Eukaryotic DNA Methylation Research Articles

Epigenetics today and tomorrow

Epigenetics is the science of the heritable properties of the organism that are not associated with changes in the DNA nucleotide sequence but can be indirectly encoded in the genome. The most well-known epigenetic mechanisms (signals) are enzymatic DNA methylation, the histone code (various enzymatic histone modifications including acetylation, methylation, phosphorylation, ubiquitination, etc.), and gene silencing mediated by small RNAs (miRNA, siRNA). All these processes are usually interconnected and even partially interchangeable. This is apparently required for the reliable implementation of epigenetic signaling. Anyway, these processes are closely associated with chromatin structural and functional organization. DNA methylation in plants and animals, performed by site-specific enzymes, cytosine DNA-methyltransferases, produces 5-methylcytosine (m5C) residues in DNA sequences such as CG, CNG, and CNN. Adenine DNA methylation also occurs in plants. The resulting m5C residues in DNA substantially affect the interaction of DNA with different proteins, including regulatory proteins. DNA methylation often prevents DNA binding to such proteins and inhibits gene transcription, but sometimes it is required for binding to other regulatory proteins. Specific m5CpG DNA-binding proteins were described. The binding of such proteins to DNA orchestrates the whole protein ensemble of the transcription machinery and induces its activity. Thus, DNA methylation can serve as a signal of positive and negative control for gene activities. DNA methylation in eukaryotes is species- and tissue-specific. It is regulated by hormones, changes with age, and is one of the mechanisms controlling cellular and sex differentiation. DNA methylation controls all genetic processes: DNA replication, repair, recombination, transcription, etc. Distortions in DNA methylation and other epigenetic signals cause premature aging, cancer, diabetes, asthma, severe mental dysfunctions, etc. Changes in the DNA methylation profile accompany carcinogenesis and are a reliable diagnostic marker of various types of cancer even at the early stages of tumorigenesis. Epigenetic parameters are very important for understanding the somaclonal variation mechanisms; characterization of clones and cell cultures, including stem cells at various differentiation stages; and determination of their differentiation directions. Directed change in the DNA methylation profile is an efficient biotechnological tool for activation of transcription of seed storage protein genes in cereals and it is used, in particular, for an inheritable increase in protein content in wheat grain. The inhibitor of DNA methylation with 5-azacytidine is used for treatment of skin cancer. Various regulators of enzymatic modifications of histones are already used in clinical practice for the treatment of some human and animal diseases. The use of specific small RNAs in the therapy of cancer and other diseases appears to be particularly promising, especially in connection with directed inhibition of transcription of the genes responsible for cell malignization and metastasis. The therapeutic effect of many small biologically active peptides can be largely determined by their action at the epigenetic level. Thus, the phenotype is the product of combined realization of the genome and epigenome. In this regard, P. Medawar’s well-known expression “genetics supposes, epigenetics disposes” sounds quite correct and very impressive. Epigenetics is a quickly developing and very promising science of the 21st century that is already ingrained in biotechnologies, medicine, and agriculture.

A methylation-blocked cascade amplification strategy for label-free colorimetric detection of DNA methyltransferase activity

DNA methyltransferase (MTase), catalyzing DNA methylation in both eukaryotes and prokaryotes, is closely related with cancer and bacterial diseases. Although there are various methods focusing on DNA MTase detection, most of them share common defects such as complicated setup, laborious operation and requirement of expensive analytical instruments. In this work, a simple strategy based on methylation-blocked cascade amplification is developed for label-free colorimetric assay of MTase activity. When DNA adenine methylation (Dam) MTase is introduced, the hairpin probe is methylated. This blocks the amplified generation of G-riched DNAzyme by nicking endonuclease and DNA polymerase, and inhibits the DNAzyme-catalyzed colorimetric reaction. Contrarily, an effective colorimetric reaction is initiated and high color signal is clearly observed by the naked eye in the absence of Dam MTase. A satisfying sensitivity and high selectivity are readily achieved within a short assay time of 77min, which are superior to those of some existing approaches. Additionally, the application of the sensing system in human serum is successfully verified with good recovery and reproducibility, indicating great potential for the practicality in high concentrations of interfering species. By using several anticancer and antimicrobial drugs as model, the inhibition of Dam MTase is well investigated. Therefore, the proposed method is not only promising and convenient in visualized analysis of MTase, but also useful for further application in fundamental biological research, early clinical diagnosis and drug discovery.

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung + host proficient in uracil excision repair.

Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum

DNA cytosine methylation is a widely conserved epigenetic mark in eukaryotes that appears to have critical roles in the regulation of genome structure and transcription. Genome-wide methylation maps have so far only been established from the supergroups Archaeplastida and Unikont. Here we report the first whole-genome methylome from a stramenopile, the marine model diatom Phaeodactylum tricornutum. Around 6% of the genome is intermittently methylated in a mosaic pattern. We find extensive methylation in transposable elements. We also detect methylation in over 320 genes. Extensive gene methylation correlates strongly with transcriptional silencing and differential expression under specific conditions. By contrast, we find that genes with partial methylation tend to be constitutively expressed. These patterns contrast with those found previously in other eukaryotes. By going beyond plants, animals and fungi, this stramenopile methylome adds significantly to our understanding of the evolution of DNA methylation in eukaryotes.

Sensitive Detection of DNA Methyltransferase Using Hairpin Probe-Based Primer Generation Rolling Circle Amplification-Induced Chemiluminescence

DNA methyltransferases (MTases) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the 5-positon of cytosine in CpG islands, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Despite the development of various methods for the MTase assay, most of them are laborious and costly with poor sensitivity. Herein, we develop a highly sensitive chemiluminescence method for the MTase assay using hairpin probe-based primer generation rolling circle amplification (PG-RCA). In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate PG-RCA reaction by hybridizing with the circular DNA template, producing a large number of horseradish peroxidase-mimicking DNAzyme chains, which can catalyze the oxidation of luminal by H2O2 in the presence of hemin to yield distinct chemiluminescence signal. While in the absence of Dam MTase, neither methylation/cleavage nor PG-RCA reaction can be initiated and no chemiluminescence signal is observed. The proposed method exhibits a wide dynamic range from 0.025 to 400 U/mL and an extremely low detection limit of 1.29 × 10(-4) U/mL, which is superior to most conventional approaches for the MTase assay. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

BackgroundGene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data.ResultsWe generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation.ConclusionsThis study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Conservation and divergence in eukaryotic DNA methylation

Cytosine methylation is a common DNA modification found in most eukaryotic organisms including plants, animals, and fungi (1, 2). The addition of a methyl group to cytosine nucleotides in DNA does not change the primary DNA sequence, but the covalent modification of DNA by methylation can impact gene expression and activity in a heritable fashion. This type of epigenetic regulation through DNA methylation appears to be a critical process, in an evolutionary sense, with highly conserved enzymes mediating the process (3). In humans, aberrant DNA methylation has been associated with diseases, including cancer (4). To study cytosine methylation patterns across the genome, researchers have used microarray hybridization or direct sequencing of bisulfite-treated DNA (5). However, mapping methylation of individual cytosines in a given genome has been a challenging task and, accordingly, comparative analysis of genome methylation patterns across species has not been performed. Several new studies have addressed this gap in our understanding of the evolution of cytosine methylation (6, 7). Feng et al. in PNAS (7) used next-generation sequencing to investigate the DNA methylation patterns in eight divergent species, including green algae, flowering plants, insects, and vertebrates. Their data allowed a comprehensive comparison of whole-genome methylation profiles across the plant and animal kingdoms, revealing both conserved and divergent features of DNA methylation in eukaryotes.

Epigenetic regulation of the honey bee transcriptome: unravelling the nature of methylated genes

BackgroundEpigenetic modification of DNA via methylation is one of the key inventions in eukaryotic evolution. It provides a source for the switching of gene activities, the maintenance of stable phenotypes and the integration of environmental and genomic signals. Although this process is widespread among eukaryotes, both the patterns of methylation and their relevant biological roles not only vary noticeably in different lineages, but often are poorly understood. In addition, the evolutionary origins of DNA methylation in multicellular organisms remain enigmatic. Here we used a new 'epigenetic' model, the social honey bee Apis mellifera, to gain insights into the significance of methylated genes.ResultsWe combined microarray profiling of several tissues with genome-scale bioinformatics and bisulfite sequencing of selected genes to study the honey bee methylome. We find that around 35% of the annotated honey bee genes are expected to be methylated at the CpG dinucleotides by a highly conserved DNA methylation system. We show that one unifying feature of the methylated genes in this species is their broad pattern of expression and the associated 'housekeeping' roles. In contrast, genes involved in more stringently regulated spatial or temporal functions are predicted to be un-methylated.ConclusionOur data suggest that honey bees use CpG methylation of intragenic regions as an epigenetic mechanism to control the levels of activity of the genes that are broadly expressed and might be needed for conserved core biological processes in virtually every type of cell. We discuss the implications of our findings for genome-scale regulatory network structures and the evolution of the role(s) of DNA methylation in eukaryotes. Our findings are particularly important in the context of the emerging evidence that environmental factors can influence the epigenetic settings of some genes and lead to serious metabolic and behavioural disorders.

