Eukaryotic DNA Methylation Research Articles

DNA cytosine methylation suppresses meiotic recombination at the sex-determining region.

Meiotic recombination between homologous chromosomes is vital for maximizing genetic variation among offspring. However, sex-determining regions are often rearranged and blocked from recombination. It remains unclear whether rearrangements or other mechanisms might be responsible for recombination suppression. Here, we uncover that the deficiency of the DNA cytosine methyltransferase DNMT1 in the green alga Chlamydomonas reinhardtii causes anomalous meiotic recombination at the mating-type locus (MT), generating haploid progeny containing both plus and minus mating-type markers due to crossovers within MT. The deficiency of a histone methyltransferase for H3K9 methylation does not lead to anomalous recombination. These findings suggest that DNA methylation, rather than rearrangements or histone methylation, suppresses meiotic recombination, revealing an unappreciated biological function for DNA methylation in eukaryotes.

Now and then in eukaryotic DNA methylation.

Since the mid-1970s, increasingly innovative methods to detect DNA methylation provided detailed information about its distribution, functions, and dynamics. As a result, new concepts were formulated and older ones were revised, transforming our understanding of the associated biology and catalyzing unprecedented advances in biomedical research, drug development, anthropology, and evolutionary biology. In this review, we discuss a few of the most notable advances, which are intimately intertwined with the study of DNA methylation, with a particular emphasis on the past three decades. Examples of these strides include elucidating the intricacies of 5-methylcytosine (5-mC) oxidation, which are at the core of the reversibility of this epigenetic modification; the three-dimensional structural characterization of eukaryotic DNA methyltransferases, which offered insights into the mechanisms that explain several disease-associated mutations; a more in-depth understanding of DNA methylation in development and disease; the possibility to learn about the biology of extinct species; the development of epigenetic clocks and their use to interrogate aging and disease; and the emergence of epigenetic biomarkers and therapies.

NanoCon: contrastive learning-based deep hybrid network for nanopore methylation detection.

5-Methylcytosine (5mC), a fundamental element of DNA methylation in eukaryotes, plays a vital role in gene expression regulation, embryonic development, and other biological processes. Although several computational methods have been proposed for detecting the base modifications in DNA like 5mC sites from Nanopore sequencing data, they face challenges including sensitivity to noise, and ignoring the imbalanced distribution of methylation sites in real-world scenarios. Here, we develop NanoCon, a deep hybrid network coupled with contrastive learning strategy to detect 5mC methylation sites from Nanopore reads. In particular, we adopted a contrastive learning module to alleviate the issues caused by imbalanced data distribution in nanopore sequencing, offering a more accurate and robust detection of 5mC sites. Evaluation results demonstrate that NanoCon outperforms existing methods, highlighting its potential as a valuable tool in genomic sequencing and methylation prediction. In addition, we also verified the effectiveness of our representation learning ability on two datasets by visualizing the dimension reduction of the features of methylation and nonmethylation sites from our NanoCon. Furthermore, cross-species and cross-5mC methylation motifs experiments indicated the robustness and the ability to perform transfer learning of our model. We hope this work can contribute to the community by providing a powerful and reliable solution for 5mC site detection in genomic studies. The project code is available at https://github.com/Challis-yin/NanoCon.

Encyclopaedia of eukaryotic DNA methylation: from patterns to mechanisms and functions.

DNA methylation is an epigenetic modification with a very long evolutionary history. However, DNA methylation evolves surprisingly rapidly across eukaryotes. The genome-wide distribution of methylation diversifies rapidly in different lineages, and DNA methylation is lost altogether surprisingly frequently. The growing availability of genomic and epigenomic sequencing across organisms highlights this diversity but also illuminates potential factors that could explain why both the DNA methylation machinery and its genome-wide distribution evolve so rapidly. Key to this are new discoveries about the fitness costs associated with DNA methylation, and new theories about how the fundamental biochemical mechanisms of DNA methylation introduction and maintenance could explain how new genome-wide patterns of methylation evolve.

