Epigenomes

22 pages, 3650 KiB

Open AccessArticle

Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres

by Susana M. Contreras, Romina T. Zambrano Siri, Elías M. Rivera, Constanza Cristaldi, Laura Kamenetzky, Kami Kim, Marina Clemente, Josefina Ocampo, Laura Vanagas and Sergio O. Angel

Epigenomes 2022, 6(3), 29; https://doi.org/10.3390/epigenomes6030029 - 15 Sep 2022

Cited by 8 | Viewed by 3189

Abstract

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize [...] Read more.

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5′ end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5′ end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes. Full article

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30 pages, 2157 KiB

Open AccessReview

Polycomb Directed Cell Fate Decisions in Development and Cancer

by Beatriz German and Leigh Ellis

Epigenomes 2022, 6(3), 28; https://doi.org/10.3390/epigenomes6030028 - 6 Sep 2022

Cited by 13 | Viewed by 7828

Abstract

The polycomb group (PcG) proteins are a subset of transcription regulators highly conserved throughout evolution. Their principal role is to epigenetically modify chromatin landscapes and control the expression of master transcriptional programs to determine cellular identity. The two mayor PcG protein complexes that [...] Read more.

The polycomb group (PcG) proteins are a subset of transcription regulators highly conserved throughout evolution. Their principal role is to epigenetically modify chromatin landscapes and control the expression of master transcriptional programs to determine cellular identity. The two mayor PcG protein complexes that have been identified in mammals to date are Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). These protein complexes selectively repress gene expression via the induction of covalent post-translational histone modifications, promoting chromatin structure stabilization. PRC2 catalyzes the histone H3 methylation at lysine 27 (H3K27me1/2/3), inducing heterochromatin structures. This activity is controlled by the formation of a multi-subunit complex, which includes enhancer of zeste (EZH2), embryonic ectoderm development protein (EED), and suppressor of zeste 12 (SUZ12). This review will summarize the latest insights into how PRC2 in mammalian cells regulates transcription to orchestrate the temporal and tissue-specific expression of genes to determine cell identity and cell-fate decisions. We will specifically describe how PRC2 dysregulation in different cell types can promote phenotypic plasticity and/or non-mutational epigenetic reprogramming, inducing the development of highly aggressive epithelial neuroendocrine carcinomas, including prostate, small cell lung, and Merkel cell cancer. With this, EZH2 has emerged as an important actionable therapeutic target in such cancers. Full article

(This article belongs to the Special Issue Polycomb Proteins)

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13 pages, 708 KiB

Open AccessCommentary

Neurogenetic and Epigenetic Aspects of Cannabinoids

by Catherine A. Dennen, Kenneth Blum, Abdalla Bowirrat, Jag Khalsa, Panayotis K. Thanos, David Baron, Rajendra D. Badgaiyan, Ashim Gupta, Eric R. Braverman and Mark S. Gold

Epigenomes 2022, 6(3), 27; https://doi.org/10.3390/epigenomes6030027 - 26 Aug 2022

Cited by 8 | Viewed by 4027

Abstract

Cannabis is one of the most commonly used and abused illicit drugs in the world today. The United States (US) currently has the highest annual prevalence rate of cannabis consumption in the world, 17.9% in individuals aged 12 or older, and it is [...] Read more.

Cannabis is one of the most commonly used and abused illicit drugs in the world today. The United States (US) currently has the highest annual prevalence rate of cannabis consumption in the world, 17.9% in individuals aged 12 or older, and it is on the rise. With increasing cannabis use comes the potential for an increase in abuse, and according to the Substance Abuse and Mental Health Services Administration (SAMHSA), approximately 5.1% of Americans had Cannabis Use Disorder (CUD) in 2020. Research has shown that genetics and epigenetics play a significant role in cannabis use and CUD. In fact, approximately 50–70% of liability to CUD and 40–48% of cannabis use initiation have been found to be the result of genetic factors. Cannabis usage and CUD have also been linked to an increased risk of psychiatric disorders and Reward Deficiency Syndrome (RDS) subsets like schizophrenia, depression, anxiety, and substance use disorder. Comprehension of the genetic and epigenetic aspects of cannabinoids is necessary for future research, treatment plans, and the production of pure cannabinoid compounds, which will be essential for FDA approval. In conclusion, having a better understanding of the epigenetic and genetic underpinnings of cannabis use, CUD, and the endocannabinoid system as a whole will aid in the development of effective FDA-approved treatment therapies and the advancement of personalized medicine. Full article

