The Human Proteins MBD5 and MBD6 Associate with
Heterochromatin but They Do Not Bind Methylated DNA
Sophie Laget1,2, Michael Joulie2, Florent Le Masson3, Nobuhiro Sasai2, Elisabeth Christians3, Sriharsa
Pradhan1, Richard J. Roberts1, Pierre-Antoine Defossez2*
1New England Biolabs, Ipswich, Massachusetts, United States of America, 2CNRS UMR7216, Universite ´ Paris-Diderot, Paris, France, 3CNRS UMR 5547, Universite ´ Toulouse
3, Toulouse, France
Background: MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding
Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA
containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential
epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic
research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental
Principal Findings: Here we report the first functional characterization of MBD5 and MBD6. We have observed that the
proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD.
However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro,
neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested.
Conclusions: Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to
the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a
possible role in epigenetic reprogramming after fertilization.
Citation: Laget S, Joulie M, Le Masson F, Sasai N, Christians E, et al. (2010) The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do
Not Bind Methylated DNA. PLoS ONE 5(8): e11982. doi:10.1371/journal.pone.0011982
Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America
Received June 1, 2010; Accepted July 7, 2010; Published August 6, 2010
Copyright: ? 2010 Laget et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: SL was supported in part by a doctoral fellowship from Centre National de la Recherche Scientifique. SL, SP and RJR acknowledge Don Comb and Jim
Ellard for encouraging basic research and New England Biolabs, Inc. for funding. MJ was supported by a doctoral fellowship from Institut National du Cancer, and
NS was supported by a postdoctoral fellowship from Institut National du Cancer. Work in the lab of PAD is supported by Association pour la Recherche contre le
Cancer and Ligue Nationale contre le Cancer (Comite ´ de Paris). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Publication charges for open access are covered by New England Biolabs.
Competing Interests: SP and RJR are employees of New England Biolabs, Inc. (NEB) and SL is a Ph.D. student supported by NEB. They have no other potential
competing commercial interests such as consultancy, patents, products in development or modified products. This employment does not alter the authors’
adherence to all the PLoS ONE policies on sharing data and materials. The other authors declare that they have no other conflicts of interest.
* E-mail: Pierre-Antoine.Defossez@univ-paris-diderot.fr
DNA methylation is an essential epigenetic mark in mammals.
It regulates the expression of imprinted genes, and possibly also
non-imprinted genes. It maintains the repression of the inactive
X in female mammals. Finally, it is essential to ensure the
transcriptional silencing of repeated sequences [1,2].
Nine different proteins are currently known to bind methylated
DNA in mammals; they are called Methyl-Binding Proteins
(MBPs), and they fall into three structural families . The first
family contains MBD1, MBD2, MBD4, and MeCP2; these
proteins share a domain called methyl-CpG binding domain
(MBD). The second family comprises UHRF1 and UHRF2, which
bind methylated DNA via a SET- and Ring finger- Associated
(SRA) domain. The third family is made up of three related Zinc-
finger proteins: Kaiso, ZBTB4, and ZBTB38.
DNMT3b are essential for mouse viability . In contrast, the
deletion of Mbd1 , Mbd2 , Mbd4 , Mecp2 [8,9], or Kaiso
, yields animals that are viable and fertile. The compound
knockouts Mbd2/Mecp2 and Kaiso/Mbd2/Mecp2 have conse-
quences similar to the single Mecp2 knockout . The only
MBP that has been shown to be essential for development so far is
There areatleastthree possible explanations forthelack ofmajor
phenotypeseen upon deletion ofMBDgenes.First,itispossible that
DNA methylation is essential, but that it does not act primarily by
recruiting MBPs. It could instead serve mostly to inhibit the
interaction of DNA-binding proteins with the genome [13,14].
Second, it is possible that there is extensive redundancy between
MBD proteins. Third, it is possible that other MBD proteins remain
to be found. In support of this last possibility, a systematic search of
the mammalian genome has uncovered 6 additional proteins with
domains related to the MBD: BAZ2A (also called TIP5) and
BAZ2B; the histone methyltransferases KMT1E (also called ESET
or SETDB1) and KMT1F (also called CLLD8 or SETDB2); and
two uncharacterized proteins, KIAA1461 and KIAA1887, that
were renamed MBD5 and MBD6 [15,16].
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The mammalian genes MBD5 and MBD6 contain an intron
within the MBD-coding region; this intron exists in the same
location in the ‘‘canonical’’ MBDs (MBD1–4 and MeCP2), but is
absent from BAZ2A, BAZ2B, SETDB1, and SETDB2 .
Therefore, from an evolutionary standpoint, MBD5 and MBD6
are more closely related to the well-characterized MBDs than
BAZ2A, BAZ2B, SETDB1, and SETDB2. This is also supported
by a phylogenetic analysis based on the amino-acid sequence of
the MBD domain .
