Figure 3.

CTCF binding differentially impacts BLV LTR promoter activities. (A) Schematic representation of the BLV LTR with the identified transcription start site for either the sense (nt +211) or the antisense (nt +460) transcription. The CTCF logo is indicated above the LTR_WT sequence. The three mutations in the CTCF binding site are highlighted in red and named as follows: LTR_m5; LTR_m10; LTR_mFull. The closest cis-regulatory element (BRE) is underlined. (B) Chromatin prepared from 293T cells stably transduced with either the wild-type BLV LTR or the mutated LTR was immunoprecipitated with a specific antibody directed against CTCF or with an IgG as background measurement. Purified DNA was then amplified with oligonucleotide primers hybridizing to either the BLV LTR or host cellular genes (GAPDH and MDM2). Results are presented as histograms indicating percentages of immunoprecipitated DNA compared to the input DNA (% IP/Input). Data are the mean ± SD from one representative of at least three independent experiments. (C) Illustration of the reporter constructs without LTR (X-luc) or with the WT or mutated LTR cloned in sense (S) or antisense (AS) orientation relative to the Firefly luciferase gene (luc). (D) The Raji B-cell line was transiently co-transfected with 600 ng of the reporter constructs and 50 ng of pRL-TK. Results are presented as histograms indicating relative luciferase activities compared to the value obtained with the LTR_WT-S-luc construct which was assigned to the value of 1. Data are the mean ± SD of at least three independent experiments. The Wilcoxon signed-rank (value of 1) or the Mann-Whitney statistical tests were used for sense and anti-sense orientations, respectively, with P > 0.05 = ns; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.001 = ***.