Methylation Of Sequences Research Articles

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight

BackgroundThe Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes.ResultsTo provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate.ConclusionsFENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Simultaneous profiling of chromatin accessibility and DNA methylation in complete plant genomes using long-read sequencing.

Epigenetic regulations, including chromatin accessibility, nucleosome positioningand DNA methylation intricately shape genome function. However, current chromatin profiling techniques relying on short-read sequencing technologies fail to characterise highly repetitive genomic regions and cannot detect multiple chromatin features simultaneously. Here, we performed Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq) of purified plant nuclei. Thanks to the use of long-read nanopore sequencing, SAM-seq enables high-resolution profiling of m6A-tagged chromatin accessibility together with endogenous cytosine methylation in plants. Analysis of naked genomic DNA revealed significant sequence preference biases of m6A-MTases, controllable through a normalisation step. By applying SAM-seq to Arabidopsis and maize nuclei we obtained fine-grained accessibility and DNA methylation landscapes genome-wide. We uncovered crosstalk between chromatin accessibility and DNA methylation within nucleosomes of genes, TEs, and centromeric repeats. SAM-seq also detects DNA footprints over cis-regulatory regions. Furthermore, using the single-molecule information provided by SAM-seq we identified extensive cellular heterogeneity at chromatin domains with antagonistic chromatin marks, suggesting that bivalency reflects cell-specific regulations. SAM-seq is a powerful approach to simultaneously study multiple epigenetic features over unique and repetitive sequences, opening new opportunities for the investigation of epigenetic mechanisms.

A new high-throughput screening methodology for the discovery of cancer-testis antigen using multi-omics data

BackgroundCancer/testis antigens (CTAs), also known as tumor-specific antigens (TSAs) are specifically expressed in cancer cells and exhibit high immunogenicity, making them promising targets for immunotherapy and cancer vaccines. MethodsA new integrated high-throughput screening methodology for CTAs was proposed in this study through combining DNA methylation and RNA sequencing data. Briefly, the genes with increased transcript level and decreased DNA methylation were identified by multi-omics analysis. RNA sequencing studies in cell lines exposed to DNA methyltransferase (DNMT) inhibitors were performed to validate the inherent causal relationship between DNA hypomethylation and gene expression upregulation. ResultsWe proposed a new integrated high-throughput screening methodology for identification of CTAs using multi-omics analysis. In addition, we tested the feasibility of this method using gastric cancer (GC) as an example. In GC, we identified over 2000 primary candidate CTAs and ultimately identified 20 CTAs with significant tissue-specificity, including a testis-specific serine protease TESSP1/PRSS41. Integrated analysis confirmed that PRSS41 expression was reactivated in gastrointestinal cancers by promoter DNA hypomethylation at the CpG site (cg08104780). Additionally, DNA hypomethylation of PRSS41 predicted a poor prognosis in GC. ConclusionWe propose a new high-throughput screening method for the identification of CTAs in cancer and validate its effectiveness. Our work emphasizes that serine protease PRSS41 is a novel TSA that is reactivated in GC due to promoter DNA hypomethylation.

Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastaticdisease.

It is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with (i) stable PCa (sPCa)postprimary or salvage RT,(ii) progressing PCa (pPCa) post-RT,and (iii) de novo metastatic PCa(mPCa). A cohort of 19 patients with diagnostic prostate biopsies (n = 6 sPCa, n = 5 pPCa, n = 8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3'RNA sequencing (3'RNAseq) (n = 19), nanoString analysis (n = 12), and Illumina 850k methylation (n = 8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes and methylated genomic regions (MGRs). Eighteen of 19 samples provided useable 3'RNAseq data. Principal component analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified ~600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p < 0.0001) in an external cohort. 3'RNAseq, nanoString and 850k-methylation analyses are each achievable from historical FFPE diagnostic pretreatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

Identification of methylation driver genes for predicting the prognosis of pancreatic cancer patients based on whole-genome DNA methylation sequencing technology