MBD5 is expressed in the human brain, and several lines of
evidence link MBD5 mutations with mental disorders. First, a
microdeletion of the MBD5 gene has recently been shown to
correlate with mental retardation in 8 human patients [17,18,19].
Additionally, 4 low-frequency missense variants in the coding
sequence were found in one or more mentally retarded patients
but not in healthy controls . Finally, the MBD5 gene is located
on chromosome 2q23.1, a region in which aberrations are
associated with epilepsy . Mutations in MECP2 cause Rett
syndrome, a neurodevelopmental disorder [8,9], and it is tempting
to speculate, by analogy, that MBD5 is also a protein that binds
methylated DNA and whose loss causes cerebral dysfunctions.
MBD6 is also expressed in the human brain, and it might be
involved in neurodegenerative diseases for the following reasons.
ATXN1 is an RNA-binding protein present in neuron nuclei; the
expansion of its polyglutamine domain causes spinocerebellar
ataxia type 1 (SCA1) . ATXN1L is related to ATX1, with
which it interacts, and it attenuates the neurotoxic effects of
mutant ATXN1 . It was found in a two-hybrid screen that
ATXN1L interacts with MBD6 .
In this study, we have initiated the characterization of the
human proteins MBD5 and MBD6. In particular, we have tested
the hypothesis that they might bind methylated DNA. Our
findings suggest that MBD5 and MBD6 associate with hetero-
chromatin, and that their MBD is involved in this association, but
that the proteins do not bind methylated DNA.
Organization of the MBD5 and MBD6 genes and
The human MBD5 gene has 15 exons (Figure 1A). The Uniprot
database describes two isoforms for MBD5 (Figure 1B): the longer
Figure 1. Organization of the human MBD5 and MBD6 genes and proteins. A- The coding exons of human MBD5 (top) and MBD6 (bottom).
Translation of exons 6–15 of MBD5 yields protein isoform 1; translation of exons 6–9 with retention of the following intron yields protein isoform 2. B-
Organization of the human MBD5 and MBD6 proteins. Amino-acid numbers are indicated. P rich: proline-rich segment; PWWP: PWWP domain; NLS:
putative nuclear localization signal. The amino-acids that are unique to MBD5 isoform 2 are depicted in blue. C- Sequence comparison of all known
human MBD domains. Periods, colons, and stars indicate increasing conservation of a given residue. The arrow shows the residue we mutated in
MBD5 for further experiments. The secondary structure of MeCP2 is shown at the top. ß: Beta-sheet; a: alpha-helix; L: loop.
MBD5, MBD6 Do Not Bind meCpG
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variant, Isoform 1 (Q9P267-1), has 1448 amino-acids, and is
encoded by exons 6 to 15; Isoform 2 (Q9P267-2) is encoded by
exons 6 to 9, with the intron following exon 9 being retained. This
results in a protein of 851 residues, with residues 1–841 shared
with Isoform 1, and residues 842–851 unique to this isoform. We
have been able to detect cDNAs corresponding to both isoforms,
and we have observed in western blotting two MBD5 bands, with
molecular weights consistent with the predicted isoforms (see
following section). As for MBD6, the expression databases suggest
that it is expressed mostly as one species, encoding a protein of
In each protein, the MBD is N-terminal (Figure 1B). Both
MBD5 isoforms contain a stretch of 80 amino-acids that is Proline-
rich (23 out of 80 residues are Proline). The C-terminus of Isoform
1 also contains a domain with a proline-tryptophan-tryptophan-
proline (PWWP) central core. This domain is found (but not
exclusively) in chromatin-associated proteins such as DNMT3A,
DNMT3B, BRD1 and BRPF1. The central portion of MBD6,
accounting for 70% of its total length, is Proline-rich (181 out of
706 residues are Proline). Finally, both MBD5 and MBD6 contain
putative Nuclear Localization Signals (NLS) , and are
therefore predicted to be nuclear proteins.
As pointed out in two earlier studies [15,16], MBD5 and MBD6
present two major differences with other human MBDs: a deletion
of 9 amino-acids in the first third of the MBD, and an insertion of
6 amino-acids in the last third (Figure 1C). The three-dimensional
structure of the MBD from MBD1  and MeCP2  has been
determined; 3 beta-sheets, an alpha-helix, and a hairpin-loop
occur at identical positions in both proteins. The insertion and the
deletion that occur in MBD5 and MBD6 are predicted not to
disrupt any of these features, suggesting that their MBD may have
an overall architecture similar to that of MBD1 and MeCP2.
Detection of two MBD5 protein isoforms in cells
We generated vectors for the expression of HA-tagged Isoform
1 or Isoform 2 of MBD5, which we transfected into human cells.
