Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA Mehrnaz Fatemi*, Andrea Hermann*, Humaira Gowher and Albert Jeltsch Institut fu ¨ r Biochemie, Justus-Liebig-Universita ¨ t, Gießen, Germany Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high pre- ference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is obser- ved if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methy- lation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increa- sed stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo. Keywords: DNA methylation; enzyme mechanism; DNA methyltransferase; Dnmt1; Dnmt3a. Cytosine residues are methylated at the 5-position for 70–80% of all CG sequences in mammalian DNA. The pattern of DNA methylation serves as an epigenetic mark in general leading to a repression of gene expression [reviews 1,2,3]. It is used to memorize developmental decisions of the cell and to control monoallelic expression of genes during imprinting [review 4] and X-chromosome inactivation [5]. Work with knockout mice has shown that DNA methylation is an absolutely essential process in mammals during late embryogenesis [6,7]. The methylation pattern is created by de novo methylation and demethyla- tion of the DNA, and maintained during mitosis by maintenance methylation [reviews 8,9]. De novo methyla- tion of DNA is most prevalent during embryogenesis, where the methylation is restored after an almost complete demethylation of the genome that takes place during the first cleavage divisions [review 10]. In addition, de novo methylation can also occur later in development and even in adult cells to silence acquired proviral DNA or to alter the developmental program of the cell. Aberrant de novo methylation may lead to hypermethylation of promotor regions of tumor suppressor genes in cancer cells and is an important mechanism for cancer progression [11–13]. The mechanism of de novo methylation of DNA is still poorly understood and requires further investigation as this process (together with specific demethylation events) cre- ates the pattern of DNA and therefore transfers the epigenetic information to the DNA. DNA methylation is introduced by DNA methyltrans- ferases (MTases) which use S-adenosylmethionine as donor for an activated methyl group [reviews 3,14,15,16]. Four candidate DNA MTases have been identified in mammals so far: Dnmt1, Dnmt2, Dnmt3a and Dnmt3b. Results obtained with Dnmt1 knock-out mice have implicated this enzyme in maintenance methylation [6], a role that is in agreement to its pronounced preference for methylation of hemimethylated DNA in vitro [17,18]. However, Dnmt1 also shows capabilities for de novo methylation of DNA [15]. Interestingly, the de novo activity of Dnmt1 is stimulated by binding of methylated DNA to an allosteric site located in the N-terminal domain of the enzyme [18,19]. However, de novo methylation activity is also associated to the Dnmt3a and Dnmt3b enzymes, which do not show a preference for methylation of hemimeth- ylated CG sites [20–23]. It has been shown that one target for the Dnmt3b enzyme are satellite sequences [7,24,25], whereas specific targets for the Dnmt3a enzyme are not yet known. In biochemical studies, it has been shown that Dnmt3a methylates DNA in a distributive mechanism [21]. This was a surprising observation, because it makes the enzyme badly adapted for a fast methylation of one domain of the DNA. However, the intriguing possibility appeared that Dnmt3a and Dnmt1 might functionally cooperate during de novo methylation of DNA. This model assumes that Dnmt3a might initiate de novo methylation by transferring methyl groups to one region of DNA. This would recruit and stimulate Dnmt1, which could then methylate the whole domain of the DNA [3,21]. Correspondence to A. Jeltsch, Institut fu ¨ r Biochemie, FB 8, Justus-Liebig-Universita ¨ t, Heinrich-Buff-Ring, 58 35392 Gießen, Germany. Fax: + 49 641 99 35409, Tel.: + 49 641 99 35410, E-mail: Albert.Jeltsch@chemie.bio.uni-giessen.de Abbreviations: Mtases, methyltransferases. Note: *These authors contributed equally to the work. (Received 21 June 2002, revised 8 August 2002, accepted 21 August 2002) Eur. J. Biochem. 269, 4981–4984 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03198.x EXPERIMENTAL PROCEDURES Dnmt1 and Dnmt3a were purified as described [18,21]. Methylation analyses were carried out with a 958mer and a 310mer PCR fragment which contain 95 and 27 CG sites, respectively. Both substrates were amplified by PCR from pAT153 plasmid. The 310mer had been selected to contain three HpaII sites at one end. In either case, one of the PCR primers carries a biotin group on its 5¢ end, such that the PCR product is also biotinylated. Substrates were purified using PCR Purification Kits (Qiagen). Concentrations were determined from the absorbance at 260 nm. Methylation kinetics were performed as described [26] using 3.7 l M [methyl- 3 H]-S-adenosylmethionine (5.55 · 10 14 BqÆmol )1 , Moravek Biochemicals, Brea, CA) in