Organization of the MBD5 and MBD6 genes and corresponding proteins
The human MBD5 gene has 15 exons (). The Uniprot database describes two isoforms for MBD5 (): the longer 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 1003 amino-acids.
Organization of the human MBD5 and MBD6 genes and proteins.
In each protein, the MBD is N-terminal (). 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 
, 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 (). 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 (). 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.
Detection of MBD isoforms 1 and 2 in cell extracts.
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 (). 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 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).
MBD5 and MBD6 are differentially expressed in mouse tissues.
qRT-PCR primers used in this study.
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 (ovary).
We note that MBD5 and MBD6 are highly expressed in organs where epigenetic reprogramming occurs: in the testis and in oocytes.
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 
. Cytologically, this compartment is easily recognizable: it stains brightly with 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 ().
MBD5 and MBD6 can colocalize with methylated regions in mouse nuclei.
We observed that MBD5 (both isoforms) and MBD6 are nuclear proteins. Isoform 1 of MBD5 was always found at the chromocenters, whereas isoform 2 never was (). To identify the determinants necessary for chromocentric localization, we introduced 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 (). We sought to introduce a point mutation in the MBD of MBD6, but our multiple attempts at inverse PCR were unsuccessful, probably 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 highly methylated pericentric heterochromatin of mouse cells, and that this function requires their respective MBDs. This finding is compatible 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 methylated 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 Dnmt1
−/− cells and matching control Dnmt1
+/+ cells (). 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 
MBD5 and MBD6 can be recruited to chromocenters in Dnmt1−/− cells.
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 chromocenters in about 25% of transfected cells. ZBTB4 was always associated with chromocenters ().
We then examined Dnmt1−/− cells. The diffuse nuclear localization of RFP-ZBTB4 (as opposed to chromocenter association) 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 experiments, 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 , is explained below), and we investigated the homologous regions of human MBD5 and MBD6 (AA 1–93 of each protein).
The MBD domain of MBD5 and MBD6 does not bind methylated DNA in vitro.
The first 3 experiments used proteins tagged with 6 Histidines at the N-terminus (6×His tag). All proteins were equally pure and soluble, and they were used at equal molar amounts (). Experiment 1 employed probe SL1, an artificial sequence that contains 2 CpGs (Probe sequences are given in ). We observed, as expected, that MeCP2 bound the methylated probe, but not the unmethylated version of the same probe (). 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 ().
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. (more ...)
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 (). 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 (). 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 DNA.
We considered the possibility that the 6×His tag interfered with the function of MBD5 and MBD6. To investigate this, we changed to a different tag, Maltose-Binding Protein (MBP). MBP-MBD5, and MBP-MBD6 were highly expressed and soluble; MBP-MeCP2 was expressed less efficiently (); the three proteins were used at the same concentration to test interaction with the CpG-rich probe SL3. MBP-MeCP2 displayed methylation-dependent interaction with the probe; MBP-MBD5 and MBP-MBD6 did not interact with either methylated or unmethylated probe, and also failed to significantly bind the probe in the absence of competitor DNA ().
Our third tagging approach was to fuse the MBD domains to GST (). 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 ().
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 ().
We conclude that, in vitro, MBD5 and MBD6 do not bind the different methylated sequences that were tested.