Automethylation of the mouse DNA(cytosine C5 ... - Wiley Online Library

3 downloads 32152 Views 476KB Size Report
Dec 16, 2010 - Biochemistry Laboratory, School of Engineering and Science, Jacobs ... of auto-methylation is increased by addition of Dnmt3L, which functions.
Auto-methylation of the mouse DNA-(cytosine C5)methyltransferase Dnmt3a at its active site cysteine residue Abu Nasar Siddique, Renata Z. Jurkowska, Tomasz P. Jurkowski and Albert Jeltsch Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Germany

Keywords auto-methylation; DNA methyltransferase; enzyme mechanism; enzyme regulation; protein methylation Correspondence T. P. Jurkowski or A. Jeltsch, Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany Fax: +49 421 200 3249 Tel: +49 421 200 3247 E-mail: [email protected] or [email protected] (Received 16 December 2010, revised 28 March 2011, accepted 6 April 2011) doi:10.1111/j.1742-4658.2011.08121.x

The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methylation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. Enzymes Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. Structured digital abstract Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) l Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction) l

Introduction Methylation of biomolecules including proteins, DNA, RNA and small molecules plays important and diverse roles in biology [1]. For these reactions, S-adenosyl-lmethionine (AdoMet) is by far the most commonly used methyl group donor. It contains the methyl group bound to a positively charged sulfonium center; consequently the methyl group is highly activated towards a nucleophilic attack and AdoMet is a highly reactive compound with high methylation capacity. Overall, following ATP, AdoMet is the second most commonly used coenzyme in nature [2] and it has been estimated

that about 3% of all enzymes listed in the EC nomenclature represent AdoMet-dependent methyltransferases [3]. Methylation substrates range in size from small compounds like catechol to biopolymers like proteins, RNA and DNA; the target atoms for methylation can be carbon, oxygen, nitrogen, sulfur or even halides [4]. DNA methylation is common to almost all living organisms. In bacteria, three kinds of methylated bases are present, 5-methylcytosine, 4-methylcytosine and 6-methyladenine, whereas only 5-methylcytosine is found in higher eukaryotes [5]. In mammals, DNA

Abbreviations AdoHcy, S-adenosyl-L-homocysteine; AdoMet, S-adenosyl-L-methionine; DNA methyltransferase, MTase.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

2055

Auto-methylation of the Dnmt3a methyltransferase

A. N. Siddique et al.

methylation is restricted mostly to CpG dinucleotides. The cell and tissue specific DNA methylation pattern is set early during embryonic development by the action of the Dnmt3a and Dnmt3b de novo DNA methyltransferases (MTases). Once established, the methylation pattern is further maintained during each DNA replication and cell division by the maintenance MTase Dnmt1 [6,7]. DNA methylation contributes to major biological processes, like epigenetic regulation of gene expression, genomic imprinting, X-chromosome inactivation, protection against selfish genomic elements and maintenance of genomic stability [6,7]. The Dnmt3a MTase comprises a large N-terminal regulatory domain and a C-terminal catalytic domain, which is active in an isolated form [8]. The catalytic domain shares a well conserved structure with all DNA MTases, called ‘AdoMet-dependent MTase fold’, which consists of a mixed seven-stranded b-sheet, formed by six parallel and the seventh anti-parallel b strands, inserted between the fifth and sixth b strands. This central b-sheet is sandwiched between six a-helices [9,10]. Because the target base is buried in the DNA helix and not readily accessible for catalysis, DNA MTases flip out the target base and insert it in a hydrophobic pocket in their active center [11]. The catalytic mechanism used by Dnmt3a is characteristic for the DNA-(cytosine C5)-MTases and it is mainly involved in activation of the substrate by increasing its nucleophilicity [5]. For that purpose, DNA-(cytosine C5)-MTases use a catalytic cysteine residue to perform a nucleophilic attack on the sixth position of the cytosine, which leads to the formation of a covalent bond between the enzyme and the substrate base. The formation of the cysteine–cytosine bond increases the negative charge density at the C5 atom of the cytosine, which then attacks the methyl group bound to AdoMet. Base flipping and the nucleophilic attack of the cysteine are facilitated by a contact of a conserved glutamate residue to the exocyclic amino group and the N3 ring nitrogen atom. In addition, a conserved arginine residue plays a role in base flipping and catalysis. Exchange of any of these residues leads to a reduction or complete loss of the catalytic activity of Dnmt3a [12,13].

