Cytosine Residues In DNA Research Articles

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

CARCINOGENICITY OF IONIZING RADIATION: A LITERATURE REVIEW

Relevance: According to WHO, malignant neoplasms rank second in population mortality structure due to a constantly increasing influence of technogenic factors that have a direct carcinogenic effect on the body and suppress defense mechanisms. Ionizing radiation plays a special role in the development of cancer. It is used in industry, agriculture, medicine, and scientific research as a diagnostic tool in modern healthcare and radiation therapy for cancer treatment. The consequences of radiation influence are not only the result of a direct effect on the body but also a delayed one through generations of parents and grandparents. According to the radiobiological hypothesis, any level of radiation, no matter how small, poses a risk of long-term consequences, including cancer, in exposed people and their descendants of the first two generations. That is, cancerous tumors are likely consequences of the influence of radiation. Despite various theories of the biological effect of low doses of ionizing radiation, most authors attach primary importance to DNA damage in the manifestation of genetic effects (the concept of non-threshold mutational action).
 The study aimed to highlight the role of ionizing radiation in tumorigenesis.
 Methods: Data from MEDLINE, Embase, Scopus, PubMed, Cochrane Central Register of Controlled Trials was analyzed to select and analyze relevant information over the past 10 years using the keywords: gamma irradiation, spontaneous oncogenesis, prevention of oncogenesis.
 Results: Radiation exposure may increase the risk of cancer development due to epigenetic changes leading to increased genomic instability (GI) and/or specific suppression of tumor suppressor genes. Changes in the TP53 gene network expression occur; the most significant genes as predictors of carcinogenesis are ST13, IER3, BRCAI, LRDD, and MRAS. Epigenetic changes also influence individual susceptibility to radiation-induced cancer. In addition to the mutagenic effects of ROS and AFN, there is also evidence that oxidative stress plays a fundamental role in epigenetic modifications.
 Conclusion: As a result of radiation exposure, damage occurs that causes genetic and epigenetic changes, leading to changes in the level of protein expression due to changes in the methylation of cytosine residues in DNA, modification of histones, and regulation of microRNA expression.

CARCINOGENICITY OF IONIZING RADIATION:
 A LITERATURE REVIEW

Relevance: According to WHO, malignant neoplasms rank second in population mortality structure due to a constantly increasing influence of
 technogenic factors that have a direct carcinogenic effect on the body and suppress defense mechanisms. Ionizing radiation plays a special role in
 the development of cancer. It is used in industry, agriculture, medicine, and scientific research as a diagnostic tool in modern healthcare and radiation therapy for cancer treatment. The consequences of radiation influence are not only the result of a direct effect on the body but also a delayed
 one through generations of parents and grandparents. According to the radiobiological hypothesis, any level of radiation, no matter how small,
 ОБЗОРЫ ЛИТЕРАТУРЫ
 Онкология и Радиология Казахстана, №4 (70) 2023 45
 poses a risk of long-term consequences, including cancer, in exposed people and their descendants of the first two generations. That is, cancerous
 tumors are likely consequences of the influence of radiation. Despite various theories of the biological effect of low doses of ionizing radiation,
 most authors attach primary importance to DNA damage in the manifestation of genetic effects (the concept of non-threshold mutational action).
 The study aimed to highlight the role of ionizing radiation in tumorigenesis.
 Methods: Data from MEDLINE, Embase, Scopus, PubMed, Cochrane Central Register of Controlled Trials was analyzed to select and analyze
 relevant information over the past 10 years using the keywords: gamma irradiation, spontaneous oncogenesis, prevention of oncogenesis,
 Results: Radiation exposure may increase the risk of cancer development due to epigenetic changes leading to increased genomic instability
 (GI) and/or specific suppression of tumor suppressor genes. Changes in the TP53 gene network expression occur; the most significant genes as
 predictors of carcinogenesis are ST13, IER3, BRCAI, LRDD, and MRAS. Epigenetic changes also influence individual susceptibility to radiation-induced cancer. In addition to the mutagenic effects of ROS and AFN, there is also evidence that oxidative stress plays a fundamental role in
 epigenetic modifications.
 Conclusion: As a result of radiation exposure, damage occurs that causes genetic and epigenetic changes, leading to changes in the level of
 protein expression due to changes in the methylation of cytosine residues in DNA, modification of histones, and regulation of microRNA expression.

