Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

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.

RNA m5C methylation modification: a potential therapeutic target for SARS-CoV-2-associated myocarditis.

The Corona Virus Disease (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has quickly spread worldwide and resulted in significant morbidity and mortality. Although most infections are mild, some patients can also develop severe and fatal myocarditis. In eukaryotic RNAs, 5-methylcytosine (m5C) is a common kind of post-transcriptional modification, which is involved in regulating various biological processes (such as RNA export, translation, and stability maintenance). With the rapid development of m5C modification detection technology, studies related to viral m5C modification are ever-increasing. These studies have revealed that m5C modification plays an important role in various stages of viral replication, including transcription and translation. According to recent studies, m5C methylation modification can regulate SARS-CoV-2 infection by modulating innate immune signaling pathways. However, the specific role of m5C modification in SARS-CoV-2-induced myocarditis remains unclear. Therefore, this review aims to provide insights into the molecular mechanisms of m5C methylation in SARS-CoV-2 infection. Moreover, the regulatory role of NSUN2 in viral infection and host innate immune response was also highlighted. This review may provide new directions for developing therapeutic strategies for SARS-CoV-2-associated myocarditis.

5-Hydroxymethylcytosine in Cell-Free DNA Predicts Immunotherapy Response in Lung Cancer.

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.

Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions.

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. TET1V and TET1HxD 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.

DNA Methylation in Ctenophores.

Epigenomic regulation and dynamic DNA methylation, in particular, are widespread mechanisms orchestrating the genome operation across time and species. Whole-genome bisulfite sequencing (WGBS) is currently the only method for unbiasedly capturing the presence of 5-methylcytosine (5-mC) DNA methylation patterns across an entire genome with single-nucleotide resolution. Bisulfite treatment converts unmethylated cytosines to uracils but leaves methylated cytosines intact, thereby creating a map of all methylated cytosines across a genome also known as a methylome. These epigenomic patterns of DNA methylation have been found to regulate gene expression and influence gene evolution rates between species. While protocols have been optimized for vertebrate methylome production, little adaptation has been done for invertebrates. Creating a methylome reference allows comparisons to be made between rates of transcription and epigenomic patterning in animals. Here we present a method of library construction for bisulfite sequencing optimized for non-bilateral metazoans such as the ctenophore, Mnemiopsis leidyi. We have improved upon our previously published method by including spike-in genomic DNA controls to measure methylation conversion rates. By pooling two bisulfite conversion reactions from the same individual, we also produced sequencing libraries that yielded a higher percentage of sequenced reads uniquely mapping to the reference genome. We successfully detected 5-mC in whole-animal methylomes at CpG, CHG, and CHH sites and visualized datasets using circos diagrams. The proof-of-concept tests were performed both under control conditions and following injury tests with changes in methylation patterns of genes encoding innexins, toxins and neuropeptides. Our approach can be easily adapted to produce epigenomes from other fragile marine animals.

Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta.

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.

Detecting DNA hydroxymethylation: exploring its role in genome regulation.

DNA methylation is one of the most extensively studied epigenetic regulatory mechanisms, known to play crucial roles in various organisms. It has been implicated in the regulation of gene expression and chromatin changes, ranging from global alterations during cell state transitions to locus-specific modifications. 5-hydroxymethylcytosine (5hmC) is produced by a major oxidation, from 5-methylcytosine (5mC), catalyzed by the ten-eleven translocation (TET) enzymes, and is gradually being recognized for its significant role in genome regulation. With the development of state-of-the-art experimental techniques, it has become possible to detect and distinguish 5mC and 5hmC at base resolution. Various techniques have evolved, encompassing chemical and enzymatic approaches, as well as thirdgeneration sequencing techniques. These advancements have paved the way for a thorough exploration of the role of 5hmC across a diverse array of cell types, from embryonic stem cells (ESCs) to various differentiated cells. This review aims to comprehensively report on recent techniques and discuss the emerging roles of 5hmC. [BMB Reports 2024; 57(3): 135-142].

The level of active DNA demethylation compounds in leukocytes and urine samples as potential epigenetic biomarkers in breast cancer patients.