Results Detection of auto-methylation of Dnmt3a-C The methylation of lysine and arginine residues of histones is an important post-translation modification involved in regulation of gene expression and chromatin biology [6,14,15]. However, recently the regulatory 2056

function of lysine methylation of non-histone proteins has moved into the focus of research [16,17]. To look into the possible regulation of Dnmt3a-C by lysine methylation, we investigated the potential lysine methylation of the Dnmt3a-C enzyme by several mammalian protein lysine methyltransferases. To this end, purified Dnmt3a-C was incubated with different protein lysine methyltransferases in the presence of S-[methyl-3H]-adenosyl-l-methionine (AdoMet) with radioactively labeled methyl group in order to detect the transfer of radioactivity to Dnmt3a-C. Afterwards samples were analyzed by SDS ⁄ PAGE electrophoresis and autoradiography. However, after incubation of Dnmt3a-C with radioactively labeled AdoMet for longer periods of time, we detected the transfer of radioactivity to the Dnmt3a-C protein even without addition of a protein methyltransferase (Fig. 1). This modification was resistant to heat (95 C for 5 min in the presence of 2% SDS and 5 mm dithiothreitol); therefore, it seemed to be of covalent nature and it most probably resulted from an intrinsic auto-methylation activity of the enzyme. A similar observation was also made with full-length Dnmt3a2 (Fig. 1C), which is the predominant isoform of Dnmt3a in embryonic stem cells and embryonal carcinoma cells [18]. Since we suspected this covalent labeling of Dnmt3a-C would inhibit the enzyme and it could have a regulatory role, we decided to study the phenomenon in more detail. Literature searches uncovered similar observations already made for some bacterial MTases, including the DNA-(cytosine C5)-MTases M.BspRI [19,20] and Dcm [21] and the DNA-(adenine N6)MTase M.EcoPI [22]. For M.BspRI, it was suggested that the methyl group can be directly transferred from the AdoMet to a cysteine residue of the protein, leading to the formation of a chemically stable S-methylcysteine and resulting in inactivation of the enzyme [19]. Kinetics and irreversible nature of the auto-methylation To follow the time course of auto-methylation, we have incubated Dnmt3a-C with radioactively labeled AdoMet and removed aliquots from the reaction mixture at different time points. Reactions were stopped by the addition of SDS to a final concentration of 2 mm, followed by heat denaturation of the protein and SDS ⁄ PAGE electrophoresis. The extent of radioactivity bound to the protein was visualized by autoradiography and quantified by densitometry. As shown in Fig. 1A and B, the radioactive signal is increasing slowly over the course of hours with a roughly linear increase for the first 4 h of the reaction. Fitting of the

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

A. N. Siddique et al.

Auto-methylation of the Dnmt3a methyltransferase

A

B

Time

3.0E + 06 kDa

0‘

5‘

15‘

30‘

1h

2h

4h

14 h

170 130 100 70

Rel. intensity (a.u.)

