Figure 4.

SIRT1 interacts with and deacetylates EZH2 (A and B) EZH2 acetylation is increased in the presence of nicotinamide. H1299 cells were co-transfected with FLAG-EZH2 and FLAG-PCAF and then treated with 3 μM TSA or 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab and immunoprecipitates were immunoblotted with the AcK Ab (A). H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab, then immunoblotted with the AcK Ab (B). (C and D) EZH2 interacts with SIRT1 in vivo. HEK-293T cells were co-transfected with Myc-EZH2, Myc-EZH2 plus FLAG-SIRT1 or Myc-EZH2 plus FLAG-SIRT2. Forty-eight hours post transfection, lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by a western blot with an anti-Myc Ab (C). HEK-293T cells were co-transfected with Myc-EZH2 and FLAG-SIRT1. The lysates were co-immunoprecipitated with an anti-Myc Ab, followed by western blot with an anti-FLAG Ab (D). (E and F) EZH2 is deacetylated by SIRT1. HEK-293T cells were transfected with FLAG-EZH2, FLAG-PCAF and FLAG-SIRT1 as indicated. Lysates were co-immunoprecipitated with an anti-FLAG Ab and detected by the AcK Ab (E). The in vitro deacetylation reaction mixtures were subjected to SDS-PAGE and immunoblotted with the anti-AcK348-EZH2 Ab. The purified GST fusion proteins were detected by Coomassie blue staining (F).