Methylation pattern of Activator transposase binding sites in maize endosperm.

The maize transposable element Activator (Ac) transposes after replication from only one of the two daughter chromatids. It has been suggested that DNA methylation in conjunction with methylation-sensitive transposase binding to DNA may control the association of Ac transposition and replication. We present here a detailed genomic sequencing analysis of the cytosine methylation patterns of the transposase binding sites within both Ac ends in the wx-m9::Ac allele, where Ac is inserted into the tenth exon of the Waxy gene. The Ac elements in wx-m9::Ac kernels exhibit intriguing methylation patterns and fall into two distinct groups. Approximately 50% of the elements are fully unmethylated at cytosine residues through the 256 nucleotides at the 5' end (the promoter end). The other half is partially methylated between Ac residues 27 and 92. In contrast, at the 3' end, all Ac molecules are heavily methylated between residues 4372 and 4554. The more internally located Ac sequences and the flanking Waxy DNA are unmethylated. Although most methylated cytosines in Ac are in the symmetrical CpG and CpNpG arrangements, nonsymmetrical cytosine methylation is also common in the hypermethylated regions of Ac. These results suggest a model in which differential activation of transposon ends by hemimethylation controls the chromatid selectivity of transposition and the association with replication.     

Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction

Cytosine methylation at CpG dinucleotides is an important control mechanism in development, differentiation, and neoplasia. Bisulfite genomic sequencing and its modifications have been developed to examine methylation at these CpG dinucleotides. To use these methods, one has to (i) manually convert the sequence to that produced by bisulfite conversion and PCR amplification, taking into account that cytosine residues at CpG dinucleotides may or may not be converted depending on their methylation status, (ii) identify relevant restriction sites that may be used for methylation analysis, and (iii) conduct similar steps with the other DNA strand since the two strands of DNA are no longer complementary after bisulfite conversion. To automate these steps, we have developed a macro that can be used with Microsoft Word. This macro (i) converts genomic sequence to modified sequence that would result after bisulfite treatment facilitating primer design for bisulfite genomic sequencing and methylation-sensitive PCR assay and (ii) identifies restriction sites that are preserved in bisulfite-converted and PCR-amplified product only if cytosine residues at relevant CpG dinucleotides are methylated (and thereby not converted to uracil) in the genomic DNA.     

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides

A large part of human genetic disease apparently arises from deamination of cytosine residues in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contains 35 CpG dinucleotides. The CpG dinucleotides in the beta and gamma-globin genes are methylated, while those in the alpha-globin genes are under-methylated. One would therefore have expected the CpG dinucleotides to be a frequent source of mutations in the beta and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG dinucleotides being a frequent source of mutations in both the alpha and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG dinucleotides are abnormally high, which conflicts with published evidence, or that there is a finite chance of some of these in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.     

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.     

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.     

Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.     

PAGE separation of hemi-methylated or unmethylated oligonucleotide substrates to distinguish between maintenance and de novo DNA methyltranferase activity

DNA methyltransferase (DNMT) enzymes catalyze the addition of a methyl group to cytosine residues in DNA. Appropriate cytosine methylation of CpG dinucleotides is required for normal mammalian development and homeostasis, and quantitative methods are necessary to assess DNMT activity in various cell extracts. The method described in this report utilizes incorporation of S-[methyl-(3)H]-adenosyl-L-methionine into hemi-methylated or unmethylated oligonucleotides to distinguish between maintenance and de novo DNMT activity, respectively. However, unlike previously described methods, this protocol uses native polyacrylamide gel electrophoresis to detect the incorporation of radioactivity into substrate oligonucleotides. This approach distinguishes between incorporation of radioactivity into target substrate oligonucleotides and incorporation into non-specific cellular DNA that often contaminates nuclear extracts, and permits the reproducible quantitation and comparison of de novo and maintenance DNMT activities in various cell lines. Electrophoretic separation of the methylated substrates is a cost-effective, specific, and reproducible approach to quantitate DNMT activities in nuclear extracts.     

