MOLECULAR AND CELLULAR BIOLOGY, Feb. 2006, p. 843–851
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 3
MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with
Different Biochemical and Functional Properties
Xavier Le Guezennec,1† Michiel Vermeulen,1† Arie B. Brinkman,1Wieteke A. M. Hoeijmakers,1
Adrian Cohen,1Edwin Lasonder,1,2and Hendrik G. Stunnenberg1*
Department of Molecular Biology, Nijmegen Center for Molecular Life Sciences, Radboud University,
6500 HB Nijmegen, The Netherlands,1and Center for Molecular and Biomolecular Informatics,
Toernooiveld 1, 6525 ED Nijmegen, The Netherlands2
Received 20 August 2005/Returned for modification 18 September 2005/Accepted 16 November 2005
The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into
a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging
and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/
NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor sup-
pressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were
identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates
the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and
MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of
PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein
complexes with different biochemical and functional properties.
Methylation of CpG dinucleotides in regulatory regions of
genes is an important mark for epigenetic regulation of tran-
scription (2). Since DNA methylation is passed on to daughter
cells during cell division, these methyl CpG marks can be
maintained during development and provide epigenetic mem-
ory (31). A number of proteins have been identified in the
human genome that can specifically bind to methylated CpG
residues via a methyl CpG binding domain (MBD) (15, 39).
Recruitment of these proteins to promoters containing meth-
ylated CpG-rich stretches—CpG islands—is thought to result
in modulation of chromatin structure and repression of tran-
scription. The human genome encodes five MBD proteins:
MeCP2 and MBD1 to -4 (6, 14, 23). Apart from MBD3, these
proteins have been shown to have specific methyl CpG binding
activity. Recently a novel protein, Kaiso, was identified as a
methyl CpG binding protein even though this protein lacks a
classical MBD but appears to bind specifically to methylated
DNA via a zinc finger domain (30).
Several MBD proteins have been reported to interact with
histone deacetylases (HDACs) as well as histone methyltrans-
ferases. MeCP2 has been described to interact with the Sin3/
HDAC corepressor complex (18) and Brahma (13), as well as
with the histone H3 lysine-9 methyltransferase Suvar 3-9 (12),
although these interactions may not be stable since MeCP2 is
mostly present inside the cell as a monomer (12, 18, 19, 26).
MBD2 and MBD3 have been identified as core subunits of the
Mi-2/NuRD complex (9, 27), whereas Kaiso is part of the
HDAC-containing N-CoR complex that plays an important
role in transcription regulation by nuclear hormone receptors
(27, 42, 44). Collectively, these findings suggest a functional
link between DNA methylation, histone deacetylation, and
histone methylation and indicate that these epigenetic events
functionally cooperate to regulate transcription and cellular
MBD2 and MBD3 have both been described as subunits of
the Mi-2/NuRD complex. It has been proposed that MBD2,
which exhibits methyl CpG binding activity, serves to recruit
the MBD3-containing Mi-2/NuRD complex to methylated pro-
moters (44). Knockout studies in mice, however, suggest that
MBD2 and MBD3 have distinct nonoverlapping functions:
whereas knocking out MBD3 results in embryonic lethality,
MBD2-knockout mice are viable and display relatively subtle
defects (16). Interestingly, Sansom and coworkers recently
showed that the absence of MBD2 protects against intestinal
tumorigenesis (32). Thus, although biochemical evidence sug-
gests that MBD2 and MBD3 are part of the same complex, the
knockout studies suggest that both proteins have specific or
maybe partially overlapping functions.
To gain insights into the protein composition and function of
MBD2 and MBD3, we generated stable cell lines expressing
tagged versions of these proteins. Purification of the protein
complexes revealed that MBD2 and MBD3 are not copurifying
but are mutually exclusive. In addition to known Mi-2/NuRD
subunits, a 12-kDa protein called DOC-1 was identified as a
novel core subunit of both the MBD3 and MBD2 complexes.
Furthermore, PRMT5 and its associated cofactor MEP50 were
found to copurify with and methylate MBD2 in vitro. Finally,
PRMT5 and its H4R3 histone methyltransferase activity were
shown to be recruited with MBD2 to CpG islands in a meth-
ylation-sensitive manner in vivo, suggesting an unexpected role
for an arginine methyltransferase in repression by MBD2. Col-
lectively, these findings provide evidence that MBD2/NuRD
and MBD3/NuRD define two distinct protein complexes with
different biochemical and functional properties.
* Corresponding author. Mailing address: Department of Molecular
Biology, NCMLS M850/3.79, Radboud University, P.O. Box 9101,
6500 HB Nijmegen, The Netherlands. Phone: 31-24-3610524. Fax:
31-24-3610520. E-mail: firstname.lastname@example.org.
† The first two authors contributed equally.
