Fig. 2: The nucleosome-free region harbors enhancer-related histone modifications and produces enhancer RNAs.

a Schematic figure of the HTLV-1 provirus structure. The 5′LTR (black plaid), CTCF-binding site (blue)¹¹, NFR (red), and the HBZ promoter (yellow)²⁰ are shown. b–d Transcriptional regulatory function of the NFR was analyzed by luciferase reporter assays in Jurkat cells. b, c The HBZ promoter²⁰ and c 5′LTR were used as a promoter. d The HBZ promoter was used as a promoter and cells were stimulated with either PMA or TNF-α. Luciferase activity was normalized to Renilla activity. Relative luciferase activity is defined as the fold change to pGL4-basic (b) or pGL4-basic-HBZ promoter (c, d). n = 3 biologically independent samples, mean ± SD. P values are calculated by a two-sided Student’s t-test. e H3K4me1 (top row), H3K4me2 (middle row), and H3K27Ac (bottom row) ChIP-seq signals near the viral integration site in ED cells. ChIP-seq signals were visualized by IGV. The location (purple square) of provirus and NFR (red square) in ED cells are shown in the upper panel. Provirus direction is represented as a white arrow. The range in square brackets indicates the range of read counts. IS, integration site. f H3K4me1 (top row), H3K4me2 (middle row), and H3K27Ac (bottom row) ChIP-seq signals within the provirus in ED cells. ChIP-seq signals were visualized by IGV. Gray-shaded areas and orange-shaded areas indicate the ChIP-seq signals mapped to LTRs and NFR, respectively. The range in square brackets indicates the range of read counts. g NET-CAGE of ED cells nuclear lysates in the sense (top row) and antisense (bottom row) orientations, demonstrating eRNAs at the NFR. The bottom panel is an enlarged image of the signals around the NFR. NET-CAGE signals were visualized by IGV.