FIG. 2.

G9a in SHP-containing complexes methylates H3K9 in vitro. (A). Nuclear extracts (NE) from HepG2 cells or mouse liver were incubated with bacterially expressed, purified GST-SHP or GST that had been bound to glutathione-Sepharose. The proteins associated with GST-SHP or GST were used as the source of enzyme in in vitro HMT assays. Proteins were separated by SDS-PAGE, followed by Coomassie staining (lower panel) of the gels and autoradiography (upper panel). The positions of histones H3 and H4 are indicated. (B) Mouse liver nuclear extracts (NE) were incubated with bacterially expressed, purified GST-SHP or GST that had been bound to glutathione-Sepharose. The proteins associated with the Sepharose beads were used as the source of enzyme in HMT assays. After the reaction, proteins were separated by SDS-PAGE, and dimethylated H3K9 histones were detected by Western blotting using dimethylated H3K9 antibody. The position of a molecular mass marker is shown at left. The position of H3 (dimethyl H3K9) is indicated at right. (C) HepG2 nuclear extracts were immunoprecipitated with either SHP antibody (Ab) or control IgG, and the immunoprecipitates were used as the source of enzyme in HMT assays. The proteins were separated by SDS-PAGE and then by Coomassie staining of the gels to detect the amounts of core histone substrate used in the assay (lower panel), followed by autoradiography to detect ³H-labeled histones (upper panel). The positions of histones H3 and H4 are indicated. (D) Experimental outline. HepG2 cells were infected with control empty adenoviral vector (Ad-empty) or adenoviral vector encoding G9a-DN (Ad-G9a-DN), and 36 h later, the infected cells were subjected to semiquantitative RT-PCR, Western blotting, or immunoprecipitation followed by in vitro HMT assays. (E) Cell extracts were subjected to Western blotting using G9a antibody and HNF-4 antibody as a control. (F) Cell extracts were immunoprecipitated with SHP antibody (Ab) or control IgG, and the immunoprecipitates were used as the source of enzyme in in vitro HMT assays. Bacterially expressed and purified GST-H3(1-84) (WT) or a GST-H3 mutant (H3R9) in which K9 is mutated to Arg as shown was used as histone substrates. The HMT reaction samples were analyzed as described in panel A.