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Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. We integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view of 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Here, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans., Competing Interests: Competing interests D. Rieke reports advisory agreement with BeiGene and Bayer, honoraria from Bristol Myers Squibb, Bayer and Roche, research support from Seagen, and personal fees from Bayer and Johnson & Johnson, all outside the submitted work. A. Picca reports personal fees from AstraZeneca and Servier, all outside the submitted work. F. Bielle reports funding of research from Abbvie, service agreement for research contracted between his institution and Treefrog Therapeutics as well as Owkin, personal fees from Bristol Myers Squibb and a next-of-kin employed by Bristol Myers Squibb, all outside the submitted work. M.L. Yaspo is COO/CSO and shareholder of Alacris Theranostics without conflict of interest with the submitted work. M. Rodrigues reports non-financial support from AstraZeneca and Merck Sharp and Dohme, grants from Daiichi Sankyo, personal fees from AstraZeneca, Immunocore, Merck Sharp and Dohme and GlaxoSmithKline, all outside the submitted work. M.-H. Stern reports grants from Immunocore and Bionano, and royalties from Myriad Genetics, all outside the submitted work. The remaining authors have no conflict of interest to declare., (© 2024. The Author(s).)

5-Methylcytosine (m 5 C) is closely associated with cancer. However, the role of m 5 C in breast cancer(BC)remains unclear. This study combined single-cell RNA sequencing (scRNA-Seq) and transcriptomics datasets to screen m 5 C regulators associated with BC progression and analyze their clinical values. Firstly, This study elucidates the mechanisms of the m 5 C landscape and the specific roles of m 5 C regulators in BC patients. we found that the dysregulation of m 5 C regulators with m 5 Cscore play the essential role of the carcinogenesis and progression in epithelial cells and myeloid cells of BC at single cell level. External validation was conducted using an independent scRNA-Seq datasets. Then, three distinct m 5 C modification patterns were identified by transcriptomics datasets. Based on the m 5 C differentially expressed regulators, the m 5 Cscore was constructed, and used to divide patients with BC into high and low m 5 Cscore groups. Patients with a high m 5 Cscore had more abundant immune cell infiltration, stronger antitumor immunity, and better prognoses. Finally, Quantitative real-time (PCR) and immunohistochemistry were used for the in vitro experimental validation, which had extensive prognostic value. In this study, we aimed to assess the expression of m 5 C regulators involved in BC and investigate their correlation with the tumor microenvironment, clinicopathological characteristics, and prognosis of BC. The m 5 C regulators could be used to effectively assess the cell specific regulation prognosis of patients with BC and develop more effective immunotherapy strategies., Competing Interests: Declarations Ethics approval and consent to participate The studies involving human participants were reviewed and approved by Ethics Committee of the First Hospital of Shanxi Medical University, the ethics number is K-K0109. The patients provided their written informed consent to participate in this study. Consent for publication The patients involved have obtained ethical approval and written informed consent for the publication of any potentially identifiable images or data included in this article. Competing interests The authors declare no competing interests. Statement All methods were performed in accordance with the relevant guidelines and regulations., (© 2024. The Author(s).)

5-Methyldeoxycytidine (5mC) is a major epigenetic marker that regulates cellular functions in mammals. Endogenous lipid peroxidation can convert 5mC into 3,N4-etheno-5-methylcytosine (ϵ5mC). ϵ5mC is structurally similar to the mutagenic analog 3,N4-ethenocytosine (ϵC), which is repaired by AlkB family enzymes in the direct reversal repair (DRR) pathway and excised by DNA glycosylases in the base excision repair (BER) pathway. However, the repair of ϵ5mC has not been reported. Here, we examined the activities against ϵ5mC by DRR and BER enzymes and TET1-3, enzymes that modify the 5-methyl group in 5mC. We found that the etheno modification of 5mC blocks oxidation by TET1-3. Conversely, three human homologs in the AlkB family, ALKBH2, 3 and FTO were able to repair ϵ5mC to 5mC, which was subsequently modified by TET1 to 5-hydroxymethylcytosine. We also demonstrated that ALKBH2 likely repairs ϵ5mC in MEF cells. Another homolog, ALKBH5, could not repair ϵ5mC. Also, ϵ5mC is not a substrate for BER glycosylases SMUG1, AAG, or TDG. These findings indicate DRR committed by ALKBH2, 3 and FTO could reduce the detrimental effects of ϵ5mC in genetics and epigenetics and may work together with TET enzymes to modulate epigenetic regulations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epigenetics plays a pivotal role in regulating gene expression and cellular differentiation. DNA methylation, involving the addition of methyl groups to specific cytosine bases, is a well-known epigenetic modification. The recent discovery of 5-hydroxymethylcytosine (5hmC) has provided new insights into cytosine modifications. 5hmC, derived from the oxidation of 5-methylcytosine (5mC), serves as both an intermediate in demethylation and a stable chemical modification in the genome. In this comprehensive review, we summarize the recent research advancements regarding the functions of 5hmC in development and disease. We discuss its implications in gene expression regulation, cellular differentiation, and its potential role as a diagnostic and prognostic marker in various diseases. Additionally, we highlight the challenges associated with accurately detecting and quantifying 5hmC and present the latest methodologies employed for its detection. Understanding the functional role of 5hmC in epigenetic regulation and further advancing our understanding of gene expression dynamics and cellular processes hold immense promise for the development of novel therapeutic strategies and precision medicine approaches.