Role of DNA Methylation and Epialleles in Eukaryotes

Epialleles that emerge due to methylation variation in genetically identical individuals are gaining more interest due to their involvement in physiological and pathological processes. These are also important for transgenerational epigenetic inheritance and evolution. Both stable and metastable epialleles have their importance because of their contribution to the alteration of gene expression that may lead to useful traits or diseases. The main aim of this work lies in a comparative study between stable and metastable epialleles and the latest advancements that are helpful for the interpretation and analysis of DNA methylation and epialleles. However, there is so much to discover and understand because of the inadequate knowledge about methylomes of species as well as the naturally occurring epialleles in the wild. We will get more opportunities to apply this knowledge if we have a complete understanding of methylomes and epialleles and their contribution towards the normal functioning of an organism.

Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

CpGmotifs: a tool to discover DNA motifs associated to CpG methylation events

BackgroundThe investigation of molecular alterations associated with the conservation and variation of DNA methylation in eukaryotes is gaining interest in the biomedical research community. Among the different determinants of methylation stability, the DNA composition of the CpG surrounding regions has been shown to have a crucial role in the maintenance and establishment of methylation statuses. This aspect has been previously characterized in a quantitative manner by inspecting the nucleotidic composition in the region. Research in this field still lacks a qualitative perspective, linked to the identification of certain sequences (or DNA motifs) related to particular DNA methylation phenomena.ResultsHere we present a novel computational strategy based on short DNA motif discovery in order to characterize sequence patterns related to aberrant CpG methylation events. We provide our framework as a user-friendly, shiny-based application, CpGmotifs, to easily retrieve and characterize DNA patterns related to CpG methylation in the human genome. Our tool supports the functional interpretation of deregulated methylation events by predicting transcription factors binding sites (TFBS) encompassing the identified motifs.ConclusionsCpGmotifs is an open source software. Its source code is available on GitHub https://github.com/Greco-Lab/CpGmotifs and a ready-to-use docker image is provided on DockerHub at https://hub.docker.com/r/grecolab/cpgmotifs.

N6-Methyladenine in Eukaryotic DNA: Tissue Distribution, Early Embryo Development, and Neuronal Toxicity.

DNA methylation is one of the most important epigenetic modifications and is closely related with several biological processes such as regulation of gene transcription and the development of non-malignant diseases. The prevailing dogma states that DNA methylation in eukaryotes occurs essentially through 5-methylcytosine (5mC) but recently adenine methylation was also found to be present in eukaryotes. In mouse embryonic stem cells, 6-methyladenine (6mA) was associated with the repression and silencing of genes, particularly in the X-chromosome, known to play an important role in cell fate determination. Here, we have demonstrated that 6mA is a ubiquitous eukaryotic epigenetic modification that is put in place during epigenetically sensitive periods such as embryogenesis and fetal development. In somatic cells there are clear tissue specificity in 6mA levels, with the highest 6mA levels being observed in the brain. In zebrafish, during the first 120 h of embryo development, from a single pluripotent cell to an almost fully formed individual, 6mA levels steadily increase. An identical pattern was observed over embryonic days 7–21 in the mouse. Furthermore, exposure to a neurotoxic environmental pollutant during the same early life period may led to a decrease in the levels of this modification in female rats. The identification of the periods during which 6mA epigenetic marks are put in place increases our understanding of this mammalian epigenetic modification, and raises the possibility that it may be associated with developmental processes.

