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Metal-organic frameworks (MOFs) show unique advantages in simulating the dynamics and fidelity of natural coordination. Inspired by zinc finger protein, a second linker was introduced to affect the homogeneous MOF system and thus facilitate the emergence of diverse functionalities. Under the systematic identification of 12 MOF species (i.e., metal ions, linkers) and 6 second linkers (trigger), a dissipative system consisting of Co-BDC-NO 2 and o -phenylenediamine ( o PD) was screened out, which can rapidly and in situ generate a high photothermal complex (η = 36.9%). Meanwhile, both the carboxylation of epigenetic modifications and metal ion (Fe 3+ , Ni 2+ , Cu 2+ , Zn 2+ , Co 2+ and Mn 2+ ) screening were utilized to improve the local coordination environment so that the adaptable Co-MOF growth on the DNA strand was realized. Thus, epigenetic modification information on DNA was converted to an amplified metal ion signal, and then o PD was further introduced to generate bimodal dissipative signals by which a simple, high-sensitivity detection strategy of 5-hydroxymethylcytosine (LOD = 0.02%) and 5-formylcytosine (LOD = 0.025‰) was developed. The strategy provides one low-cost method (< 0.01 $/sample) for quantifying global epigenetic modifications, which greatly promotes epigenetic modification-based early disease diagnosis. This work also proposes a general heterocoordination design concept for molecular recognition and signal transduction, opening a new MOF-based sensing paradigm.

Coordination of neuronal differentiation with expansion of the neuroepithelial/neural progenitor cell (NEPC/NPC) pool is essential in early brain development. Our in vitro and in vivo studies identify independent and opposing roles for two neural-specific and differentially expressed non-coding RNAs derived from the same locus: the evolutionarily conserved lncRNA Rncr3 and the embedded microRNA miR124a-1. Rncr3 regulates NEPC/NPC proliferation and controls the biogenesis of miR124a, which determines neuronal differentiation. Rncr3 conserved exons 2/3 are cytosine methylated and bound by methyl-CpG binding protein MeCP2, which restricts expression of miR124a embedded in exon 4 to prevent premature neuronal differentiation, and to orchestrate proper brain growth. MeCP2 directly binds cytosine-methylated Rncr3 through previously unrecognized lysine residues and suppresses miR124a processing by recruiting PTBP1 to block access of DROSHA-DGCR8. Thus, miRNA processing is controlled by lncRNA m 5 C methylation along with the defined m 5 C epitranscriptomic RNA reader protein MeCP2 to coordinate brain development., (© 2024. The Author(s).)

Despite extensive research on 5-methylcytosine (5mC) in relation to smoking, there has been limited exploration into the interaction between smoking and 5-hydroxymethylcytosine (5hmC). In this study, total DNA methylation (5mC+5hmC), true DNA methylation (5mC) and hydroxymethylation (5hmC) levels were profiled utilizing conventional bisulphite (BS) and oxidative bisulphite (oxBS) treatment, measured with the Illumina Infinium Methylation EPIC BeadChip. An epigenome-wide association study (EWAS) of 5mC+5hmC methylation revealed a total of 38,575 differentially methylated positions (DMPs) and 2023 differentially methylated regions (DMRs) associated with current smoking, along with 82 DMPs and 76 DMRs associated with former smoking (FDR-adjusted p < 0.05). Additionally, a focused examination of 5mC identified 33 DMPs linked to current smoking and 1 DMP associated with former smoking (FDR-adjusted p < 0.05). In the 5hmC category, eight DMPs related to current smoking and two DMPs tied to former smoking were identified, each meeting a suggestive threshold ( p < 1 × 10 -5 ). The substantial number of recognized DMPs, including 5mC+5hmC (7069/38,575, 2/82), 5mC (0/33, 1/1), and 5hmC (2/8, 0/2), have not been previously reported. Our findings corroborated previously established methylation positions and revealed novel candidates linked to tobacco smoking. Moreover, the identification of hydroxymethylated CpG sites with suggestive links provides avenues for future research.

Background: Like its parent base 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) is a direct epigenetic modification of cytosines in the context of CpG dinucleotides. 5hmC is the most abundant oxidized form of 5mC, generated through the action of TET dioxygenases at gene bodies of actively-transcribed genes and at active or lineage-specific enhancers. Although such enrichments are reported for 5hmC, to date, predictive models of gene expression state or putative regulatory regions for genes using 5hmC have not been developed., Results: Here, by using only 5hmC enrichment in genic regions and their vicinity, we develop neural network models that predict gene expression state across 49 cell types. We show that our deep neural network models distinguish high vs low expression state utilizing only 5hmC levels and these predictive models generalize to unseen cell types. Further, in order to leverage 5hmC signal in distal enhancers for expression prediction, we employ an Activity-by-Contact model and also develop a graph convolutional neural network model with both utilizing Hi-C data and 5hmC enrichment to prioritize enhancer-promoter links. These approaches identify known and novel putative enhancers for key genes in multiple immune cell subsets., Conclusions: Our work highlights the importance of 5hmC in gene regulation through proximal and distal mechanisms and provides a framework to link it to genome function. With the recent advances in 6-letter DNA sequencing by short and long-read techniques, profiling of 5mC and 5hmC may be done routinely in the near future, hence, providing a broad range of applications for the methods developed here., (© 2024. The Author(s).)

