scholarly journals The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples

2020 ◽  
Vol 8 (8) ◽  
pp. 1252
Author(s):  
Bart Cuypers ◽  
Franck Dumetz ◽  
Pieter Meysman ◽  
Kris Laukens ◽  
Géraldine De Muylder ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Leishmania Donovani ◽  
Clinical Samples ◽  
Knock Out ◽  
The Status ◽  
Genome Bisulfite Sequencing ◽  
Genomic Scale ◽  
Commercial Kits ◽  
Eukaryotic Organisms

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.

scholarly journals The absence of C-5 DNA methylation in Leishmania donovani allows DNA enrichment from complex samples

2019 ◽  
Author(s):  
B Cuypers ◽  
F Dumetz ◽  
P Meysman ◽  
K Laukens ◽  
G De Muylder ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Leishmania Donovani ◽  
Clinical Samples ◽  
Knock Out ◽  
Complex Samples ◽  
The Status ◽  
Genome Bisulfite Sequencing ◽  
Commercial Kits ◽  
Eukaryotic Organisms

AbstractCytosine C5 methylation is an important epigenetic control mechanism in a wide array of Eukaryotic organisms and generally carried out by proteins of C-5 DNA methyltransferase family (DNMTs). In several protozoans the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we show that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, of which the function is still unclear, and verified its expression at RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterised their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites and 0.0126% at CHH sites at genome scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrate that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation), allows enrichment of parasite versus host DNA using Methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62x up to 263x. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.

scholarly journals Whole genome bisulfite sequencing reveals a sparse, but robust pattern of DNA methylation in the Dictyostelium discoideum genome

2017 ◽  
Author(s):  
Jacob L. Steenwyk ◽  
James St. Denis ◽  
Jacqueline M. Dresch ◽  
Denis A. Larochelle ◽  
Robert A. Drewell
Keyword(s):  
Dna Methylation ◽  
Dictyostelium Discoideum ◽  
Dna Methyltransferase ◽  
Model Organism ◽  
Epigenetic Mark ◽  
Whole Genome ◽  
Protein Coding ◽  
Genome Bisulfite Sequencing ◽  
Chromatin Structural ◽  
And Function

AbstractDNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the slime mold Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methyl-cytosine in the genome is unclear. Here we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage, replicate sequencing of bisulfite converted gDNA extracted from post-starvation cells. We find an overall very low level of DNA methylation, occurring at only 462 out of the ~7.5 million (0.006%) cytosines in the genome. Despite this sparse profile, significant methylation can be detected at 51 of these sites in replicate experiments, suggesting they are robust targets for DNA methylation. These 5-methyl-cytosines are associated with a broad range of protein-coding genes, tRNA-encoding genes and retrotransposable elements. Our data provides evidence of a minimal, but functional, methylome in Dictyostelium, thereby making Dictyostelium a candidate model organism to further investigate the evolutionary function of DNA methylation.

scholarly journals Exploration of the Effect on Genome-Wide DNA Methylation by miR-143 Knock-Out in Mice Liver

2021 ◽  
Vol 22 (23) ◽  
pp. 13075
Author(s):  
Xingping Chen ◽  
Junyi Luo ◽  
Jie Liu ◽  
Ting Chen ◽  
Jiajie Sun ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Hepatoma Cell ◽  
Whole Genome ◽  
Phosphoribosyl Pyrophosphate ◽  
Knock Out ◽  
Spermine Synthase ◽  
Mice Liver ◽  
Acyl Coenzyme A

