Fig. 7.

YY1 overexpression causes increased transcriptional initiation and elongation from the HTLV-1 LTR. (A) Total RNA prepared from HEK-293 cells was subjected to qRT-PCR. Levels of luciferase transcripts were first normalized using the 2-ΔΔCT method to the value obtained for the alpha-tubulin gene; the obtained values were then normalized to the values obtained from HEK-293 cells transfected with EV (set to 1). Results shown are means ± SEs from three independent experiments. (B) Total RNA prepared from HEK-293 cells treated with 1 μM actinomycin D for the indicated time points was subjected to qRT-PCR as in A. Results shown are means ± SDs from two independent experiments. (C) Total RNA prepared from nuclear/cytoplasmic fractionation was subjected to qRT-PCR as in A. Results shown are means ± SDs from two independent experiments. Effectiveness of nuclear/cytoplasmic fractionation was confirmed by WB using anti-GAPDH (cytosolic control) or anti-histone H3 (nuclear control) antibodies. (D) RNA polymerase II ChIP analysis of chromatin harvested from HEK-293 cells transfected with empty vector, HA-YY1, or Tax plasmid together with HTLV-1 LTR luciferase plasmid. ChIP data are presented as percent of input DNA (means ± SDs from two independent experiments). (E) Biotin-labeled RNA prepared from nuclear run-on assay in HEK-293 cells was isolated using streptavidin-coated magnetic beads, followed by qRT-PCR to monitor transcript abundance using primers spanning the entire luciferase transcript, including the 5′ UTR. Levels of luciferase transcripts were first normalized using the 2-ΔΔCT method to the value obtained for the alpha-tubulin gene; the obtained values were then normalized to the values obtained from 5′ luciferase primer of HEK-293 cells transfected with EV (set to 1). Results shown are means ± SDs from two independent experiments.