N 6 -methyladenine (m 6 A) and 5-methylcytosine (m 5 C) are two common forms of RNA methylation that play an important role in the epigenetics of type 2 diabetes mellitus (T2DM). One type of cell death, ferroptosis, has been implicated in islet β-cell damage in T2DM. Notably, RNA methylation, an upstream regulatory mechanism of mRNAs, can regulate the expression of ferroptosis signaling molecules, thereby affecting cell proliferation and death. Here, we found that the ferroptosis signaling pathway was activated in pancreas of T2DM rats, followed by significant changes in m 6 A/m 5 C modification regulatory molecules. These detection data together with the prediction results that m 6 A and m 5 C exist in the mRNAs of ferroptosis molecules, we speculate that m 6 A and m 5 C are probably involved in pancreatic cell damage by modifying of ferroptosis signaling molecules. In short, our findings provide a new research idea for future studies on the molecular mechanisms of pancreatic cell damage and point to a new direction for exploring the mechanisms of ferroptosis from the perspective of RNA methylation modification., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5-methylcytosine (5mC) and N6-methyladenine (6mA). These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA [Formula: see text]-mer backgrounds-a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with hidden Markov models (HMMs) that cannot make successful calls for [Formula: see text]-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen [Formula: see text]-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. This type of approach is expected to be useful for calling other base modifications such as 5-hydroxymethylcytosine and for the simultaneous calling of different modifications, settings in which complete training data are not likely to be available.

Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals., (© 2024. The Author(s).)

Colon adenocarcinoma (COAD) is a common and fatal malignant tumor of digestive system with complex etiology. 5-Methylcytosine (m5C) modification of RNA by the NSUN gene family (NSUN1-NSUN7) and DNMT2 reshape cell biology and regulate tumor development. However, the expression profile, prognostic significance and function of these m5C modifiers in COAD remain largely unclear. By mining multiple integrated tumor databases, we found that NSUN1, NSUN2, NSUN5, and NSUN6 were overexpressed in COAD tumor samples relative to normal samples. Clinically, high expression of NSUN6 was significantly associated with shorter survival (including both disease-free survival and overall survival) in COAD patients. NSUN6 was further confirmed to be upregulated at both tissue and cellular levels of COAD, suggesting that NSUN6 plays a critical role in disease progression. Through comprehensive gene enrichment analysis and cell-based functional validation, it was revealed that NSUN6 promoted the cell cycle progression and cell proliferation of COAD. Mechanistically, NSUN6 upregulates the expression of oncogenic METTL3 and catalyzes its m5C modification in COAD cells. Overexpression of METTL3 significantly relieved the cell cycle inhibition of COAD caused by NSUN6 deficiency. Furthermore, NSUN6 was negatively associated with the abundance of infiltrating immune cells in COAD tumors, such as activated B cells, natural killer cells, effector memory CD8 T cells, and regulatory T cells. Importantly, pan-cancer analysis further uncovered that NSUN6 was dysregulated and heterogeneous in various tumors. Thus our findings extend the role of m5C transferase in COAD and suggest that NSUN6 is a potential biomarker and target for this malignancy., (© 2024 Wiley Periodicals LLC.)

Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multimodal single-cell data integration, enable accurate identification of neuronal subtypes and uncover context-specific regulatory effects on cell-type-specific genes by TET enzymes., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process., (© 2024 The Author(s).)

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome., (© 2024. The Author(s).)

The majority of low-grade isocitrate dehydrogenase-mutant (IDH mt ) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDH mt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDH mt astrocytoma. Five primary/malignantly progressed IDH mt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDH mt glioma malignancy., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time., Results: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by β-glucosyltransferase (β-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10 -14 to 1 × 10 -8  M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells., Significance: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis., 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.)

Cell-free DNA (cfDNA) has recently emerged as a promising minimally invasive diagnostic biomarker for various cancers. In this study, our aim was to identify cfDNA biomarkers by investigating genes that displayed significant differences between glioma patients and their corresponding controls. To accomplish this, we utilized publicly available data from the Gene Expression Omnibus, focusing on 5-hydroxymethylcytosine (5hmC) profiles in both cfDNA and genomic DNA (gDNA) from glioma patients and healthy individuals. The intersection of gene lists derived from these comparative analyses unveiled LRIG1 and ZNF703 as the two genes with elevated 5hmC levels in both the cfDNA of glioma patients and gDNA of glioma tissue compared to their respective controls. The gene expression data revealed both genes were upregulated in glioma tissue compared to normal brain tissue. Integration of 5hmC data revealed a strong positive correlation in the glioma tissue group between 5hmC and the gene expression of the LRIG1 gene. Furthermore, exploration using the AmiCa web tool indicated that LRIG1 gene expression was elevated compared to 17 other cancers included in the database, emphasizing its potential as a distinctive biomarker across multiple cancer types.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1 V and TET1 HxD rescue most hypermethylation of Tet1 -/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RNA modifications are pivotal in the development of newly synthesized structures, showcasing a vast array of alterations across various RNA classes. Among these, 5-hydroxymethylcytosine (5HMC) stands out, playing a crucial role in gene regulation and epigenetic changes, yet its detection through conventional methods proves cumbersome and costly. To address this, we propose Deep5HMC, a robust learning model leveraging machine learning algorithms and discriminative feature extraction techniques for accurate 5HMC sample identification. Our approach integrates seven feature extraction methods and various machine learning algorithms, including Random Forest, Naive Bayes, Decision Tree, and Support Vector Machine. Through K-fold cross-validation, our model achieved a notable 84.07% accuracy rate, surpassing previous models by 7.59%, signifying its potential in early cancer and cardiovascular disease diagnosis. This study underscores the promise of Deep5HMC in offering insights for improved medical assessment and treatment protocols, marking a significant advancement in RNA modification analysis., (© 2024. The Author(s).)