Enzyme-free targeted DNA demethylation using CRISPR-dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression.

DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.

Transition from Seeds to Seedlings: Hormonal and Epigenetic Aspects.

Transition from seed to seedling is one of the critical developmental steps, dramatically affecting plant growth and viability. Before plants enter the vegetative phase of their ontogenesis, massive rearrangements of signaling pathways and switching of gene expression programs are required. This results in suppression of the genes controlling seed maturation and activation of those involved in regulation of vegetative growth. At the level of hormonal regulation, these events are controlled by the balance of abscisic acid and gibberellins, although ethylene, auxins, brassinosteroids, cytokinins, and jasmonates are also involved. The key players include the members of the LAFL network—the transcription factors LEAFY COTYLEDON1 and 2 (LEC 1 and 2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (FUS3), as well as DELAY OF GERMINATION1 (DOG1). They are the negative regulators of seed germination and need to be suppressed before seedling development can be initiated. This repressive signal is mediated by chromatin remodeling complexes—POLYCOMB REPRESSIVE COMPLEX 1 and 2 (PRC1 and PRC2), as well as PICKLE (PKL) and PICKLE-RELATED2 (PKR2) proteins. Finally, epigenetic methylation of cytosine residues in DNA, histone post-translational modifications, and post-transcriptional downregulation of seed maturation genes with miRNA are discussed. Here, we summarize recent updates in the study of hormonal and epigenetic switches involved in regulation of the transition from seed germination to the post-germination stage.

Abstract 1507: Chimeric antigen receptor T cells carrying IDH1 neomorph increase CD8 positive central memory proportion without interfering with cell growth

Abstract Background: The efficacy of anti-CD19 Chimeric Antigen Receptor (CAR)-T cells in treating relapsed/refractory chronic lymphoid leukemia has proven effective, but only in a small group of cases. Our group previously reported that TET2 disruption promoted the therapeutic efficacy of CART19 cells via the enrichment of the CD8+ central-memory (Tcm) subsets. TET2 is a tumor suppressor; hence, the permanent ablation via genome engineering could result in oncogenic transformation. Since TET2 utilizes alpha-ketoglutarate as a cofactor in the removal of methylgroups from cytosine residues in DNA, the competitive inhibition of TET2 could prove safer. We here explore this strategy by expressing an R132H variant of isocitrate dehydrogenase-I (IDH1), which generates millimolar quantities of D-2-hydroxyglutarate, a competitive inhibitor of TET2. Methods: We generated a lentiviral construct carrying the full-length wildtype or mutant IDH1 open reading frame aminoterminal of a fully human, 4-1BB/CD3z-signaling CAR19, separated by a 2A element. A cell-surface expressed marker carrying the epitopes for CD20 and CD34 was added for CAR-T cells detection and purification. The above construct was transduced into healthy donors' T lymphocytes by a lentiviral transfer system. Sorted engineered cells were subsequently stimulated with artificial antigen presenting cells (aAPCs) expressing the target antigen. Cell growth was monitored and flow cytometry immunophenotyping was performed at the given time points upon several rounds of restimulation. Results: Western blot assay showed IDH1 R132H neomorph only expressed in the CAR-T cells carrying mutant IDH1. The proliferation of IDH1 neomorph-expressing CAR-T cells was similar to the IDH1 wildtype group in this repeat stimulation assay. Similar results were obtained with the anti-CD3 expressing APCs. However, CAR-T cells harboring IDH1 R132H contained a remarkable enrichment of CD8+ anti-CD19 CAR T cells after four rounds of tumor stimulations compared with the control groups. To correct for donor-to-donor variability in the proportion of CD4+ and CD8+ T cells at the start of the experiment, we seeded engineered T cells at a 2:1 ratio. The selective elevation of CD8+ proportion was only observed in CAR-T cells with IDH1 R132H. We further found CAR-T cells having mutant IDH1 displayed an increasing enrichment of CD8+ Tcm (CD45RO+CD27+) subsets after repetitive tumor challenges. In contrast, the other two groups showed a stable or reducing percentage of CD8+ Tcm. Conclusion: The above findings suggested that the introduction of IDH1[R132H] in CAR-T cells could lead to an increase of CD8+ frequency, as well as CD8+ Tcm proportion upon CAR signaling activation. Meanwhile, this introduction didn't make CAR-T cells grow wildly, indicating it is a safe strategy with the potential for further preclinical and clinical studies. Citation Format: Jie Xu, Yaroslav Kaminskiy, Jan Joseph Melenhorst. Chimeric antigen receptor T cells carrying IDH1 neomorph increase CD8 positive central memory proportion without interfering with cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1507.

