The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA

Methyl-DNA binding proteins play an important role in epigenetic gene regulation. The Drosophila genome encodes a single protein (MBD2/3) with extended homologies to the vertebrate methyl-DNA binding proteins MBD2 and MBD3. However, very little is known about its functional properties. We have now characterized an MBD2/3 null mutant allele that is viable and fertile. This mutation caused a strong dominant suppression of position-effect variegation and also resulted in a high rate of chromosome segregation defects during early embryogenesis. Confocal analysis of mutant embryos showed local displacement of MI-2 from DNA and indicated that MBD2/3 is associated with only a subset of MI-2 complexes. In addition, band shift experiments 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 was disrupted in embryos with reduced levels of DNA methylation. Our data provide novel insights into the function of MBD2/3 proteins and strongly suggest the existence of methylation-dependent chromatin structures in Drosophila.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

Methylation of genomes and genes at the invertebrate-vertebrate boundary.

Patterns of DNA methylation in animal genomes are known to vary from an apparent absence of modified bases, via methylation of a minor fraction of the genome, to genome-wide methylation. Representative genomes from 10 invertebrate phyla comprise predominantly nonmethylated DNA and (usually but not always) a minor fraction of methylated DNA. In contrast, all 27 vertebrate genomes that have been examined display genome-wide methylation. Our studies of chordate genomes suggest that the transition from fractional to global methylation occurred close to the origin of vertebrates, as amphioxus has a typically invertebrate methylation pattern whereas primitive vertebrates (hagfish and lamprey) have patterns that are typical of vertebrates. Surprisingly, methylation of genes preceded this transition, as many invertebrate genes have turned out to be heavily methylated. Methylation does not preferentially affect genes whose expression is highly regulated, as several housekeeping genes are found in the heavily methylated fraction whereas several genes expressed in a tissue-specific manner are in the nonmethylated fraction.     

Recognition of methylated DNA through methyl-CpG binding domain proteins

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines through an interplay of hydrogen bonding and cation-π interaction. Through molecular dynamics and quantum-chemistry calculations we investigate the methyl-cytosine recognition process and demonstrate that methylation enhances MBD-mDNA binding by increasing the hydrophobic interfacial area and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex.     

Characterization of Arabidopsis thaliana methyl-CpG-binding domain (MBD) proteins

Cytosine methylation at symmetrical CpG and CpNpG sequences plays a key role in the epigenetic control of plant growth and development; yet, the way by which the methylation signal is interpreted into a functional state has not been elucidated. In animals, the methylation signal is recognized by methyl-CpG-binding domain (MBD) proteins that specifically bind methylated CpG dinucleotides. In Arabidopsis thaliana, 12 putative MBD proteins were identified and classified into seven subclasses. Here, we characterized six MBD proteins representing four subclasses (II, III, IV, and VI) of the Arabidopsis MBD family. We found that AtMBD7 (subclass VI), a unique protein containing a double MBD motif, as well as AtMBD5 and AtMBD6 (subclass IV), bind specifically symmetrically methylated CpG sites. The MBD motif derived from AtMBD6, but not from AtMBD2, was sufficient for binding methylated CpG dinucleotides. AtMBD6 precipitated histone deacetylase (HDAC) activity from the leaf nuclear extract. The examined AtMBD proteins neither bound methylated CpNpG sequences nor did they display DNA demethylase activity. Our results suggest that AtMBD5, AtMBD6, and AtMBD7 are likely to function in Arabidopsis plants as mediators of the CpG methylation, linking DNA methylation-induced gene silencing with histone deacetylation.     

The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

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 retardation.

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.     

Engineering a high-affinity methyl-CpG-binding protein

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.     

Components of the DNA methylation system of chromatin control are RNA-binding proteins

The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA.protein complexes are uncharacterized. Here, we identify a novel set of RNA-binding proteins that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. We show that a subset of the DNMTs and MBD proteins can form RNA.protein complexes. We characterize the MBD protein RNA-binding activity and show that it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. We speculate that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control.     

MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties

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 suppressor, 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.     

Vertebrate genomes code excess proteins with charge periodicity of 28 residues

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.