Figure 6

H3K27me3 is enhanced upon Sug1 knockdown. (a) Sug1 siRNA efficiently decreases endogenous Sug1. HeLa cells were transfected with control or Sug1-specific siRNA, harvested and subjected to western blot analysis for (top) endogenous Sug1 and (bottom) endogenous tubulin. (b-d) H3K27me3 is elevated at cytokine-inducible genes upon diminished Sug1 expression. ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0 to 18 hours. Lysates were immunoprecipitated with control or endogenous H3K27me3 antibody. Associated DNA was isolated and analyzed as in Figure 2 using primers and probe spanning (b) CIITA pIV, (c) the MHC-II proximal promoter and (d) the GAPDH promoter. Data are presented as percentage input. IgG isotype control values were 0.003 ± 0.001 (CIITA pIV), 0.08 ± 0.02 (MHC-II promoter) and 0.01 ± 0.005 (GAPDH promoter). Values represent mean ± SEM of (n = 3) independent experiments.