Forced association of p300 with chromatin in Vpr-expressing cells. Chromosome spreads were prepared from DOX-treated cells at 48 h. Vpr (−), ΔVpr cells; Vpr (+), MIT-23 cells. (A) Histone H3 lysine 9 is strongly acetylated in the chromosomes, including centromeric regions. Chromosome spreads were immunostained with antiacetylated H3K9 antibody. (B) Typical centromere regions shown at a higher magnification. H3K9 was highly acetylated along the whole chromosome, including the centromere region, in Vpr-expressing cells (arrowheads). However, only a weak signal was detected at the primary constrictions of the chromosomes in control cells (arrows). (C) Chromosome spreads stained for p300. (D) Preextracted cells stained for p300. Arrows, cells undergoing mitosis. Insets, mitotic cells expressing Vpr. ΔVpr cells + DOX (top) and MIT-23 cells + DOX (48 h; bottom) are shown. (E) Chromosome spreads stained for Vpr. (F) Chromosome spreads from DOX-treated MIT-23 cells stained for Vpr and p300. Arrows, signals at the centromere. Two pairs of sister chromatids are shown. (G and H) Chromosome spreads stained for CENP-A and Vpr (G) and CENP-H and p300 (H). Similar results were obtained in three independent experiments. Bars: (C and E–H) 5 µm; (D) 10 µm.