MATERIALS AND METHODS
Constructs. Oligonucleotides pRAV-myc1f and pRAV-myc1r encoding a Myc
epitope with EcoRI overhangs were cloned into EcoRI-digested vector frag-
ments of pRAV-FLAG (20) to generate pRAV-myc. An EcoRI/BamHI frag-
ment from this vector containing one ProtA domain, two tobacco etch virus
(TEV) cleavage sites, and a myc epitope was then ligated with EcoRI/BamHI-
digested vector pZ-1-N (Cellzome) to generate retroviral vector pZXN. MBD3,
MBD2, and MBD2 lacking the RG stretch were PCR amplified with EcoRI and
XhoI overhangs from an MBD3 plasmid (RZPD) and an MBD2 plasmid (image
clone collection) which were then ligated into EcoRI/XhoI-digested vector pZXN.
To create a Strep-tagII (2TEV) Myc 2? hemagglutinin (HA) cassette, the
2TEV Myc cassette from pZXN was PCR amplified using a forward primer
containing an EcoRI site and a Strep-tagII epitope and a reverse primer with an
EcoRI overhang and two HA sites. This fragment was ligated into EcoRI-
digested vector psg5-HA TBP. The cassette was PCR amplified again with a
forward primer containing a BamHI restriction site and a reverse primer con-
taining one new HA epitope and a NotI restriction site. This PCR product was
digested with BamHI and NotI and ligated into BamHI/NotI-digested plasmid
pcDNA5/FRT/TO/C-TAP (kind gift from Bernard Luscher) to generate
pcDNA5/FRT/TO/stII(2TEV)myc tripleHA. MBD2 was PCR amplified using
primers containing NotI and Xho1 restriction sites and ligated into the NotI and
XhoI site of pcDNA5/FRT/TO/stII(2TEV)myc tripleHA.
A fragment encoding part of the RG stretch of MBD2 (EGARGGGRGRGR)
containing BamHI and EcoRI overhangs was cloned in plasmid pGEX2T (Amer-
sham Pharmacia Biotech). Full-length MBD2, MBD lacking the RG stretch, and
MBD3 were PCR amplified with primers containing BamHI and EcoRI over-
hangs and cloned in BamHI/EcoRI-digested pGEX2T. Primer sequences are
available upon request.
Cell culture and stable cell lines. MCF7, HEK 293, HeLa, Phoenix, and 293
FLP cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen)
supplemented with 10% fetal calf serum, 100 ?g of penicillin per ml, and 100 U
of streptomycin per ml (Invitrogen) at 37°C in 5% CO2. For 5-aza-2?-deoxycy-
tosine (AzaDc) treatment MCF7 cells were seeded at low density and treated
with 1 ?M of AzaDc for 72 h. Retroviral stable cell lines were generated
according to the following procedure. Phoenix amphotropic packaging cells
(2.5 ? 106cells) were seeded on a 9-cm dish and transfected 24 h later with 20 ?g
of retroviral plasmid pZXN-MBD2a or pZXN-MBD2 lacking the RG stretch or
pZXN-MBD3 using the calcium phosphate method. After 48 h virus-containing
supernatant was filtered through a 0.22-?m-pore-size filter. HeLa or 293 cells (105
each) were seeded in a six-well plate and transduced with 3 ml filtered virus super-
natant in the presence of 8 ?g/ml of Polybrene for two infectious rounds of 24 h.
Cells were then incubated for 24 h in normal medium. The polyclonal population of
cells was then selected with 1 ?g/ml of puromycin. Clones were then selected, grown
in isolation, and screened for recombinant protein expression.
A double stable cell line expressing tagged MBD2 and MBD3 was generated
according to the following procedure. 293 FLP cells were transfected using the
calcium phosphate method on a 9-cm dish with 2 ?g of pcDNA5/FRT/TO/
stII(2TEV)myc tripleHA-MBD2 and 18 ?g of POG44. After 36 h cells were
selected with 100 ?g/ml hygromycin. Subsequently, clones were derived and
screened for recombinant protein expression and zeocin sensitivity. One good
clone was then transduced with virus containing pZXN-MBD3 and was double
selected with 100 ?g/ml hygromycin and 1 ?g/ml of puromycin.
Protein purification. Cell pellets were resuspended in lysis buffer (420 mM
KCl, 20% glycerol, 20 mM HEPES, pH 7.9, 0.2 mM EDTA, 5 mM MgCl2, 0.1%
Triton X-100, 10 mM ?-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride
[PMSF], and complete protease inhibitors [Roche]) and homogenized by 20
strokes with a type B pestle. Extracts were then incubated for 1 hour in a rotation
wheel at 4°C to extract nuclear proteins. Lysates were subsequently clarified by
ultracentrifugation at 100,000 ? g. Whole-cell extracts were aliquoted, snap
frozen, and stored at ?80°C until further usage.
