scholarly journals Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community

2018 ◽  
Author(s):  
Satoshi Hiraoka ◽  
Yusuke Okazaki ◽  
Mizue Anda ◽  
Atsushi Toyoda ◽  
Shin-ichi Nakano ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Defense Mechanisms ◽  
Lake Biwa ◽  
Dna Methyltransferases ◽  
Phage Infection ◽  
Prokaryotic Community ◽  
Powerful Approach ◽  
Diverse Groups ◽  
Circular Consensus Sequencing

AbstractDNA methylation plays important roles in prokaryotes, such as in defense mechanisms against phage infection, and the corresponding genomic landscapes—prokaryotic epigenomes—have recently begun to be disclosed. However, our knowledge of prokaryote methylation systems has been severely limited to those of culturable prokaryotes, whereas environmental communities are in fact dominated by uncultured members that must harbor much more diverse DNA methyltransferases. Here, using single-molecule real-time and circular consensus sequencing techniques, we revealed the ‘metaepigenomes’ of an environmental prokaryotic community in the largest lake in Japan, Lake Biwa. A total of 19 draft genomes from phylogenetically diverse groups, most of which are yet to be cultured, were successfully reconstructed. The analysis of DNA chemical modifications identified 29 methylated motifs in those genomes, among which 14 motifs were novel.Furthermore, we searched for the methyltransferase genes responsible for the methylation of the detected novel motifs and confirmed their catalytic specificities via transformation experiments involving artificially synthesized genes. Finally, we found that genomes without DNA methylation tended to exhibit higher phage infection levels than those with methylation. In summary, this study proves that metaepigenomics is a powerful approach for revealing the vast unexplored variety of prokaryotic DNA methylation systems in nature.

scholarly journals The DNA Methylation Landscape of Giant Viruses

2019 ◽  
Author(s):  
Sandra Jeudy ◽  
Sofia Rigou ◽  
Jean-Marie Alempic ◽  
Jean-Michel Claverie ◽  
Chantal Abergel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Defense Mechanism ◽  
Methylation Status ◽  
Purifying Selection ◽  
Dna Methyltransferases ◽  
Epigenetic Mark ◽  
Host Defense Mechanism ◽  
Domains Of Life ◽  
Giant Viruses

AbstractDNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Prokaryotes use it through Restriction-Modification (R-M) systems as a host-defense mechanism against viruses. The recently discovered giant viruses are widespread dsDNA viruses infecting eukaryotes with gene contents overlapping the cellular world. While they are predicted to encode DNA methyltransferases (MTases), virtually nothing is known about the DNA methylation status of their genomes. Using single-molecule real-time sequencing we studied the complete methylome of a large spectrum of families: the Marseilleviridae, the Pandoraviruses, the Molliviruses, the Mimiviridae along with their associated virophages and transpoviron, the Pithoviruses and the Cedratviruses (of which we report a new strain). Here we show that DNA methylation is widespread in giant viruses although unevenly distributed. We then identified the corresponding viral MTases, all of which are of bacterial origins and subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. If some viral MTases undergo pseudogenization, most are conserved, functional and under purifying selection, suggesting that they increase the viruses’ fitness. While the Marseilleviridae, Pithoviruses and Cedratviruses DNA MTases catalyze N6-methyl-adenine modifications, some MTases of Molliviruses and Pandoraviruses unexpectedly catalyze the formation of N4-methyl-cytosine modifications. In Marseilleviridae, encoded MTases are paired with cognate restriction endonucleases (REases) forming complete R-M systems. Our data suggest that giant viruses MTases could be involved in different kind of virus-virus interactions during coinfections.

scholarly journals Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

2021 ◽  
Vol 22 (21) ◽  
pp. 11990
Author(s):  
Sook Ho Kim ◽  
Hae Jun Jung ◽  
Seok-Cheol Hong
Keyword(s):  
Dna Methylation ◽  
Anticancer Drugs ◽  
Single Molecule ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
Polymerase Chain Reaction Amplification ◽  
High Sensitivity ◽  
Inhibitory Effect ◽  
Dna Methyltransferases ◽  
Cell Stage