Rett syndrome: Methyl-CpG binding protein 2 transformation and phenotypic-genotypic characteristics

Methyl-CpG-binding protein 2 (MECP2) mutation has been coupled to Rett syndrome; X-linked neurological disorder, leading to steady slowing down of neurodevelopment that exclusively affects girls worldwide. As this gene is known for its specific localization and careful binding to methylated Cytosine residues in genomic DNA and is expected to intercede biological function of transcriptional oppression and chromatin concentration too. But a finest delivery of properly folded and tailored MECP2 protein is required for its part in directive gene transcription. The detailed research work done worldwide shows that MECP2 function is not only vital for normal brain development and neuronal pledge but is eventually requisite for establishment and upholding of normal neuronal occupation. The percentage of MECP2 mutation in classic Rett patients is up to 80% however this transformation spectrum ranges from severe to mild with congenital encephalopathy, mental retardation and various neurological side effects. Altered MECP2 shows relevance to phenotypic manifestation but the skewed X inactivation appears to influence its seriousness in a far more extensive sense. The patients with identical MECP2 mutation may show very different phenotypic features and strictness because of variation in x-inactivation prototype among individuals. This study is done to review the availability of diagnosis and treatment at molecular level. In this review the clinical spectrum and molecular findings in females with MECP2 dysfunctions are also reviewed with special reference to evaluate the sufficient available criteria for molecular diagnosis and treatment across the globe. Keywords: Genotype; MECP2 mutation; Phenotype; Rett syndrome http://dx.doi.org/10.19045/bspab.2020.90246

Clinical value of DNA methylation markers in autoimmune rheumatic diseases.

Methylation of cytosine residues in DNA, the best studied epigenetic modification, is associated with gene transcription and nuclear organization, and ultimately the function of a cell. DNA methylation can be influenced by various factors, including changes in neighbouring genomic sites such as those induced by transcription factor binding. The DNA methylation profiles in relevant cell types are altered in most human diseases compared with the healthy state. Given the physical stability of DNA and methylated DNA compared with other epigenetic modifications, DNA methylation is an ideal marker for clinical purposes. However, few DNA methylation-based markers have made it into clinical practice, with the notable exception of some markers used in the field of oncology. Autoimmune rheumatic diseases are genetically complex entities that can vary widely in terms of prognosis, subtypes, progression and treatment responses. Increasing reports showing strong links between DNA methylation profiles and different clinical outcomes and other clinical aspects in autoimmune rheumatic diseases reinforce the usefulness of DNA methylation profiles as novel clinical markers. In this Review, we provide an updated discussion on DNA methylation alterations in autoimmune rheumatic diseases and the advantages and disadvantages of using these markers in clinical practice.

DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain

Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.

Epigenetically quantified immune cells in salivary glands of Sjögren's syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology.

ObjectiveTo investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren’s and sicca syndrome and to capture immunopathology and potentially aid in diagnosis.MethodsDNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters.ResultsThe percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity.ConclusionEpigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials.