This study was based on the use of whole-genome DNA methylation sequencing technology to identify DNA methylation biomarkers in tumor tissue that can predict the prognosis of patients with pancreatic cancer (PCa). TCGA database was used to download PCa-related DNA methylation and transcriptome atlas data. Methylation driver genes (MDGs) were obtained using the MethylMix package. Candidate genes in the MDGs were screened for prognostic relevance to PCa patients by univariate Cox analysis, and a prognostic risk score model was constructed based on the key MDGs. ROC curve analysis was performed to assess the accuracy of the prognostic risk score model. The effects of PIK3C2B knockdown on malignant phenotypes of PCa cells were investigated in vitro. A total of 2737 differentially expressed genes were identified, with 649 upregulated and 2088 downregulated, using 178 PCa samples and 171 normal samples. MethylMix was employed to identify 71 methylation-driven genes (47 hypermethylated and 24 hypomethylated) from 185 TCGA PCa samples. Cox regression analyses identified eight key MDGs (LEF1, ZIC3, VAV3, TBC1D4, FABP4, MAP3K5, PIK3C2B, IGF1R) associated with prognosis in PCa. Seven of them were hypermethylated, while PIK3C2B was hypomethylated. A prognostic risk prediction model was constructed based on the eight key MDGs, which was found to accurately predict the prognosis of PCa patients. In addition, the malignant phenotypes of PANC-1 cells were decreased after the knockdown of PIK3C2B. Therefore, the prognostic risk prediction model based on the eight key MDGs could accurately predict the prognosis of PCa patients.

Repetitive element transcript accumulation is associated with inflammaging in humans.

Chronic, low-grade inflammation increases with aging, contributing to functional declines and diseases that reduce healthspan. Growing evidence suggests that transcripts from repetitive elements (RE) in the genome contribute to this "inflammaging" by stimulating innate immune activation, but evidence of RE-associated inflammation with aging in humans is limited. Here, we present transcriptomic and clinical data showing that RE transcript levels are positively related to gene expression of innate immune sensors, and to serum interleukin 6 (a marker of systemic inflammation), in a large group of middle-aged and older adults. We also: (1) use transcriptomics and whole-genome bisulfite (methylation) sequencing to show that many RE may be hypomethylated with aging, and that aerobic exercise, a healthspan-extending intervention, reduces RE transcript levels and increases RE methylation in older adults; and (2) extend our findings in a secondary dataset demonstrating age-related changes in RE chromatin accessibility. Collectively, our data support the idea that age-related RE transcript accumulation may play a role in inflammaging in humans, and that RE dysregulation with aging may be due in part to upstream epigenetic changes.

Xiaoyao Pills, a Chinese patent medicine, treats mild and moderate depression: A randomized clinical trial combined with DNA methylation analysis

BackgroundXiaoyao pills (XYP) is a commercial Chinese patent medicine used in the treatment of depression. However, the mechanisms underlying its therapeutic effects, as well as the patients who can benefit from XYP, have not been evaluated so far. ObjectivesTo this end, we conducted a double-blinded, random, and placebo-controlled clinical trial of orally administered XYP in patients with depression. MethodsThe 17-item Hamilton Depression Rating Scale (HAMD-17) scores were recorded at baseline, and every 2 weeks after the start of treatment. To further elucidate the epigenetic mechanism of XYP, we performed mRNA sequencing and genome-wide DNA methylation sequencing using peripheral blood leukocytes of patients and healthy. ResultsXYP effectively alleviated the symptoms in patients with mild or moderate depressive disorders, particularly that of psychomotor retardation. XYP restored aberrant gene expression and DNA methylation patterns associated with depression, and the normalization of DNA methylation correlated with downregulation of several genes. In addition, altered DNA methylation levels in the XYP-treated samples were attributed to increased expression of the DNA methyltransferase DNMT1. ConclusionsOur study provides new insights into the epigenetic mechanism underlying depression and the therapeutic effects of XYP, along with an experimental basis for using XYP in the treatment of depression. Trial Registration InformationThe name of the registry and number: U.S. Clinical Trials Registry. The link to the registration: ClinicalTrials.gov ISRCTN12746343 (https://www.isrctn.com/ISRCTN12746343). The name of the trial register is “Efficacy and safety of the Xiaoyao pill for improving the clinical symptoms of stagnation of liver qi (chi) and spleen deficiency”. The clinical trial registration number is ISRCTN12746343.

MTOR Signaling Promotes Rapid m6A mRNA Methylation to Regulate NK-Cell Activation and Effector Functions.