2.5E + 06

55 40 35 25 15

Autoradiography

2.0E + 06 1.5E + 06 1.0E + 06 5.0E + 05

40

0.0E + 00

35

0

5

10

15

Time (h)

25

Coomassie

C

Lane 1

Lane 2

Lane 3

Lane 1

kDa

kDa

170 130 100 70

170 130 100 70

55

55

40

40

35

35

25

Lane 2

Lane 3

25

Autoradiography

Coomassie

Fig. 1. Auto-methylation of the Dnmt3-C DNA methyltransferase. (A) Dnmt3-C protein was incubated with radioactively labeled AdoMet in the standard reaction buffer for the indicated time periods. Reactions were stopped and samples split into equal parts and both run on 15% SDS ⁄ PAGE. The first gel was fixed, sensitized, dried, and then exposed to X-ray film for 5 days (labeled Autoradiography). The second gel was stained with colloidal Coomassie to serve as loading control (labeled Coomassie). Dnmt3a-C migrates in the gel with an apparent mass of  37 kDa. (B) Quantification of the autoradiography signal coming from the Dnmt3a-C protein band. The exposed and developed X-ray films were scanned and the strength of the radioactivity signal was estimated using densitometry; the intensity values (a.u.) were plotted as a function of time and fitted to a single exponential rate equation. (C) Auto-methylation of full-length Dnmt3a. The methylation was performed for 14 h. Nine, 6 or 3 lg of the protein were loaded on polyacrylamide gels (lanes 1–3) and subjected to autoradiography or Coomassie staining.

reaction progress curve to a single exponential rate equation gave an estimate of 0.1 h)1 for the automethylation rate constant, indicating that auto-methylation is a slow process in contrast to the non-covalent AdoMet binding or exchange which happens within minutes [12]. Furthermore, non-covalently bound AdoMet will not co-migrate with Dnmt3a-C in denaturing gel electrophoresis. Hence, we conclude that indeed an auto-methylation of Dnmt3a-C occurs. To confirm the irreversible nature of the labeling of Dnmt3a-C, reactions were quenched with unlabeled AdoMet and AdoMet analogs. An initial auto-methylation reaction was performed for 1 h, allowing the formation of some auto-methylated Dnmt3a-C, and then a large excess of unlabeled AdoMet or S-adenosyl-lhomocysteine (AdoHcy) was added to the reaction and samples were taken at 1 h intervals and analyzed by autoradiography. As expected, the addition of either unlabeled AdoMet or AdoHcy inhibited the further

incorporation of radioactivity into the Dnmt3a-C protein (Fig. 2A). However, already incorporated radioactivity remained, indicating that the modification is stable and irreversible under in vitro conditions. Auto-methylation occurs at the catalytic cysteine Taking into account the catalytic mechanism of Dnmt3a-C, it seemed very likely that the catalytic cysteine residue was the methyl group acceptor because it lies in close proximity to the methyl group of AdoMet and is the most reactive residue in the catalytic center of the enzyme. To test whether the catalytic cysteine is the target for auto-methylation, we purified the alanine exchange mutant C120A of Dnmt3a-C [13] and incubated it with radioactively labeled AdoMet. As expected, the C120A mutant Dnmt3a did not get labeled (Fig. 2B), strongly suggesting that the active site cysteine is the target of modification.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

2057

Auto-methylation of the Dnmt3a methyltransferase

A

A. N. Siddique et al.

Dnmt3a-C + + AdoMet

Dnmt3a-C + 3H-AdoMet kDa

0h

1h

2h

3h

5h

Dnmt3a-C + + AdoHcy

3H-AdoMet

o/n

2h

3h

5h

3H-AdoMet

o/n

2h

3h

5h

o/n

170 130 100 70 55 40

+ AdoMet

+ AdoHcy

35 25

15

Autoradiography

B

Dnmt3a-C C120A + 3H-AdoMet

kDa MW 0 h

2h

4h

o/n

Dnmt3a-C + 3H-AdoMet 0h

2h

4h

Dnmt3a-C C120A + 3H-AdoMet o/n

kDa 0 h

170 130 100 70 55 40

170 130 100 70 55 40

35

35

25

25

15

15

2h

4h

o/n

Dnmt3a-C + 3H-AdoMet 0h

2h

4h

o/n

Autoradiography

Coomassie

C

Dnmta-C wt

5000 13.96

2934.347

4000 3000

2948.344

1000 0

Dnmta-C wt + AdoMet

2500

13.96

2000

2948.303

2934.347

Intensity (a.u.)