Whole-cell extracts derived from tandem affinity purification (TAP)-tagged
cell lines were diluted with 2 volumes binding buffer (150 mM NaCl, 20 mM
Tris-HCl, pH 8.0, 0.1% NP-40, 1 mM dithiothreitol [DTT], 1 mM PMSF, and
complete protease inhibitors [Roche]) and then incubated with immunoglobulin
G (IgG) Sepharose beads (Pharmacia) for 2 h at 4°C in a rotation wheel. Beads
were then washed three times with 10 bead volumes of wash buffer (500 mM
NaCl, 20 mM Tris-HCl, pH 8.0, 0.5% NP-40, 1 mM DTT, and 1 mM PMSF) and
twice with 10 bead volumes of TEV cleavage buffer (150 mM NaCl, 20 mM
Tris-HCl, pH 8.0, 0.1% NP-40, 1 mM DTT, and 0.5 mM EDTA). Beads were
then resuspended in 1 bead volume TEV cleavage buffer containing TEV pro-
tease and incubated overnight at 4°C in a rotation wheel. TEV eluates were
precleared with protein A beads (Pharmacia) and then subjected to immuno-
precipitation using Myc antibody (9E11). Immunoprecipitates were washed three
times with 10 bead volumes of wash buffer and twice with 10 bead volumes of
peptide elution buffer (100 mM KCl, 20% glycerol, 20 mM HEPES KOH, pH
7.9, 0.2 mM EDTA, 0.1% NP-40, 5 mM DTT, and 0.5 mM PMSF). Protein
complexes were eluted from the beads by incubation in peptide elution buffer
containing 2 mg/ml of Myc peptide at 28°C for 30 min in a thermoshaker. The
elution step was carried out twice, and both eluates were pooled.
Endogenous immunoprecipitation assays were performed with HeLa nuclear
extract in stringency conditions similar to those for the TAP tag procedure.
Antibodies used were MBD3 (IBL, Japan) and MBD2 07-198 (Upstate Biotech-
Protein analysis by liquid chromatography-tandem mass spectrometry (MS/
MS). Purified protein complexes were loaded on sodium dodecyl sulfate (SDS)-
polyacrylamide gels and run briefly to get rid of detergent and the excess of the
peptide used for the elution. The gel lane was then fixed, cut in small pieces, and
subsequently reduced and alkylated. Proteins were digested overnight with tryp-
sin (Promega) and eluted from the gel with trifluoroacetic acid. Peptide identi-
fication experiments were performed using a nano-high-pressure liquid chroma-
tography Agilent 1100 nanoflow system connected online to a 7-Tesla linear
quadrupole ion trap-Fourier transform (FT) mass spectrometer (Thermo Elec-
tron, Bremen, Germany) essentially as described previously (28).
ChIP assay. MCF7 cells were cross-linked with 1% formaldehyde for 15 min
at room temperature, and chromatin was prepared as described previously (3,
41) but excluding CsCl purification. Chromatin was sonicated to an average size
of 500 bp. Chromatin derived from 1 million cells was used for each immuno-
precipitation in incubation buffer (1% Triton X-100, 150 mM NaCl, 1 mM
EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 10 mM Tris, pH 8.0, 1 mg/ml bovine
serum albumin, and protease inhibitors). Four micrograms of the following
antibodies was used for immunoprecipitations: PRMT5 12-303 (Upstate Bio-
technology), MBD2 IMG-147 (Imgenex), MBD3 (IBL, Japan), MTA2 PC656
(Oncogene), and anti-dimethyl-histone H4 (Arg3) (07-213) (Upstate Biotech-
nology). After overnight incubation at 4°C immunoprecipitates were washed
twice with 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 0.15 M NaCl, 1 mM
EDTA, 10 mM Tris (pH 8.0), 0.5 mM EGTA; once with 0.1% SDS, 1% Triton
X-100, 0.1% deoxycholate, 0.5 M NaCl, 1 mM EDTA, 10 mM Tris (pH 8.0), 0.5
mM EGTA; once with 0.25 M LiCl, 0.5% deoxycholate, 0.5% NP-40, 1 mM
EDTA, 10 mM Tris (pH 8.0), 0.5 mM EGTA; and twice with 1 mM EDTA, 10
mM Tris (pH 8.0), 0.5 mM EGTA. Immunocomplexes were eluted from the
beads by adding 1% SDS, 0.1 M NaHCO3followed by incubation at room
temperature for 15 min. Protein-DNA cross-links were reversed in 0.2 M NaCl
at 65°C for 4 h, after which DNA was isolated by phenol-chloroform extraction.
Real-time quantitative PCR analyses were performed to assess recruitment of
the proteins to specific sites. The relative occupancy was derived from the
percent recovery of a specific CpG island against the percent recovery of a
control BMX region. Means and standard deviations were then calculated from
chromatin immunoprecipitation (ChIP) experiments performed from three in-
dependent chromatin isolations.
