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.     

Ten members of the Arabidopsis gene family encoding methyl-CpG-binding domain proteins are transcriptionally active and at least one,AtMBD11, is crucial for normal development

Animal proteins that contain a methyl-CpG-binding domain (MBD) are suggested to provide a link between DNA methylation, chromatin remodelling and gene silencing. However, some MBD proteins reside in chromatin remodelling complexes, but do not have specific affinity for methylated DNA. It has recently been shown that the Arabidopsis genome contains 12 putative genes encoding proteins with domains similar to MBD, of which at least three bind symmetrically methylated DNA. Using a bioinformatics approach, we have identified additional domains in a number of these proteins and, on this basis and extended sequence similarity, divided the proteins into subgroups. Using RT-PCR we show that 10 of the AtMBD genes are active and differentially expressed in diverse tissues. To investigate the biological significance of AtMBD proteins, we have transformed Arabidopsis with a construct aimed at RNA interference with expression of the AtMBD11 gene, normally active in most tissues. The resulting 35S::AtMBD11-RNAi plants displayed a variety of phenotypic effects, including aerial rosettes, serrated leaves, abnormal position of flowers, fertility problems and late flowering. Arabidopsis lines with reduced expression of genes involved in chromatin remodelling and transgene silencing show similar phenotypes. Our results suggest an important role for AtMBD proteins in plant development.     

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.     

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.     

DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro-methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.     

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.     

The methyl-CpG binding domain and the evolving role of DNA methylation in animals

DNA methylation occurs in bacteria, fungi, plants and animals, however its role varies widely among different organisms. Even within animal genomes, methylation patterns vary substantially from undetectable in nematodes, to global methylation in vertebrate genomes. The number and variety of proteins containing methyl-CpG binding domains (MBDs) that are encoded in animal genomes also varies, with a general correlation between the extent of genomic methylation and the number of MBD proteins. We describe here the evolution of the MBD proteins and argue that the vertebrate MBD complement evolved to exploit the benefits and protect against the dangers of a globally methylated genome.     

On how mammalian transcription factors recognize methylated DNA

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.     

PRMT11: a new Arabidopsis MBD7 protein partner with arginine methyltransferase activity

Plant methyl-DNA-binding proteins (MBDs), discovered by sequence homology to their animal counterparts, have not been well characterized at the physiological and functional levels. In order better to characterize the Arabidopsis AtMBD7 protein, unique in bearing three MBD domains, we used a yeast two-hybrid system to identify its partners. One of the interacting proteins we cloned is the Arabidopsis arginine methyltransferase 11 (AtPRMT11). Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that the two proteins interact with each other and can be co-isolated. Using GFP fluorescence, we show that both AtMBD7 and AtPRMT11 are present in the nucleus. Further analyses revealed that AtPRMT11 acts as an arginine methyltransferase active on both histones and proteins of cellular extracts. The analysis of a T-DNA mutant line lacking AtPRMT11 mRNA revealed reduced levels of proteins with asymmetrically dimethylated arginines, suggesting that AtPRMT11, which is highly similar to mammalian PRMT1, is indeed a type I arginine methyltransferase. Further, AtMBD7 is a substrate for AtPRMT11, which post-translationally modifies the portion of the protein-containing C-terminal methylated DNA-binding domain. These results suggest the existence of a link between DNA methylation and arginine methylation.     

Molecular characterization of a putative plant homolog of MBD4 DNA glycosylase

Methyl-CpG-binding domain 4 (MBD4) DNA glycosylase is involved in excision of spontaneous deamination products of cytosine and 5-methylcytosine in animals, but it is unknown whether related proteins perform similar functions in plants. We report here the isolation and biochemical characterization of a putative MBD4 homolog from Arabidopsis thaliana, designated as MBD4L (MBD4-like). The plant enzyme lacks the MBD domain present in mammalian MBD4 proteins, but conserves a DNA glycosylase domain with critical residues for substrate recognition and catalysis, and it is more closely related to MBD4 homologs than to other members of the HhH-GPD superfamily. Arabidopsis MBD4L excises uracil and thymine opposite G, and the presence of halogen substituents at C5 of the target base greatly increases its excision efficiency. No significant activity is detected on cytosine derivatives such as 5-methylcytosine or 5-hydroxymethylcytosine. The enzyme binds to the abasic site product generated after excision, which decreases its catalytic turnover in vitro. Both the full-length protein and a N-terminal truncated version retaining the catalytic domain exhibit a preference for a CpG sequence context, where most plant DNA methylation is found. Our results suggest that an important function of Arabidopsis MBD4L is to protect the plant genome from the mutagenic consequences of cytosine and 5-methylcytosine deamination.     

The SRA methyl-cytosine-binding domain links DNA and histone methylation

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.