MiR-143 play an important role in hepatocellular carcinoma and liver fibrosis via inhibiting hepatoma cell proliferation. DNA methyltransferase 3 alpha (DNMT3a), as a target of miR-143, regulates the development of primary organic solid tumors through DNA methylation mechanisms. However, the effect of miR-143 on DNA methylation profiles in liver is unclear. In this study, we used Whole-Genome Bisulfite Sequencing (WGBS) to detect the differentially methylated regions (DMRs), and investigated DMR-related genes and their enriched pathways by miR-143. We found that methylated cytosines increased 0.19% in the miR-143 knock-out (KO) liver fed with high-fat diet (HFD), compared with the wild type (WT). Furthermore, compared with the WT group, the CG methylation patterns of the KO group showed lower CG methylation levels in CG islands (CGIs), promoters and hypermethylation in CGI shores, 5′UTRs, exons, introns, 3′UTRs, and repeat regions. A total of 984 DMRs were identified between the WT and KO groups consisting of 559 hypermethylation and 425 hypomethylation DMRs. Furthermore, DMR-related genes were enriched in metabolism pathways such as carbon metabolism (serine hydroxymethyltransferase 2 (Shmt2), acyl-Coenzyme A dehydrogenase medium chain (Acadm)), arginine and proline metabolism (spermine synthase (Sms), proline dehydrogenase (Prodh2)) and purine metabolism (phosphoribosyl pyrophosphate synthetase 2 (Prps2)). In summary, we are the first to report the change in whole-genome methylation levels by miR-143-null through WGBS in mice liver, and provide an experimental basis for clinical diagnosis and treatment in liver diseases, indicating that miR-143 may be a potential therapeutic target and biomarker for liver damage-associated diseases and hepatocellular carcinoma.

scholarly journals MethHaplo: combining allele-specific DNA methylation and SNPs for haplotype region identification

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Qiangwei Zhou ◽  
Ze Wang ◽  
Jing Li ◽  
Wing-Kin Sung ◽  
Guoliang Li
Keyword(s):  
Dna Methylation ◽  
Epigenetic Modification ◽  
Critical Role ◽  
Three Dimensional ◽  
Hybrid Vigor ◽  
Nucleotide Polymorphisms ◽  
Chromosome Conformation ◽  
Genome Bisulfite Sequencing ◽  
Allele Specific ◽  
Eukaryotic Organisms

Abstract Background DNA methylation is an important epigenetic modification that plays a critical role in most eukaryotic organisms. Parental alleles in haploid genomes may exhibit different methylation patterns, which can lead to different phenotypes and even different therapeutic and drug responses to diseases. However, to our knowledge, no software is available for the identification of DNA methylation haplotype regions with combined allele-specific DNA methylation, single nucleotide polymorphisms (SNPs) and high-throughput chromosome conformation capture (Hi-C) data. Results In this paper, we developed a new method, MethHaplo, that identify DNA methylation haplotype regions with allele-specific DNA methylation and SNPs from whole-genome bisulfite sequencing (WGBS) data. Our results showed that methylation haplotype regions were ten times longer than haplotypes with SNPs only. When we integrate WGBS and Hi-C data, MethHaplo could call even longer haplotypes. Conclusions This study illustrates the usefulness of methylation haplotypes. By constructing methylation haplotypes for various cell lines, we provide a clearer picture of the effect of DNA methylation on gene expression, histone modification and three-dimensional chromosome structure at the haplotype level. Our method could benefit the study of parental inheritance-related disease and hybrid vigor in agriculture.

scholarly journals DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia

Oncogene ◽  
2015 ◽  
Vol 35 (23) ◽  
pp. 3079-3082 ◽  
Author(s):  
H J Ferreira ◽  
H Heyn ◽  
M Vizoso ◽  
C Moutinho ◽  
E Vidal ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Alternative Pathway ◽  
Missense Mutations ◽  
Cytogenetic Aberrations ◽  
Genome Bisulfite Sequencing ◽  
Acute Myeloid

Abstract Close to half of de novo acute myeloid leukemia (AML) cases do not exhibit any cytogenetic aberrations. In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.

scholarly journals A comparative analysis of 5-azacytidine and zebularine induced DNA demethylation

2016 ◽  
Author(s):  
Patrick T. Griffin ◽  
Chad E. Niederhuth ◽  
Robert J. Schmitz
Keyword(s):  
Dna Methylation ◽  
Genomic Dna ◽  
Dna Methyltransferase ◽  
Bisulfite Sequencing ◽  
Dna Demethylation ◽  
Methyltransferase Activity ◽  
Genome Wide ◽  
Construct Maps ◽  
Genome Bisulfite Sequencing ◽  
Single Base Pair

