Methylation 5mC Research Articles

Altered genomic methylation promotes Staphylococcus aureus persistence in hospital environment

Staphylococcus aureus can cause outbreaks and becomes multi-drug resistant through gene mutations and acquiring resistance genes. However, why S. aureus easily adapts to hospital environments, promoting resistance and recurrent infections, remains unknown. Here we show that a specific S. aureus lineage evolved from a clone that expresses the accessory gene regulator (Agr) system to subclones that reversibly suppressed Agr and caused an outbreak in the hospital setting. S. aureus with flexible Agr regulation shows increased ability to acquire antibiotic-resistant plasmids, escape host immunity, and colonize mice. Bacteria with flexible Agr regulation shows altered cytosine genomic methylation, including the decreased 5mC methylation in transcriptional regulator genes (pcrA and rpsD), compared to strains with normal Agr expression patterns. In this work, we discover how altered genomic methylation promotes flexible Agr regulation which is associated with persistent pathogen colonization in the hospital environment.

ALYREF recruits ELAVL1 to promote colorectal tumorigenesis via facilitating RNA m5C recognition and nuclear export

ALYREF can recognize 5-methylcytosine (m5C) decoration throughout RNAs to regulate RNA metabolism. However, its implications in cancer and precise regulatory mechanisms remain largely elusive. Here, we demonstrated that ALYREF supported colorectal cancer (CRC) growth and migration. Integrated analysis of ALYREF-RIP-Bis-seq and transcriptome profiles identified ribosomal protein S6 kinase B2 (RPS6KB2) and regulatory-associated protein of mTOR (RPTOR) as ALYREF’s possible downstream effectors. Mechanistically, ALYREF formed a complex with ELAV like RNA binding protein 1 (ELAVL1) to cooperatively promote m5C recognition and nuclear export of the two mRNAs. Moreover, ALYREF protein was highly expressed in tumor tissues of CRC patients, which predicted their poor prognosis. E2F transcription factor 6 (E2F6)-mediated transactivation gave a molecular insight into ALYREF overexpression. Collectively, ALYREF recruits ELAVL1 to collaboratively facilitate m5C recognition and nuclear export of RPS6KB2 and RPTOR transcripts for colorectal tumorigenesis, providing RNA m5C methylation as promising therapeutic targets and prognostic biomarkers for CRC.

NSUN5 promotes tumorigenic phenotypes through the WNT signaling pathway and immunosuppression of CD8+ T cells in gastric cancer

NSUN5, a key member of the M5C methylation family, plays a significant role in fundamental biological processes like cell proliferation and differentiation. However, its specific function and mechanisms in gastric cancer remain insufficiently understood. Initially, we examined NSUN5's differential expression in gastric cancer versus normal tissues, along with survival trends, associated signaling pathways, and immune infiltration using the TCGA database. Subsequently, we conducted immunohistochemistry experiments to assess NSUN5 expression in gastric cancer tissues. Gain-and loss-of-function experiments were carried out to investigate NSUN5's impact on the proliferation, stemness, and migratory capabilities of gastric cancer cells, as well as the expression of vital proteins in pertinent signaling pathways. Our findings demonstrate that NSUN5 is not only overexpressed in gastric cancer tissues, but also positively associated with tumor stage and inversely linked with patient prognosis. NSUN5 promotes the in vitro proliferation, stemness, and migration of gastric cancer cells, and the in vivo growth of these cells, chiefly through the activation of the WNT/β-catenin signaling pathway. Additionally, NSUN5 appears to diminish the infiltration of CD8+ T cells in gastric cancer, contributing to immune evasion. In conclusion, NSUN5 functions as a proto-oncogene in the progression of gastric cancer and may serve as a potential therapeutic target.

NSUN2 knockdown inhibits macrophage infiltration in diabetic nephropathy via reducing N5-methylcytosine methylation of SOCS1.