2000

1500 1000 500 0

2920

2925 2930 2935 2940 2945 2950 2955 2960

2965

m/z

Fig. 2. The Dnmt3a auto-methylation reaction is irreversible, dependent on AdoMet and occurs at the catalytic cysteine residue. (A) An automethylation reaction was incubated for 1 h to allow for creation of auto-methylated species. Then the reaction was quenched by addition of 1000-fold molar excess of either unlabeled AdoMet or AdoHcy. For reference the auto-methylation reaction was continued without addition of a quencher. Aliquots from the reactions were taken at 2 h, 3 h, 5 h and 12–14 h after the addition of quencher and run on a 15% SDS ⁄ PAGE; the amount of incorporated 3H-methyl groups was checked by autoradiography. (B) Purified C120A and wild-type Dnmt3a-C were incubated with 3H-AdoMet for 0 h, 2 h, 4 h and 12–14 h in the reaction buffer. Two aliquots from the reaction were taken at each time point and run separately on two 15% SDS ⁄ PAGE gels, from which one was stained with colloidal Coomassie G-250 and served as loading control and the other was used for autoradiography. (C) Mass spectroscopic analysis of auto-methylation of Dnmt3a-C with and without incubation with unlabeled AdoMet (1 mM). The tryptic fragment containing the active site Cys120 has a mass of 2934.3 Da (theoretical mass 2934.4 Da). After incubation with AdoMet an additional peak appears at 2948.3 Da corresponding to 2934.3 Da plus the mass of a methyl group (14 Da).

2058

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

Auto-methylation of the Dnmt3a methyltransferase

A

D3a-C

A. N. Siddique et al.

kDa

156

B

SET7/9 + histone H3.1 2.6

6.5

13

26

52

pmol of protein

170 130 100 70 55 40

SET7/9

35

Dnmt3a-C

25

Histone H3.1

15 10

Autoradiography Fig. 3. Quantification of the extent of auto-methylation. (A) Comparison of the radioactivity signal from auto-methylated Dnmt3a-C (labeled D3a-C) after overnight (14 h) incubation with 3H-AdoMet in the standard reaction conditions with signal from different amounts of SET7 ⁄ 9 methylated histone H3.1 (NEB). The total protein amounts loaded on the gel are indicated. (B) The band intensities from the autoradiography picture were extracted by densitometry and background normalized.

To confirm that auto-methylation occurs at Cys120, Dnmt3a-C was incubated with unlabeled AdoMet and subjected to tryptic digestion and MALDI-TOF mass spectrometric analysis. As shown in Fig. 2C, the peak corresponding to the unmethylated peptide containing Cys120 as well as the peak corresponding to the methylated peptide were clearly detectable. This tryptic fragment did not contain another amino acid residue that could function as nucelophile (Fig. S1). The methylated peak was not detected with a control sample that was not pre-incubated with AdoMet (Fig. 2C). As an additional control, the C120A variant was incubated with AdoMet and subjected to mass spectrometric analysis. In this experiment the peptide containing the C120A mutation was detectable, but neither a peak corresponding to the methylated C120A peptide nor a peak corresponding to the methylated C120 peptide was observed (Fig. S2). Our identification of the active site cysteine residue as the target for auto-methylation parallels literature findings with other enzymes. In the case of M.BspRI, Szilak and colleagues have identified two cysteine residues which were the targets for auto-methylation: one of them (C156) is the catalytic cysteine in M.BspRI; the other one (C181) is not conserved among DNA(cytosine-C5)-MTases [19]. In the case of Dcm only the catalytic cysteine residue was found to get modified [21]. Extent of auto-methylation In order to estimate the fraction of Dnmt3a-C which gets self-methylated, we compared the radioactivity signal generated by Dnmt3a-C after 16 h incubation under our standard reaction conditions with the signal

