Figure 2: G9a methylates ERα protein in vitro and in cells.
(a,b) G9a methylates ERα at K235 and K303 in vitro. In vitro methylation assays were performed as described in the Methods section and assessed by autoradiograph. The top panel of a shows the diagram of the ERα protein domains. AF-1: activation function 1 region; DBD: DNA-binding domain; D+H: DBD+hinge; H: hinge region; LBD: ligand-binding domain.Recombinant ERα protein fragments (a) or fragments containing the indicated ERα point mutants (b) were incubated with recombinant G9a in the presence of 3H-labelled S-adenosylmethione. GelCode blue staining was used to assess input protein levels. (c) G9a dimethylates ERα at K235. Mass spectrometric analysis of the ERα peptide (aa 227–244) with or without G9a incubation. The peptide masses are shown; a change in mass of 28 Da indicates the addition of two methyl groups. (d) The anti-ERαK235me2 antibody specifically recognizes ERα protein methylated by G9a at K235 in vitro. The anti-ERα-K235me2 antibody was assessed by western blotting using recombinant ERα protein methylated by G9a. The K235R mutant was used as a negative control. GelCode blue staining shows the relative amounts of proteins used in the methylation assay. (e,f) The anti-ERαK235me2 antibody recognizes G9a-methylated ERα protein at K235 in cells. Western blot analysis of Flag-IPed ERα from HEK-239T cells co-transfected with Flag-ERα and Myc-G9a or vector control (e) or with the Myc-G9a and either WT ERα or the ERαK235R mutant (f). ERα protein levels in whole cell extract (WCE) and IP samples were used as loading controls. (g) Methylated ERα is enriched in the chromatin fraction. Top panel, western blot analysis of Flag-ERα immunoprecipitated from the cytoplasmic (Cyto), soluble nuclear (Nu) and chromatin-associated (Chr) fractions of cells probed with the anti-ERαK235me2 antibody. HEK-239T cells were co-transfected with Flag-ERα and Myc-G9a and were cultured in regular DMEM medium containing phenol red. Cell fractionation was assessed using anti-tubulin (cytosolic marker) and anti-histone H3 (chromatin marker) antibodies. The distribution of ERα and G9a in the different cellular fractions is shown.