Western blotting revealed that HA-Isoform 1 had an apparent
molecular weight of 230 kDa, and HA-Isoform 2 an apparent
molecular weight of 110 kDa (Figure 2). We raised rabbit
polyclonal antibodies against MBD5, and used them to probe
total extracts of human cultured cells by western blotting. We
detected an intense band at 110kDa, which superimposes precisely
with the HA-Isoform 2 band (the extra 10 amino-acids of the HA
tag probably generate a molecular weight shift too small to be
detected). There was also a less intense band at 230 kDa,
superimposable with HA-Isoform 1. These results suggest that
the two isoforms of MBD5 are indeed expressed in cultured cells.
MBD5 and MBD6 are differentially expressed in mouse
tissues; MBD5 Isoform 2 is highly expressed in oocytes
We then sought to identify the expression pattern of MBD5 and
MBD6. For this, we quantified their expression in a variety of
mouse tissues by quantitative RT-PCR (Figure 3). Two pairs of
primers were used for MBD5: one spans the exon 14-exon 15
junction and is specific for Isoform 1. The other spans exon 9 and
the following intron; it detects Isoform 2 specifically (See Table 1
for primer sequences). We found that Isoform 1 was expressed in
all tissues, but with a wide range of levels: the lowest levels were
seen in E7 embryos (Embryos at day 7 of development), and the
highest levels in the brain (110-fold higher than E7 embryo) and
testis (45-fold higher than E7 embryo). Isoform 2 is conspicuously
different: its level is relatively homogeneous in the tissues we tested,
but it is very high in oocytes (100-fold higher than in E11 embryos,
the sample with lowest expression). These observations agree with
previous reports of MBD5 expression in the brain, as well as with
data present in public databases (BioGPS, http://biogps.gnf.org/;
MBD5 isoform 1: probe 1456423_at; MBD5 isoform 2: probe
gnf1m21841_at; MBD6: probe gnf1h08707_at).
We also screened for MBD6 expression. The cDNA was
detected in all tissues, with a range of expression more narrow
than for MBD5: there was a 24-fold difference between the
highest-expressing tissue (testis), and the lowest-expressing tissue
We note that MBD5 and MBD6 are highly expressed in organs
where epigenetic reprogramming occurs: in the testis and in
MBD5 and MBD6 can localize to methylated loci; this
requires the MBD
When expressed in mouse cells, most methyl-binding proteins
are found in the pericentric heterochromatin, a compartment
made up of heavily methylated repeats [27,28,29]. Cytologically,
this compartment is easily recognizable: it stains brightly with
Figure 2. Detection of MBD isoforms 1 and 2 in cell extracts.
Human MCF7 cells in culture were transfected with the indicated
expression plasmids. Total cell extracts were probed, in parallel, with
anti-HA antibodies to detect exogenous proteins, and with anti-MBD5
antibodies to detect both endogenous and exogenous proteins. Top:
long exposure of the high-molecular weight region demonstrates the
existence of Isoform 1. Middle: Isoform 2 is detected as a 110-kDa-
migrating species. Bottom: detection of Tubulin proves equal loading
between samples. The arrows indicate the two MBD5 isoforms; position
of migration markers, with their indicative weight in kDa, is indicated.
MBD5, MBD6 Do Not Bind meCpG
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DAPI or Hoechst-33342 because of its AT-rich base composition.
For this reason, we sought to determine the intracellular
localization of the two MBD5 isoforms, and of MBD6, upon
transfection in mouse cells. The proteins were tagged with
enhanced Green Fluorescent Protein (GFP) at their N-terminus,
and expressed in NIH-3T3 cells (Figure 4).
We observed that MBD5 (both isoforms) and MBD6 are nuclear
proteins. Isoform 1 of MBD5 was always found at the chromo-
centers, whereas isoform 2 never was (Figure 4A). To identify the
determinants necessary for chromocentric localization, we intro-
duced inactivating point mutations in the MBD or the PWWP
domains of MBD5 Isoform 1. Mutating either domain resulted in a
complete loss of chromocentric colocalization: the mutant proteins
showed a diffuse nuclear staining. Therefore both the MBD and
PWWP domains are necessary, but neither are sufficient, for
recruitment of MBD5 Isoform 1 to the methylated pericentric
heterochromatin. MBD6 displayed a heterogeneous subnuclear
localization in the cell population: in a quarter of the cells the
protein overlapped with the chromocenters; in the remaining cells
the protein diffused homogeneously within the nucleus (Figure 4B).
We sought to introducea point mutationinthe MBDofMBD6,but
our multiple attempts at inverse PCR were unsuccessful, probably
Figure 3. MBD5 and MBD6 are differentially expressed in mouse tissues. A-MBD5 Isoform 1 is expressed at highest levels in the brain,
whereas isoform 2 is most expressed in oocytes. B- MBD6 is expressed at highest levels in the testis. Expression levels were measured by quantitative
RT-PCR, with normalization to the RPS16 gene.
Table 1. qRT-PCR primers used in this study.
