1 Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer
Feld 580, 69120 Heidelberg, Germany
2 GSF-Forschungszentrum, Institut für Molekulare Immunologie,
Marchioninistrasse 25, 81377 München, Germany
* Author for correspondence (e-mail: f.lyko{at}dkfz.de)
Accepted 8 October 2004
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SUMMARY |
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Key words: DNA methylation, Drosophila, MBD2/3, MI-2
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Introduction |
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Methyl-DNA binding proteins provide an attractive mechanistic link between
DNA methylation and covalent histone modifications
(Bird and Wolffe, 1999). These
proteins specifically bind to methylated DNA and recruit histone-modifying
enzymes to their target sites. The mechanistic details of this process are
best understood for the vertebrate methyl-DNA binding proteins MeCP2 and MBD2.
MeCP2 has been shown to be associated with the transcriptional co-repressor
Sin3a and with histone deacetylase activity
(Jones et al., 1998
;
Nan et al., 1998
). More recent
results have also demonstrated an interaction between MeCP2 and histone
methyltransferase activity (Fuks et al.,
2003
). Together, these data indicate a close physical interaction
between methyl-DNA binding proteins and histone-modifying enzymes. Similar
interactions have also been demonstrated for the vertebrate MBD2 protein. MBD2
has been co-purified with the MI-2 complex that contains the nucleosome
remodelling enzyme MI-2 and the histone deacetylases HDAC1 and HDAC2
(Ng et al., 1999
;
Wade et al., 1999
;
Zhang et al., 1999
). In
addition, the complex also contains the histone-binding proteins RbAp46 and
RbAp48, the metastasis-associated protein 2 (MTA2), and the methyl-binding
domain containing protein MBD3. The latter protein is closely related to MBD2
but it has no detectable methyl-DNA binding activity
(Wade et al., 1999
;
Zhang et al., 1999
).
The vertebrate MBD2 and MBD3 genes are probably the
result of a gene duplication from a common MBD2/3 ancestor
(Hendrich and Tweedie, 2003).
MBD2/3 genes are widely conserved during evolution and homologues
have been described in numerous organisms
(Hendrich and Tweedie, 2003
).
The Drosophila genome also encodes a single MBD2/3 homologue, with
more than 70% amino acid similarity to vertebrate MBD2 and MBD3
(Tweedie et al., 1999
). MBD2/3
is expressed specifically in embryos and two developmentally regulated
isoforms, resulting from alternative splicing, have been described
(Ballestar et al., 2001
;
Marhold et al., 2002
;
Tweedie et al., 1999
): the
long isoform contains all functional domains, while the short isoform
(MBD2/3
) lacks part of the putative methyl-CpG binding domain and an
adjacent Drosophila-specific domain that is not found in the
vertebrate homologues. Consistent with a conserved function of MBD2/3, the
protein has been shown to be associated with some fly homologues of the
vertebrate MI-2 complex (Ballestar et al.,
2001
; Tweedie et al.,
1999
). Intriguingly, the putative methyl-CpG binding domain of
MBD2/3 contains a number of deviations from the consensus MBD that seemed to
be incompatible with a standard methyl-CpG binding activity
(Ballestar et al., 2001
;
Tweedie et al., 1999
). A very
weak association with a CpG-methylated DNA fragment could be demonstrated in
other experiments, but this interaction was observed with the short isoform
and therefore seemed to be independent of the full-length methyl-CpG binding
domain (Roder et al., 2000
).
Together, these results suggested that MBD2/3 might represent a functional
homologue of mammalian MBD3, rather than MBD2
(Ballestar et al., 2001
;
Tweedie et al., 1999
).