In vitro methylation assay. Whole-cell extracts derived from the MBD2 stable
cell line or wild-type HEK 293 cells were diluted with 3 volumes of IPP150 (150
mM NaCl, 20 mM Tris HCl, pH 8.0, 0.1% NP-40, 1 mM DTT, and 1 mM PMSF)
and incubated with IgG Sepharose beads for 2 h at 4°C in a rotation wheel. Beads
were washed three times with 10 bead volumes of IPP150 and then incubated
with 1 bead volume of PRMT5 incubation buffer (20 mM HEPES, pH 7.6, 500
mM NaCl, 1 mM MgCl2) in the presence of 0.25 ?Ci of S-[14C]adenosylmethi-
onine (Amersham). Glutathione S-transferase (GST)–PAH2, GST-MBD2,
GST-MBD2 lacking the RG stretch, GST-MBD3, and GST-EGARGGGR
GRGR were expressed and purified as described previously (22). These purified
proteins were incubated in PRMT5 incubation buffer supplemented with 0.25
?Ci of S-[14C]adenosylmethionine (Amersham) in the presence of purified
MBD2 complex or a purified Drosophila melanogaster fraction highly enriched
for PRMT5/MEP50/USP7 (37). After 2 h of incubation at 30°C products were
separated on a 12% SDS-polyacrylamide gel. The gel was then dried and exposed
on a phosphoscreen (Bio-Rad) to identify methylated proteins.
Purification of TAP-tagged MBD2 and MBD3. Human em-
bryonic kidney (HEK 293) cells stably expressing tagged ver-
sions of MBD2 and MBD3 were generated to determine their
subunit composition. Gel filtration analysis of whole-cell ex-
tracts derived from these cell lines indicated that both proteins
844 LE GUEZENNEC ET AL.MOL. CELL. BIOL.
are present in high-molecular-weight fractions of approxi-
mately 1 to 1.5 MDa (Fig. 1A).
To assess whether the purified complexes were enzymati-
cally active, we performed deacetylation assays on nucleosomal
templates acetylated by the SAGA and NuA4 complex (38).
Both MBD2 and MBD3 protein complexes displayed robust
trichostatin A-sensitive deacetylation activity towards histone
H3 and histone H4 (Fig. 1B). To further investigate the func-
tionality of the purified complexes, electrophoretic mobility
shift assays using methylated DNA probes were performed
which revealed that only the MBD2 complex was able to bind
to methylated DNA. MBD3 shows no affinity for methylated
DNA, despite the presence of a highly conserved MBD, as
reported previously (40) (Fig. 1C, compare lanes 2 and 4).
MBD2 and MBD3 are mutually exclusive. Previous studies
have reported that MBD2 and MBD3 are part of the same
complex (9). Silver staining of the purified MBD2 and MBD3
complexes revealed a protein of approximately 35 kDa that is
lacking in the MBD2 preparation (Fig. 2A, marked with an
arrow). Western blotting identified this protein as MBD3 and
revealed the absence of MBD3 in the MBD2 complex (Fig. 2B,
compare lanes 1 and 2). The absence of MBD3 in the MBD2
preparation cannot be explained by a shortage of endogenous
MBD3 in 293 cells (Fig. 2B, lane 3). These data suggest that
MBD2 and MBD3 are not part of the same complex but that
they may even be mutually exclusive. If MBD2 and MBD3 are,
however, present as a heterodimer in the Mi-2/NuRD complex,
overexpression of one of the MBDs could cause a shift from an
MBD2/MBD3 heterodimer population to an MBD2/MBD2 or
MBD3/MBD3 homodimer population. To assess this possibil-
ity, we generated a stable cell line expressing MBD2 and
MBD3 with different tag combinations. MBD3 was tagged with
a ProtA domain and a Myc epitope, whereas MBD2 was
tagged with a Strep-tagII and an HA epitope. Western blotting
shows that both of these proteins are expressed in the stable
cell line (Fig. 2C, lane 6). Purification of ProtA-Myc-MBD3 on
IgG beads resulted in purification of MBD3 (Fig. 2C, lane 3);
tagged MBD2 could not be detected in the immunoprecipitate.
Similarly, purification of Strep-tagII-HA-MBD2a on streptac-
tin beads resulted in purification of MBD2, whereas ProtA-
Myc-MBD3 did not copurify (Fig. 2C, lane 1). HDAC1 copu-
rified with tagged MBD2 as well as MBD3, indicating that both
tagged proteins assemble in a functional complex. To further
substantiate these observations, immunoprecipitation experi-
ments against endogenous MBD2 and MBD3 in HeLa cells
were performed (Fig. 2D). Immunoprecipitation of MBD2
resulted in purification of MBD2 but not of MBD3. Similarly,
immunoprecipitation of MBD3 resulted in purification of two
polypeptides whereas MBD2 did not copurify. Based on their
relative migration we presume these two polypeptides to be
MBD3 and the smaller variant MBD3L2 lacking the MBD.
Collectively these experiments strongly suggest that MBD2
and MBD3 are mutually exclusive.
FT-MS/MS analysis of the purified MBD2 and MBD3 com-
plex. To further characterize the purified MBD2 and MBD3
complex, liquid chromatography FT-ICR MS analyses were
performed (Table 1). In agreement with the results described
above, the MBD2 complex did not contain MBD3 and vice
versa, corroborating and extending our conclusion that MBD2
and MBD3 are mutually exclusive in 293 cells. To unambigu-
ously determine whether the observed mutual exclusiveness of
MBD2 and MBD3 is specific for 293 cells or whether this is
also true in other cells, a HeLa cell line stably expressing
tagged MBD3 was generated. Purification of MBD3 from this
cell line resulted in the purification of a Mi-2/NuRD complex
lacking MBD2, indicating that in HeLa cells MBD2 and
MBD3 are also mutually exclusive (unpublished data).