Target Forward sequenceReverse sequence
Mbd5 Isoform 1 GAGGCCATGAGCGAACTGTCTTCCTCCTCTTGGGTTTG
Mbd5 isoform 2 ACGTCCTCCACTCCAGTGATTTCACAATGGGGAAAGGAAC
MBD5, MBD6 Do Not Bind meCpG
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as the result of the unusual sequence characteristics of the MBD6
cDNA. We succeeded, however, in deleting this domain. The
resulting truncated protein was still nuclear, but was never enriched
at chromocenters, indicating that the MBD is necessary for
recruitment to the pericentric heterochromatin.
These results show that MBD5 and MBD6 can be recruited to the
highlymethylated pericentric heterochromatin of mouse cells,and that
with the possibility that these domains bind methylated DNA.
MBD5 and MBD6 can localize at the chromocenters
independently of Dnmt1
We then sought to find out whether the localization of MBD5
and MBD6 at chromocenters required their containing methy-
lated DNA. Mouse fibroblasts lacking DNMT1 have been
established in a p53-null background ; their chromocenters
are undermethylated to varying levels in the cell population
and, in many cells, the hypomethylation is sufficient to prevent
recruitment of methyl-binding proteins.
We transfected GFP fusions of MBD5 and MBD6 into
Dnmt12/2 cells and matching control Dnmt1+/+ cells (Figure 5).
We also included in the transfection an RFP-ZBTB4 expression
construct: as we have previously reported, this protein does not
associate with chromocenters in cells that have lost methylation
In the Dnmt1+/+ cells, we observed a situation identical to that
reported above for 3T3 cells: MBD5 was always associated with
chromocenters, whereas MBD6 was only associated with the
Figure 4. MBD5 and MBD6 can colocalize with methylated regions in mouse nuclei. The indicated proteins were transfected into mouse
3T3 cells. The distribution of the various proteins in fixed cells was recorded, and representative images are provided. When different types of
localization occurred, their approximate proportion is indicated. A-MBD5 isoform 1 always colocalizes with the chromocenters, DAPI-dense regions
that harbor hypermethylated heterochromatin; mutating either MBD or PWWP domain changes this distribution to a diffuse nuclear pattern. Isoform
2 is also incapable of chromocenter localization. B- MBD6 colocalizes with chromocenters in 25% of the observed cells. Upon deletion of the MBD, the
pattern becomes diffuse in all cells.
MBD5, MBD6 Do Not Bind meCpG
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chromocenters in about 25% of transfected cells. ZBTB4 was
always associated with chromocenters (Figure 5A).
We then examined Dnmt12/2 cells. The diffuse nuclear
localization of RFP-ZBTB4 (as opposed to chromocenter associa-
tion) was used to ensure that the transfected cells indeed had a low
level of DNA methylation. In the cells with diffuse ZBTB4, we
observed that MBD5 was still associated with chromocenters.
Similarly, MBD6 sometimes colocalized to chromocenters, even in
cells where ZBTB4 was delocalized.
These results indicate that MBD5 and MBD6 can associate with
chromocenters even in cells where the DNA is demethylated
enough to prohibit recruitment of ZBTB4. This could mean that
MBD5 and MBD6 bind methylated DNA in vivo with an affinity
greater than that of ZBTB4. Alternatively, it could mean that
MBD5 and MBD6 are attracted to pericentric heterochromatin by
a determinant that does not depend on DNA methylation.
The MBD domains of MBD5 and MBD6 do not bind
methylated DNA in vitro
To assess the DNA binding properties of MBD5 and MBD6 in
vitro we performed Electrophoretic Mobility Shift Assay (EMSA)
experiments with oligonucleotides containing cytosines that were
unmethylated or methylated. We carried out 7 different experi-
ments, using various combinations of recombinant proteins and
DNA probes. The MBD domain of human MeCP2 (AA 77–164)
was used as the positive control in most experiments (the
exception, presented in figure 6H and 6I, is explained below),
and we investigated the homologous regions of human MBD5 and
MBD6 (AA 1–93 of each protein).
The first 3 experiments used proteins tagged with 6 Histidines at
the N-terminus (66His tag). All proteins were equally pure and
soluble, and they were used at equal molar amounts (Figure 6A).
Experiment 1 employed probe SL1, an artificial sequence that
contains 2 CpGs (Probe sequences are given in Table 2). We
observed, as expected, that MeCP2 bound the methylated probe,
but not the unmethylated version of the same probe (Figure 6B).
Under the same conditions, neither MBD5 nor MBD6 bound
probe SL1. This experiment was carried out under standard
EMSA conditions: it included non-specific competitor DNA (poly
dA–dC). We hypothesized that these conditions might mask a
positive result if MBD5 and MBD6 bind DNA non-specifically. To
test this possibility, we repeated the binding experiments in the
absence of competitor. Under these conditions, as expected,
MeCP2-MBD interacted non-specifically with DNA: it shifted
both methylated and unmethylated probes. In contrast, both
MBD5 and MBD6 failed to shift the probes (Figure 6B).
