PTRE-HPRT inactivation correlates with repressive histone modifications.
ChIP analysis measuring histone H3 modifications at the PTRE-HPRT promoter in HP14 cells expressing high levels of HPRT (Untreated), reduced levels of HPRT after 1 week Dox treatment (Dox), and HP14-derived TG-resistant cell lines (TG1, TG5, and TG6). (A) ChIP analysis measuring acetylated histone H3 using a polyclonal antibody raised against a peptide corresponding to acetyl-K9 and acetyl-K14. (B) ChIP analysis measuring methylation at lysine 4 of histone H3 (methyl-K4 H3). The antibody used for immunoprecipitation recognizes all three forms of methylation at K4, mono-, di-, and tri-methyl. (C) ChIP analysis measuring the repressive modification of dimethylation at lysine 9 of histone H3 (di-methyl-K9 H3). (D) ChIP analysis measuring acetylation at lysine 9 of histone H3 (acetyl-K9 H3). Immunoprecipitated DNA levels were quantified by qRT-PCR. Specific signal was calculated by measuring fold change between pull down and input at the Hprt promoter (PTRE-HPRT), Gapdh promoter (P-Gapdh), and Mage-a promoter (P-Mage-a). For activating modifications, levels at PTRE-HPRT are displayed relative to the Gapdh promoter; for the repressive modification, dimethyl-K9 H3, results are displayed relative to the Mage promoter. Error bars indicate the SD from triplicate reactions.
