From the Laboratory of Cellular Biochemistry,
Department of Animal Resource Sciences/Veterinary Medical
Sciences, University of Tokyo, Tokyo 113-8657, Japan
Received for publication, September 27, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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During mammalian cell division, DNA methylation
patterns are transferred accurately to the newly synthesized DNA
strand. This depends on maintenance DNA methyltransferase activity. DNA
methylation can affect chromatin organization and gene expression by
recruitment of histone deacetylases (HDACs). Here we show that the
methyl-CpG binding protein, MeCP2, interacts directly with the
maintenance DNA methyltransferase, Dnmt1. The region of MeCP2 that
interacts with Dnmt1 corresponds to the transcription repressor
domain which can also recruit HDACs via a corepressor, mSin3A.
Dnmt1 can form complexes with HDACs as well as MeCP2. Surprisingly, the
MeCP2-Dnmt1 complex does not contain the histone deacetylase, HDAC1.
Thus, Dnmt1 takes the place of the mSin3A-HDAC1 complex, indicating that the MeCP2-interacting Dnmt1 does not bind to HDAC1. Further, we
demonstrate that MeCP2 can form a complex with hemimethylated as well
as fully methylated DNA. Immunoprecipitated MeCP2 complexes show DNA
methyltransferase activity to hemimethylated DNA. These results suggest
that Dnmt1 associates with MeCP2 in order to perform maintenance
methylation in vivo. We propose that genome-wide and/or -specific local DNA methylation may be maintained by the Dnmt1-MeCP2 complexes, bound to hemimethylated DNA. Dnmt1 may be recruited to
targeted regions via multiple steps that may or may not involve histone deacetylases.
DNA methylation occurs at the fifth carbon position of cytosine in
CpG dinucleotide sequences. In mammalian cells, 60-80% of CpG
dinucleotides are methylated (1). These modifications are considered to
be important for development (2, 3), genomic imprinting (4), and X
chromosome inactivation through gene silencing (5, 6). Aberrant DNA
methylation has been observed in cancer cells (7). The occurrence of
DNA methylation is closely connected with chromatin organization by
both histone acetylation and methylation, which are catalyzed by
histone acetyltransferases (HDACs)1 and
methyltransferases, respectively (8). A maintenance DNA methyltransferase, Dnmt1, preferentially methylates hemimethylated DNA
after DNA replication (9, 10). Therefore, the patterns of DNA
methylation in the genome are maintained by the maintenance DNA
methyltransferase activity of Dnmt1, allowing inheritance of
appropriate DNA methylation patterns. Targeted disruption of Dnmt1
causes partial loss of genomic methylation that leads to lethality (2).
The carboxyl-terminal region of Dnmt1 transfers methyl groups from
S-adenosyl-L-methionine to cytosines in CpG dinucleotides. It is thought that an N-terminal region of Dnmt1 regulates Dnmt1 function by protein-protein interactions with factors
such as PCNA (11), DMAP1 (12), HDAC1/2 (12, 13), and Rb (14) (15),
which are associated with DNA replication, histone deacetylation, and
gene expression during the cell cycle.
Methyl-CpG binding proteins (MBDs) bind DNA, including methylated CpG
dinucleotides. MBDs are involved in chromatin organization and gene
silencing via recruitment of HDACs (16). Individual MBDs have specific
methylated DNA binding properties and interact with transcriptional
repressors and chromatin remodeling factors (5). MeCP2, the first
cloned Methyl-CpG binding protein, binds to single CpG dinucleotides
that are symmetrically methylated (17), whereas other MBDs have little
or no binding activity to single symmetrically methylated CpG
dinucleotides (18). Thus, MeCP2 possesses higher affinity for all or
most of the methylated CpG sites than other known MBDs. MeCP2 represses
gene expression by recruiting mSin3A, which interacts with HDAC1 (19,
20). The tissue distribution of MeCP2 is ubiquitous, and its expression is relatively abundant (21, 22). Mutations of the MeCP2 gene cause loss
of body weight and a neurological disorder in the mouse, which is
consistent with MECP2 mutations in Rett syndrome (23, 24). This
indicates that MeCP2 is not essential for development but participates
in epigenetic control of neuronal function.