FIG. 1. TAP-MBD2 and TAP-MBD3 assemble into a functional
Mi-2/NuRD-like complex. (A) Superose 6 gel filtration of whole-cell
extracts derived from stable cell lines expressing TAP-MBD2 or TAP-
MBD3. Fractions were analyzed by Western blotting using a ProtA
antibody. The void of the column is indicated between fractions 7 and
8. (B) Nucleosomal templates reconstituted with recombinant histones
were acetylated by the Saccharomyces cerevisiae SAGA or NuA4 com-
plex and subsequently incubated with the TAP-MBD2 or TAP-MBD3
complex in the absence or presence of Trypticase soy agar (38). The
amount of H3 or H4 acetylation was determined by Western blotting
using antibodies against diacetylated histone H3 Lys-9,14 or tetra-
acetylated H4, respectively. The binding of the MBD2 and MBD3
complex to the nucleosomal templates was determined by Western
blotting using a Myc and an HDAC2 antibody. (C) Electrophoretic
mobility shift assays using purified MBD2 and MBD3 complex were
performed on the GAM12 probe as described previously (27). Shifted
methylated probe is indicated with an arrow. Free probe is indicated
with an asterisk.
VOL. 26, 2006MBD2 AND MBD3 DEFINE DISTINCT NuRD COMPLEXES845
In 293 cells, Mi-2? and Mi-2? were identified in both the
MBD2 and MBD3 complexes. These proteins have previously
been characterized by the Schreiber lab as Mi-2/NuRD com-
ponents CHD3 and CHD4, respectively (35). At present we do
not know whether these two isoforms are forming het-
erodimers or whether Mi-2? and Mi-2? are assembled into
distinct complexes. In both complexes, RbAp48 and -46 and
HDAC1 and -2, the catalytic module for nucleosomal deacety-
lation activity, were identified. Furthermore, the highly related
p66? and p66? proteins were identified in both complexes as
described previously (5). We did not identify peptides match-
ing the histone demethylase LSD1, which has been reported to
interact with the Mi-2/NuRD complex (34). A 12-kDa protein
called cdk2-associated protein 1 was identified as a novel Mi-
2/NuRD component of both the MBD2 and MBD3 complexes.
cdk2-associated protein 1 or DOC-1 (deleted in oral cancer 1)
is a putative tumor suppressor reported to be inactivated dur-
ing oral carcinogenesis and colon cancer (36, 43). Furthermore
the MBD2 but not the MBD3 eluate contained a large number
of peptides matching the arginine methyltransferase PRMT5
as well as its associated protein called MEP50. Finally, several
peptides matching different importin ? nuclear transport pro-
teins were identified. These proteins were absent in the MBD3
FIG. 2. MBD2 and MBD3 are mutually exclusive. (A) Silver-stained gel of purified MBD2 and MBD3 complexes from HEK 293 cells. MBD3
is indicated with an arrow. (B) Purified TAP-MBD2 and TAP-MBD3 complexes as well as whole-cell extracts derived from HEK 293 or HeLa cells
were analyzed by Western blotting using an MBD3 antibody. (C) Immunoprecipitation of MBD2 and MBD3 using Strep-tactin or IgG beads,
respectively. Lanes: 1, Strep purification on stII-3HA-MBD2 ProtA-Myc-MBD3 extract; 2, Strep purification on HEK 293 wild-type extract; 3, IgG
purification on stII-3HA-MBD2 ProtA-Myc-MBD3 extract; 4, IgG purification on HEK 293 wild-type extract; 5, input HEK 293 extract; 6, input
stII-3HA-MBD2 ProtA-Myc-MBD3 extract. Eluted proteins were analyzed by Western blotting using an HA or ProtA antibody. Probing the blot
with an HDAC1 antibody reveals coprecipitation of HDAC1 with MBD2 and MBD3. (D) Immunoprecipitation of endogenous MBD2 and MDB3.
Whole-cell extracts from HeLa cells were subjected to immunoprecipitation using antibodies against MBD3 (IBL, Japan) or MBD2 (Upstate
Biotechnology). Western blotting was performed using the same antibodies.
TABLE 1. FT-MS/MS analysis of the purified MBD2
and MBD3 complex
Purification (HEK 293 cells)
MTA3 splicing variant
Importin ?-6 subunit
Importin ?-5 subunit
Importin ?-4 subunit
846LE GUEZENNEC ET AL.MOL. CELL. BIOL.
complex, suggesting a specific interaction with MBD2. The
association between MBD2 and importins may indicate that
MBD2 shuttles between the cytoplasm and the nucleus.