Eimeria tenella is the major causative agent of chicken coccidiosis. 5-Methylcytosine (m 5 C) is a type of RNA chemical modifications reported to regulate diverse biological processes. However, the distribution and biological functions of m 5 C in E. tenella mRNAs are yet to be known. Herein, we report transcriptome-wide profiling of mRNA m 5 C in E. tenella by employing m 5 C RNA immunoprecipitation followed by a deep-sequencing approach (m 5 C-RIP-seq). Our data showed that m 5 C peaks were distributed across the whole mRNA body. Compared with unsporulated oocysts, there were 2813 hypermethylated and 1850 hypomethylated m 5 C peaks in sporulated oocysts. Generally, a positive correlation between m 5 C modification and gene expression levels was observed. The mRNA sequencing (RNA-seq) and m 5 C-RIP-seq data were consistent with the results of the quantitative reverse transcription PCR (RT-qPCR) and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), respectively. Gene Ontology (GO) and pathway enrichment analysis predicated diverse biological functions and pathways, including microtubule motor activity, helicase activity, cGMP-PKG signaling pathway, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis, and spliceosome. Meanwhile, stage-specific gene expression signatures of m 5 C-related regulators were observed. Altogether, our findings reveal the transcriptional significance of m 5 C modification in E. tenella oocysts, providing resources and clues for further in-depth research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since the mid-1970s, increasingly innovative methods to detect DNA methylation provided detailed information about its distribution, functions, and dynamics. As a result, new concepts were formulated and older ones were revised, transforming our understanding of the associated biology and catalyzing unprecedented advances in biomedical research, drug development, anthropology, and evolutionary biology. In this review, we discuss a few of the most notable advances, which are intimately intertwined with the study of DNA methylation, with a particular emphasis on the past three decades. Examples of these strides include elucidating the intricacies of 5-methylcytosine (5-mC) oxidation, which are at the core of the reversibility of this epigenetic modification; the three-dimensional structural characterization of eukaryotic DNA methyltransferases, which offered insights into the mechanisms that explain several disease-associated mutations; a more in-depth understanding of DNA methylation in development and disease; the possibility to learn about the biology of extinct species; the development of epigenetic clocks and their use to interrogate aging and disease; and the emergence of epigenetic biomarkers and therapies.

Epigenetic modifications, particularly RNA methylation and histone alterations, play a crucial role in heredity, development, and disease. Among these, RNA 5-methylcytosine (m5C) is the most prevalent RNA modification in mammalian cells, essential for processes such as ribosome synthesis, translational fidelity, mRNA nuclear export, turnover, and translation. The increasing volume of nucleotide sequences has led to the development of machine learning-based predictors for m5C site prediction. However, these predictors often face challenges related to training data limitations and overfitting due to insufficient external validation. This study introduces m5C-Seq, an ensemble learning approach for RNA modification profiling, designed to address these issues. m5C-Seq employs a meta-classifier that integrates 15 probabilities generated from a novel, large dataset using systematic encoding methods to make final predictions. Demonstrating superior performance compared to existing predictors, m5C-Seq represents a significant advancement in accurate RNA modification profiling. The code and the newly established datasets are made available through GitHub at https://github.com/Z-Abbas/m5C-Seq., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Epigenetic mechanisms play a critical role in the pathogenesis of human diseases including kidney disorders. As the erasers of DNA methylation, Ten-eleven translocation (TET) family proteins can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), thus leading to passive or active DNA demethylation. Similarly, TET family proteins can also catalyze the same reaction on RNA. In addition, TET family proteins can also regulate chromatin structure and gene expression in a catalytic activity-independent manner through recruiting the SIN3A/HDAC co-repressor complex. In 2012, we reported for the first time that the genomic 5-hydroxymethylcytosine level and the mRNA levels of Tet1 and Tet2 were significantly downregulated in murine kidneys upon ischemia and reperfusion injury. Since then, accumulating evidences have eventually established an indispensable role of TET family proteins in not only acute kidney injury but also chronic kidney disease. In this review, we summarize the upstream regulatory mechanisms and the pathophysiological role of TET family proteins in major types of kidney diseases and discuss their potential values in clinical diagnosis and treatment., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Maintenance methylation, of palindromic CpG dinucleotides at DNA replication forks, is crucial for the faithful mitotic inheritance of genomic 5-methylcytosine (5mC) methylation patterns. MBD proteins use two arginine residues to recognize symmetrically-positioned methyl groups in fully-methylated 5mCpG/5mCpG and 5mCpA/TpG dinucleotides. In contrast, C2H2 zinc finger (ZF) proteins recognize CpG and CpA, whether methylated or not, within longer specific sequences in a site- and strand-specific manner. Unmethylated CpG sites, often within CpG island (CGI) promoters, need protection by protein factors to maintain their hypomethylated status. Members of the BEN domain proteins bind CGCG or CACG elements within CGIs to regulate gene expression. Despite their overall structural diversity, MBD, ZF and BEN proteins all use arginine residues to recognize guanine, adopting either a 'straight-on' or 'oblique' conformation. The straight-on conformation accommodates a methyl group in the (5mC/T)pG dinucleotide, while the oblique conformation can clash with the methyl group of 5mC, leading to preferential binding of unmethylated sequences., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human thymine-DNA glycosylase (TDG) excises T mispaired with G in a CpG context to initiate the base excision repair (BER) pathway. TDG is also involved in epigenetic regulation of gene expression by participating in active DNA demethylation. Here we demonstrate that under extended incubation time the full-length TDG (TDGFL), but neither its isolated catalytic domain (TDGcat) nor methyl-CpG binding domain-containing protein 4 (MBD4) DNA glycosylase, exhibits significant excision activity towards T and C in regular non-damaged DNA duplex in TpG/CpA and CpG/CpG contexts. Time course of the cleavage product accumulation under single-turnover conditions shows that the apparent rate constant for TDGFL-catalysed excision of T from T•A base pairs (0.0014-0.0069 min-1) is 85-330-fold lower than for the excision of T from T•G mispairs (0.47-0.61 min-1). Unexpectedly, TDGFL, but not TDGcat, exhibits prolonged enzyme survival at 37°C when incubated in the presence of equimolar concentrations of a non-specific DNA duplex, suggesting that the disordered N- and C-terminal domains of TDG can interact with DNA and stabilize the overall conformation of the protein. Notably, TDGFL was able to excise 5-hydroxymethylcytosine (5hmC), but not 5-methylcytosine residues from duplex DNA with the efficiency that could be physiologically relevant in post-mitotic cells. Our findings demonstrate that, under the experimental conditions used, TDG catalyses sequence context-dependent removal of T, C and 5hmC residues from regular DNA duplexes. We propose that in vivo the TDG-initiated futile DNA BER may lead to formation of persistent single-strand breaks in non-methylated or hydroxymethylated chromatin regions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Manapkyzy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cervical cancer (CC), one of the most prevalent and detrimental gynaecologic cancers, evolves through genetic and epigenetic alterations resulting in the promotion of oncogenic activity and dysfunction of tumour-suppressing mechanisms. Despite medical advancement, the prognosis for advanced-stage patients remains extremely low due to high recurrence rates and resistance to existing treatments. Thereby, the search for potential prognostic biomarkers is heightened to unravel new modalities of CC pathogenesis and to develop novel anti-cancer therapies. Epitranscriptomic modifications, reversible epigenetic RNA modifications, regulate various biological processes by deciding RNA fate to mediating RNA interactions. This narrative review provides insight into the cellular and molecular roles of endogenous RNA-editing proteins and their associated epitranscriptomic modifications, especially N 6 -methyladenosine (m 6 A), 5-methylcytosine (m 5 C) and N 1 -methyladenosine (m 1 A), in governing the development, progression and metastasis of CC. We discussed the in-depth epitranscriptomic mechanisms underlying the regulation of over 50 RNAs responsible for tumorigenesis, proliferation, migration, invasion, survival, autophagy, stemness, epithelial-mesenchymal transition, metabolism (glucose, lipid, glutamate and glutamine), resistance (drug and radiation), angiogenesis and recurrence of CC. Additionally, we provided a concise overview of the therapeutic potential of targeting the altered expression of endogenous RNA-editing proteins and aberrant deposition of RNA modifications on both coding and non-coding RNAs in CC.