Immune checkpoint inhibitors (ICIs) drastically improve therapeutic outcomes for lung cancer, but accurately predicting individual patient responses to ICIs remains a challenge. We performed the genome-wide profiling of 5-hydroxymethylcytosine (5hmC) in 85 plasma cell-free DNA (cfDNA) samples from lung cancer patients and developed a 5hmC signature that was significantly associated with progression-free survival (PFS). We built a 5hmC predictive model to quantify the 5hmC level and validated the model in the validation, test, and control sets. Low weighted predictive scores (wp-scores) were significantly associated with a longer PFS compared to high wp-scores in the validation [median 7.6 versus 1.8 months; p = 0.0012; hazard ratio (HR) 0.12; 95% confidence interval (CI), 0.03-0.54] and test (median 14.9 versus 3.3 months; p = 0.00074; HR 0.10; 95% CI, 0.02-0.50) sets. Objective response rates in patients with a low or high wp-score were 75.0% (95% CI, 42.8-94.5%) versus 0.0% (95% CI, 0.0-60.2%) in the validation set ( p = 0.019) and 80.0% (95% CI, 44.4-97.5%) versus 0.0% (95% CI, 0.0-36.9%) in the test set ( p = 0.0011). The wp-scores were also significantly associated with PFS in patients receiving single-agent ICI treatment ( p < 0.05). In addition, the 5hmC predictive signature demonstrated superior predictive capability to tumor programmed death-ligand 1 and specificity to ICI treatment response prediction. Moreover, we identified novel 5hmC-associated genes and signaling pathways integral to ICI treatment response in lung cancer. This study provides proof-of-concept evidence that the cfDNA 5hmC signature is a robust biomarker for predicting ICI treatment response in lung cancer.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates β-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of β-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe 2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Aberrant DNA methylation is a vital molecular alteration commonly detected in type I endometrial cancers (EC), and tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine (5hmC) play significant roles in DNA demethylation. However, little is known about the function and correlation of TET2 and 5hmC co-expressed in EC. This study intended to investigate the clinical significance of TET2 and 5hmC in EC., Methods: The levels of TET2 and 5hmC were detected in 326 endometrial tissues by immumohistochemistry, and the correlation of their level was detected by Pearson analysis. The association between the levels of TET2 and 5hmC and clinicopathologic characteristics was analyzed. Prognostic value of TET2 and 5hmC was explored by Kaplan-Meier analysis. The Cox proportional hazard regression model was used for univariate and multivariate analyses., Results: Based on the analysis results, TET2 protein level was positively correlated with 5hmC level in EC tissues (r = 0.801, P < 0.001). TET2 + 5hmC + (high TET2 and high 5hmC) association was significantly associated with well differentiation, myometrial invasion, negative lymph node metastasis, and tumor stage in EC. Association of TET2 and 5hmC was confirmed as a prognostic factor (HR = 2.843, 95%CI = 1.226-3.605, P = 0.007) for EC patients, and EC patients with TET2 - 5hmC - level had poor overall survival., Conclusions: In summary, the association of TET2 and 5hmC was downregulated in EC tissues, and may be a potential poor prognostic indicator for EC patients. Combined detection of TET2 and 5hmC may be valuable for the diagnosis and prognosis of EC., (© 2024. The Author(s).)

Epigenetic phenomena play a central role in cell regulatory processes and are important factors for understanding complex human disease. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) in CpG dinucleotides were long known to undergo methylation at the 5-position of the pyrimidine ring (mC). Later it was found that mC can be oxidized to 5-hydroxymethylcytosine (hmC) or even further to 5-formylcytosine (fC) and to 5-carboxylcytosine (caC) by the action of 2-oxoglutarate-dependent dioxygenases of the TET family. These findings unveiled a long elusive mechanism of active DNA demethylation and bolstered a wave of studies in the area of epigenetic regulation in mammals. This review is dedicated to critical assessment of recent data on biochemical and chemical aspects of the formation and conversion of hmC in DNA, analytical techniques used for detection and mapping of this nucleobase in mammalian genomes as well as epigenetic roles of hmC in DNA replication, transcription, cell differentiation and human disease.

Objective: The genome-wide profiling of 5-hydroxymethylcytosines (5hmC) on circulating cell-free DNA (cfDNA) has revealed promising biomarkers for various diseases. The purpose of this study was to investigate 5hmC signals in serum cfDNA and identify novel predictive biomarkers for the development of chemoresistance in high-grade serous ovarian cancer (HGSOC). We hypothesized that 5hmC profiles in cfDNA reflect the development of chemoresistance and elucidate pathways that may drive chemoresistance in HGSOC. Moreover, we sought to identify predictors that would better stratify outcomes for women with intermediate-sensitive HGSOC., Methods: Women diagnosed with HGSOC and known platinum sensitivity status were selected for this study. Nano-hmC-Seal was performed on cfDNA isolated from archived serum samples, and differential 5hmC features were identified using DESeq2 to establish a model predictive of chemoresistance., Results: A multivariate model consisting of three features (preoperative CA-125, largest residual implant after surgery, 5hmC level of OSGEPL), stratified samples from intermediate sensitive, chemo-naive women diagnosed with HGSOC into chemotherapy-resistant- and sensitive-like strata with a significant difference in overall survival (OS). Independent analysis of The Cancer Genome Atlas data further confirmed that high OSGEPL1 expression is a favorable prognostic factor for HGSOC., Conclusions: We have developed a novel multivariate model based on clinico-pathologic data and a cfDNA-derived 5hmC modified gene, OSGEPL1, that predicted response to platinum-based chemotherapy in intermediate-sensitive HGSOC. Our multivariate model applies to chemo-naïve samples regardless if the patint was treated with adjuvant or neoadjuvant chemotherapy. These results merit further investigation of the predictive capability of our model in larger cohorts., Competing Interests: Declaration of competing interest Ernst Lengyel reported receiving research funding to perform experimental pre-clinical research on ovarian cancer from Arsenal Bioscience, AbbVie, outside the submitted work. Chuan He is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, Inc., and AccuraDX Inc., a scientific cofounder and equity holder of Accent Therapeutics, Inc., and a member of the scientific advisory board of Rona Therapeutics. Wei Zhang is a consultant for the biomarker discovery program of Tempus Labs, Inc. All other authors declare no relationships that may present a conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cytosine modifications at the 5-carbon position play a critical role in gene expression regulation and have been implicated in cancer development. 5-Hydroxymethylcytosine (5hmC), arising from 5-methylcytosine (5-mC) oxidation, has shown promise as a potential malignancy marker due to its depletion in various human cancers. However, its significance in thyroid tumors remains underexplored, primarily due to limited data. In our study, we evaluated 5hmC expression levels by immunohistochemistry in a cohort of 318 thyroid tumors. Our analysis revealed significant correlations between 5hmC staining extension scores and nodule size, vascular invasion, and oncocytic morphology. Nuclear 5hmC staining intensity demonstrated associations with focality, capsule status, extrathyroidal extension, vascular invasion, and oncocytic morphology. Follicular/oncocytic adenomas exhibited higher 5hmC expression than uncertain malignant potential (UMP) or noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), as well as malignant neoplasms, including papillary thyroid carcinomas (PTCs), oncocytic carcinomas (OCAs), follicular thyroid carcinomas (FTCs), and invasive encapsulated follicular variants of PTC (IEFV-PTC). TERT promoter mutation cases showed notably lower values for the 5hmC expression, while RAS (H, N, or K) mutations, particularly HRAS mutations, were associated with higher 5hmC expression. Additionally, we identified, for the first time, a significant link between 5hmC expression and oncocytic morphology. However, despite the merits of these discoveries, we acknowledge that 5hmC currently cannot segregate minimally invasive from widely invasive tumors, although 5hmC levels were lower in wi-FPTCs. Further research is needed to explore the potential clinical implications of 5hmC in thyroid tumors., (© 2024. The Author(s).)