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody’s incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

scholarly journals DNA methylation epigenetically regulates gene expression in Burkholderia cenocepacia

2020 ◽  
Author(s):  
Ian Vandenbussche ◽  
Andrea Sass ◽  
Marta Pinto-Carbó ◽  
Olga Mannweiler ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Single Molecule ◽  
Regulation Of Gene Expression ◽  
Dna Methyltransferases ◽  
Lung Damage ◽  
Burkholderia Cenocepacia ◽  
Model Organisms ◽  
Deletion Mutants ◽  
Tract Infections

AbstractRespiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation has shown to be an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2, and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, ΔBCAM0992 was less motile. B. cenocepacia wild type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-Molecule Real-Time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified and qPCR experiments showed an upregulation of several genes, including biofilm and motility related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation and motility.ImportanceCF patients diagnosed with B. cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multi-replicon genome and a lot remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to lead to new insights in how to tackle B. cenocepacia infections in CF patients.

scholarly journals Neuroblastoma and the epigenome

2021 ◽  
Author(s):  
Irfete S. Fetahu ◽  
Sabine Taschner-Mandl
Keyword(s):  
Dna Methylation ◽  
Histone Modifications ◽  
Disease Diagnosis ◽  
Dna Methyltransferases ◽  
Epigenetic Alterations ◽  
Aberrant Expression ◽  
Tet Proteins ◽  
Relative Paucity ◽  
Underlying Causes ◽  
Aberrant Dna Methylation

AbstractNeuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system and one of the most common solid tumors in infancy. Amplification of MYCN, copy number alterations, numerical and segmental chromosomal aberrations, mutations, and rearrangements on a handful of genes, such as ALK, ATRX, TP53, RAS/MAPK pathway genes, and TERT, are attributed as underlying causes that give rise to NB. However, the heterogeneous nature of the disease—along with the relative paucity of recurrent somatic mutations—reinforces the need to understand the interplay of genetic factors and epigenetic alterations in the context of NB. Epigenetic mechanisms tightly control gene expression, embryogenesis, imprinting, chromosomal stability, and tumorigenesis, thereby playing a pivotal role in physio- and pathological settings. The main epigenetic alterations include aberrant DNA methylation, disrupted patterns of posttranslational histone modifications, alterations in chromatin composition and/or architecture, and aberrant expression of non-coding RNAs. DNA methylation and demethylation are mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, respectively, while histone modifications are coordinated by histone acetyltransferases and deacetylases (HATs, HDACs), and histone methyltransferases and demethylases (HMTs, HDMs). This article focuses predominately on the crosstalk between the epigenome and NB, and the implications it has on disease diagnosis and treatment.

scholarly journals MBRS-47. RAPID MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA BASED ON DNA METHYLATION BY NANOPORE SEQUENCING

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Julien Masliah-Planchon ◽  
Elodie Girard ◽  
Philipp Euskirchen ◽  
Christine Bourneix ◽  
Delphine Lequin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Molecule ◽  
Nanopore Sequencing ◽  
Molecular Subgroup ◽  
Group Assignment ◽  
Group 4 ◽  
Methylation Assay ◽  
Tumor Group ◽  
Long Read ◽  
Group 3

Abstract Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

scholarly journals Interplay between Histone and DNA Methylation Seen through Comparative Methylomes in Rare Mendelian Disorders

2021 ◽  
Vol 22 (7) ◽  
pp. 3735
Author(s):  
Guillaume Velasco ◽  
Damien Ulveling ◽  
Sophie Rondeau ◽  
Pauline Marzin ◽  
Motoko Unoki ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Catalytic Domain ◽  
De Novo ◽  
Mutation Screening ◽  
Histone H3 ◽  
Regulatory Elements ◽  
Germline Mutations ◽  
Dna Methyltransferases ◽  
Mendelian Disorders ◽  
Epigenetic Machinery

DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.