Background: SLE is a complex autoimmune disease with heterogeneous manifestations and unpredictable outcomes. Early diagnosis is challenging due to non-specific symptoms, and current treatments only manage symptoms. Epigenetic alternations, including 5-Hydroxymethylome (5hmC) modifications, are important contributors to SLE pathogenesis. However, the 5hmC modification status in circulating cell-free DNA (cfDNA) of patients with SLE remains largely unexplored. We investigated the distribution of 5hmC in cfDNA of patients with SLE and healthy controls (HCs), and explored its potential as an SLE diagnosis marker., Methods: We used 5hmC-Seal to generate genome-wide 5hmC profiles of plasma cfDNA and bioinformatics analysis to screen differentially hydroxymethylated regions (DhMRs). In vitro mechanistic exploration was conducted to investigate the regulatory effect of CCCTC-binding factor (CTCF) in 5hmC candidate biomarkers., Results: We found distinct differences in genomic regions and 5hmC modification motif patterns between patients with SLE and HCs, varying with disease progression. Increased 5hmC modification enrichment was detected in SLE. Additionally, we screened 151 genes with hyper-5hmC, which are significantly involved in SLE-related processes, and 5hmC-modified BCL2 , CD83 , ETS1 and GZMB as SLE biomarkers. Our findings suggest that CTCF regulates 5hmC modification of these genes by recruiting TET (ten-eleven translocation) protein, and CTCF knockdown affected the protein expression of these genes in vitro., Conclusions: Our findings demonstrate the increased 5hmC distribution in plasma cfDNA in different disease activity in patients with SLE compared with HCs and relating DhMRs involved in SLE-associated pathways. Furthermore, we identified a panel of SLE relevant biomarkers, and these viewpoints could provide insight into the pathogenesis of SLE., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2) drives myeloid malignancy initiation and progression 1-3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4,5 . However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6, which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub, more open chromatin and increased transcription in stem cells. TET2-mutant human leukaemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocking proliferation of TET2-mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies., (© 2024. The Author(s).)