Accurate prognostic markers are essential for guiding effective lung cancer treatment strategies. The level of 5-hydroxymethylcytosine (5hmC) in tissue is independently associated with overall survival (OS) in lung cancer patients. We explored the prognostic value of cell-free DNA (cfDNA) 5hmC through genome-wide analysis of 5hmC in plasma samples from 97 lung cancer patients. In both training and validation sets, we discovered a cfDNA 5hmC signature significantly associated with OS in lung cancer patients. We built a 5hmC prognostic model and calculated the weighted predictive scores (wp-score) for each sample. Low wp-scores were significantly associated with longer OS compared to high wp-scores in the training [median 22.9 versus 8.2 months; p = 1.30 × 10 -10 ; hazard ratio (HR) 0.04; 95% confidence interval (CI), 0.00-0.16] and validation (median 18.8 versus 5.2 months; p = 0.00059; HR 0.22; 95% CI: 0.09-0.57) sets. The 5hmC signature independently predicted prognosis and outperformed age, sex, smoking, and TNM stage for predicting lung cancer outcomes. Our findings reveal critical genes and signaling pathways with aberrant 5hmC levels, enhancing our understanding of lung cancer pathophysiology. The study underscores the potential of cfDNA 5hmC as a superior prognostic tool for guiding more personalized therapeutic strategies for lung cancer patients.

Skin aging is characterized by wrinkle formation and increased frailty and laxity, leading to the risk of age-related skin diseases. Keratinocyte is an important component of the epidermis in skin structure, and keratinocyte senescence has been identified as a pivotal factor in skin aging development. Because epigenetic pathways play a vital role in the regulation of skin aging, we evaluated human skin samples for DNA hydroxymethylation (5-hydroxymethylcytosine; 5-hmC) and SIRT4 expressions. Results found that both 5-hmC and SIRT4 showed a significant decrease in aged human skin samples. To test the results in vitro, human keratinocytes were cultured in H 2 O 2 , which modulates skin aging in vivo. However, H 2 O 2 -induced keratinocytes showed senescence-associated protein expression and significant downregulation of 5-hmC and SIRT4 expressions. Moreover, 5-hmC-converting enzymes ten eleven translocation 2 (TET2) showed a decrease and enhanced TET2 acetylation level in H 2 O 2 -induced keratinocytes. However, the overexpression of SIRT4 in keratinocytes alleviates the senescence phenotype, such as senescence-associated protein expression, decreases the TET2 acetylation, but increases TET2 and 5-hmC expressions. Our results provide a novel relevant mechanism whereby the epigenetic regulation of keratinocytes in skin aging may be correlated with SIRT4 expression and TET2 acetylation in 5-hmC alteration. Our study may provide a potential strategy for antiskin aging, which targets the SIRT4/TET2 axis involving epigenetic modification in keratinocyte senescence., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe 2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC 50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods., (Copyright © 2024. Published by Elsevier Inc.)

Recent studies have highlighted the significant role of 5-hydroxymethylcytosine (5hmC) in carcinogenesis. However, the specific role of 5hmC in osteosarcoma (OS) remains largely unexplored. The-re-fore, this study aimed to investigate the function of 5hmC and TET3 in OS. In this study, we found a decreased total level of 5hmC in OS tissues. The expression of the TET3 protein was also decreased in OS. Importantly, the decreased levels of TET3 were associated with a decreased disease-free survival (DFS) rate in patients. To investigate the role of TET3 and 5hmC in OS, we manipulated the levels of TET3 in MG-63 cells. Silencing TET3 in these cells resulted in a twofold increase in proliferation. Additio-nally, the level of 5hmC decreased in these cells. Con-versely, over-expression of TET3 in MG-63 cells led to the expected inhibition of proliferation and invasion, accompanied by an increase in 5hmC levels. In conclusion, both 5hmC and TET3 protein levels were decreased in OS. Additionally, the over-expression of TET3 inhibited the proliferation of MG-63 cells, while the suppression of TET3 had the opposite effect. These findings suggest that decreased levels of 5hmC and TET3 may serve as potential markers for OS.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The discovery of 5-hydroxymethylcytosine (5hmC) as a common DNA modification in mammalian genomes has ushered in new areas of inquiry regarding the dynamic epigenome. The balance between 5hmC and its precursor, 5-methylcytosine (5mC), has emerged as a determinant of key processes including cell fate specification, and alterations involving these bases have been implicated in the pathogenesis of various diseases. The identification of 5hmC separately from 5mC initially posed a challenge given that legacy epigenetic sequencing technologies could not discriminate between these two most abundant modifications, a significant blind spot considering their potentially functionally opposing roles. The growing interest in 5hmC, as well as in the Ten-Eleven Translocation (TET) family enzymes that catalyze its generation and further oxidation to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), has spurred the development of versatile methods for 5hmC detection. These methods enable the quantification and localization of 5hmC in diverse biological samples and, in some cases, at the resolution of individual nucleotides. However, navigating this growing toolbox of methods for 5hmC detection can be challenging. Here, we detail existing and emerging methods for the detection, quantification, and localization of 5hmC at global, locus-specific, and base resolution levels. These methods are discussed in the context of their advantages and limitations, with the goal of providing a framework to help guide researchers in choosing the level of resolution and the associated method that could be most suitable for specific needs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma., Methods: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models., Results: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma., Conclusion: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