Gene body DNA methylation in plants: a means to an end or an end to a means?

Cytosine methylation is widely known for its role in silencing transposable elements and some genes in plants and mammals. However, whereas methylation of promoter sequences can lead to transcriptional repression, the function of gene body methylation remains elusive. This situation is particularly perplexing in the plant Arabidopsis, where many genes are methylated, specifically, over their body. A new study in this issue of The EMBO Journal shows that gene body methylation results from two conflicting activities, one imposing it at CG sites, and one preventing its extension to CHG sites (where H=A,T or C). Importantly, the latter activity is not targeted towards silent transposable elements and is likely coupled to transcription elongation, suggesting that CHG methylation hinders this step.

Genome-wide high throughput analysis of DNA methylation in eukaryotes

Cytosine methylation is the quintessential epigenetic mark. Two well-established methods, bisulfite sequencing and methyl-DNA immunoprecipitation (MeDIP) lend themselves to the genome-wide analysis of DNA methylation by high throughput sequencing. Here we provide an overview and brief review of these methods. We summarize our experience with MeDIP followed by high throughput Illumina/Solexa sequencing, exemplified by the analysis of the methylated fraction of the Neurospora crassa genome (“methylome”). We provide detailed methods for DNA isolation, processing and the generation of in vitro libraries for Illumina/Solexa sequencing. We discuss potential problems in the generation of sequencing libraries. Finally, we provide an overview of software that is appropriate for the analysis of high throughput sequencing data generated by Illumina/Solexa-type sequencing by synthesis, with a special emphasis on approaches and applications that can generate more accurate depictions of sequence reads that fall in repeated regions of a chosen reference genome.

Functional Reassembly of Split Enzymes On‐Site: A Novel Approach for Highly Sequence‐Specific Targeted DNA Methylation

In mammalian genomes, a significant fraction of the cytosine residues is methylated at the 5-position (5-methylcytosine), and this modified nucleobase is found in 5’-CG-3’ sequences (CpG sites). The methylation pattern of the genome changes during ontogenesis, depends on the tissue and can substantially differ in several diseases, notably cancer. We are only at the beginning of understanding the biological role of DNA methylation in higher organisms, but the emerging view is that methylation of the promoter region of a substantial fraction of genes leads to transcriptional inactivation (gene silencing). Recognition of the importance of DNA methylation in gene regulation raised the possibility of silencing selected genes by exogenous, targeted methylation of their promoters. Directing DNA methylation to predetermined sites, besides being a promising research tool for silencing genes of interest and studying DNA methylation in higher eukaryotes, could lead to therapeutic applications in diseases characterised by aberrant expression of one or a small number of genes. A potential advantage of DNA methylation-mediated gene silencing is that the de novo established methylation pattern is stably propagated by maintenance methylation through cycles of semiconservative replication. The silencing mechanism is likely to involve other epigenetic factors (e.g. , histone methyltransferases, histone deacetylases) recruited by the DNA methylation marks. DNA methylation is catalysed by DNA methyltransferases (MTases), which transfer the activated methyl group from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet or SAM) to their target nucleobase within short DNA recognition sequences ranging from two to eight base pairs (bps). Directing DNA MTases to a preselected recognition sequence (targeted DNA methylation) was pioneered by Xu and Bestor, who genetically fused the bacterial DNA (cytosine-C5) MTase M.SssI (recognition sequence 5’CG-3’) to a zinc finger protein (ZFP) that recognises a 9 bp DNA sequence with high specificity and serves as targeting domain. The idea was that the chimeric protein would bind to the DNA sequence specific for the targeting domain, and the DNA MTase would selectively methylate CpG sequences located close to the binding site of the fusion partner. Indeed, preferential methylation of a CpG sequence located in the vicinity of the ZFP binding site was observed in in vitro experiments. Later research demonstrated that this approach can, in principle, also be applied in vivo for targeting chromosomal as well as mitochondrial DNA. In the most extensive study so far, the catalytic domains of the murine DNA MTases Dnmt3a and Dnmt3b were fused to sequence-specific DNA-binding proteins. In transient cotransfection experiments on human cells, dense methylation of the targeted promoter regions and silencing of the targeted genes was demonstrated. In almost all such studies, ZFPs were used as targeting devices because customised ZFPs can now be engineered to bind with high affinity and sequence specificity to almost any DNA sequence. A key issue of targeted DNA methylation is specificity, that is, the difference between levels of exogenous methylation at the targeted site and nontargeted sites. Analysis of the methylation status of nontargeted sites revealed either methylation far from the target site, 8] or extensive methylation of regions flanking the binding site of the targeting domain. Although efficient gene silencing might require methylation of many CpG sites in a promoter, and thus the latter phenomenon could be an advantage in many cases, high-resolution analysis of the effect of DNA methylation would require a method suitable for the methylation of single CpG sites. Methylation of nontargeted sites is not surprising owing to the inherent affinity of DNA MTases for their recognition sequences (Scheme 1A). Nontargeted methylation limits the use of simple DNA MTase fusions as a research tool and would be a serious obstacle to therapeutic application. Therefore, novel strategies that allow highly sequence-specific targeted DNA methylation are of prime importance. One approach to improving targeting specificity employs DNA MTase variants with reduced DNA-binding affinity. Here, DNA binding of the mutant DNA MTase-ZFP fusions is dominated by the targeting ZFP domain’s improving the specificity. A recent paper by Nomura and Barbas describes another approach that appears to be a significant step towards the goal of methylating single CpG sites within whole genomes. The authors capitalised on previous observations of protein fragment complementa[a] Prof. Dr. E. Weinhold Institut f r Organische Chemie RWTH Aachen University Landoltweg 1, 52056 Aachen (Germany) Fax: (+49)241-80-92528 E-mail : elmar.weinhold@oc.rwth-aachen.de [b] Prof. A. Kiss Institute of Biochemistry Biological Research Centre of the Hungarian Academy of Sciences Temesv7ri krt. 62, 6726 Szeged (Hungary) Fax: (+36)62-433-506 E-mail : kissa@brc.hu