Early detection of pancreatic cancer has been shown to improve patient survival rates. However, effective early detection tools to detect pancreatic cancer do not currently exist. The Avantect Pancreatic Cancer Test, leveraging the 5-hydroxymethylation [5-hydroxymethylcytosine (5hmC)] signatures in cell-free DNA, was developed and analytically validated to address this unmet need. We report a comprehensive analytical validation study encompassing precision, sample stability, limit of detection, interfering substance studies, and a comparison with an alternative method. The assay performance on an independent case-control patient cohort was previously reported with a sensitivity for early-stage (stage I/II) pancreatic cancer of 68.3% (95% CI, 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). Precision studies showed a cancer classification of 100% concordance in biological replicates. The sample stability studies revealed stable assay performance for up to 7 days after blood collection. The limit of detection studies revealed equal results between early- and late-stage cancer samples, emphasizing strong early-stage performance characteristics. Comparisons of concordance of the Avantect assay with the enzymatic methyl sequencing (EM-Seq) method, which measures both methylation (5-methylcytosine) and 5hmC, were >95% for all samples tested. The Avantect Pancreatic Cancer Test showed strong analytical validation in multiple validation studies required for laboratory-developed test accreditation. The comparison of 5hmC versus EM-Seq further validated the 5hmC approach as a robust and reproducible assay., Competing Interests: Disclosure Statement All authors are employees of ClearNote Health., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aims: 5 mC methylation and hydroxymethylation (5hmC) are associated with Alzheimer's disease (AD). However, previous studies were limited by the absence of a 5hmC calculation. This study aims to find AD associated predictors and potential therapeutic chemicals using bioinformatics approach integrating 5 mC, 5hmC, and expression changes, and an AD mouse model., Methods: Gene expression microarray and 5 mC and 5hmC sequencing datasets were downloaded from GEO repository. 142 AD and 52 normal entorhinal cortex specimens were enrolled. Data from oxidative bisulfite sequencing (oxBS)-treated samples, which represent only 5 mC, were used to calculate 5hmC level. Functional analyses, random forest supervised classification and methylation validation were applied. Potential chemicals were predicted by CMap. Morris water maze, Y maze and novel object recognition behavior tests were performed using FAD 4T AD mice model. Cortex and hippocampus tissues were isolated for immunohistochemical staining., Results: C1QTNF5, UBD, ZFP106, NEDD1, AKT3, and MBP genes involving 13 promoter CpG sites with 5mc, 5hmC methylation and expression difference were identified. AKT3 and MBP were down-regulated in both patients and mouse model. Three CpG sites in AKT3 and MBP showed significant methylation difference on validation. FAD 4T AD mice showed recession in brain functions and lower AKT3 expression in both cortex and hippocampus. Ten chemicals were predicted as potential treatments for AD., Conclusions: AKT3 and MBP may be associated with AD pathology and could serve as biomarkers. The ten predicted chemicals might offer new therapeutic approaches. Our findings could contribute to identifying novel markers and advancing the understanding of AD mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Among over 170 different types of chemical modifications on RNA nucleobases identified so far, RNA methylation is the major type of epitranscriptomic modifications existing on almost all types of RNAs, and has been demonstrated to participate in the entire process of RNA metabolism, including transcription, pre-mRNA alternative splicing and maturation, mRNA nucleus export, mRNA degradation and stabilization, mRNA translation. Attributing to the development of high-throughput detection technologies and the identification of both dynamic regulators and recognition proteins, mechanisms of RNA methylation modification in regulating the normal development of the organism as well as various disease occurrence and developmental abnormalities upon RNA methylation dysregulation have become increasingly clear. Here, we particularly focus on three types of RNA methylations: N 6 -methylcytosine (m 6 A), 5-methylcytosine (m 5 C), and N 7 -methyladenosine (m 7 G). We summarize the elements related to their dynamic installment and removal, specific binding proteins, and the development of high-throughput detection technologies. Then, for a comprehensive understanding of their biological significance, we also overview the latest knowledge on the underlying mechanisms and key roles of these three mRNA methylation modifications in gametogenesis, embryonic development, immune system development, as well as disease and tumor progression., (© 2024. Science China Press.)

Asthma is characterized by aberrant airway smooth muscle (ASM) proliferation, which increases the thickness of the ASM layer within the airway wall and exacerbates airway obstruction during asthma attacks. The mechanisms that drive ASM proliferation in asthma are not entirely elucidated. Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC. In this study, TET1 activity and protein were enhanced in asthmatic human ASM cell cultures. Moreover, the concentration of 5-hmC was higher in asthmatic ASM cells than in nonasthmatic ASM cells. Knockdown (KD) of TET1, but not TET2, reduced the concentration of 5-hmC in asthmatic cells. Because the cytoskeletal protein nestin controls cell proliferation by modulating mTOR, we evaluated the effects of TET1 KD on this pathway. TET1 KD reduced nestin expression in ASM cells. In addition, TET1 inhibition alleviated the platelet-derived growth factor-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. TET1 inhibition also attenuated the proliferation of ASM cells. Taken together, these results suggest that TET1 drives ASM proliferation via the nestin-mTOR axis.

The expression of some genes regulated by their DNA methylation is involved in pathogenesis and disease progression of myelodysplastic syndrome (MDS), which is characterised by abnormal differentiation and development of myeloid cells. Therefore, it is significant for us to work on investigating what factors regulate U2AF1 expression and hydroxymethylation in MDS patients. However, the members of TET protein family can change 5-methylcytosine (5mC) into 5-hydroxymethylcytosine5-methyl cytosine (5hmC). In general, 5mC and 5hmC levels maintain dynamic equilibrium, and their imbalance is associated with the onset and progression of some tumors. In this study, the expression and 5mC and 5hmC levels of U2AF1 gene decreased significantly after the treatment by decitabine in Mutz-1 cells. The decreased degree of 5hmC is far greater than that of 5mC. IDH2 expression decreased significantly followed by U2AF1 5hmC levels. However, the expression of other hydroxymethylation-related genes such as IDH1, TET1 and TET2 also decreased, but the difference did not achieve significance. Compared with IDH2 or U2AF1 wild-type MDS patients, U2AF1 expression and 5hmC level in patients with these two gene mutations were both significantly reduced.