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11 pages, 1145 KiB

Open AccessReview

R-Loop Formation in Meiosis: Roles in Meiotic Transcription-Associated DNA Damage

by Yasuhiro Fujiwara, Mary Ann Handel and Yuki Okada

Epigenomes 2022, 6(3), 26; https://doi.org/10.3390/epigenomes6030026 - 24 Aug 2022

Cited by 3 | Viewed by 4042

Abstract

Meiosis is specialized cell division during gametogenesis that produces genetically unique gametes via homologous recombination. Meiotic homologous recombination entails repairing programmed 200–300 DNA double-strand breaks generated during the early prophase. To avoid interference between meiotic gene transcription and homologous recombination, mammalian meiosis is [...] Read more.

Meiosis is specialized cell division during gametogenesis that produces genetically unique gametes via homologous recombination. Meiotic homologous recombination entails repairing programmed 200–300 DNA double-strand breaks generated during the early prophase. To avoid interference between meiotic gene transcription and homologous recombination, mammalian meiosis is thought to employ a strategy of exclusively transcribing meiotic or post-meiotic genes before their use. Recent studies have shown that R-loops, three-stranded DNA/RNA hybrid nucleotide structures formed during transcription, play a crucial role in transcription and genome integrity. Although our knowledge about the function of R-loops during meiosis is limited, recent findings in mouse models have suggested that they play crucial roles in meiosis. Given that defective formation of an R-loop can cause abnormal transcription and transcription-coupled DNA damage, the precise regulatory network of R-loops may be essential in vivo for the faithful progression of mammalian meiosis and gametogenesis. Full article

(This article belongs to the Special Issue Chromatin Unlimited)

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26 pages, 2303 KiB

Open AccessReview

De Novo Polycomb Recruitment and Repressive Domain Formation

by Itzel Alejandra Hernández-Romero and Victor Julian Valdes

Epigenomes 2022, 6(3), 25; https://doi.org/10.3390/epigenomes6030025 - 22 Aug 2022

Cited by 4 | Viewed by 4249

Abstract

Every cell of an organism shares the same genome; even so, each cellular lineage owns a different transcriptome and proteome. The Polycomb group proteins (PcG) are essential regulators of gene repression patterning during development and homeostasis. However, it is unknown how the repressive [...] Read more.

Every cell of an organism shares the same genome; even so, each cellular lineage owns a different transcriptome and proteome. The Polycomb group proteins (PcG) are essential regulators of gene repression patterning during development and homeostasis. However, it is unknown how the repressive complexes, PRC1 and PRC2, identify their targets and elicit new Polycomb domains during cell differentiation. Classical recruitment models consider the pre-existence of repressive histone marks; still, de novo target binding overcomes the absence of both H3K27me3 and H2AK119ub. The CpG islands (CGIs), non-core proteins, and RNA molecules are involved in Polycomb recruitment. Nonetheless, it is unclear how de novo targets are identified depending on the physiological context and developmental stage and which are the leading players stabilizing Polycomb complexes at domain nucleation sites. Here, we examine the features of de novo sites and the accessory elements bridging its recruitment and discuss the first steps of Polycomb domain formation and transcriptional regulation, comprehended by the experimental reconstruction of the repressive domains through time-resolved genomic analyses in mammals. Full article

(This article belongs to the Special Issue Polycomb Proteins)

