Figure 6.
Figure 6. p300 is required for Vpr-induced PCS. (A) Expression of p300 or Vpr in immunoprecipitates (IP) measured using anti-Vpr and anti-p300 antibodies and control (Cont) IgG. Cells were transfected with pCMV-Vpr (Vpr) or control vector (Vec). (B) Vpr immunocomplexes exhibit HAT. 293T cells were transfected with Flag-EGFP, Flag-Vpr, and Flag-p300 (positive control), and HAT activity was measured. Flag-immunoprecipitated Flag-Vpr showed higher HAT activity than did Flag-EGFP, control IgG, IP-Flag-EGFP, or Flag-Vpr. IP-Flag-p300 reached maximal levels 2 h before measurement, indicating that actual p300 HAT activity may be higher than the measured value. Results shown are representative of two independent experiments. (C) HAT activity in Vpr-expressing cells. HAT activity targeting histone H3K9 in Flag-EGFP or Flag-Vpr immunoprecipitates was examined using histone H3 (HH3) as a substrate. (D) Expression of HP1-α in the isolated chromatin of Vpr-expressing MIT-23 cells (+DOX, 48 h) transfected with control or p300 siRNA. (left) Immunoblotting of the indicated proteins. β-Tubulin (βTub), whole-cell lysate loading control; HH3, isolated chromatin loading control. (right) Quantification of the HP1-α intensities shown in the immunoblot. RNAi of p300 in Vpr-expressing cells (lane 3) reversed the amount of chromatin-bound HP1-α compared with control RNAi (lane 2) with statistical significance (*, P = 0.046). Values represent the mean ± SD of three independent experiments. (E) Immunostaining of chromatin-bound HP1-α, p300, and Vpr. Typical micrographs are shown. The levels of chromatin-bound HP1-α in Vpr-expressing cells were restored when p300 was down-regulated (right panels). Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bar, 10 µm. (F) PCS frequencies in cells treated with p300 siRNA or AA (7.5 µM in DMSO) in the presence of Vpr expression. Values represent the mean ± SD of three independent experiments. *, P = 0.0009 for p300 RNAi and P = 0.0088 for AA versus control RNAi in DOX-induced MIT-23 cells.

p300 is required for Vpr-induced PCS. (A) Expression of p300 or Vpr in immunoprecipitates (IP) measured using anti-Vpr and anti-p300 antibodies and control (Cont) IgG. Cells were transfected with pCMV-Vpr (Vpr) or control vector (Vec). (B) Vpr immunocomplexes exhibit HAT. 293T cells were transfected with Flag-EGFP, Flag-Vpr, and Flag-p300 (positive control), and HAT activity was measured. Flag-immunoprecipitated Flag-Vpr showed higher HAT activity than did Flag-EGFP, control IgG, IP-Flag-EGFP, or Flag-Vpr. IP-Flag-p300 reached maximal levels 2 h before measurement, indicating that actual p300 HAT activity may be higher than the measured value. Results shown are representative of two independent experiments. (C) HAT activity in Vpr-expressing cells. HAT activity targeting histone H3K9 in Flag-EGFP or Flag-Vpr immunoprecipitates was examined using histone H3 (HH3) as a substrate. (D) Expression of HP1-α in the isolated chromatin of Vpr-expressing MIT-23 cells (+DOX, 48 h) transfected with control or p300 siRNA. (left) Immunoblotting of the indicated proteins. β-Tubulin (βTub), whole-cell lysate loading control; HH3, isolated chromatin loading control. (right) Quantification of the HP1-α intensities shown in the immunoblot. RNAi of p300 in Vpr-expressing cells (lane 3) reversed the amount of chromatin-bound HP1-α compared with control RNAi (lane 2) with statistical significance (*, P = 0.046). Values represent the mean ± SD of three independent experiments. (E) Immunostaining of chromatin-bound HP1-α, p300, and Vpr. Typical micrographs are shown. The levels of chromatin-bound HP1-α in Vpr-expressing cells were restored when p300 was down-regulated (right panels). Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bar, 10 µm. (F) PCS frequencies in cells treated with p300 siRNA or AA (7.5 µM in DMSO) in the presence of Vpr expression. Values represent the mean ± SD of three independent experiments. *, P = 0.0009 for p300 RNAi and P = 0.0088 for AA versus control RNAi in DOX-induced MIT-23 cells.

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