We then used the same proteins to carry out an EMSA
experiment in the presence of a limited amount of competitor
DNA, and with an unrelated probe, SL2, that contains 5 CpGs
(Figure 6C). Identical results were obtained: MeCP2 bound the
methylated probe, and, with less efficiency, the unmethylated
probe. MBD5 and MBD6 failed to bind either probe.
To increase the probability of detecting an interaction between
MBD5, MBD6, and DNA, we then moved on to a probe with a
very high CG proportion, SL3: it contains 11 CpG dinucleotides
(Figure 6D). MeCP2 bound the methylated probe, but not the
unmethylated probe. MBD5 and MBD6 did not bind the probe in
either condition. And, again, the removal of competitor DNA
failed to uncover an interaction between MBD5 or MBD6 and
We considered the possibility that the 66His tag interfered with
the function of MBD5 and MBD6. To investigate this, we changed
Figure 5. MBD5 and MBD6 can be recruited to chromocenters in Dnmt12 2/2 2 cells. The various GFP fusions were transfected into mouse
fibroblasts of the indicated genotype. A -ZBTB4, MBD5, and MBD6, are recruited to the chromocenters of Dnmt1+/+ cells. B- MBD5 and MBD6 can be
recruited to the demethylated chromocenters of Dnmt12/2 cells; ZBTB4 does not overlap with the chromocenters in the same cells.
MBD5, MBD6 Do Not Bind meCpG
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Figure 6. The MBD domain of MBD5 and MBD6 does not bind methylated DNA in vitro. A- The MeCP2, MBD5 and MBD6 MBDs, tagged
with 66His, were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. Apparent weights of migration markers, in
kDa, are indicated. B-Gel retardation assay with probe SL1 and the proteins of panel A. In this panel and the following, the probe is depicted as a line;
open circles represent an unmethylated CpG, and filled circles a methylated CpG. In panels 2B–2D, the dash indicates the probe-only lane (no protein
added). ‘‘MeCP2’’ : MBD domain of MeCP2, tagged with 66His; same abbreviation for MBD5 and MBD6. C-Gel retardation assay with probe SL2 and
the proteins of panel A. D-Gel retardation assay with probe SL3 and the proteins of panel A. E- The MeCP2, MBD5 and MBD6 MBDs, tagged with
Maltose-Binding Protein (MBP), were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. F-Gel retardation assay
with probe SL3, and the proteins of panel E. ‘‘MeCP2’’ : MBD domain of MeCP2 tagged with MBP; same abbreviation for MBD5 and MBD6. ‘‘MBP’’ :
Maltose-Binding Protein only lane. G- The MBDs of MBD5 and MBD6, tagged with GST, were expressed in bacteria, purified, and examined on a
MBD5, MBD6 Do Not Bind meCpG
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to a different tag, Maltose-Binding Protein (MBP). MBP-MBD5,
and MBP-MBD6 were highly expressed and soluble; MBP-
MeCP2 was expressed less efficiently (Figure 6E); the three
proteins were used at the same concentration to test interaction
with the CpG-rich probe SL3. MBP-MeCP2 displayed methyla-
tion-dependent interaction with the probe; MBP-MBD5 and
MBP-MBD6 did not interact with either methylated or unmethy-
lated probe, and also failed to significantly bind the probe in the
absence of competitor DNA (Figure 6F).
Our third tagging approach was to fuse the MBD domains to
GST (Figure 6G). Under these conditions we first tested probe
SL4. It contains a single methylated CpG, that can be recognized
by Kaiso, ZBTB4, and ZBTB38 . GST-ZBTB4 was the
positive control in this experiment, and it bound methylated SL4.
GST-MBD5 and GST-MBD6 were inactive (Figure 6H).
It is possible that MBD5 and MBD6 have specific sequence
requirements for binding, and that these requirements are
unfulfilled in any of the probes investigated so far. Therefore we
turned to probe SL5: it is a mixture of oligonucleotides that all
contain a fixed central CpG flanked by six randomized positions
on either side. ZBTB4, which binds methylated DNA in a
sequence-specific fashion, interacted with some of the labeled
oligonucleotides. In contrast, GST-MBD5 and GST-MBD6 did
not show any detectable interaction with probe SL5 (Figure 6I).
We conclude that, in vitro, MBD5 and MBD6 do not bind the
different methylated sequences that were tested.
MBD5 and MBD6 do not bind methylated DNA in vitro
We find that MBD5 and MBD6 do not bind methylated DNA
in vitro. As with any negative conclusion, this has to be carefully
First, it is possible that the proteins expressed in bacteria are not
correctly folded or lack a critical post-translational modification.