The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs' activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5-mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

5-Methylcytosine immunohistochemistry for predicting cutaneous melanoma prognosis.

There is a correlation between DNA methylation and the diseased stage and poor survival. 5-methylcytosine (5-mC) is one of the epigenetic modifications of bases that researchers focus on. Staining with 5-mC immunohistochemistry was used to examine pathological samples taken from individuals diagnosed with cutaneous melanoma. Between Breslow levels 2 and 4, there was a significant difference in the H-score of 5-mC expression (p = 0.046). A significant reduction in 5-mC expression H-scores was seen in patients who were diagnosed with ulcers (p = 0.039). It was shown that patients with low 5-mC had a significantly worse overall survival rate (p = 0.027).

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.

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 ß-glucosyltransferase (ß-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.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

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 ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-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 Fe2+ 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.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt 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 IDHmt astrocytoma. Five primary/malignantly progressed IDHmt 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 IDHmt glioma malignancy.

Molecular basis of an atypical dsDNA 5mC/6mA bifunctional dioxygenase CcTet from Coprinopsis cinerea in catalyzing dsDNA 5mC demethylation.

The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes.

NSUN2 promotes lung adenocarcinoma progression through stabilizing PIK3R2 mRNA in an m5C-dependent manner.

It is well known that 5-methylcytosine (m5C) is involved in variety of crucial biological processes in cancers. However, its biological roles in lung adenocarcinoma (LAUD) remain to be determined. The LUAD samples were used to assess the clinical value of NOP2/Sun RNA Methyltransferase 2 (NSUN2). Dot blot was used to determine global m5C levels. ChIP and dual-luciferase assays were performed to investigate the MYC-associated zinc finger protein (MAZ)-binding sites in NSUN2 promoter. RNA-seq was used to explore the downstream molecular mechanisms of NSUN2. Dual luciferase reporter assay, m5C-RIP-qPCR, and mRNA stability assay were conducted to explore the effect of NSUN2-depletion on target genes. Cell viability, transwell, and xenograft mouse model were designed to demonstrate the characteristic of NSUN2 in promoting LUAD progression. The m5C methyltransferase NSUN2 was highly expressed and caused elevated m5C methylation in LUAD samples. Mechanistically, MAZ positively regulated the transcription of NSUN2 and was related to poor survival of LUAD patients. Silencing NSUN2 decreased the global m5C levels, suppressed proliferation, migration and invasion, and inhibited activation of PI3K-AKT signaling in A549 and SPAC-1 cells. Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) was upregulated by NSUN2-mediated m5C methylation by enhancing its mRNA stabilization and activated the phosphorylation of the PI3K-AKT signaling. The present study explored the underlying mechanism and biological function of NSUN2-meditated m5C RNA methylation in LUAD. NSUN2 was discovered to facilitate the malignancy progression of LUAD through regulating m5C modifications to stabilize PIK3R2 activating the PI3K-AKT signaling, suggesting that NSUN2 could be a novel biomarker and promising therapeutic target for LUAD patients.

Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.

5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

Cell-Free DNA 5-Hydroxymethylcytosine Signatures for Lung Cancer Prognosis.

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.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Zebrafish Mbd5 binds to RNA m5C and regulates histone deubiquitylation and gene expression in development metabolism and behavior.

Complex biological processes are regulated by both genetic and epigenetic programs. One class of epigenetic modifications is methylation. Evolutionarily conserved methyl-CpG-binding domain (MBD)-containing proteins are known as readers of DNA methylation. MBD5 is linked to multiple human diseases but its mechanism of action remains unclear. Here we report that the zebrafish Mbd5 does not bind to methylated DNA; but rather, it directly binds to 5-methylcytosine (m5C)-modified mRNAs and regulates embryonic development, erythrocyte differentiation, iron metabolism, and behavior. We further show that Mbd5 facilitates removal of the monoubiquitin mark at histone H2A-K119 through an interaction with the Polycomb repressive deubiquitinase (PR-DUB) complex in vivo. The direct target genes of Mbd5 are enriched with both RNA m5C and H2A-K119 ubiquitylation signals. Together, we propose that zebrafish MBD5 is an RNA m5C reader that potentially links RNA methylation to histone modification and in turn transcription regulation in vivo.