Tubulin β-3 staining pattern and staining intensity of 5-hydroxymethyl cytosine (5-hmC) are potential diagnostic and prognostic markers in melanocytic lesions that need further evaluation. Melanocytic nevi and primary cutaneous melanomas were immunohistochemically stained for tubulin-β-3 and 5-hmC. Immunoreactivity and staining patterns were correlated with Breslow-thickness, clinical and pathological characteristics, and progression-free survival. Melanocytes showed positive tubulin β-3 staining. However, in most nevi, tubulin β-3 staining appeared as a gradient with intense cytoplasmic staining in cells of the superficial part of the lesion that faded to weak staining in the deep dermal part, while no gradient was found in deep penetrating nevi and melanomas. In 53 % of the melanomas, areas with loss of tubulin β-3 staining were found. 5-hmC staining intensity was significantly higher in melanocytic nevi compared to melanomas. Breslow thickness in combination with low 5-hmC score and loss of tubulin-β-3 staining was predictive for poor prognosis. As single markers, tubulin-β-3 and 5-hmC can be useful to distinguish between melanocytic nevi and melanoma, but staining variability limits the use of 5-hmC. In melanomas measuring >1.5 mm, combination of low 5-hmC score and loss of tubulin-β-3 staining may have prognostic value., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Farah Lubin reports financial support was provided by National Institute of Neurological Disorders and Stroke. Lawrence Ver Hoef reports was provided by National Institute of Neurological Disorders and Stroke. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression., Competing Interests: Dr. TÄ is a Director of Data Science at Covera Health. No funding from Covera Health was received for this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gioulbasani, Äijö, Valenzuela, Bettes and Tsagaratou.)

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has acquired and/or evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized that more were encoded in V. cholerae given the low diversity of phages that have been isolated, which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16-kB region of the Vibrio pathogenicity island-2, which we name TgvA and TgvB ( T ype I-embedded g mrSD -like system of V PI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modifications are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff, becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We also show that the Type I restriction-modification system that embeds the tgvAB genes protects against phage T3, secΦ18, secΦ27, and λ, suggesting that this region is a phage defense island. Our study uncovers a novel Type IV restriction system in V. cholerae , increasing our understanding of the evolution and ecology of V. cholerae, while highlighting the evolutionary interplay between restriction systems and phage genome modification.IMPORTANCEBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. In this study, we discover a Type IV restriction system encoded in V. cholerae, which we name TgvAB, and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation., Competing Interests: The authors declare no conflict of interest.

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase δ. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase ζ on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase δ or ε., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tomohiko Sugiyama. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: N6-methyladenosine (m 6 A) and 5-methylcytosine (m 5 C) play a role in modifying long non-coding RNAs (lncRNAs) implicated in tumorigenesis and progression. This study was performed to evaluate prognostic value of m 6 A- and m 5 C-related lncRNAs and develop an efficient model for prognosis prediction in cervical cancer (CC)., Methods: Using gene expression data of TCGA set, we identified m 6 A- and m 5 C-related lncRNAs. Consensus Clustering Analysis was performed for samples subtyping based on survival-related lncRNAs, followed by analyzing tumor infiltrating immune cells (TIICs). Optimal signature lncRNAs were obtained using lasso Cox regression analysis for constructing a prognostic model and a nomogram to predict prognosis., Results: We built a co-expression network of 23 m 6 A-related genes, 15 m 5 C-related genes, and 62 lncRNAs. Based on 9 m 6 A- and m 5 C-related lncRNAs significantly associated with overall survival (OS) time, two molecular subtypes were obtained, which had significantly different OS time and fractions of TIICs. A prognostic model based on six m 6 A- and m 5 C-related signature lncRNAs was constructed, which could dichotomize patients into two risk subgroups with significantly different OS time. Prognostic power of the model was successfully validated in an independent dataset. We subsequently constructed a nomogram which could accurately predict survival probabilities. Drug sensitivity analysis found preferred chemotherapeutic agents for high and low-risk patients, respectively., Conclusion: Our study reveals that m 6 A- and m 5 C-related lncRNAs are associated with prognosis and immune microenvironment of CC. The m 6 A- and m 5 C-related six-lncRNA signature may be a useful tool for survival stratification in CC and open new avenues for individualized therapies., (© 2024. The Author(s).)

Both DNA 5-methylcytosine (5mC) and RNA N6-methyladenosine (m 6 A) modifications are reported to participate in cellular stress responses including inflammation. Phosphoenolpyruvate carboxykinase 2 (PCK2) is upregulated in Kupffer cells (KCs) to facilitate the proinflammatory phosphorylation signaling cascades upon LPS stimulation, yet the role of 5mC and m 6 A in PCK2 upregulation remain elusive. Here, we report that the significantly augmented PCK2 mRNA and protein levels are associated with global 5mC demethylation coupled with m 6 A hypermethylation in LPS-activated KCs. The suppression of 5mC demethylation or m 6 A hypermethylation significantly alleviates the upregulation of PCK2 and proinflammatory cytokines in LPS-challenged KCs. Further reciprocal tests indicate 5mC demethylation is upstream of m 6 A hypermethylation. Specifically, CpG islands in the promoters of PCK2 and RNA methyltransferase (METTL3 and METTL14) genes are demethylated, while the 3'UTR of PCK2 mRNA is m6A hypermethylated, in LPS-stimulated KCs. These modifications contribute to the transactivation of the PCK2 gene as well as increased PCK2 mRNA stability and protein production via a m 6 A-mediated mechanism with IGF2BP1 as the reader protein. These results indicate that DNA 5mC and RNA m6A collaborate to upregulate PCK2 expression, respectively, at the transcriptional and post-transcriptional levels during KC activation.

Background: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous., Results: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA)., Conclusions: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening., (© 2024. The Author(s).)

