Dysregulated Recruitment of the Histone Methyltransferase EZH2 to the Class II Transactivator (CIITA) Promoter IV in Breast Cancer Cells
Figure 6
EZH2 knockdown decreases CIITApIV histone H3K27 trimethylation.
(A–B) EZH2 knockdown is efficient and specific. MDA MB 435, 435-Brain 1, and 435-Lung 2 cells were transfected with either EZH2 specific siRNA or with control siRNA. 10% of lysates were subjected to immunoblot (IB) for EZH2 and tubulin. Results reported are representative data from three independent experiments. The remaining lysates were subjected to EZH2 mRNA isolation and quantitation by Q-PCR. Q-PCR was performed in triplicate and results shown represent the mean ± SD of three independent experiments. (C) Levels of trimethylated H3K27 in breast cancer cells. ChIP assays were carried out in MDA MB 435 variants transfected with either EZH2 siRNA or control siRNA and stimulated as indicated with IFN-γ. Lysates were IP with control antibody or with antibody to H3K27me3 and associated DNA was isolated and analyzed as above via Q-PCR using primers spanning the IRF-E-GAS box of CIITApIV. Values shown represent mean ± SEM of three independent experiments. Control IP values were 1.2±0.6 ***P<0.0005, **P<0.005.