methylation buffer (20 m M Hepes pH 7.5, 50 m M KCl, 50 lgÆmL )1 BSA). Premethylation of the 310mer PCR fragment (6 pmol) was performed with 12 U M.HpaII (NEB) for 2 h at 37 °C. The DNA was then precipitated and the concentration deter- mined. To ensure complete methylation of all HpaII sites in this experiment an aliquot of the purified methylated DNA was subjected to a second round of methylation with M.HpaII using radiolabeled AdoMet. No radioactivity was incorporated into the DNA demonstrating that all HpaII sites are fully methylated. RESULTS AND DISCUSSION It was the aim of this work to experimentally investigate a model for de novo methylation of DNA that postulates functional cooperation of Dnmt1 and Dnmt3a. Purified Dnmt1 and Dnmt3a enzymes were employed to methylate a 958mer PCR product using labeled [methyl- 3 H]-S-adeno- sylmethionine. We performed a series of three methylation reactions, one with Dnmt1, the second with Dnmt3a and the third with Dnmt1 and Dnmt3a. As shown in Fig. 1, we observed a much higher activity in the presence of both enzymes, that is 4.7 ± 0.1-fold higher than the rate corresponding to the sum of the rates observed with Dnmt1 and Dnmt3a alone. We conclude that a significant stimu- lation of de novo methylation occurs if both enzymes are present at the same time. Experiments with different amounts of Dnmt1 and Dnmt3a showed that the level of stimulation increases with increasing amounts of Dnmt3a and slightly decreases with increasing amounts of Dnmt1 (data not shown). This result is reasonable, because methylation by Dnmt3a is the initial event for de novo methylation and due to its own de novo activity Dnmt1 might be able to stimulate itself in an autocatalytic fashion, iftoomuchofthisenzymeisused. We next examined if the stimulation requires a physical interaction of Dnmt1 and Dnmt3a, or if both enzymes only interact via the methyl groups transferred to the DNA. To this end, we have compared the methylation activities observed when Dnmt3a and Dnmt1 are present simulta- neously in the reaction mixture with the activities obtained when equal amounts of the two enzymes are added one after the other (first Dnmt3a then Dnmt1) with intervening ethanol precipitation of the DNA. In the first experiment a physical interaction of both enzymes is possible, whereas this is not possible in the second one. For control purposes, we have also carried out reactions in which incubation of the DNA with Dnmt1 was followed by incubation with Dnmt3a or in which the DNA was incubated twice with Dnmt3a. As shown in Fig. 2 incubation of Dnmt3a before Dnmt1 but not Dnmt1 before Dnmt3a results in stimulation of the DNA methylation activity. This finding supports the Fig. 1. Stimulation of DNA methylation by cooperation of Dnmt3a and Dnmt1. A 958mer PCR product (20 n M ) was methylated using labeled [methyl- 3 H]-AdoMet by Dnmt1 (80 n M )(j), Dnmt3a (400 n M )(d) and both enzymes present at the same time (r). All experiments were carried out in triplicate. We observed a 4.7 ± 0.1-fold higher rate of DNA methylation in the presence of Dnmt3a and Dnmt1 than the sum of the rates observed with either enzyme alone. Fig. 2. Stimulation of DNA methylation observed with different mixing protocols of Dnmt1 and Dnmt3a. In the experiment labeled D3a/D3a, methylation of 958mer (20 n M ) was carried out with Dnmt3a (400 n M ) for 30 min. Then, the DNA was precipitated with ethanol and a sec- ond methylation reaction was carried out with Dnmt3a (400 n M ). In the experiment labeled D1/D3a the first methylation reaction was carried out with Dnmt1 (80 n M ) and the second reaction with Dnmt3a (400 n M ). In the experiment labeled D3a/D1 the first methylation reaction was performed with Dnmt3a (400 n M ) and the second reac- tion with Dnmt1 (80 n M ). In the experiment labeled D1 + D3a, the DNA was incubated with Dnmt1 (40 n M ) and Dnmt3a (200 n M )inthe first reaction. As in the other experiments, the DNA was precipitated. The second methylation reaction was performed with the same amounts of Dnmt1 and Dnmt3a. All experiments were carried out in triplicate. The figure shows average values of DNA methylation, the error bars indicate the standard deviation of the individual data. 4982 M. Fatemi et al.(Eur. J. Biochem. 