Contribution of promoter DNA sequence to heterochromatin formation velocity and memory of gene repression in mouse embryo fibroblasts.

Durable gene silencing through the formation of compact heterochromatin domains plays a critical role during mammalian development in establishing defined tissues capable of retaining cellular identity. Hallmarks of heterochromatin gene repression are the binding of heterochromatin protein 1 (HP1), trimethylation of lysine 9 on histone H3 (H3K9me3) and the methylation of cytosine residues of DNA. HP1 binds directly to the H3K9me3 histone modification, and while DNA methyltransferases have been found in complex with histone methyltransferases and HP1, there remains much to be known about the relationship between DNA sequence and HP1 in differentiated mammalian cells. To further explore this interplay in a controlled system, we designed a system to test the effect of promoter CpG content on the formation kinetics and memory of an HP1-mediated heterochromatin domain in mouse embryo fibroblasts (MEF)s. To do this, we have constructed a side-by-side comparison of wild-type (CpGFull) and CpG-depleted (CpGDep) promoter-driven reporter constructs in the context of the Chromatin in vivo Assay (CiA), which uses chemically-induced proximity (CIP) to tether the chromoshadow domain of HP1α (csHP1α) to a fluorescent reporter gene in a reversible, chemically-dependent manner. By comparing the response of CpGFull and CpGDep reporter constructs, we discovered that the heterochromatin formation by recruitment of csHP1α is unaffected by the underlying CpG dinucleotide content of the promoter, as measured by the velocity of gene silencing or enrichment of H3K9me3 at the silenced gene. However, recovery from long-term silencing is measurably faster in the CpG-depleted reporter lines. These data provide evidence that the stability of the HP1 heterochromatin domain is reliant on the underlying DNA sequence. Moreover, these cell lines represent a new modular system with which to study the effect of the underlying DNA sequences on the efficacy of epigenetic modifiers.

Polyamine Metabolism and Gene Methylation in Conjunction with One-Carbon Metabolism.

Recent investigations have revealed that changes in DNA methylation status play an important role in aging-associated pathologies and lifespan. The methylation of DNA is regulated by DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) in the presence of S-adenosylmethionine (SAM), which serves as a methyl group donor. Increased availability of SAM enhances DNMT activity, while its metabolites, S-adenosyl-l-homocysteine (SAH) and decarboxylated S-adenosylmethionine (dcSAM), act to inhibit DNMT activity. SAH, which is converted from SAM by adding a methyl group to cytosine residues in DNA, is an intermediate precursor of homocysteine. dcSAM, converted from SAM by the enzymatic activity of adenosylmethionine decarboxylase, provides an aminopropyl group to synthesize the polyamines spermine and spermidine. Increased homocysteine levels are a significant risk factor for the development of a wide range of conditions, including cardiovascular diseases. However, successful homocysteine-lowering treatment by vitamins (B6, B12, and folate) failed to improve these conditions. Long-term increased polyamine intake elevated blood spermine levels and inhibited aging-associated pathologies in mice and humans. Spermine reversed changes (increased dcSAM, decreased DNMT activity, aberrant DNA methylation, and proinflammatory status) induced by the inhibition of ornithine decarboxylase. The relation between polyamine metabolism, one-carbon metabolism, DNA methylation, and the biological mechanism of spermine-induced lifespan extension is discussed.

N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle.

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified m6A site in the 5' epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

Can Reprogramming of Overall Epigenetic Memory and Specific Parental Genomic Imprinting Memory within Donor Cell-Inherited Nuclear Genome be a Major Hindrance for the Somatic Cell Cloning of Mammals? – A Review

