Strand-biased DNA methylation associated with centromeric regions in Arabidopsis

The Arabidopsis genome project assembled 15 megabases of heterochromatic sequence, facilitating investigations of heterochromatin assembly, maintenance, and structure. In many species, large quantities of methylcytosine decorate heterochromatin; these modifications are typically maintained by methyltransferases that recognize newly replicated hemimethylated DNA. We assessed the extent and patterns of Arabidopsis heterochromatin methylation by amplifying and sequencing genomic DNA treated with bisulfite, which converts cytosine, but not methylcytosine, to uracil. This survey revealed unexpected asymmetries in methylation patterns, with one helix strand often exhibiting higher levels of methylation. We confirmed these observations both by immunoprecipitating methylated DNA strands and by restriction enzyme digestion of amplified, bisulfite-treated DNA. We also developed a primer-extension assay that can monitor the methylation status of an entire chromosome, demonstrating that strand-specific methylation occurs predominantly in the centromeric regions. Conventional models for methylation maintenance do not explain these unusual patterns; instead, new models that allow for strand specificity are required. The abundance of Arabidopsis strand-specific modifications points to their importance, perhaps as epigenetic signals that mark the heterochromatic regions that confer centromere activity.     

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.     

5-Methyl-2''-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.     

An N-Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.     

DNA methylation at asymmetric sites is associated with numerous transition mutations.

We describe two unusual 5S RNA regions from Neurospora crassa that are tightly linked. Sequence analysis suggests that these genes or pseudogenes, which we designate zeta (zeta) and eta (eta), arose by a 794-base-pair tandem duplication followed by hundreds of exclusively cytosine to thymine mutations. The duplication was most likely generated by nonhomologous recombination involving a DNA segment having a striking purine-pyrimidine strand asymmetry. Restriction analysis of genomic DNA from tissue grown in the presence or absence of 5-azacytidine indicates that many, and perhaps all, cytosines in the duplicated region are methylated in most cells. This is in contrast to the situation typically observed in eukaryotes, where 5-methylcytosine is found only at positions one or two nucleotides preceding guanine residues. No DNA methylation was detected in the unique DNA flanking the zeta-eta duplication. Thus the "signal" for methylation may be the duplication itself. The numerous transition mutations in this region probably occurred by deamination of 5-methylcytosines. Our results suggest that DNA methylation can have important evolutionary consequences in eukaryotes.     

Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine.

The base-pairing interactions of promutagenic O6-methylguanine (O6-MeGua) with cytosine and thymine in deuterated chloroform were investigated by 1H NMR spectroscopy. Nucleosides were derivatized at hydroxyl positions with triisopropylsilyl groups to obtain solubility in nonaqueous solvents and to prevent the ribose hydroxyls from forming hydrogen bonds. We were able to observe hydrogen-bonding interactions between nucleic acid bases in a solvent of low dielectric constant, a condition that approximates the hydrophobic interior of the DNA helix. O6-MeGua was observed to form a hydrogen-bonded mispair with thymine. Whereas O6-MeGua did not form hydrogen bonds with cytosine (via usual, wobble, or unusual tautomeric structures), it did form a 1:1 hydrogen-bonded complex with protonated cytosine. The pairing of unprotonated cytosine in chloroform is thus consistent with the known preference of O6-MeGua for thymine over cytosine in polymerase reactions. In contrast, the pairing of protonated cytosine is consistent with the greater stability of oligonucleotide duplexes containing cytosine.O6-MeGua as compared with thymine.O6-MeGua base pairs [Gaffney, B. L., Markey, L. A. & Jones, R. A. (1984) Biochemistry 23, 5686-5691]. Our observation that cytosine must be protonated in order to pair with O6-MeGua suggests that the cytosine.O6-MeGua base pair in DNA is stabilized by protonation of cytosine. Through this mechanism, methylation at the O6 position of guanine in double-stranded DNA could promote cross-strand deamination of cytosine (or 5-methylcytosine) to produce uracil (or thymine).     

Efficient removal of uracil from G.U mispairs by the mismatch-specific thymine DNA glycosylase from HeLa cells.

