From the ¶ Wellcome/Cancer Research UK Institute
and Department of Pathology, University of Cambridge, Tennis Court
Road, Cambridge, CB2 1QR, United Kingdom and the Wellcome Trust
Center for Cell Biology, University of Edinburgh, Kings Buildings,
Edinburgh, EH9 3JR, United Kingdom
Received for publication, August 23, 2002
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ABSTRACT |
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DNA methylation plays an important role in
mammalian development and correlates with chromatin-associated
gene silencing. The recruitment of MeCP2 to methylated
CpG dinucleotides represents a major mechanism by which DNA
methylation can repress transcription. MeCP2 silences gene expression
partly by recruiting histone deacetylase (HDAC) activity, resulting in
chromatin remodeling. Here, we show that MeCP2 associates with histone
methyltransferase activity in vivo and that this activity
is directed against Lys9 of histone H3. Two
characterized repression domains of MeCP2 are involved in tethering the
histone methyltransferase to MeCP2. We asked if MeCP2 can deliver
Lys9 H3 methylation to the H19 gene, whose activity
it represses. We show that the presence of MeCP2 on nucleosomes within
the repressor region of the H19 gene (the differentially methylated
domain) coincides with an increase in H3 Lys9
methylation. Our data provide evidence that MeCP2 reinforces a
repressive chromatin state by acting as a bridge between two global
epigenetic modifications, DNA methylation and histone methylation.
Methylation of cytosines is essential for mammalian development
and is associated with gene silencing (1). DNA methylation represses
genes partly by recruitment of methyl-CpG-binding domain proteins,
which selectively recognize methylated CpG dinucleotides. MeCP2 is the
founder member of the methyl-CpG-binding domain proteins, which
consists of a single polypeptide that contains a methyl-CpG-binding domain and a transcriptional repression domain. MeCP2 is capable of
binding to a single symmetrically methylated CpG both in naked DNA and
within chromatin (2, 3).
It is now well established that MeCP2 silences transcription by
recruiting the histone deacetylase
(HDAC)1 repressive machinery,
which removes acetyl groups from histones resulting in gene silencing
(4, 5). However, the inhibition of histone deacetylase activity using
drugs such as Trichostatin A only partially relieves MeCP2-mediated
transcriptional repression. This partial relief indicates that
additional mechanisms of repression by MeCP2 likely exist aside from
the recruitment of histone deacetylase.
Beside histone deacetylation, histone methylation is emerging as
another key post-translational modification of histones and represents
an important epigenetic mechanism for the organization of chromatin
structure and the regulation of gene expression. In particular,
methylation at lysine 9 of histone H3 is associated with gene
silencing, and several enzymes that catalyze the addition of methyl
groups to lysine 9 have recently been identified (6). Interestingly,
recent data have shown that the retinoblastoma protein represses
transcription through the recruitment of HDAC activity, but in a second
step it recruits histone methylation activity specific for lysine 9 of
histone H3 (7). By analogy to retinoblastoma, we considered in the
present work whether MeCP2-mediated repression might also include,
besides histone deacetylation, a second stage involving histone methylation.
Here, we show that MeCP2 associates with histone methylation in
vitro as well as in vivo. The MeCP2-associated
methylation activity is found to be specific for lysine 9 of histone
H3. By means of chromatin immunoprecipitation (ChIP) experiments, we show that MeCP2 facilitates H3 Lys9 methylation of the H19
gene, a bona fide MeCP2-regulated gene. Our results indicate that MeCP2
acts as a mechanistic bridge between DNA methylation and histone
methylation and thus reinforce the repressive function of these two
distinct methylation events.
Plasmids--
We cloned MeCP2 1-77 into the pGEX vector
(Pharmacia) by PCR using the appropriate sets of primers. The other
pGEX expression vectors for full-length and deletion mutants of MeCP2
have been described previously (8). The HA-tagged mammalian expression construct of MeCP2 (HA-MeCP2) was generated by insertion of a synthetic
oligonucleotide encoding the HA epitope sequence into the 5' end of
MeCP2 cDNA in frame in pBluescriptSK Generation of MeCP2 Tet-Off Cell Line, Cell Culture, and
Transfections--
L929 mouse fibroblast cells (L cells) were
co-transfected with pTet-Off and pTRE-HA-MeCP2/Zeo by electroporation.
