Origin and Evolution of DNA methyltransferases (DNMT) along the tree of life: A multi-genome survey

AbstractBackgroundCytosine methylation is a common DNA modification found in most eukaryotic organisms including plants, animals, and fungi. (Cytosine-5)-DNA methyltransferases (C5-DNA MTases) belong to the DNMT family of enzymes that catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to cytosine residues of DNA. In mammals, four members of the DNMT family have been reported: DNMT1, DNMT3a, DNMT3b and DNMT3L, but only DNMT1, DNMT3a and DNMT3b possess methyltransferase activity. There have been many reports about the methylation landscape in different organisms yet there is no systematic report of how the enzyme DNA (C5) methyltransferases have evolved in different organisms.ResultDNA methyltransferases are found to be present in all three domains of life. However, significant variability has been observed in length, copy number and sequence identity when compared across kingdoms. Sequence conservation is greatly increased in invertebrates and vertebrates compared to other groups. Similarly, sequence length has been found to be increased while domain lengths remain more or less conserved. Vertebrates are also found to be associated with more conserved DNMT domains. Finally, comparison between single nucleotide polymorphisms (SNPs) prevailing in human populations and evolutionary changes in DNMT vertebrate alignment revealed that most of the SNPs were conserved in vertebrates.ConclusionThe sequences (including the catalytic domain and motifs) and structure of the DNMT enzymes have been evolved greatly from bacteria to vertebrates with a steady increase in complexity and specificity. This study provides a systematic report of the evolution of DNA methyltransferase enzyme across different lineages of tree of life.

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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

Nucleic Acids Research ◽

10.1093/nar/gkaa938 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11495-11509

Author(s):

Michael Dukatz ◽

Sabrina Adam ◽

Mahamaya Biswal ◽

Jikui Song ◽

Pavel Bashtrykov ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Flanking Sequence ◽

Cpg Sites ◽

Binding Interface ◽

Target Sites ◽

Flanking Sequences

Abstract DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

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A protein architecture guided screen for modification dependent restriction endonucleases

Nucleic Acids Research ◽

10.1093/nar/gkz755 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9761-9776 ◽

Cited By ~ 5

Author(s):

Thomas Lutz ◽

Kiersten Flodman ◽

Alyssa Copelas ◽

Honorata Czapinska ◽

Megumu Mabuchi ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Proteins ◽

Dna Methyltransferase ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Restriction Endonucleases ◽

Endonuclease Activity ◽

Protein Architecture ◽

Escherichia Coli Cells ◽

Dependent Activity

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

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Evolutionary analysis of DNA methyltransferases in microeukaryotes: Insights from the model diatom Phaeodactylum tricornutum

10.1101/2021.06.11.447926 ◽

2021 ◽

Author(s):

ANTOINE HOGUIN ◽

Ouardia Ait Mohamed ◽

Chris Bowler ◽

Auguste Genovesio ◽

Fabio RJ Vieira ◽

...

Keyword(s):

Dna Methylation ◽

Transposable Elements ◽

Phaeodactylum Tricornutum ◽

Dna Methyltransferase ◽

Transcriptional Control ◽

Dna Methyltransferases ◽

Epigenetic Mark ◽

Evolutionary Analysis ◽

And Function ◽

Dnmt Family

Cytosine DNA methylation is an important epigenetic mark in eukaryotes that is involved in the transcriptional control of mainly transposable elements in mammals, plants, and fungi. Eukaryotes encode a diverse set of DNA methyltransferases that were iteratively acquired and lost during evolution. The Stramenopiles-Alveolate-Rhizaria (SAR) lineages are a major group of ecologically important marine microeukaryotes that include the main phytoplankton classes such as diatoms and dinoflagellates. However, little is known about the diversity of DNA methyltransferases and their role in the deposition and maintenance of DNA methylation in microalgae. We performed a phylogenetic analysis of DNA methyltransferase families found in marine microeukaryotes and show that they encode divergent DNMT3, DNMT4, DNMT5 and DNMT6 enzymes family revisiting previously established phylogenies. Furthermore, we reveal a novel group of DNMTs with three classes of enzymes within the DNMT5 family. Using a CRISPR/Cas9 strategy we demonstrate that the loss of the DNMT5 gene correlates with a global depletion of DNA methylation and overexpression of transposable elements in the model diatom Phaeodactylum tricornutum. The study provides a pioneering view of the structure and function of a DNMT family in the SAR supergroup.

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

International Journal of Molecular Sciences ◽

10.3390/ijms222111990 ◽

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.