We have previously shown that MBD2/3 dynamically associates with
Drosophila chromosomes during embryogenesis and with the Y-chromosome
during spermatogenesis (Marhold et al.,
2002). This observation has been interpreted to reflect a
recruitment of MBD2/3 to epigenetically silenced loci during large-scale
genome activation processes (Marhold et
al., 2002
). We have now characterized a loss-of-function allele
for MBD2/3. Homozygous mutant flies were viable and fertile, but they showed a
high incidence of chromosome segregation defects and a strong suppression of
position-effect variegation. Mutant analysis also revealed a functional
interaction with MI-2 and with CpT/A-methylated DNA. In conclusion, our
combined data support the notion that MBD2/3 represents a functional homologue
of mammalian MBD2. In addition, they reveal novel functions of MBD2/3 in the
regulation of pericentric heterochromatin stability.
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Materials and methods |
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Antibodies
The following antibodies have been described previously: rabbit anti-MI-2
(Brehm et al., 2000), rabbit
anti-MBD2/3 (Ballestar et al.,
2001
), human anti-DNA (NatuTec)
(Kunert et al., 2003
) and
rabbit anti-NAP1 (Lankenau et al.,
2003
). For the generation of the monoclonal antibodies against
MBD2/3, Lou/C rats were immunized with a KLH-coupled peptide
(NALKRKFARSQGGNAAGAAC) that is specific for the long isoform of the protein.
After an 8-week interval, a final boost was given 3 days before fusion of the
rat spleen cells with the murine myeloma cell line P3X63-Ag8.653. Hybridoma
supernatants were tested in an ELISA using the same peptide coupled to
ovalbumin. Antibodies reacting specifically with the peptides were confirmed
by western blotting with 1:1000 diluted hybridoma supernatants. Based on the
western blot results the hybridoma supernatant MBD 8E7 (rat IgG1) was used for
further experiments.
Immunostaining of Drosophila embryos
Embryo immunostaining was performed as described previously
(Marhold et al., 2002).
Briefly, MBD1 or wild-type Oregon R embryos were collected
from a population cage. Embryos were then washed, dechorionated, fixed and
permeabilized. After mounting, embryos were analyzed by confocal
microscopy.
Band shift assays
GST-MBD2/3 and GST-MBD2/3 fusion proteins were obtained by cloning
the coding region of the two isoforms in pGEX4T1 (Amersham) and expression in
BL-21 bacteria according to the manufacturer's protocol. GST-MBD2a was
obtained from Hidetoshi Fujita (Fujita et
al., 2003
). Gel mobility shifts were performed with the DIG Gel
Shift Kit (Roche) according to the manufacturer's protocol, except that the
oligonucleotides were labelled radioactively. To analyze interactions with CpG
methylation we used the double-stranded GAM12 oligonucleotide and its
unmethylated counterpart GAC12 (Lewis et
al., 1992
). To analyze interactions with CpT/A methylation, the
following oligonucleotides were synthesized (MWG-Biotech, Germany): MATF, GAT
AGC TGM AGM TGC AGC TGM AGC TGC AGC TGC AGM TGC ATC; and MATR, CTA TCG ACG TMG
ACG TMG ACG TCG ACG TCG AMG TCG AMG TAG (M represents 5-methylcytosine). For
controls, we also synthesized the corresponding unmethylated oligonucleotides.
Prior to the binding assay, oligonucleotides were boiled for 10 minutes at
95°C in TEN buffer (10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH 8.0) and slowly
cooled down to 22°C. Annealing was verified by standard agarose gel
electrophoresis. Probes were then labelled with T4-polynucleotide kinase (New
England Biolabs) and gamma-[32P]ATP (Amersham), and column-purified
using the Qiaquick nucleotide removal kit (Qiagen). One picomol of
radiolabelled probe was mixed with 100 ng of GST-purified recombinant proteins
(GST-MBD2a, GST-MBD2/3 or GST-MBD2/3
), incubated for 15 minutes at room
temperature and then electrophoresed on a native 8% polyacrylamide gel in
0.5xTBE buffer. As competitor we used the unlabelled probe in 400-fold
concentration.