The cells were placed under selection for G418-resistance and
Zeocin-resistance after 24 h following transfection.
G418/Zeocin-resistant cell lines were tested for background and induced
expression of HA-MeCP2 in the presence and absence of tetracycline,
respectively. Expression was detected by anti-HA and anti-MeCP2
antibodies on Western blots. Clone L-4 did not express
detectable HA-MeCP2 background but was strongly inducible by
tetracycline withdrawal. Mouse MeCP2 Tet-Off fibroblasts (L cells) were
grown to 75% confluence in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% tetracycline-free fetal calf serum
(Clontech) at 37 °C, 5% CO2. Cells
were then treated or not with 5 µg/ml tetracycline (Sigma) and
harvested 1 or 3 days later. 293T cells were maintained in DMEM
supplemented with 10% fetal calf serum and grown at 37 °C, 5%
CO2. For transfection of 293T, we seeded ~2-3 × 106 cells on the evening prior to procedure. We transfected
cells with a total of 20 µg of DNA using the calcium phosphate
co-precipitation method. The precipitate was washed off 10 h
after transfection and incubated for an additional 24 h before
harvesting. Wild type and Mecp2 Glutathione S-transferase (GST) Pull Downs and
Immunoprecipitations for Histone Methyltransferase Assays--
We
expressed GST and GST fusion proteins in Escherichia coli
XA90 using the pGEX (Pharmacia) vector system and purified protein from
crude bacterial lysates according to the manufacturer's instructions. We performed pull-down assays by incubating GST or GST fusion proteins
prebound to glutathione beads (Pharmacia) with HeLa nuclear extracts
(50 µl) in radioimmune precipitation assay buffer (200 µl; 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40,
and 0.1% SDS). The incubation was performed at 4 °C for 12 h.
The beads were then washed four times in 1 ml of radioimmune
precipitation assay buffer before processing to histone
methyltransferase assays. For immunoprecipitations prior to histone
methyltransferase assays, antibodies against HA (12C5A, Roche), MeCP2
(07-013, Upstate Biotechnology), or green fluorescence protein (Abcam,
ab290) were used. Extracts from transfected 293T cells that were lysed
in IPH buffer (10) or HeLa nuclear extracts (50 µl, Computer
Cell Culture Centre, Mons, Belgium) were incubated with the
relevant antibodies overnight at 4 °C. Antibody complexes were
collected on protein A/G-Sepharose beads, washed four times in
radioimmune precipitation assay buffer, and assayed for histone
methyltransferase assays.
Histone Methyltransferase Assays and Protein
Sequencing--
Precipitations from pull downs or immunoprecipitations
from HeLa nuclear extract or 293T transfected extracts were incubated with either 10 µg of histones (Sigma) or 5 µg of recombinant H3 (gift from Karl Nightingale) and 2 µl
[3H-Me]-S-adenosyl methionine (Amersham
Biosciences, 67 Ci mmol Western Blot Analysis--
Western blotting from MeCP2 Tet-Off
cells were done as described (11) using MeCP2 antibody (07-013,
Upstate Biotechnology).
RNA Purification and RT-PCR Analysis--
Total RNA was
isolated from wild type and Mecp2 ChIP--
MeCP2 Tet-Off cells treated or not with tetracycline
were cross-linked with 0.75% formaldehyde (Sigma) at room temperature for 10 min. Cells were rinsed twice with ice-cold phosphate-buffered saline (pH 7.4) and collected in phosphate-buffered saline and centrifuged for 5 min at 1200 rpm. ChIPs were then performed as described (12, 13) with either 4 µl of anti-H3 methyl
Lys9 (MeK9, Abcam, ab7658) or 4 µl of an
affinity-purified unrelated antibody (raised against the N terminus of
the nuclear import factor RCH1) or 5 µl of anti-MeCP2 (Upstate
Biotechnology 07-013) or 5 µl of an affinity-purified unrelated
antibody (anti-HA, Upstate Biotechnology 06-831). The specificity for
anti-MeK9 H3 has been reported previously (7). Immunoprecipitations
were analyzed by PCR for the presence of the H19 DMD-encompassing
fragment. Amplification of the H19 DMD region was performed using
primer pairs at positions Silencing of transcription by MeCP2 involves the recruitment of
HDAC activity (4, 5). However, additional pathways by which MeCP2
represses transcription probably exist because HDAC inhibitors do not
completely relieve MeCP2-mediated silencing (4, 5). The retinoblastoma
protein represses transcription not only through recruitment of HDAC
activity but also through recruitment of histone methylation activity
specific for lysine 9 of histone H3 (7). These recent observations
prompted us to ask whether histone methylation might represent another
pathway by which MeCP2 silences transcription. To test this possibility we first asked if MeCP2 associates with histone methyltransferase activity. Fig. 1A shows that
MeCP2 fused to GST is able to purify from nuclear extracts, an activity
that specifically methylates histone H3 from a mixture of bulk
histones. Recombinant histone H3 can also be used as the substrate for
the methyltransferase associated with GST MeCP2 (Fig. 1B).
