analyzed by qRT-PCR, using 2 different primer sets (P1 and P2), after 72 hours of Dox treatment. Expression was normalized to that of the internal control (GAPDH) and is presented as the fold-change relative to the dimethyl sulfoxide (DMSO)-treated control: as mean of SD of duplicates. *P < .05; **P < .01; ***P < .001 by a 2-sample, 2-tailed t-test compared with the DMSO-treated control.