Analysis of MBD2/3 localization in demethylated embryos
Oregon R embryo collections (0-30 min) were dechorionated and demethylated
with 5-azacytidine, as described previously
(Kunert et al., 2003).
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Results |
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Discussion |
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MBD2/3 is the only gene in the Drosophila genome with
extensive homologies to vertebrate genes encoding methyl-DNA binding proteins
(Adams et al., 2000). This made
the protein a primary candidate for a functional link between methylated DNA
and epigenetic chromatin structures. It has been previously suggested that
MBD2/3 is associated with the Drosophila MI-2 complex
(Ballestar et al., 2001
;
Tweedie et al., 1999
). Our
data confirms this interaction on a functional level and suggests that MBD2/3
acts as a co-repressor that targets the MI-2 complex to methylated DNA. A
similar function has been proposed for vertebrate MBD2
(Feng and Zhang, 2001
). Other,
DNA methylation-independent co-repressors are involved in recruiting the MI-2
complex to a variety of target genes
(Ahringer, 2000
). For example,
the Drosophila hunchback and Tramtrack69 proteins have been implied
in targeting the complex to homeotic and neuronal-specific genes, respectively
(Kehle et al., 1998
;
Murawsky et al., 2001
).
Our results also revealed a detectable interaction between MBD2/3 and
methylated DNA. This interaction appeared to be specific for CpT/A methylation
and could not be seen with a CpG-methylated probe that effectively interacted
with the human MBD2 protein. The differential specificities of the fly and
vertebrate proteins are in agreement with the methylation patterns found in
the respective species. Vertebrates methylate their genome mainly at
symmetrical CpG sequences and human MBD2 showed a corresponding preference for
CpG-methylated DNA. Fly DNA is methylated predominantly at asymmetrical CpT/A
sequences (Kunert et al.,
2003; Lyko et al.,
2000
) and MBD2/3 showed a corresponding preference for
CpT/A-methylated DNA. This difference in specificity might involve some of the
sequences that are found in the N-terminal half of Drosophila MBD2/3,
but not in the vertebrate homologues
(Hendrich and Tweedie, 2003
;
Tweedie et al., 1999
).
Consistently, the CpT/A-binding activity of MBD2/3 was undetectable with the
short isoform of the protein, which lacks most of these non-conserved
sequences. In addition, the long isoform is expressed only during early stages
of embryogenesis and it associates with methylated DNA during the cellular
blastoderm stage, when DNA methylation appears to be most abundant
(Kunert et al., 2003
;
Marhold et al., 2002
). The
short isoform of MBD2/3 is expressed in the mid-to late stages of
embryogenesis (Marhold et al.,
2002
), when DNA methylation levels are much lower
(Kunert et al., 2003
). It is
possible that the transient expression of the long isoform creates a short
window of time for the establishment of DNA methylation-dependent chromatin
structures during Drosophila embryogenesis.
Last, our results can also be used to address the functional similarities
between MBD2/3 and mammalian MBD2/MBD3
(Table 1). The latter two
proteins are highly similar at the sequence level but distinguished by
strikingly different functional characteristics: mouse MBD2 binds methylated
DNA, while MBD3 does not (Hendrich and
Bird, 1998). MBD2 has been shown to be peripherally associated
with the MI-2 complex (Feng and Zhang,
2001
), while MBD3 is an integral component of it
(Ng et al., 1999
;
Wade et al., 1999
;
Zhang et al., 1999
). MBD2
knockout mice were shown to be viable and fertile, while loss of MBD3 resulted
in embryonic lethality (Hendrich et al.,
2001
). Our results showed that MBD2/3 binds to methylated DNA,
that the protein co-localized with only a subset of MI-2 proteins and that
MBD2/3 mutants are viable and fertile. All these characteristics show
unambiguous parallels between Drosophila MBD2/3 and mammalian MBD2
(Table 1) and therefore suggest
that MBD2/3 is a functional homolog of mammalian MBD2, rather than MBD3.
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ACKNOWLEDGMENTS |
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