Deletion analysis of MeCP2 indicates that the association of MeCP2 with
histone H3 methyltransferase activity is primarily mediated by its
N-terminal portion, which overlaps with its methyl-CpG-binding domain
(Fig. 1C). In addition, the more C-terminal region of MeCP2,
108-392, that contains the transcriptional repression domain also
contributes to the binding of enzymatic activity (Fig.
1C).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
based vector
p65
(8). pCMV-HA-MeCP2 was constructed by insertion of the
NotI/EcoRI-restricted fragment encoding HA-MeCP2
into the BamHI site of pCMV-Bam-Neo vector such that the CMV
promoter drives HA-MeCP2 expression. pTRE-MeCP2 was constructed by
insertion of the EcoRI-restricted HA-MeCP2 fragment from
pCMV-HA-MeCP2 into the EcoRI site of pTRE
(Clontech). pTRE-HA-MeCP2/Zeo was constructed by
insertion of a BamHI/BglII-restricted Zeocin
resistance cassette from pZeo/SV (Invitrogen) into the XhoI
site of pTRE-HA-MeCP2.
/y mouse cells
(9) were grown essentially as described before in modified Eagle's
medium alpha and 10% fetal calf serum and grown at 37 °C, 5%
CO2.
1) in buffer MAB (50 mM Tris, pH 8.5, 20 mM KCl, 10 mM
MgCl2, 10 mM 2-mercaptoethanol, and 250 mM w/v sucrose) at 30 °C for 4 h. The reaction
products were then resolved by SDS-PAGE, Western blotted, and
autoradiography was performed. The position of radiolabeled H3
was identified by its size using radiolabeled molecular weight markers
(Amersham Biosciences) as well as by comparing the autoradiography with
the gel stained with Ponceau. For N-terminal sequencing, radiolabeled
histone H3 was blotted to polyvinylidene difluoride (Millipore) and
sequenced by Edman degradation (Protein Sequencing Facility, University
of Cambridge, Cambridge, UK). Amino acid fractions were analyzed
for the presence of tritium by scintillation counting.
/y mouse cells (9)
following the Qiagen RNeasy Midi protocol. Purified RNA (0.5 µg) was
used for quantitative RT-PCR, following the Qiagen One Step protocol,
for 28 PCR cycles.
2892 bp versus start site to
2137. The cycle number and the amount of template were varied to
ensure that results were within the linear range of the PCR.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
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Fig. 1.
MeCP2 binds in vitro to a
histone methyltransferase activity specific for H3. A,
GST MeCP2 binds histone H3 methyltransferase activity from nuclear
extracts. Histone methyltransferase assays using bulk histones as
substrate incubated with HeLa nuclear extracts and equivalent amounts
of GST or GST MeCP2 bound to Sepharose beads. After incubation, the
beads were washed and assayed for histone methyltransferase activity.
The reaction products were then analyzed by SDS-PAGE followed by
Western blotting and autoradiography. The radiolabeled H3 is indicated
by an arrow on the left and was identified
through its molecular size as well as by Ponceau staining.
B, MeCP2-associated activity methylates recombinant histone
H3. Histone methyltransferase assays were performed as in A,
this time with recombinant H3 as a substrate instead of bulk histones.