Dnmt1 does not contain a methyl-CpG binding domain common to the MBD
family (18), raising the question of how this enzyme is recruited to
hemimethylated DNA and how it replicates the methylation pattern. We
tested the hypothesis that the pattern of DNA methylation is replicated
through recruitment of Dnmt1 by MeCP2, which binds to the methylated
cytosine of the template DNA. Here we show that Dnmt1 interacts with
MeCP2 directly, and this interaction is mainly dependent upon the
transcription repressor domain (TRD) of MeCP2. The Dnmt1-MeCP2 complex
does not include HDAC1 but has maintenance methyltransferase activity.
Moreover, MeCP2 can preferentially associate with both hemimethylated
and fully methylated DNA under a physiological salt concentration
condition. These results suggest that both Dnmt1 and MeCP2 may
contribute to maintenance of DNA methylation during DNA replication by
multiple regulatory machineries including various protein-protein interactions.
Plasmids--
We subcloned rat Dnmt1 cDNA (25) into pCS2+MT (26)
and then cloned Myc-tagged rat Dnmt1 cDNA and rat Dnmt1 cDNA into
pcDNA3 to produce pcDNA3-Myc-Dnmt1. We cloned rat MeCP2 cDNA and
internal deletion constructs ( Cell Culture and Transfections--
We maintained cell lines in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum at 37 °C with 5% CO2. For transfection of
293T, we seeded 4 × 105 cells before the procedure
and transfected them with DNA (21 µg) using a calcium phosphate
co-precipitation method (27). The culture medium was changed 6 h
after transfection, and the cells were incubated for an additional
42 h before harvest.
Immunoprecipitations, Western Blot Analysis, and
Reprobing--
We transiently transfected 293T cells in culture dishes
(60-mm diameter) with appropriate expression vectors including pME18S SR GST Fusion Proteins, Pull-down Assays, and in Vitro
Translation--
We expressed GST and GST fusion proteins in
Escherichia coli XL1-blue using the pGEX vector system and
purified protein from crude bacterial lysates according to the
manufacturer's instructions (Amersham Biosciences). We carried out
in vitro transcription/translation using the TNT system
(Promega). Myc-Dnmt1 was translated in vitro from
pCS2+MT-Dnmt1. We performed pull-down assays by preincubating GST or
GST fusion protein and glutathione-Sepharose beads in binding buffer
(50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Tween 20, 0.5% skim milk) at room temperature. After 10 min, in vitro
translated proteins were added, and the mixture was incubated for
1 h at 4 °C. The beads were then washed four times in the ice-cold binding buffer without 0.5% skim milk. Pelleted beads were
suspended in 2× SDS-PAGE sample buffer. Bound proteins were resolved
by SDS-PAGE and subjected to autoradiography. Alternatively, we
transiently transfected 293T cells in culture dishes (100-mm diameter)
with pcDNA3 (30 µg) or pcDNA3-Myc-Dnmt1 (30 µg), and the
cells were harvested 48 h after the transfection and then lysed.
GST or GST fusion protein was added into the lysate and incubated for
1 h on ice. Subsequently, glutathione-Sepharose beads were added
and incubated with a rotary for 1 h at 4 °C. The beads were
pelleted by centrifugation and washed with ice-cold Nonidet P-40 buffer
three times. Bound proteins were resolved by SDS-PAGE and subjected to
Western blot analysis using anti-Myc antibody (9E10).
DNA Pull-down Assay--
We transiently transfected 293T cells
in culture dishes (90-mm diameter) with pCMV2-FLAG vector or
pCMV2-FLAG-MeCP2 (30 µg) and collected cells 48 h
post-transfection. We lysed cells in Nonidet P-40 buffer for 30 min at
4 °C and removed debris by centrifugation. The cleared lysates
(total protein amount 1 mg) were then subjected to each DNA binding
reaction for 1 h at 4 °C using 6 µg (30 µmol) of
double-stranded oligonucleotides corresponding to E-cadherin CpG
nucleotide sequences (28) (Sigma Genosys Japan KK). These were either
nonmethylated, hemimethylated (on either the sense strand or on the
antisense strand), or fully methylated, and all were biotinylated at
the 5'-ends. The sense strand has the sequence 5'-CCAGCCCGGCCCGACCCGACCGCACCCGGCGC-3'
(portion of methylated cytosines are underlined).