A view of an elemental naturalist at the DNA world (Base composition, sequences, methylation)

The pioneering data on base composition and pyrimidine sequences in DNA of pro- and eukaryotes are considered, and their significance for the origin of genosystematics is discussed. The modern views on specificity and functional role of enzymatic DNA methylation in eukaryotes are described. DNA methylation controls all genetic functions and is a mechanism of cellular differentiation and gene silencing. A model of regulation of DNA replication by methylation is suggested. Adenine DNA methylation in higher eukaryotes (higher plants) was first observed, and it was established that one and the same gene can be methylated at both cytosine and adenine moieties. Thus, there are at least two different and seemingly interdependent DNA methylation systems present in eukaryotic cells. The first eukaryotic adenine DNA-methyltransferase is isolated from wheat seedlings and described: the enzyme methylates DNA with formation of N6-methyladenine in the sequence TGATCA-->TGm6ATCA. It is found that higher plants have endonucleases that are dependent on S-adenosyl-L-methionine (SAM) and sensitive to DNA methylation status. Therefore, as in bacteria, plants seem to have a restriction-modification (R-M) system. A system of conjugated up- and down-regulation of SAM-dependent endonucleases by SAM modulations is found in plants. Revelation of an essential role of DNA methylation in regulation of genetic processes is a fundament of materialization of epigenetics and epigenomics.

DNA Damage, Homology-Directed Repair, and DNA Methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

DNA methylation with a sting: An active DNA methylation system in the honeybee

The existence of DNA methylation in insects has been a controversial subject over a long period of time. The recently completed genome sequence of the honeybee Apis mellifera has revealed the first insect with a full complement of DNA methyltransferases. A parallel study demonstrated that these enzymes are catalytically active and that Apis genes can be methylated in specific patterns. These findings establish bees as a model to analyze the function of DNA methylation systems in invertebrate organisms and might also be important to understand evolutionary aspects of DNA methylation in higher eukaryotes.

A Histone Methylation-Dependent DNA Methylation Pathway Is Uniquely Impaired by Deficiency in Arabidopsis S-Adenosylhomocysteine Hydrolase

S-adenosylhomocysteine hydrolase (SAH) is a key enzyme in the maintenance of methylation homeostasis in eukaryotes because it is needed to metabolize the by-product of transmethylation reactions, S-adenosylhomocysteine (AdoHcy), which causes by-product inhibition of methyltransferases (MTase's). Complete loss of SAH function is lethal. Partial loss of SAH function causes pleiotropic effects including developmental abnormalities and reduced cytosine methylation. Here we describe a novel partial-function missense allele of the Arabidopsis SAH1 gene that causes loss of cytosine methylation specifically in non-CG contexts controlled by the CMT3 DNA MTase and transcriptional reactivation of a silenced reporter gene, without conferring developmental abnormalities. The CMT3 pathway depends on histone H3 lysine 9 methylation (H3 mK9) to guide DNA methylation. Our results suggest that this pathway is uniquely sensitive to SAH impairment because of its requirement for two transmethylation reactions that can both be inhibited by AdoHcy. Our results further suggest that gene silencing pathways involving an interplay between histone and DNA methylation in other eukaryotes can be selectively impaired by controlled SAH downregulation.