C, a region in the N terminus of MeCP2, which overlaps with
the methyl-CpG-binding domain and to a lesser extent the
transcriptional repression domain, mediates the association with
histone H3 methyltransferase activity. The indicated GST MeCP2
fusion fragments were tested for their association with histone
methyltransferase activity. Residues 1-77 (lane 8) were
tested in a separate experiment than the other constructs (lane
1 7). Below is shown a schematic representation of
MeCP2 with its methyl-CpG-binding domain and transcriptional repression
domain. Also shown are the GST fusion proteins of MeCP2 tested in the
histone methyltransferase assay, with the results summarized on the
right (+++, indicates strong MeCP2-associated
methyltransferase activity;
, indicates no associated
activity).
To further verify the association of MeCP2 with a methyltransferase, we
used a co-immunoprecipitation approach. We transfected mammalian cells
with an HA-tagged, full-length MeCP2 (HA-MeCP2), lysed the
cells, and carried out precipitation with anti-HA antibody. The
resulting immunoprecipitate was assayed for the presence of methyltransferase activity on histones. Fig.
2A (lane 1)
indicates that HA-MeCP2 purifies activity specific for histone H3. When endogenous MeCP2 was immunoprecipitated from HeLa nuclear extracts with
a MeCP2-specific antibody, the association with the histone H3
methyltransferase was also detected (Fig. 2B, lane
1). Control immunoprecipitation with an irrelevant antibody (green
fluorescence protein) gave background activity (Fig. 2B,
lane 2). Together, these results demonstrate that MeCP2 is
associated with a histone methyltransferase in vivo and that
this enzyme specifically methylates histone H3.
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We next wanted to identify the residue(s) of histone H3 that are
modified by the MeCP2-interacting methyltransferase. Given that a
tailless recombinant H3 did not act as a substrate (data not shown),
histone H3 radioactively methylated by the MeCP2-interacting methyltransferase was put through Edman degradation to identify the
methylation site in the H3 tail. Protein sequence analysis revealed
that MeCP2 binds histone methyltransferase activity that is specific
for lysine 9 of histone H3 (H3 Lys9; Fig.
3). The nature of the MeCP2-interacting
histone Lys9 methyltransferase is currently unknown. So far
five mammalian H3 Lys9 methyltransferases have been
identified: SuvarH1 (14), SuvarH2 (15), G9a (16), ESET (17), and
Eu-HMTase1 (18), and several others are predicted to exist (6).
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We next wanted to assess whether MeCP2 can facilitate H3
Lys9 methylation of a DNA-methylated gene that it
regulates. We chose to investigate the H19 gene because it contains a
characterized repressor domain in its upstream regulatory region, (the
DMD), which is known to be DNA-methylated (19) and associated with MeCP2 (20). To establish whether MeCP2 represses transcription of H19
in vivo, we monitored the expression of H19 in wild type and
knock-out Mecp2 /y cells (9). RNA isolated from each cell type was reverse transcribed and amplified by polymerase chain reaction
(RT-PCR). Fig. 4A
shows that H19 messenger RNA levels are elevated in the MeCP2 knock-out
cells compared with wild type cells. In contrast, the expression of an
unrelated house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase,
is unchanged (Fig. 4A). Thus, H19 is a bona fide
MeCP2-regulated gene that can be used to analyze MeCP2 functions.
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To establish if MeCP2 delivers H3 methylation to the H19 gene we next
examined the wild type and knock-out Mecp2 /y cells for
differential histone methylation at the H19 DMD. An antibody that
recognizes histone H3 when methylated at lysine 9 was used in ChIPs.
The outcome of these experiments was not conclusive because the
chromatin isolated from the Mecp2
/y knock-out cells was
qualitatively different from wild type controls. Indeed, purification followed by fragmentation (by sonication or nuclease digestion) of the
chromatin from the MeCP2 null and wild type cells consistently resulted
in different size fragments upon analysis on agarose gel
electrophoresis. So direct quantification and comparison between the
two chromatin sets was not possible. We therefore turned to an
inducible Tet-Off cell line, where the levels of MeCP2 could be
manipulated by the addition of tetracycline (Tc). In this Tet-Off cell
line, the presence of tetracycline reduces MeCP2 expression down to
endogenous levels (Fig. 4B).
