Fig. 1.

Acetylation of DNMT1 in vivo and in vitro. (A) 293T cells were transfected with HA-DNMT1 and either Flag-PCAF, HA-p300, or empty Flag vector. Cell lysates were immunoprecipitated (IP) with antibody to HA, and immunoprecipitates were immunoblotted (IB) with antibody to HA, Flag, or acetyllysine (AcK). Ac, acetylated. Direct immunoblot assays were performed with anti-Flag and anti-HA to assess expressions of Flag-PCAF and HA-p300, respectively. (B) His-DNMT1 was expressed in insect cells using the baculovirus system and purified on nickel affinity columns. Flag-PCAF was expressed in 293T cells and immunopurified from cell lysates with an anti-Flag antibody. In vitro acetylation reaction mixtures contained His-DNMT1, Flag-PCAF, and acetyl-CoA as indicated. Reaction products were detected with an antiacetyllysine antibody. Anti-DNMT1 Western blots and Ponceau S staining show equal amounts of His-DNMT1 and Flag-PCAF in reaction mixtures. (C) 293T cells expressing HA-DNMT1 were treated with 15 mM nicotinamide plus 100 ng/ml TSA overnight. HA-DNMT1 was immunoprecipitated from cell lysates with antibody to HA, and immunoprecipitated material was analyzed by mass spectrometry. Acetylated lysines identified in the analysis, plus previously reported acetylation sites (Lys111, Lys1113, Lys1115, and Lys1117), are indicated in bold type. The domains of DNMT1 are shown and are drawn in approximate scale. DMAP1, DNA methyltransferase 1-associated protein 1-binding domain; PCNA, proliferating cell nuclear antigen-binding domain; N, nuclear localization signal; replication foci, replication focus-targeting domain; KEN, KEN box (KENXXXR sequence); Zn, zinc finger region; BAH1 and BAH2, bromo-adjacent homology domains; KG, lysine-glycine repeats, also called GK linker.