Epigenetic Mark In Eukaryotes Research Articles

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

The impact of DNA methylation on CTCF-mediated 3D genome organization.

Cytosine DNA methylation is a highly conserved epigenetic mark in eukaryotes. Although the role of DNA methylation at gene promoters and repetitive elements has been extensively studied, the function of DNA methylation in other genomic contexts remains less clear. In the nucleus of mammalian cells, the genome is spatially organized at different levels, and strongly influences myriad genomic processes. There are a number of factors that regulate the three-dimensional (3D) organization of the genome, with the CTCF insulator protein being among the most well-characterized. Pertinently, CTCF binding has been reported as being DNA methylation-sensitive in certain contexts, perhaps most notably in the process of genomic imprinting. Therefore, it stands to reason that DNA methylation may play a broader role in the regulation of chromatin architecture. Here we summarize the current understanding that is relevant to both the mammalian DNA methylation and chromatin architecture fields and attempt to assess the extent to which DNA methylation impacts the folding of the genome. The focus is in early embryonic development and cellular transitions when the epigenome is in flux, but we also describe insights from pathological contexts, such as cancer, in which the epigenome and 3D genome organization are misregulated.

Characteristics and functions of DNA N(6)-methyladenine in embryonic chicken muscle development

DNA N(6)-methyladenine (DNA-6mA) is a new epigenetic mark in eukaryotes, the distribution and functions of which in genomic DNA remain unknown. Although recent studies have suggested that 6mA is present in multiple model organisms and is dynamically regulated during development, the genomic features of 6mA in avian species have yet to be elucidated. 6mA immunoprecipitation sequencing approach was used to analysis the distribution and function of 6mA in the muscle genomic DNA during embryonic chicken development. 6mA immunoprecipitation sequencing was combined with transcriptomic sequencing to reveal the role of 6mA in the regulation of gene expression and to explore possible pathways by which 6mA is involved in muscle development. We here provide evidence that 6mA modification exists widely throughout the chicken genome, and show preliminary data regarding genome-wide distribution of this epigenetic mark. Gene expression was shown to be inhibited by 6mA modification in promoter regions. In addition, the promoters of some genes related to development were modified by 6mA, indicating that 6mA may be involved in embryonic chicken development. Furthermore, 6mA may participate in muscle development and immune function by regulating HSPB8 and OASL expression. Our study improves our understanding of the distribution and function of 6mA modification in higher organisms and provide new information about differences between mammals and other vertebrates. These findings demonstrate an epigenetic role for 6mA in gene expression and potential involvement in chicken muscle development. Furthermore, the results suggest a potential epigenetic role for 6mA in avian embryonic development.

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA

DNA modifications are used to regulate gene expression and defend against invading genetic elements. In eukaryotes, modifications predominantly involve C5-methylcytosine (5mC) and occasionally N6-methyladenine (6mA), while bacteria frequently use N4-methylcytosine (4mC) in addition to 5mC and 6mA. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, tiny freshwater invertebrates with transposon-poor genomes rich in foreign genes, lack canonical eukaryotic C5-methyltransferases for 5mC addition, but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT deposits 4mC at active transposons and certain tandem repeats, and fusion to a chromodomain shapes its “histone-read-DNA-write” architecture recognizing silent chromatin marks. Furthermore, amplification of SETDB1 H3K9me3 histone methyltransferases yields variants preferentially binding 4mC-DNA, suggesting “DNA-read-histone-write” partnership to maintain chromatin-based silencing. Our results show how non-native DNA methyl groups can reshape epigenetic systems to silence transposons and demonstrate the potential of horizontal gene transfer to drive regulatory innovation in eukaryotes.

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

Background5-Methylcytosine (5mC) is an important epigenetic mark in eukaryotes. Little information about its role exists for invertebrates. To investigate the contribution of 5mC to phenotypic variation in invertebrates, alteration of methylation patterns needs to be produced. Here, we apply new non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce aleatory changes into the methylome of mollusk species.ResultsFlavanone inhibitor Flv1 was efficient in reducing 5mC in the freshwater snails Biomphalaria glabrata and Physa acuta, and to a lesser degree, probably due to lower stability in sea water, in the oyster Crassostrea gigas. Flv1 has no toxic effects and significantly decreased the 5mC level in the treated B. glabrata and in its offspring. Drug treatment triggers significant variation in the shell height in both generations. A reduced representation bisulfite-sequencing method called epiGBS corroborates hypomethylation effect of Flv1 in both B. glabrata generations and identifies seven Differential Methylated Regions (DMR) out of 32 found both in Flv1-exposed snails and its progeny, from which 5 were hypomethylated, demonstrating a multigenerational effect. By targeted bisulfite sequencing, we confirmed hypomethylation in a locus and show that it is associated with reduced gene expression.ConclusionsFlv1 is a new and efficient DNMTi that can be used to induce transient and heritable modifications of the epigenetic landscape and phenotypic traits in mollusks, a phylum of the invertebrates in which epigenetics is understudied.

Computational investigations of selected enzymes from two iron and α-ketoglutarate-dependent families.

DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.

Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis thaliana with enzymatic methyl sequencing

Background5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.ResultsUsing an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions.ConclusionWe suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

The epigenetic roles of DNA N6-Methyladenine (6mA) modification in eukaryotes.