Restriction sites containing CpG show a higher frequency of polymorphism in human DNA

Unique loci in the human genome were examined with restriction enzymes in order to detect restriction fragment length polymorphisms (RFLPs). Of 31 arbitrary loci, nine were detectably polymorphic, reflecting ten polymorphic restriction sites. Nine of the ten polymorphic sites were revealed with two restriction enzymes, Msp I and Taq I, whose recognition sequences have in common the dimer sequence CpG. The cytosines in the CpG sequence are known to be frequently methylated in mammals, and the occurrence of significant variation in Msp I and Taq I sites supports the view that methylated cytosine residues are hotspots for mutation in mammalian DNA.     

The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression.

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.     

Impact of C5-cytosine methylation on the solution structure of d(GAAAACGTTTTC)2. An NMR and molecular modelling investigation.

The solution structures of d(GAAAACGTTTTC)2 and of its methylated derivative d(GAAAAMe5CGTTTTC)2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1H NMR and 31P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Back-calculations of NOESY spectra and simulations of double-quantum-filtered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program JUMNA. The MORASS software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.     

Effect of CpG methylation on Msp I.

The restriction enzyme Msp I is inhibited by the presence of a methyl moiety at the external cytosine of the sequence CCGG, but is generally unaffected by methylation at the internal cytosine. At specific subsets of this sequence such as the hexanucleotide CCGGCC, however, methylation of the internal cytosine strongly inhibits Msp I digestion, leading to artifacts in the interpretation of DNA methylation analyses. Our results show, for instance, that the CCGG site at the 5' end of the human gamma globin gene, which was thought to be methylated at both the internal and external cytosines, is actually methylated only at the internal CpG residue.     

Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element

Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs. Binding by these proteins was determined to be cytosine methylation-independent. However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE. A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP).     

Effect of Methylation on Local Mechanics and Hydration Structure of DNA

Cytosine methylation affects mechanical properties of DNA and potentially alters the hydration fingerprint for recognition by proteins. The atomistic origin for these effects is not well understood, and we address this via all-atom molecular dynamics simulations. We find that the stiffness of the methylated dinucleotide step changes marginally, whereas the neighboring steps become stiffer. Stiffening is further enhanced for consecutively methylated steps, providing a mechanistic origin for the effect of hypermethylation. Steric interactions between the added methyl groups and the nonpolar groups of the neighboring nucleotides are responsible for the stiffening in most cases. By constructing hydration maps, we found that methylation also alters the surface hydration structure in distinct ways. Its resistance to deformation may contribute to the stiffening of DNA for deformational modes lacking steric interactions. These results highlight the sequence- and deformational-mode-dependent effects of cytosine methylation.     

Detection of DNA Methylation by Dnmt3a Methyltransferase using Methyl-Dependent Restriction Endonucleases

DNA methylation at cytosine residues in CpG sites by DNA methyltransferases (MTases) is associated with various cell processes. Eukaryotic MTase Dnmt3a is the key enzyme that establishes the de novo methylation pattern. A new in vitro assay for DNA methylation by murine MTase Dnmt3a was developed using methyl-dependent restriction endonucleases (MD-REs), which specifically cleave methylated DNA. The Dnmt3a catalytic domain (Dnmt3a-CD) was used together with KroI and PcsI MD-REs. The assay consists in consecutive methylation and cleavage of fluorescently labeled DNA substrates, then the reaction products are visualized in polyacrylamide gel to determine the DNA methylation efficiency. Each MD-RE was tested with various substrates, including partly methylated ones. PcsI was identified as an optimal MDRE. PcsI recognizes two methylated CpG sites located 7 bp apart, the distance roughly corresponding to the distance between the active centers of the Dnmt3a-CD tetramer. An optimal substrate was designed to contain two methylated cytosine residues and two target cytosines in the orientation suitable for methylation by Dnmt3a-CD. The assay is reliable, simple, and inexpensive and, unlike conventional methods, does not require radioactive compounds. The assay may be used to assess the effectiveness of Dnmt3a inhibitors as potential therapeutic agents and to investigate the features of the Dnmt3a-CD function.