Objective: Cutaneous lupus erythematosus (CLE) is a heterogenous skin disease. The two most common subtypes are discoid LE (DLE) characterized by scarring skin damage and acute CLE (ACLE) presenting with transiently reversible skin lesions. It remains unknown what causes the difference of skin lesions. Studies have shown the existence of tissue-specific 5-Hydroxymethylcytosine (5 hmC)-modified regions in human tissues, which may affect the tissue-related diseases. Here, we aim to assess the expression of 5 hmc in DLE and ACLE lesions and explore the relationship of 5 hmc with scarring damage in DLE., Methods: 84 CLE samples were included in the study. We evaluated the skin damage score and reviewed the histopathologic sections. Immunohistochemical staining was performed to detect the expression of 5 hmc in the appendage and periappendageal inflammatory cells. The 5 hmc expression in periappendageal lymphocytic cells was investigated by multi-spectrum immunohistochemistry staining., Results: Scarring/atrophy was the most significant damage in differentiating the DLE from ACLE. Perifollicular inflammatory infiltration was present in all patients with DLE scarring alopecia (DLESA). The 5 hmc expression in the appendage and periappendageal inflammatory cells was significantxly increased in DLESA than ACLE. Similar expression pattern was seen in the staining of IFN-alpha/beta Receptor (IFNAR). The expression of 5 hmc in the appendage was positively correlated with that in the periappendageal inflammatory cells. There was an increased 5 hmc expression in lymphocytes cluster around hair follicle consisting of CD4 + cells, CD8 + cells, and CD19 + cells in DLESA lesions., Conclusion: These data demonstrate a close association of the expression pattern of 5 hmc with the histopathological characteristic distribution, and with the type I interferons (IFNs) signals in DLESA, supporting the importance of 5 hmc in the amplification of appendage damage and periappendageal inflammation, thereby offering a novel insight into the scarring damage of DLE and the heterogeneity of CLE skin lesions.

Background: DNA 5-hydroxymethylcytosine (5hmC) is produced by dynamic 5mC oxidation process contributing to tissue specification, and loss of 5hmC has been reported in multiple cancers including genitourinary cancers. However, 5hmC is also cell-type specific, and its variability may exist between differentiated tumor cells and cancer stem cells. Thus, cancer-associated changes in 5hmC may be contributed by distinct sets of tumor cells within the tumor tissues., Results: Here, we applied a sensitive immunoprecipitation-based method (hMeDIP-seq) to analyze 5hmC changes during genitourinary carcinogenesis (including prostate, urothelial and kidney). We confirmed the tissue-specific distribution of 5hmC in genitourinary tissues and identified regional gain and global loss of 5hmC coexisting in genitourinary cancers. The genes with gain of 5hmC during tumorigenesis were functionally enriched in regulating stemness and hypoxia, whereas were associated with poor clinical prognosis irrespective of their differences in tumor type. We identified that gain of 5hmC occurred in soft fibrin gel-induced 3D tumor spheres with a tumor-repopulating phenotype in two prostate cancer cell lines, 22RV1 and PC3, compared with conventional two-dimensional (2D) rigid dishes. Then, we defined a malignant signature derived from the differentially hydroxymethylated regions affected genes of cancer stem-like cells, which could predict a worse clinical outcome and identified phenotypically malignant populations of cells from prostate cancer tumors. Notably, an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, restored 5hmC and killed the cancer stem cell-like cells leading to apoptosis in prostate cancer cell lines., Conclusions: Collectively, our study dissects the regional gain of 5hmC in maintaining cancer stem-like cells and related to poor prognosis, which provides proof of concept for an epigenetic differentiation therapy with vitamin C by 5hmC reprogramming., (© 2022. The Author(s).)

RNA 5-hydroxymethylcytosine (5hmC) is a kind of RNA modification, which is related to the life activities of many organisms. Studying its distribution is very important to reveal its biological function. Previously, high-throughput sequencing was used to identify 5hmC, but it is expensive and inefficient. Therefore, machine learning is used to identify 5hmC sites. Here, we design a model called R5hmCFDV, which is mainly divided into feature representation, feature fusion and classification. (i) Pseudo dinucleotide composition, dinucleotide binary profile and frequency, natural vector and physicochemical property are used to extract features from four aspects: nucleotide composition, coding, natural language and physical and chemical properties. (ii) To strengthen the relevance of features, we construct a novel feature fusion method. Firstly, the attention mechanism is employed to process four single features, stitch them together and feed them to the convolution layer. After that, the output data are processed by BiGRU and BiLSTM, respectively. Finally, the features of these two parts are fused by the multiply function. (iii) We design the deep voting algorithm for classification by imitating the soft voting mechanism in the Python package. The base classifiers contain deep neural network (DNN), convolutional neural network (CNN) and improved gated recurrent unit (GRU). And then using the principle of soft voting, the corresponding weights are assigned to the predicted probabilities of the three classifiers. The predicted probability values are multiplied by the corresponding weights and then summed to obtain the final prediction results. We use 10-fold cross-validation to evaluate the model, and the evaluation indicators are significantly improved. The prediction accuracy of the two datasets is as high as 95.41% and 93.50%, respectively. It demonstrates the stronger competitiveness and generalization performance of our model. In addition, all datasets and source codes can be found at https://github.com/HongyanShi026/R5hmCFDV., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>-5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA., 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 © 2022 Elsevier B.V. All rights reserved.)

