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

2020 ◽  
Vol 48 (7) ◽  
pp. 3949-3961 ◽  
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.

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

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 The Roles of Human DNA Methyltransferases and Their Isoforms in Shaping the Epigenome

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 172 ◽  
Author(s):  
Hemant Gujar ◽  
Daniel Weisenberger ◽  
Gangning Liang
Keyword(s):  
Dna Methylation ◽  
Cytosine Methylation ◽  
De Novo ◽  
Ring Finger ◽  
Dna Methyltransferases ◽  
Epigenetic Modifications ◽  
Hard Copy ◽  
Physiological Processes ◽  
New Gene ◽  
Cpg Dinucleotide

A DNA sequence is the hard copy of the human genome and it is a driving force in determining the physiological processes in an organism. Concurrently, the chemical modification of the genome and its related histone proteins is dynamically involved in regulating physiological processes and diseases, which overall constitutes the epigenome network. Among the various forms of epigenetic modifications, DNA methylation at the C-5 position of cytosine in the cytosine–guanine (CpG) dinucleotide is one of the most well studied epigenetic modifications. DNA methyltransferases (DNMTs) are a family of enzymes involved in generating and maintaining CpG methylation across the genome. In mammalian systems, DNA methylation is performed by DNMT1 and DNMT3s (DNMT3A and 3B). DNMT1 is predominantly involved in the maintenance of DNA methylation during cell division, while DNMT3s are involved in establishing de novo cytosine methylation and maintenance in both embryonic and somatic cells. In general, all DNMTs require accessory proteins, such as ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domain 1 (UHRF1) or DNMT3-like (DNMT3L), for their biological function. This review mainly focuses on the role of DNMT3B and its isoforms in de novo methylation and maintenance of DNA methylation, especially with respect to their role as an accessory protein.

scholarly journals Stochastic Modeling Reveals Kinetic Heterogeneity in Post-replication DNA Methylation

2019 ◽  
Author(s):  
Luis Busto-Moner ◽  
Julien Morival ◽  
Arjang Fahim ◽  
Zachary Reitz ◽  
Timothy L. Downing ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Dna Methylation ◽  
Rate Constants ◽  
Cytosine Methylation ◽  
Temporal Dynamics ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Likelihood Estimation ◽  
Dna Methyltransferases ◽  
Methylation Patterns

AbstractDNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.

scholarly journals Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

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.

scholarly journals DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3559-3569 ◽  
Author(s):  
Rita Shaknovich ◽  
Leandro Cerchietti ◽  
Lucas Tsikitas ◽  
Matthias Kormaksson ◽  
Subhajyoti De ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cells ◽  
Germinal Center ◽  
Dna Methyltransferase ◽  
Cytosine Deaminase ◽  
Dna Methyltransferases ◽  
B Cell Differentiation ◽  
Dual Roles ◽  
Germinal Center B Cell ◽  
Cellular Phenotypes

Abstract The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

scholarly journals Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4461 ◽  
Author(s):  
Jiang Li ◽  
Caili Li ◽  
Shanfa Lu
Keyword(s):  
Dna Methylation ◽  
Salicylic Acid ◽  
Gene Family ◽  
Sequence Alignment ◽  
Dna Methyltransferase ◽  
Epigenetic Modification ◽  
Salvia Miltiorrhiza ◽  
Dna Methyltransferases ◽  
Genome Wide ◽  
Wide Range

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, eight SmC5-MTase genes were divided into four subfamilies, including MET, CMT, DRM and DNMT2. Genome-wide comparative analysis of the C5-MTase gene family in S. miltiorrhiza and Arabidopsis thaliana, including gene structure, sequence features, sequence alignment and conserved motifs, was carried out. The results showed conservation and divergence of the members of each subfamily in plants. The length of SmC5-MTase open reading frames ranges widely from 1,152 (SmDNMT2) to 5,034 bp (SmMET1). The intron number of SmC5-MTases varies between 7 (SmDRM1) and 20 (SmCMT1 and SmCMT2b). These features were similar to their counterparts from Arabidopsis. Sequence alignment and conserved motif analysis showed the existence of highly conserved and subfamily-specific motifs in the C5-MTases analyzed. Differential transcript abundance was detected for SmC5-MTases, implying genome-wide variance of DNA methylation in different organs and tissues. Transcriptome-wide analysis showed that the transcript levels of all SmC5-MTase genes was slightly changed under yeast extract and methyl jasmonate treatments. Six SmC5-MTases, including SmMET1, SmCMT1, SmCMT2a, SmCMT2b, SmCMT3 and SmDRM1, were salicylic acid-responsive, suggesting the involvement of SmC5-MTases in salicylic acid-dependent immunity. These results provide useful information for demonstrating the role of DNA methylation in bioactive compound biosynthesis and Dao-di herb formation in medicinal plants.

scholarly journals The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

2016 ◽  
Vol 39 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Martin Morovic ◽  
Matej Murin ◽  
Frantisek Strejcek ◽  
Michal Benc ◽  
Dusan Paál ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Coming Out ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Early Embryos

AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

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.

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