NK cells can be rapidly activated in response to cytokines during host defense against malignant cells or viral infection. However, it remains unclear what mechanisms precisely and rapidly regulate the expression of a large number of genes involved in activating NK cells. In this study, we discovered that NK-cell N6-methyladenosine (m6A) methylation levels were rapidly upregulated upon short-term NK-cell activation and were repressed in the tumor microenvironment (TME). Deficiency of methyltransferase-like 3 (METTL3) or METTL14 moderately influenced NK-cell homeostasis, while double-knockout of METTL3/14 more significantly impacted NK-cell homeostasis, maturation, and antitumor immunity. This suggests a cooperative role of METTL3 and METTL14 in regulating NK-cell development and effector functions. Using methylated RNA immunoprecipitation sequencing, we demonstrated that genes involved in NK-cell effector functions, such as Prf1 and Gzmb, were directly modified by m6A methylation. Furthermore, inhibiting mTOR complex 1 activation prevented m6A methylation levels from increasing when NK cells were activated, and this could be restored by S-adenosylmethionine supplementation. Collectively, we have unraveled crucial roles for rapid m6A mRNA methylation downstream of the mTOR complex 1-S-adenosylmethionine signal axis in regulating NK-cell activation and effector functions.

IDNA-OpenPrompt: OpenPrompt learning model for identifying DNA methylation.

Introduction: DNA methylation is a critical epigenetic modification involving the addition of a methyl group to the DNA molecule, playing a key role in regulating gene expression without changing the DNA sequence. The main difficulty in identifying DNA methylation sites lies in the subtle and complex nature of methylation patterns, which may vary across different tissues, developmental stages, and environmental conditions. Traditional methods for methylation site identification, such as bisulfite sequencing, are typically labor-intensive, costly, and require large amounts of DNA, hindering high-throughput analysis. Moreover, these methods may not always provide the resolution needed to detect methylation at specific sites, especially in genomic regions that are rich in repetitive sequences or have low levels of methylation. Furthermore, current deep learning approaches generally lack sufficient accuracy. Methods: This study introduces the iDNA-OpenPrompt model, leveraging the novel OpenPrompt learning framework. The model combines a prompt template, prompt verbalizer, and Pre-trained Language Model (PLM) to construct the prompt-learning framework for DNA methylation sequences. Moreover, a DNA vocabulary library, BERT tokenizer, and specific label words are also introduced into the model to enable accurate identification of DNA methylation sites. Results and Discussion: An extensive analysis is conducted to evaluate the predictive, reliability, and consistency capabilities of the iDNA-OpenPrompt model. The experimental outcomes, covering 17 benchmark datasets that include various species and three DNA methylation modifications (4mC, 5hmC, 6mA), consistently indicate that our model surpasses outstanding performance and robustness approaches.

Airway epithelium respiratory illnesses and allergy (AERIAL) birth cohort: study protocol.

Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to 5 years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to 6 weeks, 1, 3, and 5 years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. The AERIAL study will provide a comprehensive longitudinal assessment of factors influencing the association between epithelial dysfunction and respiratory morbidity in early life, and hopefully identify novel targets for diagnosis and early intervention.

Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA–miRNA/protein axis

BackgroundLong noncoding RNAs (lncRNAs) play essential roles in the tumorigenesis of gastric cancer. However, the influence of lncRNA methylation on gastric cancer stem cells (GCSCs) remains unclear.MethodsThe N6-methyladenosine (m6A) levels of lncRNAs in gastric cancer stem cells were detected by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the results were validated by MeRIP-quantitative polymerase chain reaction (qPCR). Specific sites of m6A modification on lncRNAs were detected by single-base elongation- and ligation-based qPCR amplification (SELECT). By constructing and transfecting the plasmid expressing methyltransferase-like 3 (METTL3) fused with catalytically inactivated Cas13 (dCas13b) and guide RNA targeting specific methylation sites of lncRNAs, we obtained gastric cancer stem cells with site-specific methylation of lncRNAs. Reverse transcription (RT)-qPCR and Western blot were used for detecting the stemness of treated gastric cancer stem cells.ResultsThe site-specific methylation of PSMA3-AS1 and MIR22HG suppressed apoptosis and promoted stemness of GCSCs. LncRNA methylation enhanced the stability of PSMA3-AS1 and MIR22HG to suppress apoptosis of GCSCs via the PSMA3-AS1–miR-411-3p– or MIR22HG–miR-24-3p–SERTAD1 axis. Simultaneously, the methylated lncRNAs promoted the interaction between PSMA3-AS1 and the EEF1A1 protein or MIR22HG and the LRPPRC protein, stabilizing the proteins and leading to the suppression of apoptosis. The in vivo data revealed that the methylated PSMA3-AS1 and MIR22HG triggered tumorigenesis of GCSCs.ConclusionsOur study revealed the requirement for site-specific methylation of lncRNAs in the tumorigenesis of GCSCs, contributing novel insights into cancer development.