Strikingly all three MTA proteins, MTA1, -2, and -3, as well
as two MTA splice variants were identified in the MBD2 and
MBD3 complex (Table 1). It has been suggested that MTA
proteins display tissue-specific differential expression giving
rise to distinct Mi-2/NuRD complexes (4, 11, 21, 44). Surpris-
ingly, a large number of different posttranslational modifica-
tions were identified in all major NuRD subunits (unpublished
data). A previous study characterized phosphorylation sites in
Mi-2?, Mi-2?, p66?, p66?, HDAC1, and HDAC2 (1). We
confirmed the presence of these phosphorylation sites in the
NuRD complex and in addition identified a plethora of new
sites in the latter as well as in MBD2, MTA1, MTA2, and
MTA3. Several posttranslational modifications were detected
in conserved domains, and these might therefore have a role in
regulating protein-protein or protein-DNA interactions or in
fine-tuning of enzymatic activities. Furthermore ?90% of
these modifications occurred in highly conserved residues, sup-
porting a role for these modifications throughout evolution.
PRMT5 associates with and symmetrically dimethylates
MBD2. PRMT5 was detected by FT-MS/MS in the MBD2
complex but was absent from the MBD3 peptide eluate, which
was confirmed by Western blotting (Fig. 3A). Inspection of the
amino acid sequence of the subunits of the MBD2 complex
revealed that MBD2 has a long stretch of RG repeats N ter-
minal to the MBD (Fig. 3B), whereas RG repeats are not
present in MBD3 or in other subunits. Since the RG motif is a
substrate for PRMT5 (10), we tested whether the MBD2 RG
stretch is a substrate for PRMT5. Incubating purified MBD2
complex in the presence of S-[14C]adenosylmethionine re-
sulted in a single radioactive band migrating at the position of
TAP-tagged MBD2 in the gel (Fig. 3C). To substantiate these
observations, we fused full-length MBD2 or the RG stretch of
MBD2 to GST and tested whether the purified MBD2 complex
containing PRMT5 could methylate these fusion proteins. As
shown in the left panel in Fig. 3D, the MBD2 complex was able
to specifically methylate these recombinant substrates but not
a GST-PAH2 control. To evaluate whether PRMT5 specifi-
cally methylates the RG stretch of MBD2, MBD2 lacking the
RG stretch or MBD3 was fused to GST and incubated with
purified MBD2 complex. As shown in the right panel in Fig. 3D,
the MBD2 complex was able to specifically methylate the RG
stretch of MBD2 but not MBD3 or MBD2 lacking the RG
stretch. A control for the methylation reaction using a purified
PRMT5-containing fraction from Drosophila displayed activity
similar to that of the MBD2 complex, thus confirming the
specificity of PRMT5 in this assay. In addition, a search for
posttranslationally modified peptides in the FT-MS/MS run of
the purified MBD2 complex indeed revealed a peptide con-
taining three dimethyl arginine residues (unpublished data).
Taken together, these experiments strongly suggest that
PRMT5 methylates MBD2 on several arginine residues lo-
cated in the RG-rich amino acid stretch immediately upstream
of the MBD of MBD2 in vitro.
To investigate whether the RG stretch of MBD2 is required
for the interaction between PRMT5 and the MBD2 complex,
we generated a stable cell line expressing a truncated MBD2
protein starting at the second methionine in the MBD2 se-
quence, thus lacking the RG stretch. Following purification
and FT-MS/MS analysis, Mi-2/NuRD components, the novel
core subunit DOC-1, and importin ? proteins were present.
However, peptides matching either PRMT5 or MEP50 were
not identified (Fig. 3E). Western blotting confirmed that
PRMT5 was present in crude extracts but absent in the trun-
cated MBD2 eluate (data not shown). Taken together these
results strongly suggest that PRMT5 interacts with the N-ter-
minal RG-rich stretch of MBD2 and methylates this RG
PRMT5 is recruited to chromatin by MBD2. To assess whether
PRMT5 plays a role on chromatin with MBD2, we performed
teins in MCF7 breast carcinoma cells. Different CpG island tar-
gets which were previously shown to be methylated and bound by
MBD proteins in MCF7 cells were analyzed (7, 24, 25) (Fig. 4A).
Chromatin immunoprecipitation using MTA2, MBD2, and
MBD3 antibodies followed by real-time quantitative PCR
analysis revealed the recruitment of these proteins to two CpG
islands, one located close to the first exon of P14ARFand a
second CpG island located before the first exon of P16INK4a
(Fig. 4B). Several other tested CpG islands did not recruit
MTA2, MBD2, and MBD3. Next, we performed ChIPs using
an antibody against PRMT5, and this revealed the recruitment
of PRMT5 to the P14ARFand P16INK4aCpG islands. These
results provide a functional link between MBD2 and the argi-
nine methyltransferase PRMT5 in vivo.
The biochemical experiments described in this study re-
vealed that MBD2 and MBD3 are mutually exclusive and that
PRMT5 interacts with MBD2 but not with MBD3. The ChIP
experiments indicate that MBD2 and PRMT5 as well as MBD3
are recruited to the P14ARFand P16INK4aCpG islands. To
investigate whether the recruitment of MBD2, PRMT5, and
MBD3 to these loci was dependent on CpG methylation, we
treated MCF7 cells with AzaDc, a specific inhibitor of DNA
methylation, and subsequently investigated the recruitment of
MBD2, MBD3, PRMT5, and MTA2 to the P14ARFand
P16INK4aCpG islands. As shown in Fig. 4C, AzaDc treatment
resulted in a significant loss of MBD2 binding. Strikingly, a
reduction of PRMT5 binding to the loci could be observed,
indicating that MBD2 and PRMT5 are binding to the P14ARF
and P16INK4aCpG islands in a methylation-sensitive manner.
