Genes and pathways regulated by IRF4 and NF-κB in ATL cells. (A) Venn diagram showing the overlap of high-confidence IRF4 and p65 target genes. TF, transcription factor. (B) Schematic representation of TCR and tumor necrosis factor (TNF) signaling pathway, as well as genes involved other biological functions such as cytokine and chemokine signaling and cancer-related genes. IRF4-p65 high-confidence genes are boxed in red. (C) mRNA expression of several IRF4-p65 target genes were analyzed by RNA-seq in 3 healthy donors, 3 HTLV-1 carriers, and 45 primary ATL cases from the cohort by Kataoka et al.,²⁶  and primary ATL cases from the NCU cohort. See Figure 1C legend for the details. ns, nonsignificant; *P < .05; **P < .01; ***P < .001 by an unequal variances t test. (D) ChIP-seq gene tracks representing IRF4 and p65 binding and H3K27ac signals at the MYC gene locus in TL-Om1 cells. The x-axis indicates the linear sequence of the genomic DNA, and the y-axis indicates the total number of mapped reads per million reads. The black horizontal bar indicates the genomic scale in kilobases. The black boxes in the gene map represent exons, and the arrows indicate the location and direction of the transcriptional start site. (E-F) Control shRNA (sh-GFP-1) and 2 independent shRNAs targeting IRF4 (sh-IRF4-1 and sh-IRF4-2) (E) and p65 (sh-p65-1 and sh-p65-2) (F) were first transduced in 2 ATL/HTLV-1-transformed T-cell lines, TL-Om1 and MT-2, and mRNA was extracted on day 3 posttransduction. The mRNA expression of MYC was measured by qRT-PCR, using 2 different primer sets (MYC P1 and P2). Expression was normalized to that of the internal control (GAPDH) and is 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.