Background: By regulating the functions of multiple RNAs, 5-methylcytosine (m 5 C) RNA methylation, particularly mediated by NOP2, is involved in tumorigenesis and developments. However, the specific functions and potential mechanisms of m 5 C, especially involving NOP2, in clear-cell renal cell carcinoma (ccRCC), remain unclear. Methods: NOP2 expression in cell lines and patient tissues was detected using western blotting, quantitative real-time polymerase chain reaction (RT-qPCR), and immunohistochemistry. The biological effects of NOP2 on ccRCC cells were investigated through a series of in vitro and in vivo experiments. To explore the potential regulatory mechanisms by which NOP2 affects ccRCC progression, m 5 C bisulfite sequencing, RNA-sequencing, RNA immunoprecipitation and methylated RNA immunoprecipitation (RIP/MeRIP) RT-qPCR assay, luciferase reporter assay, RNA stability assay, and bioinformatic analysis were performed. Results: NOP2 expression was significantly upregulated in ccRCC tissues and was associated with poor prognosis. Moreover, loss-of-function and gain-of-function assays demonstrated that NOP2 altered ccRCC cell proliferation, migration, and invasion. Mechanistically, NOP2 stimulated m 5 C modification of apolipoprotein L1 (APOL1) mRNA, and m 5 C reader YBX1 stabilized APOL1 mRNA through recognizing and binding to m 5 C site in the 3'-untranslated regions. Silencing APOL1 expression inhibited ccRCC cell proliferation in vitro and tumor formation in vivo . Furthermore, NOP2/APOL1 affected ccRCC progression via the PI3K-Akt signaling pathway. Conclusion: NOP2 functions as an oncogene in ccRCC by promoting tumor progression through the m 5 C-dependent stabilization of APOL1, which in turn regulates the PI3K-Akt signaling pathway, suggesting a potential therapeutic target for ccRCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA's operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis., (© 2024. The Author(s).)

In the era of precision oncology, identifying abnormal genetic and epigenetic alterations has transformed the way cancer is diagnosed, managed, and treated. 5-hydroxymethylcytosine (5hmC) is an emerging epigenetic modification formed through the oxidation of 5-methylcytosine (5mC) by ten-eleven translocase (TET) enzymes. DNA hydroxymethylation exhibits tissue- and cancer-specific patterns and is essential in DNA demethylation and gene regulation. Recent advancements in 5hmC detection methods and the discovery of 5hmC in cell-free DNA (cfDNA) have highlighted the potential for cell-free 5hmC as a cancer biomarker. This review explores the current and emerging techniques and applications of DNA hydroxymethylation in cancer, particularly in the context of cfDNA.

Motivation: 5-Hydroxymethylcytosine (5hmC), a crucial epigenetic mark with a significant role in regulating tissue-specific gene expression, is essential for understanding the dynamic functions of the human genome. Despite its importance, predicting 5hmC modification across the genome remains a challenging task, especially when considering the complex interplay between DNA sequences and various epigenetic factors such as histone modifications and chromatin accessibility., Results: Using tissue-specific 5hmC sequencing data, we introduce Deep5hmC, a multimodal deep learning framework that integrates both the DNA sequence and epigenetic features such as histone modification and chromatin accessibility to predict genome-wide 5hmC modification. The multimodal design of Deep5hmC demonstrates remarkable improvement in predicting both qualitative and quantitative 5hmC modification compared to unimodal versions of Deep5hmC and state-of-the-art machine learning methods. This improvement is demonstrated through benchmarking on a comprehensive set of 5hmC sequencing data collected at four developmental stages during forebrain organoid development and across 17 human tissues. Compared to DeepSEA and random forest, Deep5hmC achieves close to 4% and 17% improvement of Area Under the Receiver Operating Characteristic (AUROC) across four forebrain developmental stages, and 6% and 27% across 17 human tissues for predicting binary 5hmC modification sites; and 8% and 22% improvement of Spearman correlation coefficient across four forebrain developmental stages, and 17% and 30% across 17 human tissues for predicting continuous 5hmC modification. Notably, Deep5hmC showcases its practical utility by accurately predicting gene expression and identifying differentially hydroxymethylated regions (DhMRs) in a case-control study of Alzheimer's disease (AD). Deep5hmC significantly improves our understanding of tissue-specific gene regulation and facilitates the development of new biomarkers for complex diseases., Availability and Implementation: Deep5hmC is available via https://github.com/lichen-lab/Deep5hmC., (© The Author(s) 2024. Published by Oxford University Press.)

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development., (© 2024. The Author(s).)

Air pollution (AP) is detrimental to pregnancies including increasing risk factors of gestational diabetes mellitus. We hypothesized that exposure to AP causes cardiovascular and metabolic disruption thereby altering placental gene expression, which in turn affects the placental phenotype and thereby embryonic/fetal development. To test this hypothesis, we investigated the impact of intra-nasal instilled AP upon gestational day 16-19 maternal mouse cardiovascular and metabolic status, placental nutrient transporters, and placental-fetal size and morphology. To further unravel mechanisms, we also examined placental total DNA 5'-hydroxymethylation and bulk RNA sequenced gene expression profiles. AP exposed pregnant mice and fetuses were tachycardic with a reduction in maternal left ventricular fractional shortening and increased uterine artery with decreased umbilical artery systolic peak velocities. In addition, they were hyperglycemic, glucose intolerant and insulin resistant, with changes in placental glucose (Glut3) and fatty acid (Fatp1 & Cd36) transporters, and a spatial disruption of cells expressing Glut10 that imports L-dehydroascorbic acid in protecting against oxidative stress. Placentas revealed inflammatory cellular infiltration with associated cellular edema and necrosis, with dilated vascular spaces and hemorrhage. Placental and fetal body weights decreased in mid-gestation with a reduction in brain cortical thickness emerging in late gestation. Placental total DNA 5'-hydroxymethylation was 2.5-fold higher, with perturbed gene expression profiles involving key metabolic, inflammatory, transcriptional, cellular polarizing and processing genes and pathways. We conclude that gestational exposure to AP incites a maternal inflammatory response resulting in features mimicking maternal gestational diabetes mellitus with altered placental DNA 5'-hydroxymethylation, gene expression, and associated injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Stunting is the result of chronic malnutrition due to the lack of micronutrient-based methyl donors required for epigenetic programming during the first 1000 days of life. Methylation studies using bisulfite conversion from blood DNA are invasive and may not be practical for large-scale epidemiological investigation or nutrition intervention programs. Buccal epithelial methylation may reflect early germline methylation. Therefore, buccal cells can serve as convenient sample sources to collect biomarkers associated with the risk of stunting. This study aims to describe the feasibility of nanopore adaptive sampling in detecting DNA methylation from children's buccal DNA. We used adaptive sampling of Oxford Nanopore Technology on barcoded samples to describe differential methylation associated with malnutrition. Overall, the level of 5-methylcytosine (5mC) was lower in stunted children than in normal children. We also found differentially methylated regions at the MIR6724 and RNA45SN1 gene loci on chromosome 21, which was higher in stunted children than in normal children. We described and detected differential DNA methylation in the locus previously not known to be associated with stunting. Interestingly, this locus on chromosome 21 has been implicated in the stunted phenotype of Down syndrome.