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9 pages, 1436 KiB

Open AccessReview

Experimental and Computational Approaches for Non-CpG Methylation Analysis

by Deepa Ramasamy, Arunagiri Kuha Deva Magendhra Rao, Thangarajan Rajkumar and Samson Mani

Epigenomes 2022, 6(3), 24; https://doi.org/10.3390/epigenomes6030024 - 16 Aug 2022

Cited by 2 | Viewed by 3351

Abstract

Cytosine methylation adjacent to adenine, thymine, and cytosine residues but not guanine of the DNA is distinctively known as non-CpG methylation. This CA/CT/CC methylation accounts for 15% of the total cytosine methylation and varies among different cell and tissue types. The abundance of [...] Read more.

Cytosine methylation adjacent to adenine, thymine, and cytosine residues but not guanine of the DNA is distinctively known as non-CpG methylation. This CA/CT/CC methylation accounts for 15% of the total cytosine methylation and varies among different cell and tissue types. The abundance of CpG methylation has largely concealed the role of non-CpG methylation. Limitations in the early detection methods could not distinguish CpG methylation from non-CpG methylation. Recent advancements in enrichment strategies and high throughput sequencing technologies have enabled the detection of non-CpG methylation. This review discusses the advanced experimental and computational approaches to detect and describe the genomic distribution and function of non-CpG methylation. We present different approaches such as enzyme-based and antibody-based enrichment, which, when coupled, can also improve the sensitivity and specificity of non-CpG detection. We also describe the current bioinformatics pipelines and their specific application in computing and visualizing the imbalance of CpG and non-CpG methylation. Enrichment modes and the computational suites need to be further developed to ease the challenges of understanding the functional role of non-CpG methylation. Full article

(This article belongs to the Special Issue Non-CpG Methylation)

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18 pages, 4420 KiB

Open AccessReview

The Role of Polycomb Proteins in Cell Lineage Commitment and Embryonic Development

by Chet H. Loh and Gert Jan C. Veenstra

Epigenomes 2022, 6(3), 23; https://doi.org/10.3390/epigenomes6030023 - 12 Aug 2022

Cited by 20 | Viewed by 6079

Abstract

Embryonic development is a highly intricate and complex process. Different regulatory mechanisms cooperatively dictate the fate of cells as they progress from pluripotent stem cells to terminally differentiated cell types in tissues. A crucial regulator of these processes is the Polycomb Repressive Complex [...] Read more.

Embryonic development is a highly intricate and complex process. Different regulatory mechanisms cooperatively dictate the fate of cells as they progress from pluripotent stem cells to terminally differentiated cell types in tissues. A crucial regulator of these processes is the Polycomb Repressive Complex 2 (PRC2). By catalyzing the mono-, di-, and tri-methylation of lysine residues on histone H3 tails (H3K27me3), PRC2 compacts chromatin by cooperating with Polycomb Repressive Complex 1 (PRC1) and represses transcription of target genes. Proteomic and biochemical studies have revealed two variant complexes of PRC2, namely PRC2.1 which consists of the core proteins (EZH2, SUZ12, EED, and RBBP4/7) interacting with one of the Polycomb-like proteins (MTF2, PHF1, PHF19), and EPOP or PALI1/2, and PRC2.2 which contains JARID2 and AEBP2 proteins. MTF2 and JARID2 have been discovered to have crucial roles in directing and recruiting PRC2 to target genes for repression in embryonic stem cells (ESCs). Following these findings, recent work in the field has begun to explore the roles of different PRC2 variant complexes during different stages of embryonic development, by examining molecular phenotypes of PRC2 mutants in both in vitro (2D and 3D differentiation) and in vivo (knock-out mice) assays, analyzed with modern single-cell omics and biochemical assays. In this review, we discuss the latest findings that uncovered the roles of different PRC2 proteins during cell-fate and lineage specification and extrapolate these findings to define a developmental roadmap for different flavors of PRC2 regulation during mammalian embryonic development. Full article

(This article belongs to the Special Issue Polycomb Proteins)

