Lysine methylation and serine phosphorylation on DNMT1. (a) The lysines targeted for methylation by SET7 in various known substrates are shown in red. The serine in the consensus AKT1 kinase sequence is aligned with histone H3 Ser10 and DNMT1 Ser143. (b) SET7 methylation reaction in the presence of various concentrations of DNMT1 peptides representing either native or phosphorylated Ser143. (c) MS of the products of in vitro methylation assays (0 min, before assay; 40 min, after assay) on DNMT1 peptides with either native (left) or phosphorylated Ser143 (right). The mass shift of 14 Da represents addition of one methyl group. (d) AKT1 kinase reaction in the presence of fixed concentrations of wild-type DNMT1 peptides or S143A mutant peptides. (e) Western blot assay showing AKT1 kinase activity on dephosphorylated recombinant full-length DNMT1 on Ser143. (f) Western blot showing Ser143 phosphorylation of full-length DNMT1 in the presence (+) or absence (−) of constitutively active AKT1 kinase. Antibodies used for probe are listed; actin is shown as a loading control. MDM2 pSer166 is a positive control. (g) S143A mutation in full-length DNMT1 facilitates SET7-mediated Lys142 methylation. Western blot demonstrating effect of Ser143 phosphorylation and/or Lys142 methylation in either wild-type (WT) DsRed-DNMT1 or the mutant DsRed-DNMT1(S143A), as indicated by plus and minus signs, when coexpressed with GFP alone (lanes 1 and 2) or GFP-SET7 (lanes 3 and 4).

DNMT1 association with AKT1 kinase. (a) Colocalization of DNMT1 and AKT1 in COS-7 cells, as shown by transiently expressed DsRed-DNMT1 (red) and endogenous AKT1 kinase (green; stained with fluorescent anti-AKT1). Nuclei are Hoechst-stained (blue). Cells were released from G1/S arrest and followed through S and G2 phase for the time indicated at left. Percentages of cells showing similar pattern of colocalization are shown in parentheses. (b) Immunoprecipitation (IP) of AKT1, DNMT1 or GFP (control) from HEK293 nuclear extracts followed by western blot with indicated antibodies. (c) Coimmunoprecipitation of DNMT1–AKT1 complex at different time points after cell synchronization. (d) Expression different post-translationally modified (or total) DNMT1 species during the cell cycle. Cells were released from G1/S arrest and followed for the time shown at top. Extracts were western blotted and probed with the antibodies indicated. Anti-DNMT1 shows total enzyme. Cyclin A was used as the cell cycle marker. A representative western blot for actin (which was used as a loading control) is shown. (e) Quantification of cell cycle–dependent expression of different DNMT1 species, normalized to actin levels. phDNMT1, DNMT1-pSer143; meDNMT1, DNMT1-Lys142me1. Data shown represent two independent western blots as in d. Error bars show s.d.

Structure of the SET7–DNMT1 complex. (a) The reaction occurred during crystallization. Omit electron densities, F_o − F_c contoured at 4σ above the mean, are shown for the monomethylated Lys142 (black mesh) and the methyl group (green mesh). (b) The substrate peptide and the reaction product AdoHcy occupy binding sites in the opposite ends of a narrow target-lysine channel. (c) Electrostatic interactions, hydrogen bonds and van der Waals interactions define SET7–DNMT1 peptide interactions (dashed lines). The network of interactions includes the following: (i) DNMT1 Arg139 forms a hydrogen bond with the main chain carbonyl oxygen atom of SET7 Gly336 (not shown) and an intramolecular interaction with the main chain carbonyl oxygen of DNMT1 Ser141; (ii) DNMT1 Arg140 forms an electrostatic salt bridge to SET7 Glu348 and a hydrogen bond with the main chain carbonyl oxygen atom of SET7 Arg258; (iii) DNMT1 Ser141 forms a serineserine interaction with SET7 Ser268 and a water-mediated network with DNMT1 His252 (not shown) and SET7 Asp256; (iv) the target nitrogen atom of DNMT1 Lys142me1 forms two hydrogen bonds with the side chain hydroxyl oxygen atoms of SET7 Tyr305 and Tyr245; and (v) DNMT1 Ser143 is involved in a polar interaction with SET7 Lys317 and a van der Waals contact with SET7 Leu267. In addition, two main chain atoms of DNMT1, the carbonyl oxygen of Arg140 and the amide nitrogen of Lys142me1, link through SET7 Thr266.

AKT1 kinase–mediated phosphorylation stabilizes DNMT1. (a) Western blotting and RNA expression analysis of extracts from HeLa cells with AKT1 knockdown (si-AKT1) or control knockdown (si-Control). Left, western blot with indicated antibodies. Marker, biotinylated protein ladder showing relative molecular mass of DNMT1 as ~200 kDa. Right, RNA expression measured from quantitative PCR; error bars represent s.d. of three independent experiments. DNMT1-pSer143 is reduced in AKT1-knockdown cells, resulting in moderately higher DNMT1-Lys142me1 levels but no change in DNMT1 RNA levels. (b) Overexpression of the AKT1 dominant-negative mutant (AKT1^DN) reduces DNMT1-pSer143 and increases DNMT1-Lys142me1 levels, as shown by western blotting with the indicated antibodies. (c) The AKT kinase inhibitor LY294002 reduces DNMT1-pSer143 levels and increases DNMT1-Lys142me1 levels in a time-dependent manner, as shown by western blotting with the indicated antibodies. (d) Effect of SET7 on DNMT1-Lys142me1 in the absence (−) or presence (+) of calyculin A, a serine/threonine phosphatase inhibitor, as shown by western blotting with indicated antibodies. Cells were co-transfected with constructs expressing GFP alone with DsRed-DNMT1 (lanes 1 and 2) or GFP-SET7 with DsRed-DNMT1 (lanes 3 and 4). (e) Degradation of DNMT1 modified and unmodified species after cells were treated with cycloheximide (CHX). At indicated times, cells were lysed and western blotted with indicated antibodies. (f) Ubiquitinylation of total DNMT1 and DNMT1-Lys142me1. Wild-type DNMT1 or the DNMT1(S143A) mutant was overexpressed in cells, along with HA-ubiquitin and GFP-SET7, in the presence (+) or absence (−) of the proteasome inhibitor MG132. Total DNMT1 or DNMT1-Lys142me1 was immunoprecipitated (IP) from the nuclear extract and western blotted with antibody to HA. (g) A proposed model of cross-talk between DNMT1 (black), SET7 and AKT1 in mammalian cells. The other interactors that can reverse the post-translational modifications are an unknown phosphatase and an unknown lysine-specific demethylase (KDMT). One such KDMT may be LSD1 (ref. 17).