Fig. 2: Molecular characterization of the silencer complex on the OCR.
From: Intragenic viral silencer element regulates HTLV-1 latency via RUNX complex recruitment
a,b, ChIP-seq signals with HTLV-1 DNA-capture analysis for RUNX1, CBFβ, GATA3, ETS1, HDAC3 and Sin3A in HTLV-1-infected Jurkat T cell clones (wt39 and wt51) (a) and PBMCs of patients with ATL (Al-5 and AI-9) (b). c, Effect of overexpression of RUNX1, GATA3 or ETS1 on the 5′-LTR promoter activity with the OCR in 293T cells. d, Effect of overexpression of RUNX1 and/or ETS1 on the 5′-LTR promoter activity with the OCR in 293T cells. e, Changes in OCR-mediated silencing by mutating RUNX, GATA3 and ETS1 binding sites within the OCR in Jurkat T cells. f, Transcriptional regulation of RUNX1 mutants (S67I, W79C and R174Q) in OCR-mediated silencing of HTLV-1 5′-LTR in 293T cells. Protein expression of RUNX1 mutants was confirmed twice by western blot. g, Representative RT-qPCR result of tax mRNA expression in MT1 and TBX-4B cells transduced with RUNX1. h, Measurement of Tax protein levels in RUNX1-overexpressed MT1 cells. RUNX1 was transduced using a retroviral vector system. One representative result from each flow cytometry assay (left) and the cumulative Tax positivity values from a triplicate assay (right) are shown. i, Effect of RUNX1 knockdown via shRNA on OCR-mediated silencing of the HTLV-1 5′-LTR. Molt4 cells carrying shRNA for RUNX1 were used for luciferase assay. Luciferase reporter assays were performed 48 h after transfection. The results are representative of at least two independent experiments. The bars and error bars represent the mean ± s.d. of the results of triplicate experiments. P values were calculated using a two-sided, unpaired Student’s t-test.