coming from histone H3.1 monomethylated at lysine 4 by recombinant SET7 ⁄ 9 histone lysine MTase [23–25]. As shown in Fig. 3, the autoradiography signal of the Dnmt3a-C protein is faint in comparison with the signal of histone H3. Taking into consideration the relative strength of the autoradiography signals and the total protein amounts of Dnmt3a-C and H3.1 loaded on the gel, we estimated that about 2.6% of the Dnmt3a-C got modified during this 16 h incubation. It is interesting that the extent of auto-methylation observed in the mass spectrometric analysis (Fig. 2C) was much higher than the extent of methylation observed in Fig. 3. Although this observation needs to be interpreted carefully, since mass spectroscopy is not a fully quantitative method, the higher methylation may be related to the fact that the concentration of AdoMet was much higher in the mass spectrometric experiment (1 mm in the assay) than in the methylation with radioactively labeled AdoMet (0.76 lm in the assay). Effect of DNA and Dnmt3L on auto-methylation of Dnmt3a It is known that Dnmt3L, an activator of Dnmt3a and Dnmt3b, stimulates the DNA methylation reaction catalyzed by these enzymes [26]. As shown in Fig. 4, larger amounts of radioactivity were transferred to the Dnmt3a-C protein after adding Dnmt3L-C to the auto-methylation reaction compared with the reaction mixture with Dnmt3a alone. This result can be explained because Dnmt3L stabilizes the conformation of the active site loop of Dnmt3a-C and it increases AdoMet binding [9,26], which in turn will lead to increased formation of self-methylated Dnmt3a-C.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

2059

Auto-methylation of the Dnmt3a methyltransferase

Dnmt3a-C kDa 2 h 170 130 100 70 55 40

6h

12 h

35

Dnmt3a-C + Dnmt3L

Dnmt3a-C + CG DNA

2h

2h

6h

12 h

6h

A. N. Siddique et al.

12 h

Dnmt3a-C

25

15

DNA

10

7.66 Å

Autoradiography Fig. 4. Effect of Dnmt3L and DNA on the auto-methylation reaction. Comparison of efficiencies of auto-methylation reactions carried out in the presence of 7 lM Dnmt3L C-terminal domain or 20 lM CG containing dsDNA with the standard auto-methylation reaction. Whereas Dnmt3L increases the level of auto-methylation of Dnmt3a-C, the methylation of the dsDNA substrate is efficiently competing with the Dnmt3a-C auto-methylation. Note that oligonucleotides are not fully denatured in SDS gels and run as a mixture of single-stranded and double-stranded form. In addition, binding of Dnmt3a-C to the DNA causes the appearance of an additional retarded oligonucleotide band.

To test the effect of DNA on the auto-methylation reaction of Dnmt3a-C, we added a 20 bp doublestranded DNA containing a single CG target site and followed the incorporation of radioactivity from AdoMet into DNA and Dnmt3a-C. As can be seen in Fig. 4, addition of a double-stranded DNA substrate abolished the tritium incorporation into the MTase but at the same time the DNA got efficiently methylated. This result illustrates that after binding both substrates (DNA and AdoMet) the enzyme has a high specificity for the transfer of the methyl group to the DNA and it efficiently avoids auto-methylation.

Discussion We show here that the mammalian Dnmt3a enzyme undergoes auto-methylation in vitro at its catalytic cysteine by transferring the methyl group from its natural cofactor AdoMet to the cysteine residue. Analogous reactions were already observed for the bacterial DNA-(adenine N6)-MTase M.EcoPI [22] and the two DNA-(cytosine C5)-MTases Dcm [21] and M.BsuRI [19,20], but not for a mammalian DNA MTase. This observation highlights one interesting detail in the catalytic mechanism of DNA-(cytosine C5)-MTases. On one hand, these enzymes employ AdoMet as the donor for methyl groups, which is a highly activated coenzyme with very large methyl group transfer potential. 2060

Fig. 5. Positioning of AdoHcy and the catalytic cysteine in the active center of Dnmt3a-C, in the crystal structure of Dnmt3a ⁄ Dnmt3L complex with AdoHcy [9] (PDB: 2QRV). AdoHcy is shown in orange, Dnmt3a colored by atom type. The distance between the sulfhydryl atom of the catalytic cysteine side chain and sulfur atom of AdoHcy (7.66 A˚) is indicated.