N5-methylcytosine (m5C) methylation is involved in various disease progression; however, its role in diabetic nephropathy (DN) has not been studied. The aim of this study was to investigate the role of NSUN2 in DN and the underlying mechanism. Streptozotocin-induced experimental mouse model was generated to analyze the role of NSUN2 in vivo, and high glucose (HG)-treated Raw264.7 cells were used to assess the effect of NSUN2 on macrophage infiltration in vitro. The regulation of NSUN2 on SOCS1 m5C methylation was evaluated using m5C methylated RNA immunoprecipitation, luciferase reporter analysis, and RNA stability determination assay. The results indicated that NSUN2 was highly expressed in the blood and kidney of DN mice. Knockdown of NSUN2 alleviated kidney damage, reduced blood glucose and urine albumin, and suppressed macrophage infiltration in DN mice. Moreover, NSUN2 interacted with SOCS1, and silenced NSUN2 inhibited m5C levels of SOCS1 to reduce SOCS1 mRNA stability. Additionally, interference with NSUN2 suppressed macrophage migration, invasion, and infiltration by positively regulating SOCS1 expression under HG conditions. In conclusion, silencing of NSUN2 inhibits macrophage infiltration by reducing m5C modification of SOCS1, and thereby attenuates renal injury. The findings suggest a novel regulatory mechanism between NSUN2-mediated m5C modification and DN.

RNA methyltransferase NSUN2-mediated m5C methylation promotes Cr(VI)-induced malignant transformation and lung cancer by accelerating metabolism reprogramming

Hexavalent chromium [Cr(VI)], one common environmental contaminant, has long been recognized as a carcinogen associated with lung cancer, but roles and mechanisms of Cr(VI)-induced epigenetic dysregulations in carcinogenesis remain to be investigated. In this study, we identified that RNA m5C methyltransferase NSUN2 was significantly upregulated in Cr(VI)-transformed cells and lung tissues of Cr(VI)-exposed mice. Inhibition of NSUN2 reduced cell proliferation, migration, colony formation and tube formation abilities. We found NSUN2-mediated m5C modification induced metabolic reprogramming and cell cycle by promoting the mRNA stabilities of ME1, GLUT3 and CDK2. In addition, knockdown of NSUN2 attenuated tumorigenesis and angiogenesis in vivo. RNA m5C reader ALYREF was identified to be involved in NSUN2-mediated m5C modification in Cr (VI)-induced carcinogenesis. Further study showed that EP300 induced NSUN2 upregulation through transcriptional activation by inducing histone modification at H3K27ac site for regulating Cr(VI) carcinogenesis. Our findings demonstrated novel role and mechanism of NSUN2 and epigenetic changes by increasing the RNA m5C modification that are important for Cr (VI)-induced carcinogenesis through NSUN2/ALYREF pathway. NSUN2, ALYREF, ME1, GLUT3 or/and CDK2 may be used as potential new biomarkers or/and therapeutic target(s) in the future.

DNA-binding proteins from MBD through ZF to BEN: recognition of cytosine methylation status by one arginine with two conformations.

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

Characterization of the Rat Osteosarcoma Cell Line UMR-106 by Long-Read Technologies Identifies a Large Block of Amplified Genes Associated with Human Disease.

The rat osteosarcoma cell line UMR-106 is widely used for the study of bone cancer biology but it has not been well characterized with modern genomic methods. To better understand the biology of UMR-106 cells we used a combination of optical genome mapping (OGM), long-read sequencing nanopore sequencing and RNA sequencing.The UMR-106 genome was compared to a strain-matched Sprague-Dawley rat for variants associated with human osteosarcoma while expression data were contrasted with a public osteoblast dataset. Using the COSMIC database to identify the most affected genes in human osteosarcomas we found somatic mutations in Tp53 and H3f3a. OGM identified a relatively small number of differences between the cell line and a strain-matched control animal but did detect a ~45 Mb block of amplification that included Myc on chromosome 7 which was confirmed by long-read sequencing. The amplified region showed several blocks of non-contiguous rearranged sequence implying complex rearrangements during their formation and included 14 genes reported as biomarkers in human osteosarcoma, many of which also showed increased transcription. A comparison of 5mC methylation from the nanopore reads of tumor and control samples identified genes with distinct differences including the OS marker Cdkn2a. This dataset illustrates the value of long DNA methods for the characterization of cell lines and how inter-species analysis can inform us about the genetic nature underlying mutations that underpin specific tumor types. The data should be a valuable resource for investigators studying osteosarcoma, in general, and specifically the UMR-106 model.