With ∼50% recurrent pregnancy loss cases being termed idiopathic (iRPL), understanding of contribution of male factors to iRPL is still lacking. Higher prevalence of sperm DNA fragmentation index (DFI) and lower sperm 5-methylcytosine (5-mC) levels have been previously reported in male partners of iRPL couples and shed light on importance of the male gamete in maintenance of a successful pregnancy. The present study aimed to determine the serum sex steroid hormone levels, sperm DFI and 5-mC and correlation between them in male partners of fertile and iRPL couples. Further, correlation between sperm DFI and 5-mC with semen parameters and paternal age in both groups were determined. 36 male partners of fertile couples and 45 male partners of women experiencing iRPL were enrolled for this study and semen and blood samples were collected. Serum testosterone and estradiol levels were measured by ELISA; sperm DFI and global 5-mC were determined by TUNEL assay and ELISA respectively. Significantly higher serum testosterone levels were noted in the iRPL group ( p  = 0.028). Incidence of sperm DNA fragmentation was found to be higher in the iRPL study group but with no significance difference. No significant differences in sperm 5-mC values were noted. Upon correlation analysis within both groups, strong significant negative correlation of sperm DFI % and 5-mC % was observed in the control group ( p  < 0.001) but not the iRPL group ( p  = 0.249). Hence, we infer that with lower 5-mC levels in sperm genome, there is a higher incidence of sperm DFI in fertile men. However, this trend is not noted in men of iRPL group which could possibly be due to other underlying epigenetic alterations in genomic regions probably unsusceptible to fragmentation. On the other hand, no significant correlations of semen parameters, testosterone, estradiol and paternal age with sperm DFI and 5-mC were noted in both groups.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype ( Tet1 WT), a novel Tet1 catalytically dead mutant ( Tet1 HxD), and Tet1 knockout ( Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted ( p  < 0.0001) at CpG islands (CGI), and enriched ( p  < 0.0001) in 'open sea' regions (CpG >4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively ( p  < 0.05). We identified 499 significant (empirical-p <0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS ( p  < 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, p  < 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia (SCA) caused by a polyglutamine expansion in the ataxin-3 protein, which initiates a cascade of pathogenic events, including transcriptional dysregulation. Genotype-phenotype correlations in MJD are incomplete, suggesting an influence of additional factors, such as epigenetic modifications, underlying the MJD pathogenesis. DNA methylation is known to impact the pathophysiology of neurodegenerative disorders through gene expression regulation and increased methylation has been reported for other SCAs. In this work we aimed to analyse global methylation in MJD carriers. Global 5-mC levels were quantified in blood samples of 33 MJD mutation carriers (patients and preclinical subjects) and 33 healthy controls, matched by age, sex, and smoking status. For a subset of 16 MJD subjects, a pilot follow-up analysis with two time points was also conducted. No differences were found in median global 5-mC levels between MJD mutation carriers and controls and no correlations between methylation levels and clinical or genetic variables were detected. Also, no alterations in global 5-mC levels were observed over time. Our findings do not support an increase in global blood methylation levels associated with MJD.

As the most common and aggressive primary malignant brain tumor, glioblastoma is still lacking a satisfactory curative approach. The standard management consisting of gross total resection followed by radiotherapy and chemotherapy with temozolomide only prolongs patients' life moderately. In recent years, many therapeutics have failed to give a breakthrough in GBM treatment. In the search for new treatment solutions, we became interested in the repurposing of existing medicines, which have established safety profiles. We focused on the possible implementation of well-known drugs, metformin, and arginine. Metformin is widely used in diabetes treatment, but arginine is mainly a cardiovascular protective drug. We evaluated the effects of metformin and arginine on total DNA methylation, as well as the oxidative stress evoked by treatment with those agents. In glioblastoma cell lines, a decrease in 5-methylcytosine contents was observed with increasing drug concentration. When combined with temozolomide, both guanidines parallelly increased DNA methylation and decreased 8-oxo-deoxyguanosine contents. These effects can be explained by specific interactions of the guanidine group with m 5 CpG dinucleotide. We showed that metformin and arginine act on the epigenetic level, influencing the foreground and potent DNA regulatory mechanisms. Therefore, they can be used separately or in combination with temozolomide, in various stages of disease, depending on desired treatment effects.

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichia coli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N-acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes., Competing Interests: Declaration of interests J.D.P., S.R.L., K.H.O., and L.S. are current employees of New England Biolabs, a commercial producer and supplier of molecular biology reagents. This affiliation does not impact the adherence to ethical standards of the scientific community, experimental impartiality of the authors, or availability of the data presented. All authors are named inventors on a patent application related to the work described in this manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML) and possibly β-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC) and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Obstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia (IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive function remains unclear., Methods: C57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation 1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments, pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects were studied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis., Results: IH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with the control group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of three hydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levels of Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3, IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal., Conclusion: Activation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction induced by IH., (© 2024. The Author(s).)