On the other hand, they harbor a cysteine residue in their active centers that is activated towards performing a nucleophilic attack. This cysteine residue could easily react with AdoMet because the DG for the transfer of the methyl group from AdoMet to cysteine is of the order of )70 kJÆmol)1 [2,4]. Therefore, it is essential for the enzyme that a close approximation of these two groups is avoided to prevent auto-methylation and inactivation of the enzyme. Indeed, in the Dnmt3a-C structure with AdoHcy, the sulfhydryl and AdoHcy sulfur atoms are separated by 7.66 A˚, which would correspond to a distance of about 6 A˚ between the sulfhydryl sulfur and the methyl group of AdoMet, if AdoMet were to replace AdoHcy without conformational change (Fig. 5). This suggests that a conformational change of about 3 A˚ has to occur before auto-methylation can happen, which may explain why the process of auto-methylation is slow. We show here that the conformation of Dnmt3a prevents auto-methylation efficiently but not entirely, similar to what has been observed with the bacterial enzymes mentioned above. However, if DNA is bound, no auto-methylation is happening, suggesting that the reaction occurs in binary Dnmt3a-C AdoMet complexes but in ternary

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

A. N. Siddique et al.

complexes the transfer of the methyl group to the flipped target base is much preferred. It is unclear whether this slow auto-methylation of Dnmt3a could have a biological function in cells. Since the methylcysteine is chemically stable, auto-methylation would inactivate the enzyme by causing steric constraints and interference with the reaction mechanism of DNA-(cytosine C5)-MTases. It is possible that, in cells, Dnmt3a which is in an idle state may lose its activity via auto-methylation, thereby protecting the genome against aberrant methylation. In this respect it is important to note that we observed much higher levels of auto-methylation after incubation of the Dnmt3a-C at higher concentration of AdoMet. The intracellular concentrations of AdoMet have been reported in the range of 50–250 lm [27–30], which is much higher than the AdoMet concentration used here in the radioactive methylation (0.76 lm) and may allow for more efficient auto-methylation in the cell. There also might be additional factors in the cells, which could stimulate Dnmt3a for auto-methylation and therefore inactivation. On the other hand, the auto-methylation of Dnmt3a and other MTases may simply be a side reaction caused by the high methyl group transfer potential of AdoMet.

Experimental procedures The His6-tagged full-length Dnmt3a2 and the catalytic domain of mouse Dnmt3a wild-type and catalytically inactive C120A mutant (corresponding to C706A in full-length Dnmt3a) and the His6-tagged fusion of the C-terminal part of human DNMT3L were expressed and purified as described previously [26,31]. The human SET7 ⁄ 9 protein lysine methyltransferase which among other sites monomethylates histone H3 at Lys4 [24,25] was purified as described earlier [32]. Auto-methylation reaction mixtures contained 2–5 lm Dnmt3a-C protein, 0.76 lm [methyl-3H]-AdoMet (PerkinElmer) in methylation buffer (20 mm Hepes pH 7.2, 1 mm EDTA, 50 mm KCl) in a 20 lL reaction volume. Reactions were incubated at room temperature for various time intervals and stopped by addition of 20 lL of Laemmli sample buffer (130 mm Tris ⁄ HCl pH 6.8, 20% glycerol, 4% SDS, 10 mm dithiothreitol, 0.02% bromophenol blue) and heating to 95 C for 5 min. Afterwards, the samples were analyzed on a 15% SDS ⁄ PAGE gel and either stained with colloidal Coomassie (33) or fixed with 10% methanol ⁄ 10% acetic acid, immersed in Amplify solution (GE Healthcare, Freiburg, Germany) for 1 h at room temperature with shaking, dried on a 3 mm Whatman paper and exposed to an X-ray film for 3–7 days. Signal intensities were analyzed by densitometry (AIDA v4; raytest GmbH, Straubenhardt, Germany). Auto-methylation reactions with unlabeled Ado-