scholarly journals DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors

2020 ◽  
Author(s):  
Jing Wei ◽  
Jia Cheng ◽  
Nicholas J Waddell ◽  
Zi-Jun Wang ◽  
Xiaodong Pang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Epigenetic Modification ◽  
Substantial Reduction ◽  
Dna Methyltransferases ◽  
Male Rats ◽  
Neural Pathways ◽  
Dnmt Inhibitors ◽  
Synaptic Functions

Abstract Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

scholarly journals Impact of DNA methylation on 3D genome structure

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diana Buitrago ◽  
Mireia Labrador ◽  
Juan Pablo Arcon ◽  
Rafael Lema ◽  
Oscar Flores ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromatin Structure ◽  
Chromatin Condensation ◽  
Genome Structure ◽  
Dna Methyltransferases ◽  
Model System ◽  
Structure And Function ◽  
3D Genome ◽  
Cellular Machinery ◽  
And Function

AbstractDetermining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5’ end of the gene increasing gradually toward the 3’ end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal.

Decreased expression of DNA methyltransferases in the testes of patients with non-obstructive azoospermia leads to changes in global DNA methylation levels

2019 ◽  
Vol 31 (8) ◽  
pp. 1386 ◽  
Author(s):  
Fatma Uysal ◽  
Gokhan Akkoyunlu ◽  
Saffet Ozturk
Keyword(s):  
Dna Methylation ◽  
Male Infertility ◽  
De Novo ◽  
Biological Effects ◽  
Testicular Biopsy ◽  
Round Spermatid ◽  
Dna Methyltransferases ◽  
Global Dna Methylation ◽  
Obstructive Azoospermia ◽  
Spermatogenic Cells

DNA methylation plays key roles in epigenetic regulation during mammalian spermatogenesis. DNA methyltransferases (DNMTs) function in de novo and maintenance methylation processes by adding a methyl group to the fifth carbon atom of the cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. Azoospermia is one of the main causes of male infertility, and is classified as obstructive (OA) or non-obstructive (NOA) azoospermia based on histopathological characteristics. The molecular background of NOA is still largely unknown. DNA methylation performed by DNMTs is implicated in the transcriptional regulation of spermatogenesis-related genes. The aim of the present study was to evaluate the cellular localisation and expression levels of the DNMT1, DNMT3A and DNMT3B proteins, as well as global DNA methylation profiles in testicular biopsy samples obtained from men with various types of NOA, including hypospermatogenesis (hyposperm), round spermatid (RS) arrest, spermatocyte (SC) arrest and Sertoli cell-only (SCO) syndrome. In the testicular biopsy samples, DNMT1 expression and global DNA methylation levels decreased gradually from the hyposperm to SCO groups (P<0.05). DNMT3A expression was significantly decreased in the RS arrest, SC arrest and SCO groups compared with the hyposperm group (P<0.05). DNMT3B expression was significantly lower in the RS arrest and SCO groups than in the hyposperm group (P<0.05). Although both DNMT1 and DNMT3A were localised in the cytoplasm and nucleus of the spermatogenic cells, staining for DNMT3B was more intensive in the nucleus of spermatogenic cells. In conclusion, the findings suggest that significant changes in DNMT expression and global DNA methylation levels in spermatogenic cells may contribute to development of male infertility in the NOA groups. Further studies are needed to determine the molecular biological effects of the altered DNMT expression and DNA methylation levels on development of male infertility.

scholarly journals On the origin and evolutionary consequences of gene body DNA methylation

2016 ◽  
Vol 113 (32) ◽  
pp. 9111-9116 ◽  
Author(s):  
Adam J. Bewick ◽  
Lexiang Ji ◽  
Chad E. Niederhuth ◽  
Eva-Maria Willing ◽  
Brigitte T. Hofmeister ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Recombinant Inbred Lines ◽  
Dna Methyltransferases ◽  
Inbred Lines ◽  
Gene Body ◽  
Gene Body Methylation ◽  
Eutrema Salsugineum ◽  
And Function ◽  
Evolutionary Consequences

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

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