Abstract LB249: Multi-omics cancer risk assessment of Barrett’s esophagus with DNA methylation sequencing from endoscopic brush samples

Abstract Introduction: Barrett’s esophagus (BE) confers increased cancer risk. Endoscopic surveillance is recommended to distinguish non-dysplastic BE (NDBE), low grade dysplasia (LGD), high grade dysplasia (HGD), or adenocarcinoma (EAC) for endoscopic intervention. However, this paradigm is limited by sampling error, leading to missed/interval EAC. Herein, we aimed to classify these samples more accurately by performing whole genome methylation sequencing (WGMS) and utilizing multiple genomic features on endoscopic brush samples. Materials and methods: Endoscopic brush samples from patients with NDBE (n=18), HGD (18), EAC (18), and normal controls (18) were collected and underwent WGMS (discovery dataset). Sequence reads were processed through internal pipeline to extract CpG methylation and read level methylation ratio (alpha value). Copy number aberration (CNA) detection was performed using both CNVkit and ichorCNA. Differential methylated regions (DMRs) were identified through comparing EAC/HGD with NDBE and normal samples. Targeted methylation sequencing for selected DMRs and shallow WGMS were performed on EAC (41), HGD (26), LGD (23), NDBE (42), and normal controls (37) as validation. Ratio of the reads with alpha value&amp;gt;0.6 for each DMR was used for further analysis. Detected CNA segments were summarized into chromosome arm loss or gain (aneuploidy score or AS representing the number of chromosome arms gained or lost in a sample) and trimmed median absolute deviation (tMAD). Classification models were developed using the discovery set and the best performing one was applied to the validation dataset. Results: Genome-wide DNA methylation largely segregated different groups of samples as diagnosed; however, some unexpected group crossings were observed. Top 200 DMRs were identified between EAC/HGD and NDBE and normal samples. CNA changes were widespread in EAC, very common in HGD but rare in NDBE (17/18 EACs,14/18 HGDs, 5/18 NDBEs with AS&amp;gt;0). With tMAD &amp;gt; 0.015, 16/18 EACs, 8/18 HGDs but none of 18 NDBEs passed the threshold. EAC or HGD samples without detectable CNAs had very low “tumor” fraction as determined by methylation-based cell type deconvolution or ichorCNA. Similar results were found in the validation dataset. A multi-omics (AS, tMAD, estimated tumor fraction, and DMR methylation) model could predict EAC, HGD, or NDBE with 0.81 accuracy through cross validation (10X) and 0.73 in the independent dataset. This increased to 0.98 and 0.90 in separating high risk EAC/HGD from NDBE, respectively. Conclusion: Multi-omics data obtained from single WGMS allow for a comprehensive analysis of BE samples with varying dysplasia levels. Using classification modeling, these characteristics can be effectively used to accurately identify and distinguish between EAC and HGD from NDBE, suggesting their utility as adjuncts to endoscopic surveillance biopsies. Citation Format: Zhifu Sun, Caroline L. Matchett, Seth W. Slettedahl, William R. Taylor, Panwen Wang, Calise Berger, Caryn E. Anderson, Melissa A. Passe, Ramona Lansing, Collin E. Chalmers, Patrick H. Foote, Jeanette E. Eckel Passow, Doug W. Mahoney, John B. Kisiel, Prasad G. Iyer. Multi-omics cancer risk assessment of Barrett’s esophagus with DNA methylation sequencing from endoscopic brush samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB249.

YBX1 Promotes Esophageal Squamous Cell Carcinoma Progression via m5C‐Dependent SMOX mRNA Stabilization

The modification and recognition of 5-methylcytosine (m5C) are involved in the initiation and progression of various tumor types. However, the precise role and potential mechanism of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, it is found that YBX1 is frequently upregulated in ESCC compared with matched nontumor tissues. Gain- and loss-of-function assays show that YBX1 promoted the proliferation and metastasis of ESCC cells both in vitro and in vivo. Functional studies revealed that NOP2/Sun RNA methyltransferase family member 2 (NSUN2) is a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, integrated analysis based on RNA immunoprecipitation sequencing (RIP-seq) and m5C methylated RNA immunoprecipitation and sequencing (MeRIP-seq) assays identified spermine oxidase (SMOX) as a target gene containing an m5C site in its coding sequence (CDS) region, which coincided well with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restored the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Moreover, YBX1 activated the mTORC1 signaling pathway by stabilizing SMOX mRNA. The study reveals that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, thus providing a valuable therapeutic target for ESCC.