Auto-methylation of the Dnmt3a methyltransferase

Met were carried out in the same buffer but in the presence of 1 mm AdoMet (Sigma-Aldrich) for 16 h. Unlabeled AdoMet was dissolved in 10 mm sulfuric acid, stored in aliquots at )20 C and used only once after thawing. Quenching reactions were prepared essentially like automethylation reactions; however, the auto-methylation reactions were incubated for 1 h allowing the formation of some initial auto-methylated species and then quenched by the addition of either 1 mm non-radioactive AdoMet (Sigma-Aldrich), 1 mm AdoHcy (Sigma-Aldrich) or 20 lm of double-stranded CG-DNA (GAA GCT GGG ACT TCC GGA GGA GAG TGC AA). The samples were collected at various time points and analyzed as described above. To study the effect of Dnmt3L on the auto-methylation reaction of Dnmt3a-C, auto-methylation reactions were supplemented with 7 lm recombinant Dnmt3L and analyzed as described above. To calibrate the extent of auto-methylation of Dnmt3a, human recombinant histone H3.1 (NEB) was methylated with 1.22 lm recombinant SET7 ⁄ 9. H3 methylation was performed in methylation buffer for SET7 ⁄ 9 (50 mm Tris ⁄ HCl pH 9.0, 5 mm MgCl2, 4 mm dithiothreitol) using 0.76 lm 3H-AdoMet. The reaction was incubated for 12 h to run to completion and different amounts of the methylated H3 methylation reaction mixtures were separated on a 15% SDS ⁄ PAGE gel together with the Dnmt3a-C after overnight incubation with labeled AdoMet. The amount of radioactivity incorporated in the protein bands was determined from scanned autoradiography pictures by densitometry.

Acknowledgement This work was supported by DFG (JE 252-6) and DAAD.

References 1 Cheng X & Roberts RJ (2001) AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res 29, 3784–3795. 2 Cantoni GL (1975) Biological methylation: selected aspects. Annu Rev Biochem 44, 435–451. 3 Kagan RM & Clarke S (1994) Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch Biochem Biophys 310, 417–427. 4 Schubert HL, Blumenthal RM & Cheng X (2003) Many paths to methyltransfer: a chronicle of convergence. Trends Biochem Sci 28, 329–335. 5 Jeltsch A (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem 3, 274–293.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

2061

Auto-methylation of the Dnmt3a methyltransferase

A. N. Siddique et al.

6 Bonasio R, Tu S & Reinberg D (2010) Molecular signals of epigenetic states. Science 330, 612–616. 7 Jurkowska RZ, Jurkowski TP & Jeltsch A (2011) Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206–222. 8 Gowher H & Jeltsch A (2002) Molecular enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA methyltransferases. J Biol Chem 277, 20409– 20414. 9 Jia D, Jurkowska RZ, Zhang X, Jeltsch A & Cheng X (2007) Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248–251. 10 Cheng X & Blumenthal RM (2008) Mammalian DNA methyltransferases: a structural perspective. Structure 16, 341–350. 11 Roberts RJ & Cheng X (1998) Base flipping. Annu Rev Biochem 67, 181–198. 12 Gowher H, Loutchanwoot P, Vorobjeva O, Handa V, Jurkowska RZ, Jurkowski TP & Jeltsch A (2006) Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. J Mol Biol 357, 928–941. 13 Reither S, Li F, Gowher H & Jeltsch A (2003) Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme. J Mol Biol 329, 675–684. 14 Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447, 407–412. 15 Goldberg AD, Allis CD & Bernstein E (2007) Epigenetics: a landscape takes shape. Cell 128, 635–638. 16 Huang J & Berger SL (2008) The emerging field of dynamic lysine methylation of non-histone proteins. Curr Opin Genet Dev 18, 152–158. 17 Rathert P, Dhayalan A, Ma H & Jeltsch A (2008) Specificity of protein lysine methyltransferases and methods for detection of lysine methylation of nonhistone proteins. Mol Biosyst 4, 1186–1190. 18 Chen T, Ueda Y, Xie S & Li E (2002) A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem 277, 38746– 38754. 19 Szilak L, Finta C, Patthy A, Venetianer P & Kiss A (1994) Self-methylation of BspRI DNA-methyltransferase. Nucleic Acids Res 22, 2876–2881. 20 Szilak L, Finta C, Patthy A, Venetianer P & Kiss A (1995) Self-methylation of the M.BspRI methyltransferase. Gene 157, 105. 21 Hanck T, Schmidt S & Fritz HJ (1993) Sequencespecific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to