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16 pages, 2868 KiB

Open AccessEditor’s ChoiceReview

Nucleosome Structures Built from Highly Divergent Histones: Parasites and Giant DNA Viruses

by Shoko Sato, Mariko Dacher and Hitoshi Kurumizaka

Epigenomes 2022, 6(3), 22; https://doi.org/10.3390/epigenomes6030022 - 2 Aug 2022

Cited by 3 | Viewed by 4435

Abstract

In eukaryotes, genomic DNA is bound with histone proteins and packaged into chromatin. The nucleosome, a fundamental unit of chromatin, regulates the accessibility of DNA to enzymes involved in gene regulation. During the past few years, structural analyses of chromatin architectures have been [...] Read more.

In eukaryotes, genomic DNA is bound with histone proteins and packaged into chromatin. The nucleosome, a fundamental unit of chromatin, regulates the accessibility of DNA to enzymes involved in gene regulation. During the past few years, structural analyses of chromatin architectures have been limited to evolutionarily related organisms. The amino acid sequences of histone proteins are highly conserved from humans to yeasts, but are divergent in the deeply branching protozoan groups, including human parasites that are directly related to human health. Certain large DNA viruses, as well as archaeal organisms, contain distant homologs of eukaryotic histone proteins. The divergent sequences give rise to unique and distinct nucleosome architectures, although the fundamental principles of histone folding and DNA contact are highly conserved. In this article, we review the structures and biophysical properties of nucleosomes containing histones from the human parasites Giardia lamblia and Leishmania major, and histone-like proteins from the Marseilleviridae amoeba virus family. The presented data confirm the sharing of the overall DNA compaction system among evolutionally distant species and clarify the deviations from the species-specific nature of the nucleosome. Full article

(This article belongs to the Special Issue Chromatin Unlimited)

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15 pages, 522 KiB

Open AccessReview

Epigenomic Approaches for the Diagnosis of Rare Diseases

by Beatriz Martinez-Delgado and Maria J. Barrero

Epigenomes 2022, 6(3), 21; https://doi.org/10.3390/epigenomes6030021 - 27 Jul 2022

Cited by 7 | Viewed by 4001

Abstract

Rare diseases affect more than 300 million people worldwide. Diagnosing rare diseases is a major challenge as they have different causes and etiologies. Careful assessment of clinical symptoms often leads to the testing of the most common genetic alterations that could explain the [...] Read more.

Rare diseases affect more than 300 million people worldwide. Diagnosing rare diseases is a major challenge as they have different causes and etiologies. Careful assessment of clinical symptoms often leads to the testing of the most common genetic alterations that could explain the disease. Patients with negative results for these tests frequently undergo whole exome or genome sequencing, leading to the identification of the molecular cause of the disease in 50% of patients at best. Therefore, a significant proportion of patients remain undiagnosed after sequencing their genome. Recently, approaches based on functional aspects of the genome, including transcriptomics and epigenomics, are beginning to emerge. Here, we will review these approaches, including studies that have successfully provided diagnoses for complex undiagnosed cases. Full article

(This article belongs to the Special Issue The Use of Epigenetic Biomarkers as Diagnostic and Therapeutic Options 2.0)

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13 pages, 2498 KiB

Open AccessEditor’s ChoiceReview

Making Mitotic Chromosomes in a Test Tube

by Keishi Shintomi

Epigenomes 2022, 6(3), 20; https://doi.org/10.3390/epigenomes6030020 - 20 Jul 2022

Cited by 2 | Viewed by 3460

Abstract

Mitotic chromosome assembly is an essential preparatory step for accurate transmission of the genome during cell division. During the past decades, biochemical approaches have uncovered the molecular basis of mitotic chromosomes. For example, by using cell-free assays of frog egg extracts, the condensin [...] Read more.

