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.. To this
Trang 1methyltransferase 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
Introduction
Methylation of biomolecules including proteins, DNA,
RNA and small molecules plays important and diverse
roles in biology [1] For these reactions,
S-adenosyl-l-methionine (AdoMet) is by far the most commonly
used methyl group donor It contains the methyl group
bound to a positively charged sulfonium center;
conse-quently 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 nomen-clature 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
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: t.jurkowski@jacobs-university.de or
a.jeltsch@jacobs-university.de
(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-methyl-ation 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
l Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction)
l Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction)
Abbreviations
AdoHcy, S-adenosyl- L -homocysteine; AdoMet, S-adenosyl- L -methionine; DNA methyltransferase, MTase.
Trang 2methylation 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
ele-ments 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
cyto-sine, which leads to the formation of a covalent bond
between the enzyme and the substrate base The
for-mation of the cysteine–cytosine bond increases the
neg-ative charge density at the C5 atom of the cytosine,
which then attacks the methyl group bound to
Ado-Met Base flipping and the nucleophilic attack of the
cysteine are facilitated by a contact of a conserved
glu-tamate residue to the exocyclic amino group and the
N3 ring nitrogen atom In addition, a conserved
argi-nine 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
chroma-tin biology [6,14,15] However, recently the regulatory
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 meth-ylation of the Dnmt3a-C enzyme by several mamma-lian 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-methyla-tion activity of the enzyme A similar observaauto-methyla-tion 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, lead-ing to the formation of a chemically stable S-methylcy-steine 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 mix-ture 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 radio-activity bound to the protein was visualized by autora-diography 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
Trang 3reaction progress curve to a single exponential rate
equation gave an estimate of 0.1 h)1 for the
auto-methylation rate constant, indicating that
auto-methyl-ation is a slow process in contrast to the non-covalent
AdoMet binding or exchange which happens within
minutes [12] Furthermore, non-covalently bound
Ado-Met 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-methyl-ation reaction was performed for 1 h, allowing the
for-mation of some auto-methylated Dnmt3a-C, and then
a large excess of unlabeled AdoMet or
S-adenosyl-l-homocysteine (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 pro-tein (Fig 2A) However, already incorporated radioac-tivity 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 cys-teine 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 incu-bated 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
Coomassie
kDa 0‘ 5‘ 15‘ 30‘ 1 h 2 h 4 h 14 h
Autoradiography
B A
15
25
35
40
55
70
100
170
25
35
40
Time
C
kDa
25 35 40 55 70 100
170
kDa
25 35 40 55 70 100 170
Autoradiography Coomassie
0
3.0E + 06
2.5E + 06
2.0E + 06
1.5E + 06
1.0E + 06
5.0E + 05
0.0E + 00
5
Time (h)
Lane 1 Lane 2 Lane 3 Lane 1 Lane 2 Lane 3
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.
Trang 425 35 40 55 70 100 170
Dnmt3a-C +
3 H-AdoMet
Dnmt3a-C +
3 H-AdoMet + AdoMet
Dnmt3a-C +
3 H-AdoMet + AdoHcy
0 h
Autoradiography Coomassie
Dnmt3a-C C120A +
3 H-AdoMet
Dnmt3a-C +
3 H-AdoMet
15
25
35
40
55
100
170
Dnmt3a-C C120A +
3 H-AdoMet
Dnmt3a-C +
3 H-AdoMet
kDa
kDa
15 25 35 40 55 100 170 Autoradiography
B
A
kDa
Dnmta-C wt + AdoMet
5000
4000
3000
2000
1000
0
2500
2000
1500
1000
500
0
m/z
13.96
13.96
2920 2925
2930 2935 2940 2945 2950 2955 2960 2965
Fig 2 The Dnmt3a methylation reaction is irreversible, dependent on AdoMet and occurs at the catalytic cysteine residue (A) An auto-methylation 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 with3H-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 sep-arately 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 m M ) 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).
Trang 5To 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
sam-ple that was not pre-incubated with AdoMet (Fig 2C)
As an additional control, the C120A variant was
incu-bated with AdoMet and subjected to mass
spectromet-ric 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
resi-dues 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
sig-nal 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 sig-nal of histone H3 Taking into consideration the rela-tive 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 methyla-tion 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
SET7/9
Histone H3.1
Dnmt3a-C
10 25 35 40 55 70 100 170
156
SET7/9 + histone H3.1
kDa
B A
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.