269) Ó FEBS 2002 model that Dnmt3a transfers some methyl groups to the DNA, which in turn stimulates Dnmt1 for de novo methy- lation of this region. In addition, it clearly demonstrates that a physical interaction of both enzymes is not required for stimulation to occur. We repeatedly observed a slightly higher activity if both enzymes are present at the same time. However, this effect is only small and close to the error margins of our experiment. This difference could be due to a direct interaction of Dnmt1 and Dnmt3a, which has recently been reported to occur [27]. It is known that Dnmt3a creates hemimethylated target sites during DNA methylation [28]. As the activity of Dnmt1 on hemimethylated DNA is much higher [18], generation of one hemimethylated site will immediately lead to a second methylation in the unmodified strand of this site in the presence of Dnmt1. Kinetically this reaction scheme means that two methyl groups are always introduced, one slowly by Dnmt3a or by de novo methylation catalyzed by Dnmt1 and the second one very fast. Therefore, this effect can only double the rate of DNA methylation, because every turnover by Dnmt3a can trigger only one turnover by Dnmt1 in the opposite strand of the DNA. Consequently, this effect cannot account for the fivefold stimulation observed in our experiments and additional stimulation of Dnmt1 by pre-existing methylation must occur. To show this effect we used a 310mer PCR fragment that contains three fully methylated CG sites produced by methylation with M.HpaII. As shown in Fig. 3 the premethylated DNA is modified significantly faster by Dnmt1 confirming similar results by other groups [29,30]. Furthermore, it is in agreement with the allosteric activation of Dnmt1 observed after binding of the enzyme to methylated oligonucleotides [18,19,31,32]. A similar effect is not observed with Dnmt3a showing that at least under these conditions no allosteric activation occurs in Dnmt3a, although this enzyme like Dnmt1 contains at least one additional DNA binding site in its N-terminal part [33]. The observation that Dnmt3a is not activated by pre-exiting methylation agrees with the fact that the rates of methylation of hemimethylated substrates by this enzyme are not higher than rates observed with unmethylated DNA. The results presented in this paper complement recent in vivo data also demonstrating a functional cooperation of Dnmt1 with Dnmt3a and Dnmt3b in maintenance as well as in de novo methylation of DNA [34]. In this work it was shown using Dnmt1, Dnmt3a and Dnmt3b knock-out cell lines that Dnmt1 alone is not able to maintain methylation levels at certain repeat sequences, but that the presence of Dnmt3a and or Dnmt3b in addition to Dnmt1 is required for accurate maintenance methylation in these regions. On the other hand, Dnmt3a and Dnmt3b are not capable of efficient de novo methylation of DNA in the absence of Dnmt1. Whereas the maintenance methylation is not an issue of our work, we present a mechanistic basis for the functional cooperation of Dnmt3a and Dnmt1 in de novo methylation of DNA that can only be provided by in vitro experiments. In this model Dnmt3a is targeted to a domain of the DNA that is subject to de novo methylation. As this enzyme does not work processively, it can introduce only a few methyl groups to the DNA. These attract Dnmt1 to the DNA, and activate it for de novo methylation. Then, Dnmt1 spreads the methylation over the whole domain of the DNA. Following this mechanism only a few targeting events are required to achieve sufficient methylation of one DNA domain whereas without the cooperation with Dnmt1, Dnmt3awouldhavetorebindtotheDNAaftereach turnover to achieve complete methylation of one domain of the DNA. It should be noticed that the molecular mechan- ism of these targeting events is still unknown, although the interaction of Dnmt3a with some other proteins has been shown that might be the mechanistic basis for the process [35]. Finally, it is possible that maintenance methylation at sequences that are not efficiently methylated by Dnmt1 alone also follows this mechanism. Our data contribute to the view that DNA methylation is a complicated process, in which different enzymes are involved: We show here that Dnmt1 and Dnmt3a can cooperate in de novo methylation in vitro, a similar phenomenon has also been observed in vivo [34]. These authors also have shown that the Dnmt3a and Dnmt3b enzymes (in addition to Dnmt1) are required for mainten- ance of the methylation at certain DNA sequences. A role of Fig. 3. Stimulation of Dnmt1 and Dnmt3a by fully methylated CpG sites. In this experiment the rates of methylation of the 310mer substrate were compared after premethylation of the substrate on 3 HpaII sites (CCGG) located at the 5¢ end of the molecule (j) and without premethylation (d). In the left panel 40 n M DNA were methylated using 120 n M Dnmt1, in the right panel using 400 n M Dnmt3a. In repeated experiments a 2.9 ± 0.2- fold stimulation was observed with Dnmt1, whereas no significant difference was observed with Dnmt3a. Ó FEBS 2002 Cooperation of Dnmt1 and Dnmt3a (Eur. J. Biochem. 269) 4983 Dnmt3a and Dnmt3b in maintenance methylation also has been concluded from studies with cell lines which do not express functional Dnmt1 [36] and, finally, a functional interdependence of the Dnmt3a and Dnmt3b enzymes has been demonstrated in mouse knock out studies [20]. 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We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because. late embryogenesis [6,7]. The methylation pattern is created by de novo methylation and demethyla- tion of the DNA, and maintained during mitosis by maintenance