Narrative review of research progress of RNA m5C methylation in head and neck malignancies.

Head and neck malignancies encompass a spectrum of malignant tumors occurring in the head and neck region, characterized by rapid progression, high recurrence rates, and dismal prognoses. Despite significant advancements in comprehensive surgery-based therapies, the 5-year survival rate for patients has not shown substantial improvement. There is an urgent need to investigate novel targeted therapies. With the advancements in epigenetics, RNA 5-methylcytosine (m5C) methylation, a prevalent form of RNA modification, has been identified by numerous studies as playing a pivotal role in the pathological processes of tumorigenesis and development. However, a comprehensive review within the realm of head and neck malignancies is currently lacking. This study aims to comprehensively review the biological implications of RNA m5C methylation regulators in the pathogenesis and progression of various systemic malignant tumors, with a specific focus on exploring the potential impact of RNA m5C methylation on head and neck malignancies. A literature search on RNA m5C methylation and head and neck malignancies was conducted using PubMed, resulting in the inclusion of 46 relevant articles. The Cancer Genome Atlas (TCGA) database was utilized to analyze the correlation between m5C regulatory factors and clinicopathological features in patients with head and neck squamous cell carcinoma (HNSCC). Aberrant expression of RNA m5C methylation regulators is observed in head and neck malignancies, displaying a correlation with the clinicopathological grading of tumors. RNA m5C methylation may contribute to the progression of head and neck malignancies and could be associated with an unfavorable prognosis for patients. These findings offer valuable insights for the development of targeted treatments for head and neck malignancies.

B-Cell Epigenetic Modulation of IgA Response by 5-Azacytidine and IgA Nephropathy.

IgA nephropathy is an important global cause of kidney failure. Dysregulation of IgA production is thought to play a key role in IgA nephropathy pathogenesis, however, little is known about the epigenetic mechanisms such as RNA 5- methylcytosine (5mC) modification in regulating IgA synthesis. To decipher the role of RNA 5mC in regulation of IgA class switch, the miR-23b-/- and LCWE induced Kawasaki disease mice were treated with 5-azacytidine. Trdmt1-/- and double Trdmt1-/-/ miR-23b-/- mice, Aid-/- mice or Aid-/-/ miR-23b-/- mice were also employed. We showed that miR-23b down regulated expression of Transfer RNA Aspartic Acid Methyltransferase 1 (Trdmt1) and consequently reduced 5-methylcytosine (m5C) RNA modification and IgA synthesis in B cells. Inhibition of m5C RNA modification normalised serum IgA levels and ameliorated progression of the IgA nephropathy-like kidney disease in miR-23b-/- and Kawasaki disease mice while mesangial IgA and C3 deposition failed to develop in Trdmt1-/-miR-23b-/- mice. By contrast, increased m5C RNA modification resulted in an exaggerated IgA nephropathy phenotype. miR-23b regulation of serum IgA levels and the development of an IgA nephropathy-like kidney disease in miR-23b-/- and Kawasaki disease mice is likely mediated through TRDMT1 driven 5-methylcytosine RNA modification in B cells, resulting in impaired activation-induced cytidine deaminase activity and IgA class switch recombination. This study revealed TRDMT1 induced RNA 5mC methylation regulate IgA class switch and inhibition of RNA 5mC by 5-Azacytidine could ameliorate progression of IgA nephropathy.

Epitranscriptomic m5C methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection.

While the significance of N6-methyladenosine (m6A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m5C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m5C is more abundant than m6A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m5C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m5C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m5C-MeRIP-seq identified the NSUN2-dependent m5C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m5C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m5C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m5C methylation to SARS-CoV-2 RNA.

Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4C and m6A methylation is well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on viral DNA and compared to host DNA, strengthening evidence for virus-host interaction. Applied to three different microbiomes, the method unveils over 1900 motifs that were deposited in REBASE. The motifs include a novel 8-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights to bacterial biology and biotechnological applications.

NOP2 facilitates EZH2-mediated epithelial–mesenchymal transition by enhancing EZH2 mRNA stability via m5C methylation in lung cancer progression

NOP2, a member of the NOL1/NOP2/SUN domain (NSUN) family, is responsible for catalyzing the posttranscriptional modification of RNA through 5-methylcytosine (m5C). Dysregulation of m5C modification has been linked to the pathogenesis of various malignant tumors. Herein, we investigated the expression of NOP2 in lung adenocarcinoma (LUAD) tissues and cells, and found that it was significantly upregulated. Moreover, lentivirus-mediated overexpression of NOP2 in vitro resulted in enhanced migration and invasion capabilities of lung cancer cells, while in vivo experiments demonstrated its ability to promote the growth and metastasis of xenograft tumors. In contrast, knockdown of NOP2 effectively inhibited the growth and metastasis of lung cancer cells. RNA-sequencing was conducted to ascertain the downstream targets of NOP2, and the findings revealed a significant upregulation in EZH2 mRNA expression upon overexpression of NOP2. Subsequent validation experiments demonstrated that NOP2 exerted an m5C-dependent influence on the stability of EZH2 mRNA. Additionally, our investigations revealed a co-regulatory relationship between NOP2 and the m5C reader protein ALYREF in modulating the stability of EZH2 mRNA. Notably, the NOP2/EZH2 axis facilitated the malignant phenotype of lung cancer cells by inducing epithelial–mesenchymal transition (EMT) both in vitro and in vivo. Mechanistically, ChIP analysis proved that EZH2 counteracted the impact of NOP2 on the occupancy capacity of EZH2 and H3K27me3 in the promoter regions of E-cadherin, a gene crucial for regulating EMT. In a word, our research highlights the significant role of NOP2 in LUAD and offers novel mechanistic insights into the NOP2/ALYREF/EZH2 axis, which holds promise as a potential target for lung cancer therapy.

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

HCV 5-Methylcytosine Enhances Viral RNA Replication through Interaction with m5C Reader YBX1.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV's life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.

M5C methylated lncRncr3–MeCP2 interaction restricts miR124a-initiated neurogenesis

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 m5C methylation along with the defined m5C epitranscriptomic RNA reader protein MeCP2 to coordinate brain development.

NSUN4 mediated RNA 5-methylcytosine promotes the malignant progression of glioma through improving the CDC42 mRNA stabilization

5-Methylcytosine (m5C) methylation is a significant post-transcriptional modification that play a crucial role in the development and progression of numerous cancers. Whereas the functions and molecular mechanisms underlying m5C methylation in gliomas remain unclear. This study dedicated to explore changes of m5C levels and the clinical significance of the m5C writer NSUN4 in gliomas. We found that high m5C levels were negatively related to prognosis of patients with glioma. Moreover, gain- and loss-of-function experiments revealed the role of NSUN4 in enhancing m5C modification of mRNA to promote the malignant progression of glioma. Mechanistically speaking, NSUN4-mediated m5C alterations regulated ALYREF binding to CDC42 mRNA, thereby impacting the mRNA stability of CDC42. We also demonstrated that CDC42 promoted glioma proliferation, migration, and invasion by activating the PI3K-AKT pathway. Additionally, rescue experiments proved that CDC42 overexpression weaken the inhibitory effect of NSUN4 knockdown on the malignant progression of gliomas in vitro and in vivo. Our findings elucidated that NSUN4-mediated high m5C levels promote ALYREF binding to CDC42 mRNA and regulate its stability, thereby driving the malignant progression of glioma. This provides theoretical support for targeted the treatment of gliomas.