The uracil DNA glycosylases (EC 3.2.2.3)characterized to date remove uracil from DNA irrespective of whether it is basepaired with adenine or mispaired with guanine in double-stranded substrates orwhether it is found in single-stranded DNA. We report here the characterizationof uracil glycosylase activity that can remove the base solely from a mispairwith guanine. It does not recognize uracil either in A.U pairs or insingle-stranded substrates. The enzyme, a 55-kDa polypeptide, was previouslycharacterized as a mismatch-specific thymine DNA glycosylase and was thought tobe responsible solely for the correction (to G.C) of G.T mispairs that arise asa result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine.Given the broader substrate specificity of the enzyme (in addition to uracil andthymine, the protein can also remove 5-bromouracil from mispairs with guanine),we propose that its biological role in vivo may also include the correction of asubset of G.U mispairs inefficiently removed by the more abundant ubiquitousuracil glycosylases, such as those arising from cytosine deamination in G+C-richregions of the genome.     

Microarray-based method for detecting methylation changes of p16^Ink4a gene 5＇-CpG islands in gastric carcinomas

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.     

Efficient detection of point mutations on color-coded strands of target DNA.

Presently available methods for screening largegenetic regions for unknown point mutations are neither flawless norparticularly efficient. We describe an approach, especially well suited toidentifying mutations present in the heterozygous state, that combines severalimprovements in a protocol called fluorescence-assisted mismatch analysis(FAMA). Appropriate gene regions of the wild-type and the putative mutant alleleare simultaneously amplified from genomic DNA by using the polymerase chainreaction, and large DNA fragments, so far up to 800 bp, are end labeled withstrand-specific fluorophores. Aliquots are denatured and reannealed to formheteroduplexes and subjected to conventional cytosine- and thymine-specificmodifications. Cleavages occurring on opposite strands are detected bydenaturing gel electrophoresis using an automated DNA sequencer. Since the DNAfragments derived from the mutant allele are also end labeled, the number ofinformative mispaired bases is doubled compared to conventional searches usingwild-type probes. The sensitivity of detection is also increased, becausedifferential fluorescent end labelling allows the identification and measurementof strand-specific background cleavages at matched cytosine or thymine residues.Automatic superimposition of tracings from different subjects allows mismatchdetection at sites that, because of the nature of the bases involved and of theneighboring sequence, are known to be less susceptible to cleavage. The effectsof the latter parameters have been studied quantitatively on a series of pointmutations found in the human C1-inhibitor gene in patients affected byhereditary angioedema. Dilution experiments have demonstrated that mostmutations are detected even when the mutant chromosome is diluted 10-fold ormore compared with the normal one.     

Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.     

DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning.

The Mov-3 substrain of mice carries Moloney murine leukemia virus as a Mendelian gene in its germ line. All mice segregating the Mov-3 locus activate virus and develop viremia and leukemia. The integrated provirus (i.e., Mov-3 locus) was molecularly cloned from Mov-3 liver DNA as a 16.8 kilobase long EcoRI fragment. Comparison of the cloned and genomic Mov-3 specific EcoRI fragment by restriction enzyme analysis showed no differences in the size of the fragments, indicating that no major sequence rearrangements occurred during cloning. The genomic and cloned Mov-3 DNAs were compared for methylation and infectivity. Analysis with Hha I showed that the genomic proviral and the flanking mouse sequences were methylated at cytosine residues, in contrast to the cloned Mov-3 locus. The cloned Mov-3 locus, however, was highly infectious in a transfection assay (1 x 10(-3) plaque-forming unit per viral genome) in contrast to the genomic Mov-3 DNA (less than 10(-7) per viral genome). Our results suggest that genes containing 5-methylcytosine are not expressed after transfection into susceptible cells and that removal of the methyl groups by molecular cloning in prokaryotes leads to expression generating infectious proviral DNA. If gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, this suggests that DNA methylation may play a causative role in eukaryotic gene regulation.     

Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed "hairpin-bisulfite PCR" to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of approximately 0.96 per site per cell division. For de novo methylation efficiency (E(d)), remarkably different estimates were obtained for the hypermethylated CpG island (E(d) = 0.17), compared with the hypomethylated island on the active-X chromosome (E(d) < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.     

Cloning and nucleotide sequence of the gene for protein X from Saccharomyces cerevisiae.