5-Methylcytosine (m5C) methylation is a significant post-transcriptional modification that play a crucial role in the development and progression of numerous cancers. Whereas the functions and molecular mechanisms underlying m5C methylation in gliomas remain unclear. This study dedicated to explore changes of m5C levels and the clinical significance of the m5C writer NSUN4 in gliomas. We found that high m5C levels were negatively related to prognosis of patients with glioma. Moreover, gain- and loss-of-function experiments revealed the role of NSUN4 in enhancing m5C modification of mRNA to promote the malignant progression of glioma. Mechanistically speaking, NSUN4-mediated m5C alterations regulated ALYREF binding to CDC42 mRNA, thereby impacting the mRNA stability of CDC42. We also demonstrated that CDC42 promoted glioma proliferation, migration, and invasion by activating the PI3K-AKT pathway. Additionally, rescue experiments proved that CDC42 overexpression weaken the inhibitory effect of NSUN4 knockdown on the malignant progression of gliomas in vitro and in vivo. Our findings elucidated that NSUN4-mediated high m5C levels promote ALYREF binding to CDC42 mRNA and regulate its stability, thereby driving the malignant progression of glioma. This provides theoretical support for targeted the treatment of gliomas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

While the significance of N6-methyladenosine (m 6 A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m 5 C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m 5 C is more abundant than m 6 A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m 5 C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m 5 C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m 5 C-MeRIP-seq identified the NSUN2-dependent m 5 C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m 5 C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m 5 C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m 5 C methylation to SARS-CoV-2 RNA.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m 5 C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m 5 C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release., Competing Interests: Declaration of interests K.Y. is an employee at Xaira Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

5-methylcytosine (m 5 C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m 5 C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m 5 C-TAC-seq, a bisulfite-free approach combining TET-assisted m 5 C-to-f 5 C oxidation with selective chemical labeling, therefore enabling direct base-resolution m 5 C detection through pre-enrichment and C-to-T transitions at m 5 C sites. With m 5 C-TAC-seq, we comprehensively profiled the m 5 C methylomes in human and mouse cells, identifying a substantially larger number of confident m 5 C sites. Through perturbing potential m 5 C methyltransferases, we deciphered the responsible enzymes for most m 5 C sites, including the characterization of NSUN5's involvement in mRNA m 5 C deposition. Additionally, we characterized m 5 C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m 5 C-TAC-seq allow m 5 C detection in chromatin-associated RNAs. The accurate and robust m 5 C-TAC-seq will advance research into m 5 C methylation functional investigation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tamoxifen, a selective estrogen receptor modulator (SERM), exhibits dual agonist or antagonist effects contingent upon its binding to either G-protein-coupled estrogen receptor (GPER) or estrogen nuclear receptor (ESR). Estrogen signaling plays a pivotal role in initiating epigenetic alterations and regulating estrogen-responsive genes in breast cancer. Employing three distinct breast cancer cell lines-MCF-7 (ESR+; GPER+), MDA-MB-231 (ESR-; GPER-), and SkBr3 (ESR-; GPER+)-this study subjected them to treatment with two tamoxifen derivatives: 4-hydroxytamoxifen (4-HT) and endoxifen (Endox). Through 2D high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS), varying levels of 5-methylcytosine (5-mC) were found, with MCF-7 displaying the highest levels. Furthermore, TET3 mRNA expression levels varied among the cell lines, with MCF-7 exhibiting the lowest expression. Notably, treatment with 4-HT induced significant changes in TET3 expression across all cell lines, with the most pronounced increase seen in MCF-7 and the least in MDA-MB-231. These findings underscore the influence of tamoxifen derivatives on DNA methylation patterns, particularly through modulating TET3 expression, which appears to be contingent on the presence of estrogen receptors. This study highlights the potential of targeting epigenetic modifications for personalized anti-cancer therapy, offering a novel avenue to improve treatment outcomes.

Epigenetic modifications, such as 5-methylcytosine (5mC), can sometimes be transmitted between generations, provoking speculation that epigenetic changes could play a role in adaptation and evolution. Here, we use experimental evolution to investigate how 5mC levels evolve in populations of biparental insect (Nicrophorus vespilloides) derived from a wild source population and maintained independently under different regimes of parental care in the lab. We show that 5mC levels in the transcribed regions of genes (gene bodies) diverge between populations that have been exposed to different levels of care for 30 generations. These changes in 5mC do not reflect changes in the levels of gene expression. However, the accumulation of 5mC within genes between populations is associated with reduced variability in gene expression within populations. Our results suggest that evolved change in 5mC could contribute to phenotypic evolution by influencing variability in gene expression in invertebrates., (© 2024. The Author(s).)

Inheritance of 5-methylcytosine from one cell generation to the next by DNA methyltransferase 1 (DNMT1) plays a key role in regulating cellular identity. While recent work has shown that the activity of DNMT1 is imprecise, it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides to find that the fidelity of DNMT1-mediated maintenance methylation is related to the local density of DNA methylation and the landscape of histone modifications. To gain deeper insights into methylation/demethylation turnover dynamics, we first extended Dyad-seq to quantify all combinations of 5-methylcytosine and 5-hydroxymethylcytosine at individual CpG dyads. Next, to understand how cell state transitions impact maintenance methylation, we scaled the method down to jointly profile genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from single cells (scDyad&T-seq). Using scDyad&T-seq, we demonstrate that, while distinct cell states can substantially impact the activity of the maintenance methylation machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)