Nanopore sequencing demonstrates the roles of spermatozoal DNA N6-methyladenine in mediating transgenerational lipid metabolism disorder induced by excessive folate consumpton

Increased consumption of folic acid is prevalent due to its beneficial effects, but growing evidence emphasizes the side effects pointing to excessive dietary folate intake. The effects of excessive paternal folic acid consumption on offspring and its transgenerational inheritance mechanism have not been elucidated. We hypothesize that excessive folic acid consumption will alter sperm DNA N6-methyladenine (6mA) and 5-methylcytosine (5mC) methylation and heritably influence offspring metabolic homeostasis. Here, we fed roosters either folic acid-control or folic acid-excess diet throughout life. Paternal chronic folic acid excessive supplementation increased hepatic lipogenesis and lipid accumulation but reduced lipolysis both in the roosters and their offspring, which was further confirmed to be induced by one-carbon metabolism inhibition and gene expression alteration associated with the Peroxisome proliferator-activated receptor pathway. Based on the spermatozoal genome-wide DNA methylome identified by Nanopore sequencing, multi-omics association analysis of spermatozoal and hepatic DNA methylome, transcriptome, and metabolome suggested that differential spermatozoal DNA 6mA and 5mC methylation could be involved in regulating lipid metabolism-related gene expression in offspring chickens. This model suggests that sperm DNA N6-methyladenine and 5-methylcytosine methylation were involved in epigenetic transmission and that paternal dietary excess folic acid leads to hepatic lipid accumulation in offspring.

NSUN2/YBX1 promotes the progression of breast cancer by enhancing HGH1 mRNA stability through m5C methylation

BackgroundRNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC.MethodsTumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC.ResultsAs the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells.ConclusionsThis study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.

Single-base resolution quantitative genome methylation analysis in the model bacterium Helicobacter pylori by enzymatic methyl sequencing (EM-Seq) reveals influence of strain, growth phase, and methyl homeostasis

BackgroundBacterial epigenetics is a rapidly expanding research field. DNA methylation by diverse bacterial methyltransferases (MTases) contributes to genomic integrity and replication, and many recent studies extended MTase function also to global transcript regulation and phenotypic variation. Helicobacter pylori is currently one of those bacterial species which possess the highest number and the most variably expressed set of DNA MTases. Next-generation sequencing technologies can directly detect DNA base methylation. However, they still have limitations in their quantitative and qualitative performance, in particular for cytosine methylation.ResultsAs a complementing approach, we used enzymatic methyl sequencing (EM-Seq), a technology recently established that has not yet been fully evaluated for bacteria. Thereby, we assessed quantitatively, at single-base resolution, whole genome cytosine methylation for all methylated cytosine motifs in two different H. pylori strains and isogenic MTase mutants. EM-Seq reliably detected both m5C and m4C methylation. We demonstrated that three different active cytosine MTases in H. pylori provide considerably different levels of average genome-wide single-base methylation, in contrast to isogenic mutants which completely lost specific motif methylation. We found that strain identity and changed environmental conditions, such as growth phase and interference with methyl donor homeostasis, significantly influenced quantitative global and local genome-wide methylation in H. pylori at specific motifs. We also identified significantly hyper- or hypo-methylated cytosines, partially linked to overlapping MTase target motifs. Notably, we revealed differentially methylated cytosines in genome-wide coding regions under conditions of methionine depletion, which can be linked to transcript regulation.ConclusionsThis study offers new knowledge on H. pylori global and local genome-wide methylation and establishes EM-Seq for quantitative single-site resolution analyses of bacterial cytosine methylation.

NSUN2 affects diabetic retinopathy progression by regulating MUC1 expression through RNA m5C methylation

BackgroundDiabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m5C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene MUC1, specifically through RNA m5C methylation, on the progression of DR.MethodUtilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2.ResultsThe levels of both NSUN2 and RNA m5C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted MUC1 expression by binding to the RNA m5C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover, MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing.ConclusionsNSUN2 regulates the expression of MUC1 through ALYREF-mediated RNA m5C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.