The gene encoding the protein X component of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been cloned and sequenced. A 487-base fragment of yeast genomic DNA encoding the amino-terminal region of protein X was amplified by the polymerase chain reaction using synthetic oligonucleotide primers based on amino-terminal and internal amino acid sequences. This DNA fragment was used as a probe to select two genomic DNA restriction fragments, which were cloned and sequenced. A 2.1-kilobase insert contains the complete sequence of the protein X gene. This insert has an open reading frame of 1230 nucleotides encoding a presequence of 30 amino acid residues and a mature protein of 380 amino acid residues (Mr, 42,052). Hybridization analysis showed that there is a single copy of the protein X gene and that the size of the mRNA is approximately 1.5 kilobases. Comparison of the deduced amino acid sequences of yeast protein X and dihydrolipoamide acetyltransferase indicates that the two proteins evolved from a common ancestor. The amino-terminal part of protein X (residues 1-195) resembles the acetyltransferase, but the remainder is quite different. There is strong homology between protein X and the acetyltransferase in the amino-terminal region (residues 1-84) that corresponds to the putative lipoyl domain. Protein X lacks the highly conserved sequence His-Xaa-Xaa-Xaa-Asp-Gly near the carboxyl terminus, which is thought to be part of the active site of all dihydrolipoamide acyltransferases.     

Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo.

Gene-specific methylation patterns in mammals play a role in a variety of biological processes in the embryo and adult tissues. These patterns are established during embryo development by a process that involves genome-wide demethylation in the morula and de novo methylation in the pregastrula. To elucidate the mechanism of demethylation in the early mouse embryo, we have injected mouse zygotes with gene sequences that were methylated in vitro by Hpa II methylase and analyzed the methylation status of specific sites in blastocyst DNA. Because it had been propagated in Escherichia coli, the DNA used for these injections was also methylated at adenine residues in GATC sites. This allowed us to eliminate fully methylated, unintegrated DNA by Dpn I digestion and fully unmethylated, integrated DNA that underwent several rounds of replication by Mbo I digestion. The integrated, originally injected DNA strands were in a hemimethylated state and survived this treatment. The methylation status of Hpa II sites in these molecules was analyzed by Hpa II digestion of the genomic DNA isolated from blastocysts, followed by PCR amplification using appropriate primers. The results demonstrate that demethylation is achieved by an active mechanism and that specific sites in imprinted genes escape demethylation, maintaining a methylated state throughout preimplantation development.     

Role of methylation in the modification and restriction of chloroplast DNA in Chlamydomonas.

The different metabolic paths followed by homologous chloroplast DNAs of maternal and paternal origins in zygotes of Chlamydomonas were examined by prelabeling parental cells, before mating them, with [3H]adenine, [3H]thymidine, and [3H]deoxycytidine. Within 6 hr after mating, maternal chloroplast DNA was extensively methylated to 5-methylcytosine and its bouyant density decreased. Paternal chloroplast DNA was largely degraded. Some radioactivity from deoxycytidine of maternal origin reappeared in thymine, and residual paternal DNA contained radioactivity in a base tentatively identified as uracil. These results confirm and extend our previous findings and support our hypothesis that modification (methylation) and restriction enzymes determine maternal inheritance of chloroplast DNA and that the two parental DNAs have different metabolic fates within the zygote.     

Tissue specificity and clustering of methylated cystosines in bovine satellite I DNA.

The positions of all 5-methylcytosine (mC) residues in bovine satellite I DNA were determined by sequence analysis of native purified satellite I DNAs from three bovine tissues as well as from cloned DNA. The EcoRI cleavage units from thymus and liver were found to contain 1,402 residues; that from brain contained 1,401 residues. Satellite I DNA from thymus contained a total of 5.0% mC, whereas that from liver and brain contained 4.4% and 2.6% mC, respectively. Thus, the extent of methylation of this DNA is tissue-specific. So is the location. In each tissue, the location of mCs is nonrandom, consisting of three clusters of heavily methylated regions, each of about 200 bases. However, the extent of methylation within each cluster is tissue-specific. The mCs are located entirely in C-G doublets and primarily in palindromic sequences, C-C-G-G sequences (10 methylatable sites) are almost completely methylated in all tissues examined, but T-G-G-A sequences (16 methylated in all tissues examined, but T-G-G-A sequences (16 metylatable sites) are methylated to different extents in each tissue. Neither the tissue specificity of methylation nor the clustering pattern is detectable by examining only G-C-G-G sites, leading us to emphasize the importance of total sequence determination for genomic DNAs in studies of methylation. The clustering pattern, which is preserved despite a 2-fold difference in mC content between brain and thymus, may indicate a role for DNA methylation in chromatin structure.