Methylated oligonucleotides were generated at specific portions by
using methylated precursors. Streptavidin-magnetic beads (Roche
Molecular Biochemicals) were added to collect the biotinylated
oligonucleotides with binding proteins, and incubation was continued
with a rotary for 1 h at 4 °C. The beads were washed three
times with Nonidet P-40 buffer (1 ml) and were subjected to Western
blot analysis using anti-FLAG antibody (M2).
Methyltransferase Assay--
We transfected 293T cells with
either Myc-Dnmt1 and FLAG-MeCP2 expression vectors, empty vector, and
FLAG-MeCP2 expression vectors, empty vector alone, or FLAG-Dnmt1
expression vector. Cells were lysed using Nonidet P-40 buffer as
described above. After immunoprecipitation using anti-FLAG antibody,
beads were washed three times with Nonidet P-40 buffer, and then the
precipitates were assayed for methyltransferase activity in a 100-µl
reaction solution containing a 32-bp hemimethylated oligonucleotide
substrate (500 ng (25 pmol)), which is the same as described above
(28), S-adenosyl-L-[methyl-3H]methionine
(2 µl; 77 Ci/mmol; Amersham Biosciences), 50 mM Tris-Cl, pH 7.5, 5 mM EDTA, 50% glycerol, 5 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
After incubation for 1 h at 37 °C or 4 °C, we removed
unincorporated nuclides with Sephadex G-50 spin column (Amersham
Biosciences) and determined incorporation of radioactivity in ASCII
solvent (Amersham Biosciences) by liquid scintillation counting
(13).
The majority of MeCP2 is concentrated on heterochromatin in the
genome (29). Lysis under high salt concentration conditions results in
extraction of all MeCP2 in the cell (17). However, a high salt
extraction also disrupts weaker affinity interactions. The extraction
conditions used here take advantage of excluding MeCP2 associated with
tightly condensed chromatin such as heterochromatin and of selecting
MeCP2 associated with relatively loose chromatin such as during DNA
replication. In previous reports, an interaction of Dnmt1 with PCNA
(11), DMAP1 (12), HDACs (19), and Rb (15) has been shown under the same
physiological salt condition. To test an interaction of Dnmt1 with
MeCP2, we co-transfected 293T cells with plasmid encoding Myc-tagged,
full-length rat Dnmt1 (Myc-Dnmt1) and FLAG-tagged, full-length rat
MeCP2 (FLAG-MeCP2). Then we lysed the cells using a lysis buffer with a
typical physiological salt concentration and carried out
immunoprecipitation with the anti-FLAG antibody, followed by Western
blot analysis using the anti-Myc antibody. Dnmt1 and MeCP2 interacted
in vivo (Fig. 1, A
and B, lanes 4 and 8), and
Dnmt1 was not immunoprecipitated in the absence of MeCP2 (Fig. 1,
A and B, lanes 3 and
7). Endogenous MeCP2 was detectable under the same salt
condition in mouse primary cells using an anti-MeCP2 antibody (data not
shown). We then determined whether MeCP2 can bind directly to Dnmt1
in vitro by means of a GST pull-down assay using seven kinds
of bacterially expressed and purified GST fusion MeCP2 proteins; GST
1-76, 77-161 (MBD), 162-206 (CRID), 207-310 (TRD), 311-403,
404-491, and wild type (residues 1-491) (Fig. 1C). Three
separate domains of MeCP2, as well as full-length MeCP2 protein, were
able to bind to in vitro translated full-length Dnmt1 (Fig.
1D, lanes 4-6 and 9).