Trang 6To test the effect of DNA on the auto-methylation
reaction of Dnmt3a-C, we added a 20 bp
double-stranded DNA containing a single CG target site and
followed the incorporation of radioactivity from
Ado-Met 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
methylat-ed This result illustrates that after binding both
sub-strates (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
cys-teine 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
cat-alytic mechanism of DNA-(cytosine C5)-MTases On
one hand, these enzymes employ AdoMet as the donor
for methyl groups, which is a highly activated
coen-zyme with very large methyl group transfer potential
On the other hand, they harbor a cysteine residue in their active centers that is activated towards perform-ing 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-methyla-tion and inactivaauto-methyla-tion 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 conforma-tional change (Fig 5) This suggests that a confor-mational 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-meth-ylation efficiently but not entirely, similar to what has been observed with the bacterial enzymes mentioned above However, if DNA is bound, no auto-methyla-tion is happening, suggesting that the reacauto-methyla-tion occurs
in binary Dnmt3a-C AdoMet complexes but in ternary
10
15
25
35
40
70
100
170
Dnmt3a-C Dnmt3a-C +
Dnmt3L
Dnmt3a-C +
CG DNA
Dnmt3a-C
DNA
kDa
Autoradiography
Fig 4 Effect of Dnmt3L and DNA on the auto-methylation
reac-tion Comparison of efficiencies of auto-methylation reactions
car-ried out in the presence of 7 l M Dnmt3L C-terminal domain or
20 l M 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
oligonu-cleotides 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.
7.66 Å
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.
Trang 7complexes 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-methyla-tion would inactivate the enzyme by causing steric
con-straints 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
lev-els 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
inac-tive 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
monome-thylates 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
(PerkinEl-mer) 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
inter-vals 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
heat-ing to 95C for 5 min Afterwards, the samples were
ana-lyzed 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-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 auto-methylation reactions; however, the auto-auto-methylation reac-tions 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 col-lected at various time points and analyzed as described above To study the effect of Dnmt3L on the auto-methyla-tion reacauto-methyla-tion of Dnmt3a-C, auto-methylaauto-methyla-tion reacauto-methyla-tions 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 methy-lated 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-adenosylmethio-nine-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 meth-yltransferases Chembiochem 3, 274–293
Trang 86 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
methyl-transferases 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)
Muta-tional 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
chroma-tin regulation during transcription Nature 447,
407–412
15 Goldberg AD, Allis CD & Bernstein E (2007)
Epigenet-ics: 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
non-histone 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
local-izes 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-methyltransfer-ase Nucleic Acids Res 22, 2876–2881
20 Szilak L, Finta C, Patthy A, Venetianer P & Kiss A
(1995) Self-methylation of the M.BspRI
methyltransfer-ase Gene 157, 105
21 Hanck T, Schmidt S & Fritz HJ (1993)
Sequence-specific and mechanism-based crosslinking of Dcm
DNA cytosine-C5 methyltransferase of E coli K-12 to
synthetic oligonucleotides containing 5-fluoro-2¢-deoxy-cytidine Nucleic Acids Res 21, 303–309
22 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
23 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
24 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
25 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 methyl-transferase SET7⁄ 9 Cell 111, 105–115
26 Gowher H, Liebert K, Hermann A, Xu G & Jeltsch A (2005) Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-meth-yltransferases by Dnmt3L J Biol Chem 280, 13341– 13348
27 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 deoxyribo-nucleic acid methylation J Clin Endocrinol Metab 94, 1017–1025
28 Hermes M, Osswald H, Riehle R, Piesch C & Kloor D (2008) S-Adenosylhomocysteine hydrolase overexpres-sion in HEK-293 cells: effect on intracellular adenosine levels, cell viability, and DNA methylation Cell Physiol Biochem 22, 223–236
29 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 hypomethyla-tion in HUVEC J Mol Med 83, 831–836
30 Hoffman DR, Marion DW, Cornatzer WE & Duerre
JA (1980) S-Adenosylmethionine and S-adenosylhomo-cystein metabolism in isolated rat liver Effects of
l-methionine, l-homocystein, and adenosine J Biol Chem 255, 10822–10827
31 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
32 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
Trang 933 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
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