MDR: an integrative DNA N6-methyladenine and N4-methylcytosine modification database for Rosaceae

Eukaryotic DNA methylation has been receiving increasing attention for its crucial epigenetic regulatory function. The recently developed single-molecule real-time (SMRT) sequencing technology provides an efficient way to detect DNA N6-methyladenine (6mA) and N4-methylcytosine (4mC) modifications at a single-nucleotide resolution. The family Rosaceae contains horticultural plants with a wide range of economic importance. However, little is currently known regarding the genome-wide distribution patterns and functions of 6mA and 4mC modifications in the Rosaceae. In this study, we present an integrated DNA 6mA and 4mC modification database for the Rosaceae (MDR, http://mdr.xieslab.org). MDR, the first repository for displaying and storing DNA 6mA and 4mC methylomes from SMRT sequencing data sets for Rosaceae, includes meta and statistical information, methylation densities, Gene Ontology enrichment analyses, and genome search and browse for methylated sites in NCBI. MDR provides important information regarding DNA 6mA and 4mC methylation and may help users better understand epigenetic modifications in the family Rosaceae.

Mutations in the DNMT3A DNA methyltransferase in acute myeloid leukemia patients cause both loss and gain of function and differential regulation by protein partners

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3α (DNMT3A) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B (CDKN2B or p15) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes.

Epigenetics with special reference to the human X chromosome inactivation and the enigma of Drosophila DNA methylation

Epigenetics confers adaptability and survival advantage to an organism. Most epigenetic processes demonstrate memory and heritability. DNA methylation is an epigenetic process that adds imprints which can be inherited during cell division and across generations. DNA methylation adds an additional level of information to the basic DNA sequence and can influence chromatin organization and the function of the DNA sequence. In bacteria, it works as a defence strategy and preserves genome integrity. DNA methylation in eukaryotes has been implicated in a large number of cellular regulatory processes and is implied in development, differentiation, life style diseases and cancer. Mammals have an intricate DNA methylation machinery with dNMT1, 3A and 3B enzymes. The human X chromosome inactivation, an example of differential regulation of homologous chromosomes, is known to involve many epigenetic processes with intricate interactions of lncRNAs, miRNAs and DNA methylation. Drosophila possesses very low levels of DNA methylation with only dNMT2 gene. Since Drosophila is an important model organism for study of development and differentiation, the implications of this sparse DNA methylation and the lack of DNA methylation machinery in Drosophila is discussed.

Circularly permuted variants of two CG-specific prokaryotic DNA methyltransferases.

The highly similar prokaryotic DNA (cytosine-5) methyltransferases (C5-MTases) M.MpeI and M.SssI share the specificity of eukaryotic C5-MTases (5’-CG), and can be useful research tools in the study of eukaryotic DNA methylation and epigenetic regulation. In an effort to improve the stability and solubility of complementing fragments of the two MTases, genes encoding circularly permuted (CP) variants of M.MpeI and M.SssI were created, and cloned in a plasmid vector downstream of an arabinose-inducible promoter. MTase activity of the CP variants was tested by digestion of the plasmids with methylation-sensitive restriction enzymes. Eleven of the fourteen M.MpeI permutants and six of the seven M.SssI permutants had detectable MTase activity as indicated by the full or partial protection of the plasmid carrying the cpMTase gene. Permutants cp62M.MpeI and cp58M.SssI, in which the new N-termini are located between conserved motifs II and III, had by far the highest activity. The activity of cp62M.MpeI was comparable to the activity of wild-type M.MpeI. Based on the location of the split sites, the permutants possessing MTase activity can be classified in ten types. Although most permutation sites were designed to fall outside of conserved motifs, and the MTase activity of the permutants measured in cell extracts was in most cases substantially lower than that of the wild-type enzyme, the high proportion of circular permutation topologies compatible with MTase activity is remarkable, and is a new evidence for the structural plasticity of C5-MTases. A computer search of the REBASE database identified putative C5-MTases with CP arrangement. Interestingly, all natural circularly permuted C5-MTases appear to represent only one of the ten types of permutation topology created in this work.