In contrast, recruitment of MBD3 to these loci was only mod-
erately affected. MTA2, a protein present in both the MBD2
and MBD3 complexes, was reduced about 50%. Thus, MBD2
and PRMT5 are recruited to the CpG islands in a methylation-
dependent manner, whereas MBD3 is only partially affected,
supporting the notion that MBD2/PRMT5 and MBD3 are at
least to some extent assembling on the CpG islands as distinct
complexes. PRMT5 recruitment to promoters is known to cor-
relate with arginine methylation of histones (8). To investigate
whether PRMT5 recruitment to the P14ARFand P16INK4a
CpG islands correlates with arginine methylation of histone
H4, we performed chromatin immunoprecipitation experi-
ments on MCF7 cells treated with or without AzaDc using
an antibody against dimethylated histone H4R3. As shown in
Fig. 4C recruitment of PRMT5 to the P14ARFand P16INK4a
CpG islands correlates with an enrichment in the level of
arginine dimethylated histone H4R3. Strikingly, treatment of
MCF7 cells with AzaDc resulted in a reduction of the level
VOL. 26, 2006 MBD2 AND MBD3 DEFINE DISTINCT NuRD COMPLEXES847
of dimethylated histone H4R3. Thus, PRMT5 recruitment to
the P14ARFand P16INK4aCpG islands correlates with histone
In conclusion the experiments described in this study in-
dicate that MBD2 and MBD3 assemble in distinct Mi-2/
NuRD-like complexes and are mutually exclusive. Further-
more, PRMT5 binds to and methylates MBD2 and is recruited
together with an MBD2-containing Mi-2/NuRD complex to
CpG islands in a methylation-dependent manner in vivo.
In this study we set out to gain insights into the function of
MBD2 and MBD3. We applied a protein tagging approach to
FIG. 3. PRMT5 interacts with and methylates the N-terminal RG-rich repeat of MBD2. (A) Purified TAP-MBD2 and TAP-MBD3 complexes
were analyzed by Western blotting using a PRMT5 antibody. (B) Sequence of MBD2 with the RG repeats being underlined. (C) In vitro
methylation of purified MBD2 complex upon incubation with S-[14C]adenosylmethionine. Methylated protein is indicated with an arrow. Free label
is indicated with an asterisk. MBD2 complex was purified using IgG beads. The left panel depicts a Western blot analysis of the purified MBD2
complex or a 293 control purification using anti-ProtA-horseradish peroxidase antibody. The arrow shows TAP-tagged MBD2. (D) (Left panels)
In vitro methylation of recombinant GST-RG(n) and GST-MBD2 in the presence of S-[14C]adenosylmethionine and a purified PRMT5/MEP50
fraction (middle panel) or the purified MBD2 complex (top panel). GST-PAH2 was used as a negative control. (Right panels) In vitro methylation
of recombinant GST-RG(n), GST-MBD2 lacking the RG stretch, and GST-MBD3 in the presence of S-[14C]adenosylmethionine and a purified
PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). Free label is indicated with an asterisk. Loading controls for
GST-PAH2, GST-RG(n), GST-MBD2, GST MBD2 lacking the RG stretch, and GST-MBD3 are shown in the bottom panels. (E) Silver-stained
gel of purified N-terminally truncated MBD2 complex from HEK 293 cells. The table shows the FT-MS/MS analysis with all identified proteins
and their respective peptide numbers and percent sequence coverage.
848 LE GUEZENNEC ET AL.MOL. CELL. BIOL.
purify MBD2 and MBD3 complexes from mammalian cells.
Strikingly, although these proteins have been described to
be part of the same complex, we found them to reside in
distinct complexes. MBD2 could not be detected in the
purified MBD3 complex and vice versa. Independent puri-
fication of MBD2 and MBD3 from a double stable cell line
expressing MBD2 and MBD3 with different tags to similar
levels confirmed their mutual exclusiveness. In addition, en-
dogenous MBD2 did not immunoprecipitate MBD3 and vice
versa. Finally, MBD2 could also not be detected in an MBD3
complex purified from HeLa cells stably expressing tagged
MBD3 (unpublished data). Feng and Zhang used conventional
chromatography to purify the MBD2-containing MeCP1 com-
plex from HeLa nuclear extracts and found MBD3 to copurify,
and they argued that these proteins are indeed part of the same
complex (9). However, this purified fraction may in fact be a
mixture of Mi-2/NuRD complexes, some containing MBD2
and others MBD3. As they likely have a very similar binding
affinity for the different chromatographic resins, they would
end up in the same fractions throughout the purifications.