Central neurocytoma exhibits radial glial cell signatures with FGFR3 hypomethylation and overexpression

We explored the genomic events underlying central neurocytoma (CN), a rare neoplasm of the central nervous system, via multiomics approaches, including whole-exome sequencing, bulk and single-nuclei RNA sequencing, and methylation sequencing. We identified FGFR3 hypomethylation leading to FGFR3 overexpression as a major event in the ontogeny of CN that affects crucial downstream events, such as aberrant PI3K-AKT activity and neuronal development pathways. Furthermore, we found similarities between CN and radial glial cells based on analyses of gene markers and CN tumor cells and postulate that CN tumorigenesis is due to dysregulation of radial glial cell differentiation into neurons. Our data demonstrate the potential role of FGFR3 as one of the leading drivers of tumorigenesis in CN.

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Methylation of microRNA genes and its effect on secondary xylem development of stem in poplar.

MicroRNAs (miRNAs) and DNA methylation are both vital regulators of gene expression. DNA methylation can affect the transcription of miRNAs, just like coding genes, through methylating the CpG islands in the gene regions of miRNAs. Although previous studies have shown that DNA methylation and miRNAs can each be involved in the process of wood formation, the relationship between the two has been relatively little studied in plant wood formation. Studies have shown that the second internode (IN2) (from top to bottom) of 3-month-old poplar trees can represent the primary stage of poplar stem development and IN8 can represent the secondary stage. There were also significant differences in DNA methylation patterns and miRNA expression patterns obtained from PS and SS. In this study, we first interactively analyzed methylation and miRNA sequencing data to identify 43 differentially expressed miRNAs regulated by differential methylation from the primary stage and secondary stage, which were found to be involved in multiple biological processes related to wood formation by enrichment analysis. In addition, six miRNA/target gene modules were finally identified as potentially involved in secondary xylem development of poplar stems through degradome sequencing and functional analysis. In conclusion, this study provides important reference information on the mechanism of interaction between different regulatory pathways of wood formation.

Fluorimetric detection of DNA methylation by cerium oxide nanoparticles for early cancer diagnosis

In this study, a very sensitive fluorescence nano-biosensor was developed using CeO2 nanoparticles for the rapid detection of DNA methylation. The characteristics of CeO2 nanoparticles were determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) spectroscopy, UV–visible spectroscopy, and fluorescence spectroscopy. The CeO2 nanoparticles were reacted with a single-stranded DNA (ssDNA) probe, and then methylated and unmethylated target DNAs hybridized with an ssDNA probe, and the fluorescence emission was measured.Upon adding the target unmethylated and methylated ssDNA, the fluorescence intensity increased in the linear range of concentration from 2 × 10−13 - 10−18 M. The limit of detection (LOD) was 1.597 × 10−6 M for methylated DNA and 1.043 × 10−6 M for unmethylated DNA. The fluorescence emission intensity of methylated sequences was higher than of that unmethylated sequences. The fabricated DNA nanobiosensor showed a fluorescence emission at 420 nm with an excitation wavelength of 280 nm. The impact of CeO2 binding on methylated and unmethylated DNA was further demonstrated by agarose gel electrophoresis.Finally, the actual sample analysis suggested that the nanobiosensor could have practical applications for detecting methylation in the human plasma samples.

