Figure 1. Suv39h1 Methylates Pc2 at K191.

(A) Pc2 is a non-histone substrate for Suv39h1. In vitro methylation assay was performed by incubating GST-Suv39h1 with recombinant His-Pc2, His-E2F1, histone H3, E1, E2, and SUMO1 in the presence of [³H]-SAM. (B) The enzymatic activity of Suv39h1 is required for Pc2 methylation. In vitro methylation assay was performed by incubating GST-Suv39h1 (wt, H320R or H324L mutant) with His-Pc2 in the presence of [³H]-SAM. (C) Suv39h1 methylates Pc2 at lysine 191 in vivo. FLAG-Pc2 immunoprecipitated from 293T cells (indicated by red arrow in left panel) was subjected to MOLDI-TOF analysis (right panel) and modified peptides are indicated by the red box. (D) Suv39h1 methylates Pc2 at K191 in vitro. In vitro methylation assay was performed by incubating GST-Suv39h1 with wt Pc2 or K191R mutant in the presence of [³H]-SAM. (E) Validation of Pc2K191me² and Pc2K191 antibodies. Methylated and unmethylated His-Pc2 was subjected to autoradiography (top panel) or immunoblotting with antibodies indicated (middle panels). (F and G) Suv39h1 methylates Pc2 at K191 in vivo. Cell lysates from HeLa cells transfected with siRNAs (F) or expression vectors (G) as indicated were immunoblotted with antibodies indicated. For A, B and D, the reaction products were separated by SDS-PAGE followed by Coomassie blue staining (CBB) (left panel) or autoradiography (right panel).

See also Figure S1.