Abstract Successful cloning of animals by somatic cell nuclear transfer (SCNT) requires epigenetic transcriptional reprogramming of the differentiated state of the donor cell nucleus to a totipotent embryonic ground state. It means that the donor nuclei must cease its own program of gene expression and restore a particular program of the embryonic genome expression regulation that is necessary for normal development. Transcriptional activity of somatic cell-derived nuclear genome during embryo pre- and postimplantation development as well as foetogenesis is correlated with the frequencies for spatial remodeling of chromatin architecture and reprogramming of cellular epigenetic memory. This former and this latter process include such covalent modifications as demethylation/re-methylation of DNA cytosine residues and acetylation/deacetylation as well as demethylation/re-methylation of lysine residues of nucleosomal core-derived histones H3 and H4. The main cause of low SCNT efficiency in mammals turns out to be an incomplete reprogramming of transcriptional activity for donor cell-descended genes. It has been ascertained that somatic cell nuclei should undergo the wide DNA cytosine residue demethylation changes throughout the early development of cloned embryos to reset their own overall epigenetic and parental genomic imprinting memories that have been established by re-methylation of the nuclear donor cell-inherited genome during specific pathways of somatic and germ cell lineage differentiation. A more extensive understanding of the molecular mechanisms and recognition of determinants for epigenetic transcriptional reprogrammability of somatic cell nuclear genome will be helpful to solve the problems resulting from unsatisfactory SCNT effectiveness and open new possibilities for common application of this technology in transgenic research focused on human biomedicine.

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample.

BackgroundDNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity.MethodsA magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.ResultsUnder the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits.ConclusionThese indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples.

Arginine CGA codons as a source of nonsense mutations: a possible role in multivariant gene expression, control of mRNA quality, and aging.

Methylation of cytosine residues in DNA of higher eukaryotes, including humans, creates "hot spots" of C→T transitions in the genome. The predominantly methylated sequence in mammalian DNAs is CG (CpG). Among CG-containing codons, CGA codons for arginine are unique due to their ability to create stop codons TGA (UGA in mRNA) upon epigenetic-mediated mutation. As such nonsense mutations can have a strong adverse effect on the cell and organism, we have performed a study, on the example of human genes, aimed to characterise the anticipated effects of epigenetic-mediated nonsense mutations CGA→TGA in somatic cells. It is commonly accepted that premature termination codons (PTCs) lead to the biosynthesis of truncated and usually inactive proteins. In addition, transcripts with PTC can be destroyed by a nonsense-mediated mRNA decay (NMD) machinery. We have considered the cell potentialities (gene families, diploidy, and alternative splicing) to overcome the worst consequences of nonsense mutation. As a special case, in the biosynthesis of a particular group of proteins called selenoproteins, the mutation CGA→UGA would not lead to the premature translation termination and NMD but rather to the insertion of selenocysteine or cysteine instead of former arginine. The finding of SECIS (Sel insertion sequence)-like structures in a variety of mRNAs allowed us to postulate the existence of facultative selenoproteins, whose biosynthesis might be coupled with the redefinition of premature UGA stop codons arising upon mutations, as in the case of "classic" selenoproteins. Nevertheless, a detailed structural analysis of 165 transcripts has shown that roughly 80-90% of functional human mRNAs are potential substrates for NMD upon the PTC emergence. A hypothesis was put forward highlighting a role of arginine CGA codons together with glutamine CAA and CAG codons in the control of mRNA quality and life span. According to this hypothesis, the conversion of the ribonucleic codons CGA, CAA, or CAG into stop codons UGA, UAA or UAG owing to spontaneous or enzymatic cytosine deamination might serve as a trigger for the transcript destruction by NMD (C→U control). Thus, the consequences of epigenetic-mediated nonsense mutations are diverse and may largely depend on the structure of the transcript (CGA codon position, the presence and position of introns and SECIS elements, and splicing potential) of the cognate gene. However, this diversity and the presumable role of CGA codons in performing the everyday function by controlling whether genes are expressed correctly do not exclude their long-term role as limiters of the cell and organism life span. Thus, the presumable role of CGA codons in genome functioning and stability opens new perspectives to influence aging and concomitant deceases by codon editing.