Abstract 7565: Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing

Abstract Background: Cell-free DNA (cfDNA) detected in proximal body fluids has demonstrated potential for cancer detection using minimally invasive methodology. Our past work showed that tumor cfDNA is present in the cerebrospinal fluid (CSF) and other body fluids of patients with inconclusive standard of care testing. However, past work measuring copy number aberrations or somatic mutations was limited in cancer classification. To facilitate reliable classification, even at low tumor fractions and with fragmented DNA, we developed XR-methylSeq, a methylation sequencing platform to enrich for cell type-specific markers. Methods: We benchmarked XR-methylSeq with the K562 cell line and correlated the methylation values with gold standard measurement − whole genome bisulfite sequencing (WGBS). Methylation classifiers were applied for at least 22 cytology-positive body fluids, incorporating methylation array data from public references. T-distributed stochastic neighbor embedding (t-SNE) analysis was used for visualization in R. Deconvolution of cell type fractions for at least 29 (seven cytology-negative) body fluids and plasma samples was conducted using wgbstools. Cell type-specific markers were identified from a human DNA methylation atlas. Results: Benchmarks: XR-methylSeq has a 5-fold enrichment of the cell type-specific markers compared with WGBS. XR-methylSeq at 20 ng input highly correlates with WGBS at 2 μg (Pearson’s r = 0.97). Body Fluids: Thirteen cytology-positive CSF samples had copy number aberrations, 77% of them had concordant tumor classification, while the remaining 23% clustered with low tumor fraction samples. All nine lung primaries, including a low tumor fraction case that did not originally classify, showed a consistent cell-of-origin through deconvolution, as indicated by increased contributions from lung alveolar epithelial cells. Among six other body fluids, three exhibited the highest fractions aligning with the clinically identified cancer cell-of-origin. Additionally, the plasma cfDNA of a patient with acute liver injury had a higher fraction of hepatocyte signatures (22%) than the healthy control (8%). Conclusions: This research highlights the potential of XR-methylSeq as an enriched methylation profiling method useful for liquid biopsy applications. Citation Format: Jingru Yu, Lauren S. Ahmann, Yvette Y. Yao, Angus Toland, Alicia Snowden, Chandler Ho, Benjamin Pinsky, Hannes Vogel, Ruben Y. Luo, Linlin Wang, Brooke Howitt, Brittany Holmes, Alarice C. Lowe, Wei Gu. Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7565.

Abstract 5019: Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers

Abstract Early detection of tumor activity in clinically asymptomatic patients has the potential to improve quality of life and prevent unnecessary exposure to toxic therapies. DNA methylation is one of the most studied, stable, and fundamental epigenetic marks and represents a prominent alteration in most cancers. Analysis of DNA methylation, particularly in liquid biopsies has been introduced in cancer diagnosis and risk stratification. Despite indisputable achievements, translation into clinical practice is lagging behind, primarily due to 1) challenges associated with the analysis of DNA methylation, 2) the fragmented nature of cell-free DNA (cfDNA), aggravated by bisulfite treatment, 3) lack of clinical validation of reported cfDNA methylation marks, and 4) the paucity of knowledge about the functional consequences of cfDNA methylation marks within tumors. Using a sample pooling strategy, complemented with robust whole genome and reduced representation methylation sequencing, we establish a pan-cancer cfDNA methylation resource encompassing 12 cancer entities with diverse clinical phenotypes and treatment modalities. We built this compendium using a robustly performing whole genome and reduced representaion enzymatic methylation sequencing of more than 150 cfDNA and tumor DNA. We complement our data by analyzing DNA methylation data from 15 cancer entities (more than 7,000 patients) within the TCGA. We developed an enzymatic digital PCR (dPCR) approach for pan-cancer but also entity-specific analysis of cfDNA methylation at single base-resolution. Using this dPCR assay, we analyzed cfDNA and tumor DNA samples from more than 400 patients and compared enzymatic digestion with bisulfite treatment. We demonstrate that this resource can allow for the identification of entity-agnostic and specific markers, and that cfDNA methylation patterns are mirrored in tumor samples. The approach can be applied for methylation profiling of yet unanalyzed entities. Finally, we demonstrate unconventional epigenetic regulation of gene expression by methylated DNA-binding transcription factors whose activities are tissue- and context-specific and dosage-dependent. This work, therefore, provides a reference resource to identify minimally invasive, entity-specific and pan-cancer markers applicable for diagnostics, surveillance and patient stratification. It also sets the stage for further investigation of tissue- and context-specific epigenetic regulation of gene expression in health and disease by methylated DNA-binding transcription factors. Citation Format: Smiths Sengkwawoh Lueong, Martin Metzenmacher, Gregor Zaun, Gina Lauren Mayer, Erik Jan Hemmer, Katharina Lückerath, Kelsey Pomykala, Balazs Hegedüs, Peter A. Horn, Marija Trajkovic-Arsic, Tibor Szarvas, Renáta Váraljai, Corinna Keup, Ingeborg Tinhofer, Stephen George, Sabine Kasimir-Bauer, Samuel Peña-Llopis, Alexander Roesch, Cornelius Kürten, Lukas Boosfeld, Kirsten Bruderek, Christopher Darr, Thomas Hilser, Viktor Grünwald, Sven Brandau, Boris Hadaschik, Hans Neubauer, Irene Irene Esposito Irene Esposito, Tanja Fehm, Csilla Oláh, Anita Csizmarik, Fabinshy Thangarajah, Laura Reetz, Ghanam Jamal, Basant Kumar Thakur, Halime Kalkavan, Alexander Schramm, Martin Schuler, Jens T. Siveke. Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5019.