2062

22

23

24

25

26

27

28

29

30

31

32

synthetic oligonucleotides containing 5-fluoro-2¢-deoxycytidine. Nucleic Acids Res 21, 303–309. Hornby DP, Muller M & Bickle TA (1987) High level expression of the EcoP1 modification methylase gene and characterisation of the gene product. Gene 54, 239– 245. Wang H, Cao R, Xia L, Erdjument-Bromage H, Borchers C, Tempst P & Zhang Y (2001) Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Mol Cell 8, 1207–1217. Xiao B, Jing C, Wilson JR, Walker PA, Vasisht N, Kelly G, Howell S, Taylor IA, Blackburn GM & Gamblin SJ (2003) Structure and catalytic mechanism of the human histone methyltransferase SET7 ⁄ 9. Nature 421, 652–656. Wilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackburn GM, Gamblin SJ & Xiao B (2002) Crystal structure and functional analysis of the histone methyltransferase SET7 ⁄ 9. Cell 111, 105–115. Gowher H, Liebert K, Hermann A, Xu G & Jeltsch A (2005) Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L. J Biol Chem 280, 13341– 13348. Chiang EP, Wang YC, Chen WW & Tang FY (2009) Effects of insulin and glucose on cellular metabolic fluxes in homocysteine transsulfuration, remethylation, S-adenosylmethionine synthesis, and global deoxyribonucleic acid methylation. J Clin Endocrinol Metab 94, 1017–1025. Hermes M, Osswald H, Riehle R, Piesch C & Kloor D (2008) S-Adenosylhomocysteine hydrolase overexpression in HEK-293 cells: effect on intracellular adenosine levels, cell viability, and DNA methylation. Cell Physiol Biochem 22, 223–236. Castro R, Rivera I, Martins C, Struys EA, Jansen EE, Clode N, Graca LM, Blom HJ, Jakobs C & de Almeida IT (2005) Intracellular S-adenosylhomocysteine increased levels are associated with DNA hypomethylation in HUVEC. J Mol Med 83, 831–836. Hoffman DR, Marion DW, Cornatzer WE & Duerre JA (1980) S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of l-methionine, l-homocystein, and adenosine. J Biol Chem 255, 10822–10827. Jurkowska RZ, Anspach N, Urbanke C, Jia D, Reinhardt R, Nellen W, Cheng X & Jeltsch A (2008) Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L. Nucleic Acids Res 36, 6656–6663. Dhayalan A, Kudithipudi S, Rathert P & Jeltsch A (2011) Specificity analysis based identification of new methylation targets of the SET7 ⁄ 9 protein lysine methyltransferase. Chem Biol 18, 111–120.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

A. N. Siddique et al.

33 Dyballa N & Metzger S (2009) Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp 30, 1431.

Supporting information The following supplementary material is available: Fig. S1. Sequence of the tryptic peptide containing Cys120 and its position in the Dnmt3a structure. Fig. S2. Absence of auto-methylation with the Dnmt3a-C C120A variant.

Auto-methylation of the Dnmt3a methyltransferase

This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS

2063