DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.     

Position effect takes precedence over target sequence in determination of adenine methylation patterns in the nuclear genome of a eukaryote, Tetrahymena thermophila.

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N 6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5'-NAT-3', but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation.     

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Effects of DNA binding proteins on DNA methylation in vitro

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.     

DNA methylation in mammalian nuclei

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.     

Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida.

A considerable proportion of cytosine residues in plants are methylated at carbon 5. According to a well-accepted rule, cytosine methylation is confined to symmetrical sequences such as CpG and CpNpG, which provide the signal for faithful transmission of symmetrical methylation patterns by maintenance methylase. Using a genomic sequencing technique, we have analysed cytosine methylation patterns within a hypermethylated and a hypomethylated state of a transgene in Petunia hybrida. Examination of a part of the transgene promoter revealed that in both states m5C residues located within non-symmetrical sequences could be detected. Non-symmetrical C residues in the two states were methylated at frequencies of 5.9 and 31.9%, respectively. Methylation appeared to be distributed heterogeneously, but some DNA regions were more intensively methylated than others. Our results show that at least in a transgene, a heterogeneous methylation pattern, which does not depend on symmetry of target sequences, can be established and conserved.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the 'immediate' methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the 'delayed' methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.     

Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases.

Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.     

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.     

Genome methylation patterns across castes and generations in a parasitoid wasp

Environmental influences shape phenotypes within and across generations, often through DNA methylations that modify gene expression. Methylations were proposed to mediate caste and task allocation in some eusocial insects, but how an insect's environment affects DNA methylation in its offspring is yet unknown. We characterized parental effects on methylation profiles in the polyembryonic parasitoid wasp Copidosoma koehleri, as well as methylation patterns associated with its simple caste system. We used methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) to compare methylation patterns, among (1) reproductive and soldier larvae; and (2) offspring (larvae, pupae, and adults) of wasps that were reared at either high or low larval density and mated in the four possible combinations. Methylation frequencies were similar across castes, but the profiles of methylated fragments differed significantly. Parental rearing density did not affect methylation frequencies in the offspring at any developmental stage. Principal coordinate analysis indicated no significant differences in methylation profiles among the four crossbreeding groups and the three developmental stages. Nevertheless, a clustering analysis, performed on a subset of the fragments, revealed similar methylation patterns in larvae, pupae, and adults in two of the four parental crosses. Nine fragments were methylated at two cytosine sites in all larvae, and five others were methylated at two sites in all adults. Thus, DNA methylations correlate with within‐generation phenotypic plasticity due to caste. However, their association with developmental stage and with transgenerational epigenetic effects is not clearly supported.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the ‘immediate’ methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the ‘delayed’ methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.