Fig. 3: SRF and ELK-1 bind to the NFR in a DNA sequence-dependent manner.

a Prediction of transcription factors that bind to the NFR was performed using TFBIND (http://tfbind.hgc.jp/)⁵⁶. Candidate transcription factor binding sites are shown. 8000 and 8160 are nucleotide positions in HTLV-1 provirus. b Figure shows the localization of SRF (left panel) and ELK-1 (right panel) to the NFR in cell lines and PBMCs of ATL patients determined by ChIP-seq with HTLV-1 DNA-capture. ChIP-seq signals were visualized by IGV. The range in square brackets indicates the range of read counts. Orange-shaded region indicates the NFR. c Binding ability of SRF and ELK-1 to the NFR oligonucleotides was analyzed by EMSA. Biotinylated DNA probes of 120 bp for the NFR (red) and negative control regions (gray) were incubated with nuclear extract (N.E.) of 293 T cells transfected with SRF and ELK-1 expression vectors. NFR-SRF/ELK-1 complexes and super-shifted complexes, which were detected with the anti-SRF and the anti-ELK-1 antibody, are indicated by arrowheads. The data shown are representative of two independent experiments. d Figure shows the mutations introduced to generate three different NFR mutants. The mutated nucleotides are shown in color in comparison to the wt sequence and colored as green, orange, red, and blue for the nucleotides A, G, T, and C respectively. Nucleotide sequences are shown in Supplementary Fig. 3a. e EMSA competition analysis with NFR-wt and mutant sequences. Unlabeled competitor NFR-wt or -mut probes were added to the mixture of nuclear lysates and biotin-labeled NFR-wt probe. Competition of unlabeled probes against biotin-labeled NFR-wt probes was evaluated at different doses (x100, x200, and x300). The sequence for each non-labeled competitor is shown in (d) and Supplementary Fig. 3a. Red, dots pattern, lattice pattern, and diagonal stripe pattern triangles are indicated as wt, mut_1, mut_2, and mut_3 respectively. For more details, see the method. The data shown were representative of two independent experiments. f Transcriptional regulatory function of the NFR-wt (black) or -mut_3 (diagonal stripe pattern) was analyzed using the HBZ promoter (yellow) in Jurkat cells by luciferase assay. Luciferase activity was normalized to Renilla activity. Relative luciferase assay is defined as the fold change compared to a pGL4-basic-HBZ promoter. n = 3 biologically independent samples, mean ± SD. P values are calculated by a two-sided Student’s t-test.