These associations were specific because Dnmt1 failed to bind to GST alone (Fig. 1D, lane 2). To confirm
that the GST-fused individual domains of MeCP2 and full-length MeCP2
can bind to Dnmt1 prepared from mammalian cells, we transfected 293T
cells with an empty vector or Myc-Dnmt1 expression vector. We lysed the
cells and carried out GST pull-down assays using the lysate, followed
by Western blot analysis using the anti-Myc antibody. Three domains (MBD, CRID, and TRD) of MeCP2 and full-length MeCP2 bound full-length Dnmt1 (Fig. 1E). These associations were specific because
GST alone failed to bind to Dnmt1 (Fig. 1E, lane
4). Dnmt1 was not detectable from the lysate transfected
with empty vector (Fig. 1E, odd-numbered lanes).
These results indicate that Dnmt1 binds directly to MeCP2 in
vitro (Fig. 1, D and E). Moreover, three different functional domains of MeCP2 could bind to Dnmt1 (Fig. 1,
D (lanes 4-6) and E
(lanes 8, 10, and 12)).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MBD,
CRID, and
TRD) into
pCMV2-FLAG (Sigma). Individual domains of rat MeCP2 were cloned into
pGEX 4T-3 (Amersham Biosciences) by PCR using appropriate sets of
primers. We cloned mouse HDAC1 cDNA into pHM (Roche Molecular
Biochemicals) using PCR from pCI-HDAC1-myc (gift from Dr. C. Seiser) to
produce pHM-HA-mouse HDAC1. We verified all constructs by DNA sequencing.
-Rb (gift from Dr. T. Yamamoto) and harvested cells 48 h
after transfection. The cells were pelleted by centrifugation at 5,000 rpm at 4 °C. Nonidet P-40 buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM NaF, 1 mM
Na3VO4, 1% aprotinin, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride) was
added to the cell pellets, and the cells were lysed by pipetting and
then placed for 30 min on ice. Cleared whole cell extracts were
prepared by centrifugation at 15,000 rpm for 30 min at 4 °C. The
cleared extracts were then subjected to immunoprecipitation for 1 h on ice. Immunoprecipitation and Western blot analysis were performed
using anti-Myc (9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
anti-FLAG (M2; Sigma), anti-Rb (C-15; Santa Cruz Biotechnology), or
anti-HA antibody (12CA5; Roche Molecular Biochemicals). Protein
G-Sepharose beads (Amersham Biosciences) were added, and the mixture
was rotated for 1 h at 4 °C. Beads were washed three times with
Nonidet P-40 buffer (1 ml). The precipitates or cleared extracts were
then subjected to Western blot analysis. Blots were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1%
Tween 20 and 5% skim milk. We incubated the membrane at room
temperature for 1 h with appropriate antibodies described above in
TBS containing 0.1% Tween 20 and 0.5% skim milk. The blots were then
incubated for 1 h at room temperature with secondary antibody and
washed in TBS containing 0.1% Tween 20. Immunoreactive bands were
revealed using an ECL Kit (PerkinElmer Life Sciences) according to the
manufacturer's instructions. For reprobing, blotted membrane was
treated with stripping buffer (62.5 mM Tris-Cl, pH 6.8, 2%
SDS, 100 mM 2-mercaptoethanol) for 30 min at 50 °C. The
membrane was washed three times with TBS containing 0.1% Tween 20 for
30 min and then blocked for 1 h at room temperature with TBS
containing 0.1% Tween 20 and 5% skim milk. The membrane was subjected
to antibody reactions as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Dnmt1 directly interacts with MeCP2 in
vivo. A and B, Western blots
illustrating co-immunoprecipitation of full-length rat Dnmt1 with
full-length rat MeCP2. 293T cells were co-transfected with 21 µg of
pcDNA3-Myc-Dnmt1, pCMV2-FLAG-MeCP2, and empty vector as indicated.