One argument against this possibility is that the MBD of MeCP2,
expressed in parallel using the same systems, was functional in our
assays. In addition, a large number of published data show that
bacterially expressed MBD1, MBD2, MBD4, and MeCP2 are
active for binding methylated DNA [25,28]; this establishes that
their MBD need not be post-transcriptionally modified to be
active, and argues that the same is likely to hold for MBD5 and
MBD6. Nevertheless, the identification of a ligand that binds
MBD5 or MBD6, and that could be used as a positive control for
their activity in vitro, will be necessary to formally rule out the
possibility that the bacterially produced proteins are inactive.
A second theoretical possibility is that MBD5 and MBD6 bind
methylated cytosine in a very particular sequence context, which
was not present in the probes we used. Again, this seems unlikely
for the two following reasons. First, MeCP2 favors certain binding
sites in vitro , but changing the ratio of protein, probe, and non-
specific competitor can easily reveal binding to suboptimal sites;
placing multiple CpGs on a probe also easily overrides the
sequence specificity. An illustration of this is provided by the fact
that none of the probes used here contain optimal MeCP2 binding
sites, yet they were clearly bound in vitro. By analogy, if MBD5 and
MBD6 were methyl-binding proteins with specific sites, it seems
likely that we should have detected some binding under the rather
relaxed conditions that were used. Second, we have included in
our tests a randomized probe, which is sufficiently complex to
permit binding of ZBTB4, a methyl-binding protein that requires
a defined consensus around the methylated CpG .
The simplest explanation for our results is that MBD5 and
MBD6 do not bind methylated DNA. This possibility had been
predicted by Hendrich and Tweedie based upon sequence
examination . We concur with their idea that this behavior
results, at least in part, from the 9 amino-acid deletion that
removes a region homologous to loop L1 of MBD1 and MeCP2.
In these two proteins, loop L1 enters the major groove of DNA
and interacts with the DNA backbone; it is critical for recognition
of methylated DNA [25,26].
At least one other MBD protein does not bind methylated
DNA: MBD3 . Interestingly, this loss of function is rather
recent in the course of evolution, as Xenopus MBD3 does bind
methylated DNA, whereas mammalian MBD3 does not . Is it
possible that MBD5 and MBD6 also recently lost their methyl-
binding activity? Database searches readily reveal proteins
containing MBDs related to that of MBD5 and/or MBD6 in
mammals, non-mammalian vertebrates (including Xenopus and
Zebrafish), as well as invertebrate animals (including Amphioxus
and insects). In all of these cases, the MBDs have the same
insertion and deletion as human MBD5 and MBD6. Our
prediction, therefore, is that MBD5 and MBD6 are not methyl-
binding proteins in other species either.
How are MBD5 and MBD6 recruited to pericentric
We report that MBD5 and MBD6 can be recruited to
pericentric heterochromatin even in dnmt12/2 mutant cells. It
could be argued that the residual DNA methylation existing in the
chromocenters of dnmt12/2 cells is sufficient to attract MBD5
and MBD6, however this would imply that both proteins have a
higher affinity for methylated DNA than ZBTB4 and several other
previously known MBPs — a possibility difficult to reconcile with
our in vitro results. A simpler explanation would be that MBD5 and
MBD6 are recruited to chromocenters by a component of
heterochromatin other than methylated DNA. Three hypotheses
are attractive. First: an interaction with RNA. The MBD-
containing protein BAZ2A/TIP5 is recruited to methylated rDNA
Table 2. Oligonucleotides used for EMSA analyses. The
reverse strand is not shown; at the underlined positions either
cytosine (for unmethylated probes), or 5-methyl-cytosine (for
methylated probes), was incorporated during synthesis of the
oligonucleotides. The only exception is probe SL5, which was
methylated in vitro, using SssI.
SL559- GTTTTCCCAGTCACTAC(N6)CG(N6)GTCATAGCTGTTTCCTG -39
Coomassie-stained SDS-PAGE gel. A region of MBD5 larger than just the MBD was included in this experiment, explaining the higher molecular
weight. H-Gel retardation assay with probe SL4. ‘‘ZBTB4’’: Zinc fingers of ZBTB4 fused to GST; ‘‘MBD5’’ : MBD domain of MBD5 fused to GST, same
abbreviation for MBD6. ‘‘GST’’: GST-only lane. I-Gel retardation assay with probe SL5 and the proteins used in panel H. Probe SL5 contains 6
randomized positions (‘‘N’’), on either side of the CpG. Legend as in panel H.
MBD5, MBD6 Do Not Bind meCpG
PLoS ONE | www.plosone.org8August 2010 | Volume 5 | Issue 8 | e11982
repeats by an RNA . The chromocenters produce non-coding
RNA [35,36], so it is possible that a similar mechanism is at work
here. Another possibility would be the interaction with modified
histones: H3K27me1, H3K9me3, and H4K20me3 are enriched in
chromocenters , and the PWWP domain has been shown in
certain proteins to recognize modified histones [38,39].