Mitotic chromosome assembly is an essential preparatory step for accurate transmission of the genome during cell division. During the past decades, biochemical approaches have uncovered the molecular basis of mitotic chromosomes. For example, by using cell-free assays of frog egg extracts, the condensin I complex central for the chromosome assembly process was first identified, and its functions have been intensively studied. A list of chromosome-associated proteins has been almost completed, and it is now possible to reconstitute structures resembling mitotic chromosomes with a limited number of purified factors. In this review, I introduce how far we have come in understanding the mechanism of chromosome assembly using cell-free assays and reconstitution assays, and I discuss their potential applications to solve open questions. Full article

(This article belongs to the Special Issue Chromatin Unlimited)

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4 pages, 180 KiB

Open AccessConference Report

Clinical Epigenetics on the Baltic Coast

by Ryszard Maleszka

Epigenomes 2022, 6(3), 19; https://doi.org/10.3390/epigenomes6030019 - 20 Jul 2022

Cited by 1 | Viewed by 2368

Abstract

This report summarizes the proceedings of the inaugural Clinical Epigenetics Conference that was held in Szczecin, Poland, from 8 June 2022. With focus on epigenetic diseases whose causes, progression, and prognosis are associated with aberrant epigenomic alterations, the meeting was a timely forum [...] Read more.

This report summarizes the proceedings of the inaugural Clinical Epigenetics Conference that was held in Szczecin, Poland, from 8 June 2022. With focus on epigenetic diseases whose causes, progression, and prognosis are associated with aberrant epigenomic alterations, the meeting was a timely forum to discuss recent progress in this rapidly evolving field and consider avenues for converting experimental data into clinical reality that would be beneficial for patients. The wealth of the presented data was an impressive showcase of the enormous challenges faced by researchers in their quest for understanding the benefits and limitations of the available information in the medical context. A shared view among the participants was that merging the current state of knowledge with clinical applications will be promptly achieved. Full article

3 pages, 181 KiB

Open AccessEditorial

Epidrugs: Toward Understanding and Treating Diverse Diseases

by Takashi Umehara

Epigenomes 2022, 6(3), 18; https://doi.org/10.3390/epigenomes6030018 - 12 Jul 2022

Cited by 4 | Viewed by 2568

Abstract

Epigenomic modifications are unique in the type and amount of chemical modification at each chromosomal location, can vary from cell to cell, and can be externally modulated by small molecules. In recent years, genome-wide epigenomic modifications have been revealed, and rapid progress has [...] Read more.

Epigenomic modifications are unique in the type and amount of chemical modification at each chromosomal location, can vary from cell to cell, and can be externally modulated by small molecules. In recent years, genome-wide epigenomic modifications have been revealed, and rapid progress has been made in the identification of proteins responsible for epigenomic modifications and in the development of compounds that regulate them. This Special Issue on “Epidrugs: Toward Understanding and Treating Diverse Diseases” aims to provide insights into various aspects of the biology and development of epigenome-regulating compounds. Full article

(This article belongs to the Special Issue Epidrugs: Toward Understanding and Treating Diverse Diseases)

14 pages, 7403 KiB

Open AccessArticle

DNA Hypomethylation May Contribute to Metabolic Recovery of Frozen Wood Frog Brains

by Tighe Bloskie and Kenneth B. Storey

Epigenomes 2022, 6(3), 17; https://doi.org/10.3390/epigenomes6030017 - 12 Jul 2022

Cited by 2 | Viewed by 2856

Abstract

Transcriptional suppression is characteristic of extreme stress responses, speculated to preserve energetic resources in the maintenance of hypometabolism. In recent years, epigenetic regulation has become heavily implicated in stress adaptation of many animals, including supporting freeze tolerance of the wood frog (Rana [...] Read more.