RNA 5-hydroxymethylcytosine (5 hmC) is an important RNA modification, which plays vital role in several biological processes. Currently, it is a hot topic to identify 5 hmC sites due to its benefit in understanding its biological functions. Therefore, in this study, we developed a predictor called iRNA5 hmC-HOC, which is based on a high-order correlation information method to identify 5 hmC sites. To build the model, 22 different classes of dinucleotide physicochemical (PC) properties were employed to represent RNA sequences, and the least absolute shrinkage and selection operator (LASSO) algorithm was adopted to select the most discriminative features. In the jackknife test, the proposed method achieved 89.80% classification accuracy based on support vector machine (SVM). As compared with the state-of-the-art predictors, our proposed method has significant improvement on the classification performance. It indicates that the proposed method might be a promising tool in identifying RNA 5 hmC modification sites. The dataset and source codes are available at https://figshare.com/articles/online&#95;resource/iRNA5hmC-HOC/15177450.

Background: Diabetic kidney disease (DKD), one of the main complications of diabetes mellitus (DM), has become a frequent cause of end-stage renal disease. A clinically convenient, non-invasive approach for monitoring the development of DKD would benefit the overall life quality of patients with DM and contribute to lower medical burdens through promoting preventive interventions., Methods: We utilized 5hmC-Seal to profile genome-wide 5-hydroxymethylcytosines in plasma cell-free DNA (cfDNA). Candidate genes were identified by intersecting the differentially hydroxymethylated genes and differentially expressed genes from the GSE30528 and GSE30529. Then, a protein interaction network was constructed for the candidate genes, and the hub genes were identified by the MCODE and cytoHubba algorithm. The correlation analysis between the hydroxymethylation level of the hub genes and estimated glomerular filtration rate (eGFR) was carried out. Finally, we demonstrated differences in expression levels of the protein was verified by constructing a mouse model of DKD. In addition, we constructed a network of interactions between drugs and hub genes using the Comparative Toxicogenomics Database., Results: This study found that there were significant differences in the overall distribution of 5hmC in plasma of patients with DKD, and an alteration of hydroxymethylation levels in genomic regions involved in inflammatory pathways which participate in the immune response. The final 5 hub genes, including (CTNNB1, MYD88, CD28, VCAM1, CD44) were confirmed. Further analysis indicated that this 5-gene signature showed a good capacity to distinguish between DKD and DM, and was found that protein levels were increased in renal tissue of DKD mice. Correlation analysis indicated that the hydroxymethylation level of 5 hub genes were nagatively correlated with eGFR. Toxicogenomics analysis showed that a variety of drugs for the treatment of DKD can reduce the expression levels of 4 hub genes (CD44, MYD88, VCAM1, CTNNB1)., Conclusions: The 5hmC-Seal assay was successfully applied to the plasma cfDNA samples from a cohort of DM patients with or without DKD. Altered 5hmC signatures indicate that 5hmC-Seal has the potential to be a non-invasive epigenetic tool for monitoring the development of DKD and it provides new insight for the future molecularly targeted anti-inflammation therapeutic strategies of DKD., Competing Interests: The 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 © 2022 Chu, Bi, He, Ma, Wan, Wang, Zhang, Zheng, Yang, Du, Maimaiti, Biekedawulaiti, Duolikun, Chen, Chen, Li, Tie and Lin.)

Aberrant changes in 5-hydroxymethylcytosine (5hmC) are a unique epigenetic feature in many cancers including acute myeloid leukemia (AML). However, genome-wide analysis of 5hmC in plasma cell-free DNA (cfDNA) remains unexploited in AML patients. We used a highly sensitive and robust nano-5hmC-Seal technology and profiled genome-wide 5hmC distribution in 239 plasma cfDNA samples from 103 AML patients and 81 non-cancer controls. We developed a 5hmC diagnostic model that precisely differentiates AML patients from controls with high sensitivity and specificity. We also developed a 5hmC prognostic model that accurately predicts prognosis in AML patients. High weighted prognostic scores (wp-scores) in AML patients were significantly associated with adverse overall survival (OS) in both training (P = 3.31e-05) and validation (P = 0.000464) sets. The wp-score was also significantly associated with genetic risk stratification and displayed dynamic changes with varied disease burden. Moreover, we found that high wp-scores in a single gene, BMS1 and GEMIN5 predicted OS in AML patients in both the training set (P = 0.023 and 0.031, respectively) and validation set (P = 9.66e-05 and 0.011, respectively). Lastly, our study demonstrated the genome-wide landscape of DNA hydroxymethylation in AML and revealed critical genes and pathways related to AML diagnosis and prognosis. Our data reveal plasma cfDNA 5hmC signatures as sensitive and accurate markers for AML diagnosis and prognosis. Plasma cfDNA 5hmC analysis will be an effective and minimally invasive tool for AML management., (© 2022. The Author(s).)