MBD5 and MBD6: potential roles in epigenetic
MBD5 and MBD6 associate with heterochromatin, and it is
tempting to speculate that they play a role in the epigenetic
regulation of cellular identity. We have found that MBD5 and
MBD6 are expressed at high levels in a few tissues, including brain
and testis. It might be that MBD5 and MBD6 contribute to the
unique epigenetic machinery of neurons or to the global
reorganization of chromatin during spermatogenesis [40,41].
Along similar lines, we report that Isoform 2 of MBD5 is
expressed at very high levels in mouse oocytes. This expression
pattern is reminiscent of other proteins, including Stella, that play
important roles in the epigenetic remodeling that occurs after
fertilization [42,43]. If this specific isoform of MBD5 is also
involved in this process, female Mbd5 mutants might be sterile.
This prediction will have to be tested by experimental work,
including the examination of Mbd52/2 mouse, which are
currently being generated.
Materials and Methods
A cDNA clone containing the Isoform 1 of human MBD5 was
obtained from Origene (Clone reference: SC113547). To obtain
isoform 2, we PCR-amplified this clone with two nested primers
adding the 11 amino-acids that are specific to isoform 2. A cDNA
clone for human MBD6 was obtained from ATCC (Clone
reference: 10437624). Site-directed mutagenesis was performed by
inverse PCR. For imaging, MBD5 (both isoforms), MBD6, and
their mutant derivatives were cloned into peGFP-C2 (Clontech).
The mutants were sequenced, and we verified by western blotting
that the proteins expressed had the expected size. HA-tagged
versions for expression in mammalian cells were constructed by
nested PCR and cloned into pcDNA 3.1 (Invitrogen).
For bacterial protein expression, clones encoding the MBD of
MBD5 and MBD6 were generated by assembly of oligonucleotides
with optimized codons for expression in E. coli; they were cloned
into pET21a (Novagen), and bear an N-terminal 66His tag. We
additionally cloned the MBD of MBD6 and MBD6 into an MBP
fusion plasmid (pMALp2X, New England Biolabs) and a GST
fusion fusion plasmid (pGEX-5X-1, GE Heathcare). The clone
encoding the MBP fusion of MBD of MeCP2 and His-tagged
MBD domain of MeCP2 were a kind gift of Drs. Priscilla Too and
Shuang-Yong Xu, respectively (New England Biolabs).
New England Biolabs supplied all the enzymes and their buffers,
protein and DNA markers, plasmids and competent cells. All PCR
used Phusion Hot start (Finnzyme, Finland). Plasmids were
purified with QiaprepH spin columns (Qiagen, USA), and PCR
products with the WizardHSV Gel and PCR Clean-Up System
(Promega, USA). All plasmid names and origins are given in
Reverse transcription and quantitative PCR
Work with mice and the corresponding protocols have received
the agreement # 5314 from Ministe `re de l’Enseignement
Supe ´rieur et de la Recherche (Paris, France). We extracted RNAs
from mouse placenta and ovaries using Trizol (Invitrogen), and
performed reverse-transcription with Superscript III (Invitrogen)
and oligo-dT. Oocyte cDNAs were prepared as described
Table 3. Plasmids used in this study.
pET21a Vector for bacterial expression of proteinsNovagen
pEGFP-C2 Mammalian expression vector for EGFP Clontech
pCDNA3.1 Mammalian expression vectorInvitrogen
PAD665ZBTB4 cloned into pmRFP-C2Filion 2006
PAD1358 Human MBD5 cDNA (clone SC113547)Origene
PAD1359 Human MBD6 cDNA (clone 10437624)ATCC
PAD1360 MBD5 Isoform 1 cloned into pEGFP-C2 This study
PAD1361MBD5 isoform 2 cloned into pEGFP-C2This study
PAD1362MBD5 Isoform 1 with MBD mutation cloned into pEGFP-C2 This study
PAD1363MBD5 Isoform 1 with PWWP mutation cloned into pEGFP-C2This study
PAD1364 HA-tagged MBD5 Isoform 1 cloned into pCDNA3.1This study
PAD1365HA-tagged MBD5 isoform 2 cloned into pCDNA3.1This study
PAD1366 MBD6 cloned into pEGFP-C2This study
PAD1367MBD6 with deletion of the MBD cloned into pEGFP-C2 This study
PAD1368Bacterial expression vector for 66His-tagged MBD of MBD5This study
PAD1369Bacterial expression vector for 66His-tagged MBD of MBD6This study
PAD1370 Bacterial expression vector for Maltose-Binding-Protein-tagged MBD of MBD5This study
PAD1371 Bacterial expression vector for Maltose-Binding-Protein-tagged MBD of MBD6This study
PAD1372 Bacterial expression vector for GST-tagged MBD of MBD5This study
PAD1373Bacterial expression vector for GST-tagged MBD of MBD6This study
MBD5, MBD6 Do Not Bind meCpG
PLoS ONE | www.plosone.org9August 2010 | Volume 5 | Issue 8 | e11982
previously . We verified that our cDNA preparations were not
contaminated by genomic DNA by performing qPCR in the
absence of reverse transcription. cDNAs isolated from the other
mouse tissues were purchased from Clontech, and were tested for
contamination by the manufacturer. qRT-PCR primer sequences
are in Table 1. We verified that they gave linear amplifications,
and we measured values only within the validated range. In each
sample we measured the abundance of the housekeeping gene
RPS16, and normalized the data using the 2ˆ-(DDCt) method
. The tissue with lowest expression of MBD5 or MBD6 was
arbitrarily set to a value of 1, to permit easier comparisons. The
error bars in figure 3 represent the standard deviation between
three technical replicates in one representative experiment. All
experiments were carried out at least three times. Placental cDNA
was included in all the experiments as an internal control.