DNMT3A mutations in AML patients cause loss and gain of function and differential regulation by protein partners

Eukaryotic DNA methylation, an integral component of the epigenetic machinery, prevents genomic instability by regulating the expression of oncogenes and tumor suppressor genes. The importance of dysregulated DNA methylation in diverse blood cancers including Acute Myeloid Leukemia (AML) pathogenesis is highlighted by the strong correlation between mutations in the de novo DNA methyltransferase gene, DNMT3A, and adult patients with poor prognoses. We show that clinically observed DNMT3A mutations result in dramatic changes in enzyme activity, including mutations that lead to 6‐fold hypermethylation and 3‐fold hypomethylation on the p15 human promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN‐AML) constitutes 40–50% of all AML cases, is the most epigenetically diverse AML subtype and has pronounced changes in DNA methylation in non‐CpG regions. We identified a subset of mutations in DNMT3A that lead to a 2–8 fold enhancement in the enzyme's ability to perform non‐CpG methylation. Many of the mutations studied map to regions on the protein that are well known to interact with partner proteins, which themselves contribute to AML, such as the DNA base excision repair (BER) enzyme Thymine DNA glycosylase (TDG). Here we show that DNMT3A mutations cause significant and diverse changes in the ability of these regulatory partner proteins to affect DNMT3A function. Our results present a link between DNMT3A mutations and the disruption of the epigenetic landscape in AML.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genome-wide DNA methylomes from discrete developmental stages reveal the predominance of non-CpG methylation in Tribolium castaneum.

Cytosine DNA methylation is a vital epigenetic regulator of eukaryotic development. Whether this epigenetic modification occurs in Tribolium castaneum has been controversial, its distribution pattern and functions have not been established. Here, using bisulphite sequencing (BS-Seq), we confirmed the existence of DNA methylation and described the methylation profiles of the four life stages of T. castaneum. In the T. castaneum genome, both symmetrical CpG and non-CpG methylcytosines were observed. Symmetrical CpG methylation, which was catalysed by DNMT1 and occupied a small part in T. castaneum methylome, was primarily enriched in gene bodies and was positively correlated with gene expression levels. Asymmetrical non-CpG methylation, which was predominant in the methylome, was strongly concentrated in intergenic regions and introns but absent from exons. Gene body methylation was negatively correlated with gene expression levels. The distribution pattern and functions of this type of methylation were similar only to the methylome of Drosophila melanogaster, which further supports the existence of a novel methyltransferase in the two species responsible for this type of methylation. This first life-cycle methylome of T. castaneum reveals a novel and unique methylation pattern, which will contribute to the further understanding of the variety and functions of DNA methylation in eukaryotes.

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa.

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.

DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles

DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery.

The Influence of DNA Methylation on Bone Cells.

DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain.

Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins.

SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm+), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg2+, Mn2+ and Co2+ and the cleavage activity was suppressed by Zn2+. Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.

One-pot synthesis of GO/AgNPs/luminol composites with electrochemiluminescence activity for sensitive detection of DNA methyltransferase activity

DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the target adenine or cytosine, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Herein, we developed a novel electrochemiluminescence biosensor to quantify DNA adenine methylation (Dam) methyltransferase (MTase) employing signal amplification of GO/AgNPs/luminol composites to enhance the assay sensitivity. The method was developed by designing a capture probe DNA, which was immobilized on gold electrode surface, to hybridize with azide complementary DNA to form the azide-terminated dsDNA. Then, alkynyl functionalized GO/AgNPs/luminol composites as the signal probe were immobilized to azide-terminated dsDNA modified electrode via click chemistry, resulting in a high electrochemiluminescence (ECL) signal. Once the DNA hybrid was methylated (under catalysis of Dam MTase) and further cleaved by Dpn I endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5′-G-Am-T-C-3′), GO/AgNPs/luminol composites release from the electrode surface to the solution, leading to significant reduction of the ECL signal. The change of the ECL intensity is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination. This novel strategy can be further used as a universal method for other transferase determination by designing various transferase-specific DNA sequences. In addition, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

Mechanism and Function of Oxidative Reversal of DNA and RNA Methylation

The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N(6)-methyladenosine (m(6)A) in RNA, indicating that members of this subfamily of dioxygenases have a general function in demethylating nucleic acids. In this review, we discuss recent advances in this emerging field, focusing on the mechanism and function of TET-mediated DNA demethylation.