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 β-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

Objective: To clarify the application value of 5-hydroxymethylcytosine (5hmC) in evaluating the progression of chronic hepatitis B (CHB) to hepatocellular carcinoma (HCC) based on difference analysis., Methods: A total of 180 patients were enrolled. Among them, 84 patients with chronic hepatitis B virus (HBV) infection while no progression to hepatocellular carcinoma (HCC) were included in the control group (CG), and 96 patients with HCC developed from HBV infection were included in the research group (RG). Two-thirds of the samples were used in the training set and 1/3 samples in the validation set to detect the level of 5hmC in both groups based on the modified nano-hmC-Seal technique. The expression levels of 5hmC-related genes TET2 and TET3 were quantified by qPCR, and the correlation between TET3 and 5hmC was analyzed by Pearson's correlation coefficients. Receiver operating characteristic (ROC) curves were drawn to evaluate the application value of the TET3-based 5hmC prediction model in the early diagnosis of HCC., Results: (i) The expression of 5hmC in RG was lower than that in CG, no matter in the training set or the validation set. (ii) 5hmC was significantly enriched in the region between the transcription initiation site and the transcription end site but was depleted in the flanking region. (iii) 5hmC-related genes TET2 and TET3 were significantly downregulated in HCC patients, whether in the training set or the validation set. (iv) In both the training and validation sets, TET3 showed a positive association with 5hmC. (v) ROC analysis results showed that the 5hmC prediction model could be used to predict the progression of CHB to HCC (training set: AUC = 0.81, 0.729-0.893; validation set: AUC = 0.84, 0.739-0.936)., Conclusions: TET3 expression based on 5hmC sequencing is a landmark molecule for evaluating the progression of HCC in CHB patients, which is worthy of further study and promotion., Competing Interests: The authors declare no competing interests., (Copyright © 2022 Jiao Yu et al.)

DNA methylation is a key regulatory event controlling a variety of physiological processes and can have dramatic effects on gene transcription. Methylated cytosine (5-methylcytosine) can be oxidized by the TET family of enzymes to 5-hydroxymethylcytosine (5-hmC), a key intermediate in the demethylation cycle, and 5-hmC levels are reduced in malignancies such as acute myeloid leukemia and melanoma. We constructed a tissue microarray of human cutaneous squamous cell carcinoma tumors and found a global reduction in 5-hmC levels compared with that in the adjacent skin. Using a murine K14-CreER system, we have found that loss of Tet2 promotes carcinogen-induced squamous cell carcinoma and cooperates with loss of Tp53 to drive spontaneous squamous cell carcinoma tumors in epithelial tissues. Analysis of changes in 5-hmC and gene expression after loss of Tet2 in the epidermis revealed focal alterations in 5-hmC levels and an increase in hair follicle transient amplifying cell genes along with a reduction in epidermal differentiation genes. These results show a role for TET2 in epidermal lineage specification, consistent with reported roles for TET enzymes in controlling lineage commitment in hematopoietic stem cells and embryonic stem cells and establishing TET2 as a bone fide tumor suppressor in squamous cell carcinoma., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Punicalagin exerts neuroprotective activity by improving AMP-activated kinase (AMPK) and mitochondrial Krebs cycle. AMPK and Krebs cycle metabolites regulate 5-hydroxymethylcytosine (5hmC) via acting on ten-eleven translocation (TET) enzymes. Therefore, we hypothesized that punicalagin inhibits diabetes-related neuronal apoptosis by upregulating 5hmC in the diabetic mouse brain. C57BL/6J mice aged 8 weeks were randomly separated into five groups ( n = 10), normal control (NC), diabetes mellitus (DM), resveratrol (RES), low-dose punicalagin (LPU), and high-dose punicalagin (HPU). Compared with other groups, the neuronal apoptosis rate was significantly higher and the 5hmC level of the cerebral cortex was significantly lower in the DM group. The levels of TET2 and P-AMPKα/AMPKα were significantly lower in the DM group than in both LPU and HPU groups. The ratio of (succinic acid + fumaric acid)/α-ketoglutarate was significantly higher in the DM group than in other groups. The present results suggest that punicalagin upregulates 5hmC via activating AMPK and maintaining Krebs cycle homeostasis, thus inhibiting neuronal apoptosis in the diabetic mouse brain.

Objective: Universal noninvasive genomic screening to detect cancer and/or fetal DNA in plasma at all stages of development is highly warranted. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome. In the current study, we demonstrate that discrete 5hmC sites within 80 bp hotspot regions exist in a greater proportion of cancer versus normal cells., Result: 5hmC was detected in 16 of 17 known hotspots having C to T or G to A mutations. The results show the presence of two characteristically distinct 5hmC groups: Tier 1 Group with 3 to eightfold more 5hmCs detected in tumor-cells than in normal-cell derived DNA (as observed in 6 of 11 CpG sites). Tier 2 group with equal allele frequency of 5hmC among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Thus, detection and quantification of the Tier 1 group of 5hmC sites or its prevalence at or near cancer mutation hot spots in cells may enable early detection, screening and potentially prediction of the likelihood of cancer occurrence or the severity of the cancer., (© 2022. The Author(s).)

Decreased 5-hydroxymethylcytosine (5hmC) levels caused by mitochondrial dysfunction in the brain are closely associated with the development of neurodegenerative disease. It has been reported that n-3 polyunsaturated fatty acids (PUFAs) prevent cognitive dysfunction by improving mitochondrial function in the brain. However, whether n-3 PUFA prevents cognitive dysfunction by increasing the levels of 5hmC in the brain is undisclosed. Mice were randomly divided into six groups ( n = 10), injected with D-galactose (200 mg kg -1 day -1 ) for the model group and given different oils [0.1 mL per 10 g body weight per day, fish oil (FO), peony seed oil (PSO), corn oil (CO) and olive oil (OO)] for the prevention groups, and injected with the same dose of saline for the normal control group (NC) for 10 weeks, respectively. Peony seed oil and fish oil have shown preventive effects on D-galactose-induced cognitive dysfunction in behavioral tests. The content of docosahexaenoic acid (C22:6n-3, DHA content) in the brain was significantly higher in FO and PSO groups than in the other groups. Brain oxidative stress and neuronal apoptosis were significantly lower in PSO and FO groups than in the other groups. RNA-seq results showed that the different genes between PSO and FO compared with the model group were involved in the DNA demethylation process and the 5-methylcytosine metabolic process. The brain levels of 5hmC and the ten-eleven translocation family of dioxygenases (TETs) were significantly higher in FO and PSO groups compared with the model group, as analyzed by dot-blot and western blot. In conclusion, peony seed oil and fish oil increased the C22:6n-3 content, which activated the TET activity, led to up-regulation of the 5hmc level, resulted in inhibition of neuronal apoptosis, and then improved the cognitive function in D-gal-induced mice.

