C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.     

Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading

The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1.     

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.     

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family

Summary Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element ofXenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified CT and GA substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.     

The effect of cytosine methylation on the structure and geometry of the Holliday junction: the structure of d(CCGGTACm5CGG) at 1.5 A resolution

The single crystal structure of the methylated sequence d(CCGGTACm(5)CGG) has been solved as an antiparallel stacked X Holliday junction to 1.5 A resolution. When compared with the parent nonmethylated d(CCGGTACCGG) structure, the duplexes are translated by 3.4 A along the helix axis and rotated by 10.8 degrees relative to each other, rendering the major grooves more accessible overall. A Ca(2+) complex is seen in the minor groove opposite the junction but is related to the B conformation of the stacked arms. At the junction itself, the hydrogen bond from the N4 nitrogen of cytosine C8 to the C7 phosphate at the crossover in the parent structure has been replaced by a water bridge. Thus, this direct interaction is not absolutely required to stabilize the junction at the previously defined ACC trinucleotide core. The more compact methylated junction forces the Na(+) of the protected central cavity of the nonmethylated junction into a solvent cluster that spans the space between the junction crossover and the stacked arms. A series of void volumes within the methylated and the nonmethylated structures suggests that small monovalent cations can fill and vacate this central cavity without the need to unfold the four-stranded Holliday junction completely.     

A molecular understanding of mitoxantrone-DNA adduct formation: effect of cytosine methylation and flanking sequences

When mitoxantrone is activated by formaldehyde it can form adducts with DNA. These occur preferentially at CpG and CpA sequences and are enhanced 2-3-fold at methylated CpG sequences compared with non-methylated sites. We sought to understand the molecular factors involved in enhanced adduct formation at these methylated sites. This required, first, clarification of factors that contributed to the formation of adducts at CpG sites. For this purpose mass spectrometry of an oligonucleotide duplex (containing a single CpG adduct site) was used to confirm the presence of an additional carbon atom (derived from formaldehyde) on the drug-DNA complex. The effect of 3'-flanking sequences was revealed by electrophoretic analysis of oligonucleotide-drug adducts, and the preferred adduct-forming site was identified as 5'-CGG-3'. Radiolabeled studies of drug-DNA adducts confirmed that the site of attachment involved the exocyclic amino of guanine. Molecular modeling analysis of the relative stability of the intercalated form of mitoxantrone was consistent with observed adduct-forming potential of CG sites with varying flanking sequences. The known preference for adduct formation at methylated CG sites was confirmed by energetics calculations and shown to be due to a shift of equilibrium of the intercalated form of the drug from the major groove (at CG sites) to the minor groove (at methylated CG sites). This increases the relative amount of drug that is located adjacent to the N-2 exocyclic amino of guanine in the minor groove, where covalent linkage is facilitated. These results account for the enhanced covalent binding of mitoxantrone to methylated CG sequences and provide a molecular model of the interactions.     

5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases

In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.     

Specific targeting of cytosine methylation to DNA sequences in vivo

Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions.     

An inverted repeat triggers cytosine methylation of identical sequences in Arabidopsis

The Wassilewskija (WS) strain of Arabidopsis has four PAI genes at three sites: an inverted repeat at one locus plus singlet genes at two unlinked loci. These four genes are methylated over their regions of DNA identity. In contrast, the Columbia (Col) strain has three singlet PAI genes with no methylation. To test the hypothesis that the WS inverted repeat locus triggers methylation of unlinked identical sequences, we introduced this locus into the Col background by genetic crosses. The inverted repeat induced de novo methylation of all three unmethylated Col PAI genes, with methylation efficiency varying with the position of the target locus. These results, plus results with inverted repeat transgenes, show that methylation is communicated by a DNA/DNA pairing mechanism.     

N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation

4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.     

The mechanism of target base attack in DNA cytosine carbon 5 methylation

We measured the tritium exchange reaction on cytosine C(5) in the presence of AdoMet analogues to investigate the catalytic mechanism of the bacterial DNA cytosine methyltransferase M.HhaI. Poly(dG-dC) and poly(dI-dC) substrates were used to investigate the function of the active site loop (residues 80-99), stability of the extrahelical base, base flipping mechanism, and processivity on DNA substrates. On the basis of several experimental approaches, we show that methyl transfer is the rate-limiting pre-steady-state step. Further, we show that the active site loop opening contributes to the rate-limiting step during multiple cycles of catalysis. Target base activation and nucleophilic attack by cysteine 81 are fast and readily reversible. Thus, the reaction intermediates involving the activated target base and the extrahelical base are in equilibrium and accumulate prior to the slow methyl transfer step. The stability of the activated target base depends on the active site loop closure, which is dependent on the hydrogen bond between isoleucine 86 and the guanine 5' to the target cytosine. These interactions prevent the premature release of the extrahelical base and uncontrolled solvent access; the latter modulates the exchange reaction and, by implication, the mutagenic deamination reaction. The processive catalysis by M.HhaI is also regulated by the interaction between isoleucine 86 and the DNA substrate. Nucleophilic attack by cysteine 81 is partially rate limiting when the target base is not fully stabilized in the extrahelical position, as observed during the reaction with the Gln(237)Trp mutant or in the cytosine C(5) exchange reaction in the absence of the cofactor.     

Methylated site display (MSD)-AFLP,a sensitive and affordable method for analysis of CpG methylation profiles

Background

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP.

Results

Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5′ end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans.

Conclusion

MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

     

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.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.     

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin–biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.