Cell extracts were then precipitated with anti-FLAG antibody, and the
presence of Myc-Dnmt1 in the whole cell extracts (WCE) and
the immunoprecipitates (IP) was analyzed by Western blot
analysis (WB) using anti-Myc antibody. Molecular weight
(MW) is indicated on the left. A, blot
probed with anti-Myc antibody; Myc-Dnmt1 is indicated by an
arrow. B, blot probed with anti-FLAG antibody;
FLAG-MeCP2 is indicated by arrows. C, schematic
representation of functional domains in MeCP2, and GST fusion
constructs used to determine which domain binds to Dnmt1 in the GST
pull-down assays. Construct names are shown to the left
(residues 1-491 (full-length), 1-76, 77-161, 162-206, 207-310,
311-403, and 404-491). A summary of the binding activities is
shown to the right (19). His-Pro, histidine- and
proline-rich region. The black box indicates
nuclear localization sequence (NLS). D,
autoradiogram of in vitro translated full-length rat Dnmt1
pulled down by GST or GST fusion proteins that include different
regions of MeCP2 (see C). The bound in vitro
translated Myc-Dnmt1 is indicated by an arrow on the
left. Lane 1, 35S-labeled
in vitro translated Dnmt1 input (10%). E,
Western blot analysis of Myc-Dnmt1 pulled down from lysate of 293T
cells transfected with pcDNA3 (V) or
pcDNA3-Myc-Dnmt1 (F) by GST or GST fusion proteins that
include different regions of MeCP2 (see C). The bound
Myc-Dnmt1 is indicated by an arrow on the left.
Lanes 1 and 2, whole cell extracts
(WCE) of 293T transfected with pcDNA3 or
pcDNA-Myc-Dnmt1 (1.67%).
Dnmt1 possesses regulatory domains in the N-terminal region (10), which
can form complexes with many proteins (e.g. DMAP1 (12), PCNA
(11), Rb (14), and RFTS (30)). We next examined which domain of Dnmt1
binds to MeCP2 by GST pull-down assay using several Dnmt1 constructs
with serial deletions from the C terminus. A bipartite nuclear
localization sequence has previously been identified in the N-terminal
region (NLS; residues 171-201, rat Dnmt1) (Fig.
2A) (30). All Dnmt1 constructs
could be localized in the nucleus (Fig. 2A). Lysates from
293T cells transfected with the mutant Dnmt1 constructs were provided
to the assay. Most Dnmt1 fragments (residues 1-1,622, 1-1,414,
1-1,203, 1-1,014, 1-807, 1-608, 1-428, and 1-327) bound to
GST-full-length MeCP2, and the three individual domains of MeCP2 (Fig.
2B). The binding activity of fragment 1-327 to MBD was
weaker than others. In contrast, Dnmt1 constructs 1-263 and 1-201
bound incompletely to the MeCP2 protein (Fig. 2B). These
results suggest that residues 264-326 of Dnmt1 are necessary for the
interaction with MeCP2.
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The MBD, CRID, and TRD domains of MeCP2 can bind independently to Dnmt1
(Fig. 2B). To examine which domain plays a central role for
an interaction with Dnmt1 in vivo, we generated a series of
FLAG-tagged internal deletion mutants of MeCP2 (FLAG-MeCP2, -MBD,
-
CRID, and -
TRD), all of which localize in the nucleus (Fig.
3A). We co-transfected 293T
cells with Myc-Dnmt1 and with empty vector, FLAG-MeCP2, FLAG-
MBD,
FLAG-
CRID, or FLAG-
TRD. We lysed the cells and carried out
immunoprecipitation with the anti-FLAG antibody, followed by Western
blot analysis using the anti-Myc antibody. Full-length Dnmt1 and MeCP2
interacted as in Fig. 1A (Fig. 3B,
lanes 1 and 2, column
1). Dnmt1 interacted specifically with
MBD and
CRID
(Fig. 3B, lanes 3 and 4),
but the interaction of Dnmt1 with
TRD decreased strikingly (Fig.
3B, lane 5, column 4). These results indicate that the TRD plays a central role
for an interaction of Dnmt1 with MeCP2 in vivo. Deletion of
MBD reduced the binding activity to Dnmt1 (Fig. 3B,
lane 3, column 2). This may suggest
that MBD also contributes for an interaction of MeCP2 and Dnmt1. A
previous study has reported that Dnmt1 associates with HDAC1 (13).
MeCP2 also interacts with HDAC1 by forming a corepressor complex
including mSin3A (19, 20). In our system, Dnmt1, MeCP2, and Rb all
interacted with HDAC1 (Fig. 3C). To examine whether a
Dnmt1-MeCP2 complex includes HDAC1, we co-transfected 293T cells with
plasmids containing FLAG-MeCP2, Myc-Dnmt1, and HA-HDAC1.