Antibodies and western-blotting
The MBD5 rabbit polyclonal antibody was raised against
several peptides of the human protein. For western-blotting, the
purified antibody was used at a dilution of 1:1000 overnight at 4uC
in PBST-5% milk, followed by standard washing and revelation
Transfection and microscopy
NIH-3T3, p532/2, and p532/2;Dnmt12/2 MEFs were
grown on coverslips in 24-well plates with DMEM/10% FBS, and
were transfected using Lipofectamine 2000 (Invitrogen). To
observe GFP, the cells were collected twenty-four hours after
transfection, rinsed with PBS, fixed with 2% paraformaldehyde for
10 minutes, permeabilized with 0.5% Triton X-100 for 4 minutes,
then DNA was stained with Hoechst 33342 or DAPI.
Protein expression and purification
T7 Express fresh transformants of E. coli were grown on agar
plates overnight and used to inoculate a 10 mL preculture, which
was then used to start a full-size culture (generally 1 liter). The
culture was incubated at 37uC until the Optical Density at 600 nm
reached 0.6, then expression was induced with 0.5 mM IPTG at
16uC overnight. After this the cells were pelleted and resuspended
in lysis buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 5%
glycerol, 10 mM Imidazole, 0.1% Triton X-100, 10 mM b-
mercaptoethanol and 1 mM PMSF). The cells were lysed by
sonication, the lysate clarified, and the supernatant applied to Ni-
NTA beads (Qiagen) preequilibrated with 5 bed volumes of Histag
binding buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 5%
glycerol, 10 mM Imidazole). After washing, proteins were eluted
using 2 bed volumes of the Histag elution buffer (20 mM Tris-HCl
pH 8, 500 mM NaCl, 5% glycerol, 200 mM Imidazole, 10 mM
DTT). Proteins were concentrated and buffer exchanged using a
Millipore 3,000 MWCO spin column into the heparin column
binding buffer (20 mM Tris-HCl pH 8, 0.1 mM EDTA, 5%
glycerol, 1 mM DTT). Heparin purifications were performed
using Fast Performance Liquid Chromatography (FPLC) system
and prepacked HiTrapTMHeparin resin (Amersham, UK). While
the MeCP2-MBD protein eluted around 500 mM NaCl, the
MBD of both MBD5 and MBD6 was collected in the flowthrough.
After purification, proteins were stored at 220uC in a 50%
glycerol storage buffer. To prepare GST and MBP proteins, we
used similar steps. The proteins were purified in one step using
GST and MBP resin respectively.
Gel retardation assay
The sequences of the oligonucleotides used are given in Table 2.
Probes SL1, SL2, and SL3, were labeled at the synthesis step with
the fluorescent marker FAM. Probes SL4 and SL5 were
In a typical reaction, 1 nM of probe was incubated for 20 min at
25uC with 1 mM of protein in the following binding buffer: 50 mM
TrisHCl pH 8, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5%
glycerol. For EMSA in panel 6B, C, D and F, non-specific
competitor DNA was poly(dAdC) (6B: 200 ng, 6C: 500 ng, 6D, 6F:
1 ug). In panel 6H–6I, we used 1 ug of poly(dIdC). Protein-DNA
complexes were analyzed on 6% polyacrylamide TBE gels run in
0.5 TBE at 4uC for 1 hour at 100 V. Images were acquired with a
Typhoon imager (for fluorescent probes), or a Phosphor-Imager (for
We thankIttai Ben-PorathandHowardCedarforthegift ofthednmt12/2
cells, Priscilla Too and Shuang-Yong Xu and for the gift of MeCP2 plasmids
and Ryma Abane and Vale ´rie Mezger for the mouse tissues.
Conceived and designed the experiments: SL RJR PAD. Performed the
experiments: SL MJ FLM NS. Analyzed the data: SL MJ FLM NS ESC SP
RJR PAD. Wrote the paper: SL PAD.
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