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Evolution of CG Methylation Maintenance Machinery in Plants

Epigenomes ◽

10.3390/epigenomes5030019 ◽

2021 ◽

Vol 5 (3) ◽

pp. 19

Author(s):

Louis Tirot ◽

Pauline E. Jullien ◽

Mathieu Ingouff

Keyword(s):

Genome Stability ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

De Novo ◽

Regulation Of Gene Expression ◽

Ring Finger ◽

Dna Methyltransferases ◽

Epigenetic Mark ◽

Ring Finger Domain ◽

Eukaryotic Genomes

Cytosine methylation is an epigenetic mark present in most eukaryotic genomes that contributes to the regulation of gene expression and the maintenance of genome stability. DNA methylation mostly occurs at CG sequences, where it is initially deposited by de novo DNA methyltransferases and propagated by maintenance DNA methyltransferases (DNMT) during DNA replication. In this review, we first summarize the mechanisms maintaining CG methylation in mammals that involve the DNA Methyltransferase 1 (DNMT1) enzyme and its cofactor, UHRF1 (Ubiquitin-like with PHD and RING Finger domain 1). We then discuss the evolutionary conservation and diversification of these two core factors in the plant kingdom and speculate on potential functions of novel homologues typically observed in land plants but not in mammals.

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An Evaluation of DNA Methyltransferase 1 (DNMT1) Single Nucleotide Polymorphisms and Chemotherapy-Associated Cognitive Impairment: A Prospective, Longitudinal Study

Scientific Reports ◽

10.1038/s41598-019-51203-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Alexandre Chan ◽

Angie Yeo ◽

Maung Shwe ◽

Chia Jie Tan ◽

Koon Mian Foo ◽

...

Keyword(s):

Breast Cancer ◽

Cognitive Impairment ◽

Cognitive Function ◽

Cancer Patients ◽

Dna Methyltransferase ◽

Dna Methyltransferases ◽

Breast Cancer Patients ◽

Prospective Longitudinal Study ◽

Cognitive Domains ◽

Prospective Longitudinal

Abstract Strong evidence suggests that genetic variations in DNA methyltransferases (DNMTs) may alter the downstream expression and DNA methylation patterns of neuronal genes and influence cognition. This study investigates the association between a DNMT1 polymorphism, rs2162560, and chemotherapy-associated cognitive impairment (CACI) in a cohort of breast cancer patients. This is a prospective, longitudinal cohort study. From 2011 to 2017, 351 early-stage breast cancer patients receiving chemotherapy were assessed at baseline, the midpoint, and the end of chemotherapy. DNA was extracted from whole blood, and genotyping was performed using Sanger sequencing. Patients’ self-perceived cognitive function and cognitive performance were assessed at three different time points using FACT-Cog (v.3) and a neuropsychological battery, respectively. The association between DNMT1 rs2162560 and cognitive function was evaluated using logistic regression analyses. Overall, 33.3% of the patients reported impairment relative to baseline in one or more cognitive domains. Cognitive impairment was observed in various objective cognitive domains, with incidences ranging from 7.2% to 36.9%. The DNMT1 rs2162560 A allele was observed in 21.8% of patients and this was associated with lower odds of self-reported cognitive decline in the concentration (OR = 0.45, 95% CI: 0.25–0.82, P = 0.01) and functional interference (OR = 0.48, 95% CI: 0.24–0.95, P = 0.03) domains. No significant association was observed between DNMT1 rs2162560 and objective cognitive impairment. This is the first study to show a significant association between the DNMT1 rs2162560 polymorphism and CACI. Our data suggest that epigenetic processes could contribute to CACI, and further studies are needed to validate these findings.

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Expanding the Structural Diversity of DNA Methyltransferase Inhibitors

Pharmaceuticals ◽

10.3390/ph14010017 ◽

2020 ◽

Vol 14 (1) ◽

pp. 17

Author(s):

K. Eurídice Juárez-Mercado ◽

Fernando D. Prieto-Martínez ◽

Norberto Sánchez-Cruz ◽

Andrea Peña-Castillo ◽

Diego Prada-Gracia ◽

...

Keyword(s):

Histone Deacetylases ◽

Dna Methyltransferase ◽

Structural Diversity ◽

Dna Methyltransferases ◽

Dna Methyltransferase Inhibitors ◽

Epigenetic Drug ◽

Ic50 Values ◽

Dietary Component ◽

Approved Drugs ◽

Epigenetic Processes

Inhibitors of DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug discovery. They are also chemical tools to understand the biochemistry of epigenetic processes. Herein, we report five distinct inhibitors of DNMT1 characterized in enzymatic inhibition assays that did not show activity with DNMT3B. It was concluded that the dietary component theaflavin is an inhibitor of DNMT1. Two additional novel inhibitors of DNMT1 are the approved drugs glyburide and panobinostat. The DNMT1 enzymatic inhibitory activity of panobinostat, a known pan inhibitor of histone deacetylases, agrees with experimental reports of its ability to reduce DNMT1 activity in liver cancer cell lines. Molecular docking of the active compounds with DNMT1, and re-scoring with the recently developed extended connectivity interaction features approach, led to an excellent agreement between the experimental IC50 values and docking scores.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22073735 ◽

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.

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Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

Biology ◽

10.3390/biology9050091 ◽

2020 ◽

Vol 9 (5) ◽

pp. 91 ◽

Cited By ~ 1

Author(s):

Miryam Pérez-Cañamás ◽

Elizabeth Hevia ◽

Carmen Hernández

Keyword(s):

Gene Silencing ◽

Ribosomal Dna ◽

Plant Virus ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

De Novo ◽

Methylation Pattern ◽

Transcriptional Gene Silencing ◽

Post Transcriptional Gene Silencing ◽

The Impact

DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.

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