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.

DNA methylation predominantly occurs within the CpG dinucleotide, which is the main epigenetic form of gene expression regulation in mammals. Genomic DNA with CpG sites has different sequence lengths and complex secondary structures, resulting in the complexity and diversity of the samples. Therefore, highly efficient quantification of DNA methylation in complex samples remains challenging. Herein, the regulatable DNAzyme motor triggered by strand displacement amplification (SDA) was designed to quantify 5-hydroxymethylcytosine (5hmC) signatures as a model. Briefly, the 5hmC sites as primary target were specifically labeled with DNA primers and converted into a large number of single-stranded DNA (secondary target) via the SDA reaction which could activate the DNAzyme motor. With the increase of secondary target, the DNAzyme motor gradually recovered its activity and could continuously cleave the track strands labeled quenching probes, causing electrochemiluminescence signal recovery and detection limit down to 0.49 fM for 5hmC. This strategy provides a new route to quantify natural base modifications in DNA and would hold promising potential for the early diagnosis of cancer and other diseases related to 5hmC.

Dysregulation of epigenetic mechanisms, reflected by loss of expression of 5-hydroxymethylcytosine (5-hmC) is being increasingly recognized as a marker of aggressive behavior in several neoplasms; however, the role of such epigenetic modifiers in pancreatic neuroendocrine tumors (PanNETs) has not been studied. Annotated cohort of 60 PanNETs was evaluated for 5-hmC expression using immunohistochemistry. Univariable and multivariable analyses were performed. To determine intratumor heterogeneity of 5-hmC expression, 26 additional synchronous metastatic deposits of PanNETs from 8 patients were evaluated for 5-hmC expression. 5-hmC level showed significant association with the presence of distant metastases (P=0.02), female sex (P=0.04), and Ki-67 proliferation index (P=0.002). A multivariate model created using the stepwise logistic regression analysis showed the presence of nodal metastases (odds ratio=6.15), lymphovascular invasion (odds ratio=4.07) and lack of 5-hmC expression (odds ratio=5.34) were predictive of the risk of distant metastasis in PanNETs with a c-statistic of 0.845. Epigenetic intratumoral heterogeneity of 5-hmC expression was seen in 37.5% cases (3/8). Our work provides evidence that epigenetic regulators are involved in the pathobiology of PanNETs and immunohistochemical analysis of 5-hmC may be able to refine prognostic evaluation of these tumors., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Mouse knockouts of Cntnap2 show altered neurodevelopmental behavior, deficits in striatal GABAergic signaling, and a genome-wide disruption of an environmentally sensitive DNA methylation modification (5-hydroxymethylcytosine [5hmC]) in the orthologs of a significant number of genes implicated in human neurodevelopmental disorders. We tested adult Cntnap2 heterozygous mice ( Cntnap2 +/- ; lacking behavioral or neuropathological abnormalities) subjected to a prenatal stress and found that prenatally stressed Cntnap2 +/- female mice show repetitive behaviors and altered sociability, similar to the homozygote phenotype. Genomic profiling revealed disruptions in hippocampal and striatal 5hmC levels that are correlated to altered transcript levels of genes linked to these phenotypes (e.g., Reln , Dst , Trio , and Epha5 ). Chromatin immunoprecipitation coupled with high-throughput sequencing and hippocampal nuclear lysate pull-down data indicated that 5hmC abundance alters the binding of the transcription factor CLOCK near the promoters of these genes (e.g., Palld , Gigyf1 , and Fry ), providing a mechanistic role for 5hmC in gene regulation. Together, these data support gene-by-environment hypotheses for the origins of mental illness and provide a means to identify the elusive factors contributing to complex human diseases., (© 2022 Papale et al.; Published by Cold Spring Harbor Laboratory Press.)

Aims: DNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of genes related to DNA repair system, chronic immune response and cell cycle., Methods: We evaluated the tissue DNA methylation/hydroxymethylation in bone marrow trephine biopsies of 73 patients with MDS, trying to correlate with the mRNA expression of 21 genes ( POLH, POLL, REV3L, POLN, POLQ, POLI, POLK, IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF6, IRF-7, IRF-8,IRF-9, MAD2 , CDC20 , AURKA , AURKB and TPX2 )., Results: The M-score (5mC ) was significantly higher in patients with chromosomal abnormalities than patients with normal karyotype (95% CI -27.127779 to -2.368020; p=0.022). We observed a higher 5mC/5hmC ratio in patients classified as high-risk subtypes compared with low-risk subtypes (95% CI -72.922115 to -1.855662; p=0.040) as well as patients with hypercellular bone marrow compared with patients with normocellular/hypocellular bone marrow (95% CI -69.189259 to -0.511828; p=0.047) and with the presence of dyserythropoiesis (95% CI 17.077703 to 51.331388; p=0.001). DNA pols with translesion activity are significantly influenced by methylation. As 5mC immunoexpression increases, the expressions of POLH (r=-0.816; r 2 =0.665; p=0.000) , POLQ (r=-0.790; r 2 =0.624; p=0.001) , PCNA (r=-0.635; r 2 =0.403; p=0.020) , POLK (r=-0.633; r 2 =0.400; p=0.036 and REV1 (r=-0.578; r 2 =0.334; p=0.049) decrease., Conclusions: Our results confirm that there is an imbalance in the DNA methylation in MDS, influencing the development of chromosomal abnormalities which may be associated with the low expression of DNA polymerases with translesion synthesis polymerases activity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)