The DNA N6-methyladenine (6mA) modification is a prevalent epigenetic mark in prokaryotes, but the low abundance of 6mA in eukaryotes has recently received attention. The possible role of 6mA as an epigenetic mark in eukaryotes is starting to be recognized. This review article addresses the epigenetic roles of 6mA in eukaryotes. The existence of 6mA in metazoans and plants, the correlation of 6mA with gene expression, the enzymes catalyzing the deposition and removal of the 6mA modification, the relationship of 6mA to nucleosome positioning, the 6mA interaction with chromatin, its role in tumorigenesis and other physiological conditions/diseases and technical issues in 6mA detection/profiling and bioinformatics analysis are described. New directions and unresolved issues (e.g., the base-pair-resolution 6mA-sequencing method and gene activation vs. repression) in 6mA research are discussed.

DNA N6-Methyladenine Modification in Plant Genomes-A Glimpse into Emerging Epigenetic Code.

N6-methyladenine (6mA) is a DNA base modification at the 6th nitrogen position; recently, it has been resurfaced as a potential reversible epigenetic mark in eukaryotes. Despite its existence, 6mA was considered to be absent due to its undetectable level. However, with the new advancements in methods, considerable 6mA distribution is identified across the plant genome. Unlike 5-methylcytosine (5mC) in the gene promoter, 6mA does not have a definitive role in repression but is exposed to have divergent regulation in gene expression. Though 6mA information is less known, the available evidences suggest its function in plant development, tissue differentiation, and regulations in gene expression. The current review article emphasizes the research advances in DNA 6mA modifications, identification, available databases, analysis tools and its significance in plant development, cellular functions and future perspectives of research.

N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation.

N6-methyldeoxyadenine (6mA), a major type of DNA methylation in bacteria, represents a part of restriction-modification systems to discriminate host genome from invader DNA1. With the recent advent of more sensitive detection techniques, 6mA has also been detected in some eukaryotes2-8. However, the physiological function of this epigenetic mark in eukaryotes remains elusive. Heritable changes in DNA 5mC methylation have been associated with transgenerational inheritance of responses to a high-fat diet9, thus raising the exciting possibility that 6mA may also be transmitted across generations and serve as a carrier of inheritable information. Using Caenorhabditis elegans as a model, here we report that histone H3K4me3 and DNA 6mA modifications are required for the transmission of mitochondrial stress adaptations to progeny. Intriguingly, the global DNA 6mA level is significantly elevated following mitochondrial perturbation. N6-methyldeoxyadenine marks mitochondrial stress response genes and promotes their transcription to alleviate mitochondrial stress in progeny. These findings suggest that 6mA is a precisely regulated epigenetic mark that modulates stress response and signals transgenerational inheritance in C. elegans.

DNA methylation on N6-adenine in lepidopteran Bombyx mori

Increasing knowledge of DNA methylation that occurs on the sixth position of adenine (N6-methyladenine, 6mA) has emerged as a novel epigenetic mark in eukaryotes and plays an important role in regulating gene transcription, DNA replication and repair, transposable activities, and others. Here, we show DNA 6mA methylation is present in Bombyx mori, a lepidopteran model insect, and identify the 6mA methyltransferase, METTL4, and 6mA demethylase, NMAD, which regulate the levels of 6mA in embryogenesis and cultured cells of B. mori. Importantly, RNAi knockdown of METTL4 and NMAD not only induce cell cycle arrest at G1 phase but also result in defects of chromosome alignments at metaphase. We further demonstrate that 6mA methylation is widely distributed across the genome of B. mori by 6mA-Seq and primarily enriched in the regulatory regions as well as gene bodies. Integrated analysis of 6mA-Seq and RNA-Seq reveals that 6mA methylation in B. mori is preferentially related with lowly expressed genes and negatively correlated with active gene transcription, which provides a novel regulatory mechanism of DNA 6mA methylation on target genes. Altogether, these data identify 6mA methylation in B. mori and demonstrate a crucial role of 6mA signaling in controlling cell cycle progression.

Evolutionary analysis indicates that DNA alkylation damage is a byproduct of cytosine DNA methyltransferase activity

Methylation at the 5 position of cytosine in DNA (5meC) is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication by the activity of ‘maintenance’ DNA methyltransferases (DNMTs). Despite their ancient origin, DNA methylation pathways differ widely across animals, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. We used comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode Caenorhabditis elegans lacks DNA methylation, more basal nematodes retain cytosine DNA methylation, which is targeted to repeat loci. We found that DNA methylation coevolved with the DNA alkylation repair enzyme ALKB2 across eukaryotes. In addition, we found that DNMTs introduced the toxic lesion 3-methylcytosine into DNA both in vitro and in vivo. Alkylation damage is therefore intrinsically associated with DNMT activity, and this may promote the loss of DNA methylation in many species.

DNA N(6)-methyladenine: a new epigenetic mark in eukaryotes?

DNA N(6)-adenine methylation (N(6)-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

N6-methyladenine functions as a potential epigenetic mark in eukaryotes.

N(6)-methyladenine (6mA) is one of the most abundant types of DNA methylation, and plays an important role in bacteria; however, its roles in higher eukaryotes, such as plants, insects, and mammals, have been considered less important. Recent studies highlight that 6mA does indeed occur, and that it plays an important role in eukaryotes, such as worm, fly, and green algae, and thus the regulation of 6mA has emerged as a novel epigenetic mechanism in higher eukaryotes. Despite this intriguing development, a number of important issues regarding its biological roles are yet to be addressed. In this review, we focus on the 5mC and 6mA modifications in terms of their production, distribution, and the erasure of 6mA in higher eukaryotes including mammals. We perform an analysis of the potential functions of 6mA, hence widening understanding of this new epigenetic mark in higher eukaryotes, and suggesting future studies in this field.

Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies.

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.

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.