Butyrates and decitabine cooperate to induce histone acetylation and granulocytic maturation of t(8;21) acute myeloid leukemia blasts

Core histones are proteins organized in octamers, to which DNA is wrapped more or less tightly, depending on their acetylation status. Gene transcription is regulated by a complex series of epigenetic modifications, i.e., histone modification such as methylation and acetylation, events determined by the enzymatic activity of histone methyltransferases, and histone acetyltransferases, respectively, the latter counterbalanced by histone deacetylases (HDAC). Acetylation of histones facilitates destabilization of DNA-nucleosome interaction and renders DNA more accessible to transcription factors. Methylation of different specific lysine residues of histones is differently linked to euchromatin (transcripted DNA) or heterochromatin (silenced DNA). On the other hand, methylation of the promoter regions of some genes by DNA methyltransferases (DNMT) leads to transcriptional silencing and is a common mechanism to regulate gene expression. In normal eukaryotic cells, DNA methylation and histone acetylation are interdependent and maintain equilibrium, allowing temporal expression of genes. In neoplastic cells, this balance is frequently disrupted. In leukemic cells, hypermethylation of CpG islands in the promoter region of genes critical for cell cycle and maturation is frequent, and DNMTs were found to be overexpressed, findings paralleled by evidence of transcriptional repression of downstream genes. Therefore, the combination of HDAC and DNMT inhibitors has been considered to be a possible therapeutic approach to restore normal gene expression in acute myeloid leukemia (AML) and other diseases. Human AML1/ETO Kasumi cells were exposed to the HDAC inhibitor D1 (O-n-butanoil-2,3-O-isopropylidene-alpha-D: -mannofuranoside) and 5-aza-deoxycytidine (decitabine) alone and in combination. Histone acetylation as measured by flow cytometry was increased following treatment with D1 and the combination of D1 and decitabine. Addition of D1 alone or in combination with decitabine also led to inhibition of cell proliferation and induction of apoptosis. Thus, treatment of AML with HDAC inhibitors such as D1 and DNMT inhibitors such as decitabine might have clinical benefit for patients, especially these presenting subtypes of AML, like AML1/ETO, in which the leukemogenic mechanism involves corepressor protein complexes containing HDAC and DNMT.

DNA-[Adenine] Methylation in Lower Eukaryotes

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase (MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.

DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila

The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methylation programs in general.

Short TpA-rich segments of the ζ-η region induce DNA methylation in Neurospora crassa

The mechanisms that establish DNA methylation in eukaryotes are poorly understood. In principle, methylation in a particular chromosomal region may reflect the presence of a “signal” that recruits methylation, the absence of a signal that prevents methylation, or both. Experiments were carried out to address these possibilities for the 1.6 kb zeta-eta (ζ-η) region, a relict of repeat-induced point mutation (RIP) in the fungus Neurospora crassa. The ζ-η region directs its own de novo methylation at a variety of chromosomal locations. We tested the methylation potential of a nested set of fragments with deletions from one end of the ζ-η region, various internal fragments of this region, chimeras of η and the homologous unmutated allele, theta (θ), and various synthetic variants, integrated precisely in single copy at the am locus on linkage group (LG) VR or the his-3 locus on LG IR. We found that: (1) the ζ-η region contains at least two non-overlapping methylation signals; (2) different fragments of the region can induce different levels of methylation; (3) methylation induced by ζ-η sequences can spread far into flanking sequences; (4) fragments as small as 171 bp can trigger methylation; (5) methylation signals behave similarly, but not identically, at different chromosomal sites; (6) mutation density, per se, does not determine whether sequences become methylated; and (7) neither A:T-richness nor high densities of TpA dinucleotides, typical attributes of methylated sequences in Neurospora, are essential features of methylation signals, but both promote de novo methylation. We conclude that de novo methylation of ζ-η sequences does not simply reflect the absence of signals that prevent methylation; rather, the region contains multiple, positive signals that trigger methylation. These findings conflict with earlier models for the control of DNA methylation, including the simplest version of the collapsed chromatin model.