Issue Information

Genes to CellsVolume 22, Issue 2 p. 125-129 Issue InformationFree Access Issue Information First published: 07 February 2017 https://doi.org/10.1111/gtc.12423AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Front cover: Walking monozygotic twin sisters in the snow are sharing an umbrella. They share the same genotype; however, they seem to have a different personality and taste as their kimono. It has been pointed out that the difference in epigenome would cause such difference in personality. The data printed on their sleeves that was obtained with the bisulfite sequencing (a method that can detect methylation of individual cytosine residues in DNA) and is showing their methylation patterns at a certain locus of the genome suggests that there are epigenetic differences between them. Our knowledge of the relationship between presumed hereditary traits or diseases and epigenetics will increase through studies of monozygotic twin-pairs of which one twin has such traits or diseases. Designed by TRAIS Co., Ltd. (Kobe, Japan). Volume22, Issue2February 2017Pages 125-129 RelatedInformation

The p53 protein plays a central role in the mechanism of action of epigentic drugs that alter the methylation of cytosine residues in DNA.

Both normal and cancerous cells, treated with drugs that block cytosine methylation of DNA, are preferentially killed by these drugs when they have p53 mutations and survive if they have a wild type protein. It appears that the wild type p53 protein functions to eliminate cells that undergo large epigenetic alterations and save other cells from death by this drug treatment. This has now been observed in cancerous cells in culture, tumors in animals and tumors in humans. AML cells with p53 mutations in humans treated with decitabine are killed by differentiation or senescense, but then relapse at a high rate becoming drug resistant. The mechanism of resistance to epigenetic drugs in p53 mutant cells, by possibly restoring a wild type p53 gene or restoring a defective p53 pathway, is now an interesting hypothesis to explore.

Microsphere-Based Assessment of DNA Methylation for AML Prognosis.

Epigenetic dysregulation, including aberrant methylation of cytosine residues in DNA, is a hallmark of cancer and clearly results in oncogenic cellular alterations such as transcriptional attenuation of tumor suppressors and genomic instability. A number of studies have examined DNA methylation alterations in patients with acute myeloid leukemia (AML) and have shown that analysis of multilocus methylation patterns can identify biologically distinct AML subclasses and can predict patient prognosis. In order to utilize the prognostic capability of methylation analysis in a clinical setting, we have developed a microsphere-based HpaII tiny fragment enrichment by ligation-mediated PCR (xMELP) assay to interrogate the methylation state of genomic multiple loci along with a random forest-based classification algorithm that correlates DNA methylation status with patient prognosis. These tools can be easily implemented in a clinical molecular pathology laboratory and can be utilized for more accurate risk stratification of AML patients.

Biofluids, cell mechanics and epigenetics: Flow-induced epigenetic mechanisms of endothelial gene expression

Epigenetics is the regulation of gene expression (transcription) in response to changes in the cell environment through genomic modifications that largely involve the non-coding fraction of the human genome and that cannot be attributed to modification of the primary DNA sequence. Epigenetics is dominant in establishing cell fate and positioning during programmed embryonic development. However the same pathways are used by mature postnatal and adult mammalian cells during normal physiology and are implicated in disease mechanisms. Recent research demonstrates that blood flow and pressure are cell environments that can influence transcription via epigenetic pathways. The principal epigenetic pathways are chemical modification of cytosine residues of DNA (DNA methylation) and of the amino tails of histone proteins associated with DNA in nucleosomes. They also encompass the post-transcriptional degradation of mRNA transcripts by non-coding RNAs (ncRNA). In vascular endothelium, epigenetic pathways respond to temporal and spatial variations of flow and pressure, particularly hemodynamic disturbed blood flow, with important consequences for gene expression. The biofluid environment is linked by mechanotransduction and solute transport to cardiovascular cell phenotypes via signaling pathways and epigenetic regulation for which there is an adequate interdisciplinary infrastructure with robust tools and methods available. Epigenetic mechanisms may be less familiar than acute genomic signaling to Investigators at the interface of biofluids, biomechanics and cardiovascular biology. Here we introduce a biofluids / cellular biomechanics readership to the principal epigenetic pathways and provide a contextual overview of endothelial epigenetic plasticity in the regulation of flow-responsive transcription.