β-Aminobutyric acid promotes stress tolerance, physiological adjustments, as well as broad epigenetic changes at DNA and RNA nucleobases in field elms (Ulmus minor).

Background: β-Aminobutyric acid (BABA) has been successfully used to prime stress resistance in numerous plant species; however, its effectiveness in forest trees has been poorly explored thus far. This study aimed to investigate the influence of BABA on morphological, physiological, and epigenetic parameters in field elms under various growth conditions. Epigenetic changes were assessed in both DNA and RNA through the use of reversed-phase ultra-performance liquid chromatography (UPLC) coupled with sensitive mass spectrometry.

Non-canonical bases differentially represented in the sex chromosomes of the dioecious plant Silene latifolia.

The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin.

Global DNA 5-hydroxymethylcytosine level and its chromosomal distribution in four rye species.

The rye genome has a large size with a high level of cytosine methylation, which makes it particularly convenient for studying the occurrence of potential cytosine demethylation intermediates. Levels of global 5-hydroxymethylcytosine (5hmC) were analysed by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry in four rye species: Secale cereale, Secale strictum, Secale sylvestre, and Secale vavilovii. The amount of 5hmC showed interspecific variation, and was also variable among organs, i.

Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions.

In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila.

Detection and Quantification of RNA Modifications on RNA-DNA Hybrids Using SID-UPLC-MS/MS.

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and an unpaired strand of nontemplate DNA that represent a major source of genomic instability and are involved in regulation of several important biological processes in eukaryotic cells. A growing body of experimental evidence suggests that RNA moieties of RNA-DNA hybrids may convey RNA modifications influencing various aspects of R-loop biology. Here we present a protocol for quantitative analysis of RNA modifications on RNA-DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS).

Diagnostic and Prognostic Power of Active DNA Demethylation Pathway Intermediates in Acute Myelogenous Leukemia and Myelodysplastic Syndromes.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA. The active DNA demethylation process may be linked with aberrant methylation and can be involved in leukemogenesis. The levels of 5-methylcytosine oxidation products were analyzed in minimally invasive material: the cellular DNA from peripheral blood cells and urine of patients with AML and MDS along with the control group, using isotope-dilution two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry.

The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response.

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2'-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC-MS/MS).

Preparation of Internal Standards for 2D-UPLC-MS/MS Quantification of Noncanonical DNA Bases.

Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [C,N]-5-methyl-2'-deoxycytidine from [C,N]-2'-deoxythymidine.

Quantification of DNA Modifications Using Two-Dimensional Ultraperformance Liquid Chromatography Tandem Mass Spectrometry (2D-UPLC-MS/MS).

Our hereby presented methodology is suitable for reliable assessment of the most common DNA modifications which arise as a product of fundamental metabolic processes. 8-oxoguanine, one of the oxidatively modified DNA bases is a typical biomarker of oxidative stress. A noncanonical base, uracil, may also be present in small quantities in DNA.

Mass spectrometry reveals the presence of specific set of epigenetic DNA modifications in the Norway spruce genome.

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous.

N-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells. Here we show that N-methyladenosine (mA) modification, contributing to different aspects of messenger RNA metabolism, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that mA-containing R-loops accumulate during G/M and are depleted at G/G phases of the cell cycle, and that the mA reader promoting mRNA degradation, YTHDF2 (ref.

In vivo evidence of ascorbate involvement in the generation of epigenetic DNA modifications in leukocytes from patients with colorectal carcinoma, benign adenoma and inflammatory bowel disease.

Background: A characteristic feature of malignant cells, such as colorectal cancer cells, is a profound decrease in the level of 5-hydroxymethylcytosine, a product of 5-methylcytosine oxidation by TET enzymes. Recent studies showed that ascorbate may upregulate the activity of TET enzymes in cultured cells and enhance formation of their products in genomic DNA.

Methods: The study included four groups of subjects: healthy controls (n = 79), patients with inflammatory bowel disease (IBD, n = 51), adenomatous polyps (n = 67) and colorectal cancer (n = 136).

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

Background: Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies.

Correction: Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

[This corrects the article DOI: 10.1371/journal.pone.

Oxidation Products of 5-Methylcytosine are Decreased in Senescent Cells and Tissues of Progeroid Mice.

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo).

Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.

Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA.

Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA.

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells.

Enigmatic 5-hydroxymethyluracil: Oxidatively modified base, epigenetic mark or both?

The aim of this review is to describe the reactions which lead to generation of 5-hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-hydroxymethyluracil mispaired with guanine.

Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships.

Background: Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate.

Urinary 5-hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.

Context: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer.

Objectives: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-hmUra).

Materials And Methods: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited.