A human DNA-binding protein is methylation-specific and sequence-specific.

A nuclear protein isolated from human placenta, methylated DNA-binding protein (MDBP), binds selectively to DNA enriched in 5-methylcytosine. We now demonstrate that MDBP is a sequence-specific, as well as methylation-specific, DNA-binding protein. From ten restriction fragments of pBR322 DNA methylated with human DNA methyltransferase, one was bound to MDBP very much more strongly than any of the others. For this preferential binding to MDBP, the DNA had to be methylated. By a DNase I protection experiment (DNase I footprinting), a 22-base sequence within this methylated restriction fragment was shown to be specifically protected by MDBP. The sequence-specificity of MDBP coupled with its dependence on DNA methylation suggests that this is one of the proteins which modulates important functions of human DNA methylation in vivo.     

Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.     

Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster

The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.     

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

The DNA-binding domain of human c-Abl tyrosine kinase promotes the interaction of a HMG chromosomal protein with DNA.

The biological activity of the c-Abl protein is linked to its tyrosine kinase and DNA-binding activities. The protein, which plays a major role in the cell cycle response to DNA damage, interacts preferentially with sequences containing an AAC motif and exhibits a higher affinity for bent or bendable DNA, as is the case with high mobility group (HMG) proteins. We have compared the DNA-binding characteristics of the DNA-binding domain of human c-Abl and the HMG-D protein from Drosophila melanogaster. c-Abl binds tightly to circular DNA molecules and potentiates the interaction of DNA with HMG-D. In addition, we used a series of DNA molecules containing modified bases to determine how the exocyclic groups of DNA influence the binding of the two proteins. Interfering with the 2-amino group of purines affects the binding of the two proteins similarly. Adding a 2-amino group to adenines restricts the access of the proteins to the minor groove, whereas deleting this bulky substituent from guanines facilitates the protein-DNA interaction. In contrast, c-Abl and HMG-D respond very differently to deletion or addition of the 5-methyl group of pyrimidine bases in the major groove. Adding a methyl group to cytosines favours the binding of c-Abl to DNA but inhibits the binding of HMG-D. Conversely, deleting the methyl group from thymines promotes the interaction of the DNA with HMG-D but diminishes its interaction with c-Abl. The enhanced binding of c-Abl to DNA containing 5-methylcytosine residues may result from an increased propensity of the double helix to denature locally coupled with a protein-induced reduction in the base stacking interaction. The results show that c-Abl has unique DNA-binding properties, quite different from those of HMG-D, and suggest an additional role for the protein kinase.     

The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine

DNA cytosine methylation is a widespread epigenetic mark. Biological effects of DNA methylation are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two different structural mechanisms have been established for 5mC recognition in eukaryotes; however, it is still unknown how discrimination of the 5mC modification is achieved in prokaryotes. Here we report the crystal structure of the N-terminal DNA-binding domain (McrB-N) of the methyl-specific endonuclease McrBC from Escherichia coli. The McrB-N protein shows a novel DNA-binding fold adapted for 5mC-recognition. In the McrB-N structure in complex with methylated DNA, the 5mC base is flipped out from the DNA duplex and positioned within a binding pocket. Base flipping elegantly explains why McrBC system restricts only T4-even phages impaired in glycosylation [Luria, S.E. and Human, M.L. (1952) A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol., 64, 557-569]: flipped out 5-hydroxymethylcytosine is accommodated in the binding pocket but there is no room for the glycosylated base. The mechanism for 5mC recognition employed by McrB-N is highly reminiscent of that for eukaryotic SRA domains, despite the differences in their protein folds.     

An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm⁺), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg²⁺, Mn²⁺ and Co²⁺ and the cleavage activity was suppressed by Zn²⁺. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.     

Bovine thymus satellite I DNA sequences, which are highly methylated, are preferentially located in H1-rich chromatin

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5-7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.     

Intragenome distribution of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants (Gossypium hirsutum L.)

Fractionation of DNA of healthy and wilt-infected cotton plants has been carried out according to the reassociation kinetics and the content of GC and 5-methylcytosine in the resulting fractions has been studied. The genome of cotton plant was found to be methylated quite unevenly. The GC rich (GC=64.7 mole%) fraction of highly reiterated sequences (C ₀ t=0–3.7×10^-2) has a high content of 5-methylcytosine (5.8 mole%), whereas the methylation degree of the fraction of unique sequences (C ₀ t487) is very low (the 5-methylcytosine content is about 0.5 mole%). In plants being infected with wilt, the 5-methylcytosine content in DNA of cotton leaves decreases two-fold; no other changes in the structure and molecular population of DNA has been found. The sharp change in the 5-methylcytosine content in DNA of infected plants takes place at the expense of the decrease in the 5-methylcytosine content in fractions of highly reiterated sequences. The methylation degree of unique sequences (structural genes) remains unchanged.