Jiang and coworkers performed yeast two-hybrid assays and
GST pull-downs and found MBD2 and MBD3 to interact di-
rectly (17). Although we cannot exclude that a small fraction of
MBD2 and MBD3 are interacting, our experiments clearly
FIG. 4. MBD2 recruits PRMT5 to chromatin. (A) Schematic representation of the primer sets used in the ChIP experiments. Exons are
indicated with black rectangles. CpG islands are indicated in gray. Primer pairs are indicated with arrows. (B) ChIP analysis of MBD2, MBD3,
PRMT5, and MTA2 in MCF7 cells. Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of
the results from ChIP experiments from three independent chromatin isolations. (C) ChIP analysis of MCF7 cells treated with 5-azacytidine.
Immunoprecipitations were performed with antibodies against MBD2, MBD3, MTA2, PRMT5, and anti-dimethyl-histone H4 (Arg3). Relative
occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three
independent chromatin isolations.
VOL. 26, 2006 MBD2 AND MBD3 DEFINE DISTINCT NuRD COMPLEXES 849
show that the vast majority of MBD2 and MBD3 proteins
independently assemble in distinct Mi-2/NuRD-like com-
plexes, which we propose to call MBD2/NuRD and MBD3/
NuRD, respectively. Based on the different subunit composi-
tions, in particular, the presence of PRMT5, MEP50, and the
importin complex in MBD2 but not MBD3, we hypothesize
that these complexes have different functions inside the cell.
This hypothesis is supported by the AzaDc ChIP experiments
described in Fig. 4 showing a methylation-dependent MBD2/
PRMT5 recruitment to the P14ARFand P16INK4aCpG islands
whereas MBD3 recruitment to these loci is largely indepen-
dent of methylation.
PRMT5 and the Mi-2/NuRD complex. Liquid chromatogra-
phy-MS/MS and Western blot analyses revealed the associa-
tion of the arginine methyltransferase PRMT5 and its associ-
ated protein MEP50 with the MBD2/NuRD complex, whereas
these proteins were lacking in the MBD3/NuRD complex.
Whether PRMT5 is a core subunit of the MBD2/NuRD com-
plex or a protein strongly interacting with the MBD2/NuRD
complex remains to be determined. PRMT5 is recruited to the
MBD2/NuRD complex via the RG-rich N terminus of MBD2.
In addition we provided evidence that PRMT5 can methylate
this RG stretch of MBD2. Therefore, we hypothesize that the
RG stretch of MBD2 might serve a dual purpose as a substrate
and as a docking site for PRMT5. PRMT5 has been shown to
function in repression of tumor suppressor genes, presumably
by adding repressive arginine methyl marks to the histone H3
and H4 tails (29). In agreement with this we found PRMT5 to
colocalize with MBD2 on P14ARFand P16INK4aCpG islands,
and this correlates with histone H4R3 dimethylation, thus pro-
viding a functional link between PRMT5 and MBD2 in vivo.
Mi-2/NuRD, a family of protein complexes. Since its first
description some 7 years ago, the Mi-2/NuRD complex has
generally been regarded as one biochemical entity containing a
number of core polypeptides. However, our study clearly re-
veals the presence of MBD2/NuRD and MBD3/NuRD com-
plexes with distinct subunit compositions. Previous observa-
tions from a number of labs have revealed the existence of
additional Mi-2/NuRD complexes defined for example by dif-
ferent MTA variants, which may allow for a further fine-tuning
of different Mi-2/NuRD complexes (4, 11, 21, 33, 44). Finally,
in addition to altering protein composition, posttranslational
modifications of different Mi-2/NuRD subunits (unpublished
data) also may play an important role in regulating its function.
The results described in this study lead us to propose a
feed-forward mechanism of repression by different Mi-2/
NuRD complexes. The different enzymatic activities gathered
within a single protein complex may act synergistically to reg-
ulate repression of MBD2 target genes: deacetylation of nu-
cleosomes surrounding the targeting site in combination with
the addition of transcriptional repressive arginine methyl
marks in the H4 tail by the associated PRMT5 (29). Further-
more, chromatin remodeling catalyzed by the ATPase Mi-2
may occur. The hypoacetylated and arginine methylated nu-
cleosomes surrounding the MBD2/PRMT5 targeting site in
turn may provide a binding scaffold for the MBD3/NuRD
complex, a complex which has a high affinity for hypoacetylated
nucleosomes (data not shown). This results in the co-occur-
rence of the MBD2/NuRD and MBD3/NuRD complexes on
some CpG islands. Further deacetylation of nucleosomes by
the MBD3/NuRD complex can then facilitate spreading of
deacetylation and maintenance of transcriptional repression.
Unraveling the functions unique to each Mi-2/NuRD complex
is a challenging task that lies ahead.
We thank Jan van der Knaap for the PRMT5/MEP50 protein frac-
tion, Elly van Tiel for active involvement during a Molecular Biology
practical course, and Colin Logie for technical assistance with the FLP
recombinase. Furthermore we acknowledge A. Bird, B. Luscher, R.
Bernards, and M Knuesel for plasmids; J. Conaway for the 293 FLP
cells; and R. Delwel for MBD3 antibody. Finally we thank members of
the Stunnenberg lab for discussions and critical reading of the manu-
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