Methyl-DNA binding proteins play an important role in epigenetic gene regulation. The Drosophila genome encodes a single protein (MBD-like, a.k.a. MBD2/3) with extended homologies to the vertebrate methyl-DNA binding proteins MBD2 and MBD3. However, very little is known about its functional properties. An MBD2/3 null mutant allele has been characterized that is viable and fertile. This mutation causes a strong dominant suppression of position-effect variegation and also results in a high rate of chromosome segregation defects during early embryogenesis. Confocal analysis of mutant embryos shows local displacement of MI-2 from DNA and indicates that MBD2/3 is associated with only a subset of MI-2 complexes. In addition, band shift experiments have demonstrated a specific binding of MBD2/3 to CpT/A-methylated DNA, which reflects the endogenous DNA methylation pattern of Drosophila. Consistently, the localization of MBD2/3 is disrupted in embryos with reduced levels of DNA methylation. These data provide novel insights into the function of MBD2/3 proteins and strongly suggest the existence of methylation-dependent chromatin structures in Drosophila (Marhold, 2004b).

Epigenetic regulation is mediated by DNA methylation and by covalent histone modifications. Both mechanisms are intricately linked at the molecular level. DNA methylation has been shown to be dependent on defined histone modification patterns in several organisms. However, it has also been demonstrated that histone modification patterns can depend on DNA methylation. In particular, a mutual relationship has been demonstrated between DNA methylation and histone methylation in Drosophila (Kunert, 2003; Weissmann, 2003). These results strongly suggest a cooperative action of distinct epigenetic mechanisms (Marhold, 2004b).

Methyl-DNA binding proteins provide an attractive mechanistic link between DNA methylation and covalent histone modifications (Bird, 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, 1998; Nan, 1998). More recent results have also demonstrated an interaction between MeCP2 and histone methyltransferase activity (Fuks, 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, 1999; Wade, 1999; Zhang, 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, 1999; Zhang, 1999; Marhold, 2004 and references therein).

The vertebrate MBD2 and MBD3 genes are probably the result of a gene duplication from a common MBD2/3 ancestor (Hendrich, 2003). MBD2/3 genes are widely conserved during evolution and homologues have been described in numerous organisms (Hendrich, 2003). The Drosophila genome also encodes a single MBD2/3 homologue, with more than 70% amino acid similarity to vertebrate MBD2 and MBD3 (Tweedie, 1999). Drosophila MBD2/3 is expressed specifically in embryos and two developmentally regulated isoforms, resulting from alternative splicing, have been described (Ballestar, 2001; Marhold, 2002; Tweedie, 1999): the long isoform contains all functional domains, while the short isoform (MBD2/3Delta) 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, 2001; Tweedie, 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, 2001; Tweedie, 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, 2000). Together, these results suggested that MBD2/3 might represent a functional homologue of mammalian MBD3, rather than MBD2 (Ballestar, 2001; Tweedie, 1999: Marhold, 2004 and references therein).

MBD2/3 dynamically associates with Drosophila chromosomes during embryogenesis and with the Y-chromosome during spermatogenesis (Marhold, 2002). This observation has been interpreted to reflect a recruitment of MBD2/3 to epigenetically silenced loci during large-scale genome activation processes (Marhold, 2002). This study has characterized a loss-of-function allele for MBD2/3. Homozygous mutant flies were viable and fertile, but they show 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, the 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 (Marhold, 2004b).

The presence of a functional DNA methylation system in Drosophila has been questioned for a long time. The fly homologues of central vertebrate DNA methylation factors have initially been interpreted to be evolutionary remnants with little or no functional significance (Tweedie, 1999). This view has been challenged by the recent demonstration of catalytic activity for the Drosophila DNA methyltransferase homologue Dnmt2 (Kunert, 2003). This study has used a mutant allele for the putative Drosophila methyl-DNA binding protein MBD2/3 to analyze its function. The results showed a strong suppressor effect of MBD2/3 on pericentric position-effect variegation. This indicates a role of the protein in the organization of pericentric heterochromatin. Consistent with this finding, MBD2/3 mutants also showed a high incidence of chromosome segregation defects. A mechanistic link between the stability of pericentric heterochromatin and proper chromosome segregation has also been demonstrated in other organisms, and has been explained by the structural requirements of mitotic spindle attachment sites. MBD2/3 does not localize to pericentric regions in Drosophila embryonic nuclei (Marhold, 2002) and is therefore unlikely to be a structural component of pericentric heterochromatin. However, the protein might play a more indirect role and could be involved in the regulation of genes encoding heterochromatin-associated proteins. In this respect, it is also worth mentioning that MBD2/3 adds to the growing list of epigenetic mediators that play an important role in the modulation of chromosome architecture (Marhold, 2004b).