Immunoprecipitates using anti-FLAG antibody from lysates including
FLAG-MeCP2, Myc-Dnmt1, and HA-HDAC1 did not include HA-HDAC1 (Fig.
3D, lane 7), whereas
immunoprecipitates from lysates with FLAG-MeCP2 and HA-HDAC1 included
HA-HDAC1 (Fig. 3D, lane 6). These
results suggest that an interaction of Dnmt1 with MeCP2 is stronger
than that of mSin3A with MeCP2, and the Dnmt1-MeCP2 complex excludes
HDAC1 from both Dnmt1-HDAC1 and MeCP2-mSin3A-HDAC1 components.
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If the interaction of Dnmt1 with MeCP2 is involved in replicating
patterns of methylation, MeCP2 would be predicted to bind to
hemimethylated DNA as well as to fully methylated DNA. We used synthetic biotinylated oligonucleotides corresponding to the in vivo CpG island of the E-cadherin gene (28) to investigate this. We confirmed that hemimethylated double-stranded oligonucleotides do
not form a hairpin loop structure in the methylated strand using DNA
electrophoresis under nondenaturing conditions and by oligonucleotide
secondary structure predictions using Vector NTI software (data not
shown). By means of an in vitro DNA pull-down assay (Fig.
4A), we found that MeCP2 bound
not only to fully methylated oligonucleotides but also to
hemimethylated ones (Fig. 4B). These associations were
specific because MeCP2 failed to bind to the nonmethylated
oligonucleotides of the same sequence (Fig. 4B) (17).
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This interaction led us to consider that the Dnmt1-MeCP2 complex could
be associated with maintenance DNA methyltransferase activity. To test
this idea, we performed transfections with either FLAG-MeCP2 and empty
vector or FLAG-MeCP2 and Myc-Dnmt1 expression vectors. We lysed the
cells, immunoprecipitated FLAG-MeCP2 with anti-FLAG antibody from the
lysates, and then assayed the immunoprecipitates for maintenance
methyltransferase activity. Immune complexes from cells transfected
with FLAG-MeCP2 and Myc-Dnmt1 expression vectors possessed higher
methyltransferase activity than cells transfected with FLAG-MeCP2
expression and empty vectors (Fig. 4C, columns 1 and 2). Immunoprecipitates of Dnmt1 were also
assayed as a positive control and indicated sufficient enzymatic
activity in this system (Fig. 4C, columns
3 and 4). Methyltransferase activity of Dnmt1 to
hemimethylated DNA is ~50-fold higher than de novo
methyltransferase activity (9). Thus, these results indicate that
MeCP2-interacting Dnmt1 has significant maintenance DNA
methyltransferase activity and that MeCP2 does not vanish Dnmt1
enzymatic activity. When this enzymatic reaction was also tested at
4 °C, maintenance DNA methyltransferase activity was equal to that
of cells transfected with an empty vector (data not shown). DNA
pull-down assay and methyltransferase assay were performed using the
same hemimethylated double-stranded oligonucleotides (see
"Experimental Procedures"). These results indicate that
hemimethylated double-stranded oligonucleotides do not become fully
methylated by endogenous MeCP2 under the DNA pull-down condition.
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DISCUSSION |
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Our data indicate a direct interaction between Dnmt1 and MeCP2. This interaction prevents the association of HDAC1 with MeCP2. MeCP2 associates with hemimethylated as well as fully methylated DNA in vitro. The MeCP2-Dnmt1 complex possesses maintenance DNA methyltransferase activity that is dependent upon Dnmt1, demonstrating that the Dnmt1-MeCP2 complex could be involved in maintenance of DNA methylation. Histone deacetylase activity associates with Rb (31), Dnmt1 (13), and MBD proteins such as MeCP2 (19), MBD1 (32), and MBD2 (33). Thus, Rb, Dnmt1, and MBDs complexes have histone deacetylase activity. Our findings suggest that histone deacetylation at least partly mediates various nucleosomal events such as chromatin integrity and gene regulation. We propose that histone deacetylase is not essential for maintenance DNA methyltransferase activity in the Dnmt1-MeCP2 complex.
