Figure 2. Growth Control Gene Activation Requires KDM4C-Mediated Pc2K191me² Demethylation.

(A) KDM4C demethylates Pc2 in vitro. Histone Demethylase assay was performed by incubating purified FLAG-histone demethylases with in vitro methylated His-Pc2. Changes in substrate methylation levels were analyzed by autoradiography (upper panel) and Pc2K191me² antibodies (middle panel). Immunoblotting with antibodies against His tag was used to demonstrate equal loading (bottom panel). (B) Enzymatic activity of KDM4C is required for Pc2K191me² demethylation. His-KDM4C wt or H190G/E192A mutant was incubated with in vitro methylated His-Pc2 in the presence or absence of Fe (II). The reactions were separated by SDS-PAGE followed by autoradiography (upper panel), immunoblotting with Pc2K191me² antibodies (middle panel) or Coomassie blue staining (lower panel). (C) Serum-induced demethylation of Pc2K191me² in vivo. HeLa cells were serum-starved followed by restimulation and the cell lysates were immunoprecipitated with Pc2K191 or Pc2K191me² antibodies followed by immunoblotting with pan Pc2 antibodies. (D) Serum-induced demethylation of Pc2 on growth control gene promoters. HeLa cells were serum-starved followed by restimulation and ChIP analyses were performed using Pc2K191, Pc2K191me² and pan Pc2 antibodies on indicated regions. For each primer pair, Pc2K191me² (left panel) and Pc2K191 ChIP values (right panel) were normalized to the corresponding total pan Pc2 ChIP values. (E and F) Serum-induced KDM4C recruitment is responsible for Pc2K191me² demethylation on growth control gene promoters. HeLa cells transfected with blank vector, FLAG-KDM4C wt or H190G/E192A mutant were serum-starved followed by restimulation and ChIP analyses using FLAG antibody (E) or Pc2K191me² antibody (F) were performed on indicated regions. (G) KDM4C is required for serum-induced growth control gene expression. HeLa cells transfected with control or validated KDM4C siRNAs were serum-starved followed by restimulation and relative mRNA levels of indicated genes were determined by qRT-PCR. Mean±SEM, *p<0.05.

See also Figure S2.