MBD2/3 is the only gene in the Drosophila genome with extensive homologies to vertebrate genes encoding methyl-DNA binding proteins. This made the protein a primary candidate for a functional link between methylated DNA and epigenetic chromatin structures. It has been suggested that MBD2/3 is associated with the Drosophila MI-2 complex (Ballestar, 2001; Tweedie, 1999). The current 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, 2001). Other, DNA methylation-independent co-repressors are involved in recruiting the MI-2 complex to a variety of target genes. For example, the Drosophila hunchback and Tramtrack69 proteins have been implied in targeting the complex to homeotic and neuronal-specific genes, respectively (Marhold, 2004b).

The current results also reveal a detectable interaction between MBD2/3 and methylated DNA. This interaction appears to be specific for CpT/A methylation and could not be seen with a CpG-methylated probe that effectively interacts 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 shows a corresponding preference for CpG-methylated DNA. Fly DNA is methylated predominantly at asymmetrical CpT/A sequences (Kunert, 2003; Lyko, 2000) and MBD2/3 shows 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, 2003; Tweedie, 1999). Consistently, the CpT/A-binding activity of MBD2/3 is 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, 2003; Marhold, 2002). The short isoform of MBD2/3 is expressed in the mid- to late-stages of embryogenesis (Marhold, 2002), when DNA methylation levels are much lower (Kunert, 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 (Marhold, 2004b).

Last, the current results can also be used to address the functional similarities between MBD2/3 and mammalian MBD2/MBD3. 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, 1998). MBD2 has been shown to be peripherally associated with the MI-2 complex (Feng, 2001), while MBD3 is an integral component of it (Ng, 1999; Wade, 1999; Zhang, 1999). MBD2 knockout mice are viable and fertile, while loss of MBD3 results in embryonic lethality (Hendrich, 2001). The results show that MBD2/3 binds to methylated DNA, that the protein co-localizes 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 and therefore suggest that MBD2/3 is a functional homolog of mammalian MBD2, rather than MBD3 (Marhold, 2004b).

GENE STRUCTURE

cDNA clone length - 872 (AF171098)

Bases in 5' UTR - 51

Exons - 3

Bases in 3' UTR - 140

PROTEIN STRUCTURE

Amino Acids - 226

Structural Domains

C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m(5)CpG. This study reports the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Delta. Interestingly, in vitro binding experiments show that as was the case for vertebrate MBD proteins, dMBD2/3Delta preferentially recognizes m(5)CpG-containing DNA through its MBD. Furthermore, dMBD2/3Delta as well as one of its orthologs in mouse, MBD2b, can function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appear to be dispensable for transcriptional repression by dMBD2/3Delta. Finally, dMBD2/3Delta also can repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions (Roder, 2000).

A search for Drosophila sequences similar to vertebrate methyl-CpG-binding proteins (MBDs) yielded multiple candidates. With the exception of MBD-like, the Drosophila proteins are similar to vertebrate MBD proteins only in the putative methyl-CpG-binding domain. The solution structure of this motif has been solved for MeCP2. It consists of a wedge-shaped structure composed of four antiparallel beta strands on one face and an alpha helix and hairpin loop on the other (Ballestar, 2001).

Drosophila MBD-like, identified as a sequence relative of vertebrate MBD2 and MBD3, is similar to these proteins throughout its length and is encoded by a single gene. Two mRNAs are generated from this locus, one of 1115 bases, a second of 842 bases. The protein products of these alternatively spliced mRNAs differ in the amino acids encoded by exon 2. The two MBD protein homologs have been named MBD-like (product of the 1115-base mRNA) and MBD-likedelta (product of the 842-base mRNA). Both MBD-like isoforms, particularly the portion encoded by the third exon of the Drosophila gene, share extensive sequence similarity with the recently described forms of MBD3 from Xenopus. However, there are several gaps and nonconserved amino acids in the region corresponding to the MBD. The two MBD-like proteins have an opa-like repeat inserted in the loop between strands beta2 and beta3, the region predicted to interact with DNA. In addition, MBD-like lacks the distal portion of the alpha helix making up one face of the wedge. The shorter isoform, MBD-likedelta, completely lacks the fourth beta strand, the alpha helix, and the hairpin loop. Finally, there are numerous amino-acid changes at positions predicted to be crucial for DNA interaction and structural integrity of the domain (Ballestar, 2001).

date revised: 1 June 2005

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