Transcriptional suppression is characteristic of extreme stress responses, speculated to preserve energetic resources in the maintenance of hypometabolism. In recent years, epigenetic regulation has become heavily implicated in stress adaptation of many animals, including supporting freeze tolerance of the wood frog (Rana sylvatica). However, nervous tissues are frequently lacking in these multi-tissue analyses which warrants investigation. The present study examines the role of DNA methylation, a core epigenetic mechanism, in the response of wood frog brains to freezing. We use immunoblot analysis to track the relative expression of DNA methyltransferases (DNMT), methyl-CpG-binding domain (MBD) proteins and ten-eleven-translocation (TET) demethylases across the freeze-thaw cycle in R. sylvatica brain, including selected comparisons to freeze-associated sub-stresses (anoxia and dehydration). Global methyltransferase activities and 5-hmC content were also assessed. The data show coordinated evidence for DNA hypomethylation in wood frog brains during freeze-recovery through the combined roles of depressed DNMT3A/3L expression driving lowered DNMT activity and increased TET2/3 levels leading to elevated 5-hmC genomic content (p < 0.05). Raised levels of DNMT1 during high dehydration were also noteworthy. The above suggest that alleviation of transcriptionally repressive 5-mC DNA methylation is a necessary component of the wood frog freeze-thaw cycle, potentially facilitating the resumption of a normoxic transcriptional state as frogs thaw and resume normal metabolic activities. Full article

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11 pages, 1728 KiB

Open AccessCommunication

Global m6A RNA Methylation in SARS-CoV-2 Positive Nasopharyngeal Samples in a Mexican Population: A First Approximation Study

by Jorge Luis Batista-Roche, Bruno Gómez-Gil, Gertrud Lund, César Alejandro Berlanga-Robles and Alejandra García-Gasca

Epigenomes 2022, 6(3), 16; https://doi.org/10.3390/epigenomes6030016 - 29 Jun 2022

Cited by 5 | Viewed by 3374

Abstract

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is the causal agent of COVID-19 (Coronavirus Disease-19). Both mutation and/or recombination events in the SARS-CoV-2 genome have resulted in variants that differ in transmissibility and severity. Furthermore, RNA methylation of the N6 position of adenosine (m6A) [...] Read more.

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is the causal agent of COVID-19 (Coronavirus Disease-19). Both mutation and/or recombination events in the SARS-CoV-2 genome have resulted in variants that differ in transmissibility and severity. Furthermore, RNA methylation of the N6 position of adenosine (m6A) is known to be altered in cells infected with SARS-CoV-2. However, it is not known whether this epitranscriptomic modification differs across individuals dependent on the presence of infection with distinct SARS-CoV-2 variants, the viral load, or the vaccination status. To address this issue, we selected RNAs (n = 60) from SARS-CoV-2 sequenced nasopharyngeal samples (n = 404) of 30- to 60-year-old outpatients or hospitalized individuals from the city of Mazatlán (Mexico) between February 2021 and March 2022. Control samples were non-infected individuals (n = 10). SARS-CoV-2 was determined with real-time PCR, viral variants were determined with sequencing, and global m6A levels were determined by using a competitive immunoassay method. We identified variants of concern (VOC; alpha, gamma, delta, omicron), the variant of interest (VOI; epsilon), and the lineage B.1.1.519. Global m6A methylation differed significantly across viral variants (p = 3.2 × 10⁻⁷). In particular, we found that m6A levels were significantly lower in the VOC delta- and omicron-positive individuals compared to non-infected individuals (p = 2.541236 × 10⁻² and 1.134411 × 10⁻⁴, respectively). However, we uncovered no significant correlation between global m6A levels and viral nucleocapsid (N) gene expression or age. Furthermore, individuals with complete vaccination schemes showed significantly lower m6A levels than unvaccinated individuals (p = 2.6 × 10⁻⁴), and differences in methylation levels across variants in unvaccinated individuals were significant (p = 3.068 × 10⁻³). These preliminary results suggest that SARS-CoV-2 variants show differences in global m6A levels. Full article

(This article belongs to the Special Issue The Use of Epigenetic Biomarkers as Diagnostic and Therapeutic Options 2.0)

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Epigenomes, Volume 6, Issue 3 (September 2022) – 14 articles

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