AbstractThe non-methylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had a slightly greater demethylating effect across the genome at higher concentrations. Transcriptome analyses revealed a substantial number of up-regulated genes, with an overrepresentation of transposable element genes, in particular CACTA-like elements. This demonstrates that chemical demethylating agents have a disproportionately large effect on loci that are otherwise silenced by DNA methylation.

scholarly journals Dnmt3a is an epigenetic mediator of adipose insulin resistance

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Dongjoo You ◽  
Emma Nilsson ◽  
Danielle E Tenen ◽  
Anna Lyubetskaya ◽  
James C Lo ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Human Subjects ◽  
Human Adipose Tissue ◽  
Gene Profiling ◽  
Knock Out ◽  
Unbiased Gene

Insulin resistance results from an intricate interaction between genetic make-up and environment, and thus may be orchestrated by epigenetic mechanisms like DNA methylation. Here, we demonstrate that DNA methyltransferase 3a (Dnmt3a) is both necessary and sufficient to mediate insulin resistance in cultured mouse and human adipocytes. Furthermore, adipose-specific Dnmt3a knock-out mice are protected from diet-induced insulin resistance and glucose intolerance without accompanying changes in adiposity. Unbiased gene profiling studies revealed Fgf21 as a key negatively regulated Dnmt3a target gene in adipocytes with concordant changes in DNA methylation at the Fgf21 promoter region. Consistent with this, Fgf21 can rescue Dnmt3a-mediated insulin resistance, and DNA methylation at the FGF21 locus was elevated in human subjects with diabetes and correlated negatively with expression of FGF21 in human adipose tissue. Taken together, our data demonstrate that adipose Dnmt3a is a novel epigenetic mediator of insulin resistance in vitro and in vivo.

scholarly journals DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

2015 ◽  
Vol 112 (3) ◽  
pp. 911-916 ◽  
Author(s):  
Xuehua Zhong ◽  
Christopher J. Hale ◽  
Minh Nguyen ◽  
Israel Ausin ◽  
Martin Groth ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Noncoding Rna ◽  
Dna Methyltransferase ◽  
Transcript Abundance ◽  
Transcriptional Elongation ◽  
Rna Transcripts ◽  
Pol V ◽  
Genome Defense ◽  
Eukaryotic Organisms

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

scholarly journals The evolution of DNA methylation and its relationship to sociality in insects

2016 ◽  
Author(s):  
Adam J. Bewick ◽  
Kevin J. Vogel ◽  
Allen J. Moore ◽  
Robert J. Schmitz
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Duplication Event ◽  
Neutral Evolution ◽  
Dna Methyltransferase 1 ◽  
Functional Studies ◽  
Genome Bisulfite Sequencing ◽  
Enzymatic Machinery ◽  
Eusocial Species ◽  
Solitary Species

ABSTRACTDNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of flexible traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. Furthermore, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among insects. We also predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary dependency between sociality and DNA methylation. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some hymenopteran, and paralogs have experienced divergent, non-neutral evolution. This diversity and non-neutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Altogether, DNA methylation is highly variable in insects and is not a universal driver of social behavior. Future, functional studies are required to advance our understanding of DNA methylation in insects.

DNA methyltransferase inhibitors modulate histone methylation: epigenetic crosstalk between H3K4me3 and DNA methylation during sperm differentiation

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Liliana Burlibaşa ◽  
Alina-Teodora Nicu ◽  
Carmen Domnariu
Keyword(s):  
Dna Methylation ◽  
Histone Methylation ◽  
Dna Methyltransferase ◽  
Integrated Approach ◽  
Regulatory Mechanisms ◽  
Temporal Expression ◽  
Dna Methyltransferase Inhibitors ◽  
Expression Of Genes ◽  
Species Survival ◽  
Advanced Stages

Summary The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate ‘tuning’ of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial–temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

Sign in / Sign up

Export Citation Format

Share Document