An N-terminal domain of Dnmt1 interacts with cell cycle-related molecules such as PCNA (11) and Rb (14, 15). The RFTS domain of Dnmt1 supports foci formation in the nucleus (30). The MeCP2 interaction domain of Dnmt1 is near the RFTS, and deletion of the RFTS (amino acids 328-378) reduces, but does not eliminate, interaction with MeCP2 (Fig. 2). These results suggest that interaction of the two may be supported by the RFTS. Subcellular localization of Dnmt1 may be regulated by interplay of the MeCP2 interaction domain and RFTS.
The connection between DNA methylation and histone acetylation will
mainly affect chromatin organization by maintaining a stable epigenetic
state and remodeling chromatin, but the enzymatic activity of DNA
methyltransferase itself may be regulated by molecules such as MeCP2
that bind to DNA. Some studies suggest that the Dnmt1 enzymatic
reaction may take place at classical replication foci (30), but little
is known about the molecular mechanism of how CpG methylation is
maintained. The discovery that Dnmt1 directly interacts with MeCP2 will
help us to understand this issue. In proliferating cells, expression of
Dnmt1 is induced as DNA synthesis approaches (34). Recent studies and
our data imply a mechanism by which DNA methylation is maintained as
described below (Fig. 5). First, Dnmt1 is
recruited to replication foci by DMAP1 and/or PCNA (11, 12). When
replication forks with the PCNA clamp structure progress (35), MeCP2
bound to symmetrically methylated DNA is released. Free MeCP2
reassociates with hemimethylated, newly synthesized DNA. Dnmt1
interacts with MeCP2, recognizes the complementary nonmethylated CpG
dinucleotides, and transfers a methyl group by the enzymatic reaction.
Then MeCP2 may change its conformation promptly. Finally, MeCP2 would
bind to fully methylated CpG dinucleotides to maintain gene silencing
and chromatin structures by recruitment of HDACs (33). In our model of
maintenance of DNA methylation, these properties of DNA binding
activity and interaction with some molecules may be regulated by
post-translational modifications such as phosphorylation during DNA
replication. In this regard, we found that both Dnmt1 and MeCP2 are
phosphorylated at multiple sites in
vivo.2 MeCP2 possesses
at least two kinds of modification states in vivo.2 Thus, Dnmt1 might require MeCP2 to carry out
genome-wide and/or local maintenance DNA methylation.
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Although mutations of DNA methyltransferase and methyl-CpG-binding
protein family genes affect loss of DNA methylation in development (36)
and/or cellular function (23, 24), little is understood regarding which
protein complexes are involved in those processes. Our discovery
implicates that Dnmt1 and MeCP2 may interplay in related pathways in
both DNA methylation and histone modification. An interaction of Dnmt1
and MeCP2 is dependent upon the TRD in the C-terminal region of MeCP2
in vivo. Therefore, the MBD of MeCP2 within the complex
could bind hemimethylated DNA, targeting Dnmt1 activity to
MeCP2-responsive genes. One important question that remains to be
answered about the Dnmt1-MeCP2 interaction is whether there is a
specific target for the genome or other molecules to be involved in DNA
methylation and chromatin remodeling. Future studies of the functions
of complexes formed by DNA methyltransferases and methyl-CpG binding
domain proteins will be required to determine which complexes are
responsible for tissue-specific organization and remodeling of
chromatin structure and gene regulation by DNA methylation and histone
modification in normal development and epigenetic diseases.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. Seiser and T. Yamamoto for plasmids. We also thank Drs. Tom Curran and Jared Ordway for critical reading of the manuscript and helpful comments.
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FOOTNOTES |
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* This work was supported by Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) (to K. S.) and Japan Society for the Promotion Science (to H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Developmental Neurobiology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.
¶ To whom correspondence should be addressed. E-mail: ashiota@ mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M209923200
2 H. Kimura and K. Shiota, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; CRID, co-repressor-interacting domain; TRD, transcription repressor domain; NLS, nuclear localization sequence; RFTS, replication foci targeting sequence; MBD, methyl-CpG binding domain; TBS, Tris-buffered saline; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen.
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REFERENCES |
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