Using this Tet-Off MeCP2 cell line, we performed ChIPs analysis on the H19 DMD using the MeCP2 antibody and the methyl lysine 9 H3-specific antibody. Fig. 4C shows that in the absence of Tc, when MeCP2 expression is high, MeCP2 binds to nucleosomes associated with the DMD (Fig. 4C, lane 1). This MeCP2 binding is specific because immunoprecipitations of the cross-linked chromatin with an irrelevant antibody or with the beads only gave background signal (Fig. 4C, lanes 2 and 3). In contrast, when MeCP2 levels are low, in the presence of Tc, the binding of MeCP2 is reduced considerably (Fig 4C, lane 4). These results indicate that the manipulation of MeCP2 levels in the Tet-Off cell line results in differential binding of MeCP2 to the H19 DMD.
We then monitored the levels of Me-Lys9 H3 on the
nucleosomes associated with the H19 DMD. As shown in Fig. 4D
(lane 1), it is clear that the DMD is methylated at
Lys9 when MeCP2 levels are high. If MeCP2 was required for
H3 Lys9 methylation on the H19 DMD, then down-regulation of
MeCP2 cellular level should result in a reduced histone H3 methylation.
Fig. 4D shows that under conditions in which the MeCP2 level
is low (i.e. in the presence of Tc), nucleosomes within the
H19 DMD show a significant reduction in histone H3 Lys9
methylation (Fig. 4D, compare lane 4 with
lane 1). These results indicate that the presence of MeCP2
on the H19 gene correlates with the appearance of histone H3
Lys9 methylation at the differentially DNA-methylated and
repressive DMD region of the H19 gene.
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DISCUSSION |
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The present study provides evidence that the targeting of histone methylation to DNA methylated regulatory regions is part of the mechanism by which MeCP2 functions. To date, MeCP2 action is known to involve the delivery of histone deacetylase activity. However, this mechanism cannot account for the full repressive potential of MeCP2 because inhibitors of histone deacetylase activity only relieve repression partially (4, 5). Thus the delivery of histone methylating activity by MeCP2 may represent a repressive event that follows the targeting of deacetylases. This order of events is suggested by the fact that deacetylation of histone H3 at lysine 9 is necessary for methylation to take place on this residue (21). In this way, deacetylation of histone 3 at lysine 9 would be followed by methylation, which in turn may result in the recruitment of proteins such as HP1 (14, 22). The identity of the lysine methyltransferase associated with MeCP2 is unknown, but members of a family of lysine 9-methylating proteins can be considered candidates (6).
The data presented here identify MeCP2 as a protein that can connect a
repressive modification on DNA to a repressive modification on
histones. Recent evidence from Neurospora crassa and
Arabidopsis thaliana indicate that the reverse is also
possible: methylation of histone H3 at lysine 9 leads to methylation of
DNA (23, 24). If we assume that this latter mechanism is also
operational in mammalian cells, our data suggest that MeCP2 may set up
a self-reinforcing cycle of repression, which may be necessary for the
maintenance and heritability of the repressed state. In other words, by
promoting further rounds of DNA methylation following histone
methylation, MeCP2 would maintain the methylation status at the DMD and
thus distinguish the "permanent" repressive state of imprinted
promoters from the "regulated" repressive state of other promoters.
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ACKNOWLEDGEMENTS |
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We thank R. Schneider and A. Bannister for technical guidance on ChIP experiments and H. Santos-Rosa for advice on histone methyltransferase assays. We also thank K. Nightingale for providing recombinant histone H3.
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FOOTNOTES |
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* The research in the Tony Kouzarides lab is supported by a program grant from the Cancer Research Campaign.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.
These authors contributed equally to this work.
§ Supported by the Belgian Fonds National de la Recherche Scientifique (Chargé de recherches du FNRS). Present address: Free University of Brussels, Faculty of Medicine, Laboratory of Molecular Virology, 808 route de Lennik, 1070 Brussels, Belgium.
** To whom correspondence should be addressed. Tel.: 44-1223-334112; Fax: 44-1223-334089; E-mail: tk106@mole.bio.cam.ac.uk.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M210256200
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; ChIP, chromatin immunoprecipitation; HA, hemagglutinin; GST, glutathione S-transferase; RT, reverse transcription; DMD, differentially methylated domain; Tc, tetracycline.
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