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. 2008 Jul 28;28(19):5837–5850. doi: 10.1128/MCB.00535-08

FIG. 2.

FIG. 2.

Sug1 knockdown decreases MHC-II promoter histone H3 acetylation. (A) Sug1 siRNA specifically and efficiently decreases Sug1 protein expression in the presence or absence of IFN-γ stimulation. HeLa cells transfected with control or Sug1-specific siRNA were harvested and subjected to Western blot analysis of the endogenous expression of 19S ATPases Sug1, S6a, and S7. Western blotting shows >90% knockdown of Sug1 (top) and stable expression of S6a and S7 (middle and bottom). (B) Sug1 knockdown decreases histone H3 acetylation at the MHC-II proximal promoter. ChIP assays were carried out with HeLa cells stimulated with IFN-γ for 0 to 18 h (left) or with HeLa cells transfected with scrambled siRNA control or Sug1-specific siRNA duplexes and 24 h later stimulated with IFN-γ for 0 to18 h (right). Lysates were subjected to IP with control antibody or with antibody to endogenous acetylated histone H3. Associated DNA was isolated and analyzed via real-time PCR using primers spanning the W-X-Y box of the MHC-II HLA-DRA promoter. Real-time PCR values were normalized to the total amount of HLA-DRA promoter DNA added to the reaction (input). Input values represent 5% of the total cell lysate. IP values are presented as increases in the MHC-II promoter DNA relative to unstimulated acetylated histone H3 IP sample values. Control IP values were (1.2 ± 0.3)-fold. Control and acetylated histone H3 IP values represent the mean ± the standard error of the mean (SEM) of three independent experiments. (C) Sug1 knockdown decreases histone H3 acetylation at the MHC-II proximal promoter in the presence of HDAC inhibition. ChIP assays were carried out with untransfected (left) or siRNA-transfected (right) HeLa cells which were treated with HDAC inhibitors (20 h) and stimulated with IFN-γ for 0 to 18 h. Lysates were subjected to IP with control antibody or with antibody to endogenous acetylated histone H3, and associated DNA was isolated and analyzed via real-time PCR as described for panel B. IP values are presented as increases in the MHC-II promoter DNA relative to unstimulated acetylated histone H3 IP sample values. Control IP values were (2.7 ± 0.8)-fold. Control and acetylated histone H3 IP values represent the mean ± SEM of three independent experiments. ***, P < 0.001 versus control siRNA.