Methylation Patterns Research Articles

Genome organization and stability in mammalian pre-implantation development

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.

Impact of cannabinoids on synapse markers in an SH-SY5Y cell culture model

Patients suffering from schizophrenic psychosis show reduced synaptic connectivity compared to healthy individuals, and often, the use of cannabis precedes the onset of schizophrenic psychosis. Therefore, we investigated if different types of cannabinoids impact methylation patterns and expression of schizophrenia candidate genes concerned with the development and preservation of synapses and synaptic function in a SH-SY5Y cell culture model. For this purpose, SH-SY5Y cells were differentiated into a neuron-like cell type as previously described. Effects of the cannabinoids delta-9-THC, HU-210, and Anandamide were investigated by analysis of cell morphology and measurement of neurite/dendrite lengths as well as determination of methylation pattern, expression (real time-qPCR, western blot) and localization (immunocytochemistry) of different target molecules concerned with the formation of synapses. Regarding the global impression of morphology, cells, and neurites appeared to be a bit more blunted/roundish and to have more structures that could be described a bit boldly as resembling transport vesicles under the application of the three cannabinoids in comparison to a sole application of retinoic acid (RA). However, there were no obvious differences between the three cannabinoids. Concerning dendrites or branch lengths, there was a significant difference with longer dendrites and branches in RA-treated cells than in undifferentiated control cells (as shown previously), but there were no differences between cannabinoid treatment and exclusive RA application. Methylation rates in the promoter regions of synapse candidate genes in cannabinoid-treated cells were in between those of differentiated cells and untreated controls, even though findings were significant only in some of the investigated genes. In other targets, the methylation rates of cannabinoid-treated cells did not only approach those of undifferentiated cells but were also valued even beyond. mRNA levels also showed the same tendency of values approaching those of undifferentiated controls under the application of the three cannabinoids for most investigated targets except for the structural molecules (NEFH, MAPT). Likewise, the quantification of expression via western blot analysis revealed a higher expression of targets in RA-treated cells compared to undifferentiated controls and, again, lower expression under the additional application of THC in trend. In line with our earlier findings, the application of RA led to higher fluorescence intensity and/or a differential signal distribution in the cell in most of the investigated targets in ICC. Under treatment with THC, fluorescence intensity decreased, or the signal distribution became similar to the dispersion in the undifferentiated control condition. Our findings point to a decline of neuronal differentiation markers in our in vitro cell-culture system under the application of cannabinoids.

The epigenetic impact of daily diet food choices on human health and chronic diseases

Nutrition, certain lifestyle behaviors (smoking, drug, alcohol addictions, etc.) and environment all contribute to cancer and other lines development. Similarly, epigenetic pathways are known to occur at the intersection between the generally reversible effects of lifestyle or ecological factors and the irreversible alterations that explain numerous diseases. Chemoprevention is the process of intervening in the epigenome to mitigate the detrimental effects of environmental factors or certain lifestyles before they lead to significant consequences. DNA is permanently exposed to various substances modifying both its genetic and epigenetic configuration. It’s controlled by various agents (methyl donors, sophisticated enzymes, etc.). Pesticides, artificial food additives, drugs and environmental toxins penetrating the placenta, can result in developmental program alterations and epigenetic changes in a fetus. In nutrition context, the research revealed that several foods can alter epigenetic markers. Some vitamins may alter DNA methylation patterns and histone alterations, possibly influencing gene expression and disease risk. Thus, dietary treatments may provide an epigenetic regulation, emphasizing nutrition role in preserving health and preventing illness via epigenetics Keywords: human epigenetics, epigenetic regulation of diseases, daily food ration, healthy food

Unraveling the Complexity of Chikungunya Virus Infection Immunological and Genetic Insights in Acute and Chronic Patients.

Background: The chikungunya virus (CHIKV), transmitted by infected Aedes mosquitoes, has caused a significant number of infections worldwide. In Brazil, the emergence of the CHIKV-ECSA genotype in 2014 posed a major public health challenge due to its association with more severe symptoms. Objectives/Methods: This study aimed to shed new light on the host immune response by examining the whole-blood transcriptomic profile of both CHIKV-acute and chronically infected individuals from Feira de Santana, Bahia, Brazil, a region heavily affected by CHIKV, Dengue, and Zika virus epidemics. Results: Our data reveal complex symptomatology characterized by arthralgia and post-chikungunya neuropathy in individuals with chronic sequelae, particularly affecting women living in socially vulnerable situations. Analysis of gene modules suggests heightened metabolic processes, represented by an increase in NADH, COX5A, COA3, CYC1, and cap methylation in patients with acute disease. In contrast, individuals with chronic manifestations exhibit a distinct pattern of histone methylation, probably mediated by NCOA3 in the coactivation of different nuclear receptors, KMT2 genes, KDM3B and TET2, and with alterations in the immunological response, majorly led by IL-17RA, IL-6R, and STAT3 Th17 genes. Conclusion: Our results emphasize the complexity of CHIKV disease progression, demonstrating the heterogeneous gene expression and symptomatologic scenario across both acute and chronic phases. Moreover, the identification of specific gene modules associated with viral pathogenesis provides critical insights into the molecular mechanisms underlying these distinct clinical manifestations.

A DNA Polymerase Variant Senses the Epigenetic Marker 5-Methylcytosine by Increased Misincorporation.

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

An integrative analysis to identify pancancer epigenetic biomarkers

Integrating and analyzing the pancancer data collected from different experiments is crucial for gaining insights into the common mechanisms in the molecular level underlying the development and progression of cancers. Epigenetic study of the pancancer data can provide promising results in biomarker discovery. The genes that are epigenetically dysregulated in different cancers are powerful biomarkers for drug-related studies. This paper identifies the genes having altered expression due to aberrant methylation patterns using differential analysis of TCGA pancancer data of 12 different cancers. We identified a comprehensive set of 115 epigenetic biomarker genes out of which 106 genes having pancancer properties. The correlation analysis, gene set enrichment, protein–protein interaction analysis, pancancer characteristics analysis, and diagnostic modeling were performed on these biomarkers to illustrate the power of this signature and found to be important in different molecular operations related to cancer. An accuracy of 97.56% was obtained on TCGA pancancer gene expression dataset for predicting the binary class tumor or normal. The source code and dataset of this work are available at https://github.com/panchamisuneeth/EpiPanCan.git.

Evaluation of a multimodal ctDNA-based assay for detection of aggressive cancers lacking standard screening tests.

Aim: Cancers lacking standard screening (LSS) options account for approximately 70% of cancer-related deaths due to late-stage diagnosis. Circulating tumor DNA (ctDNA) is a promising biomarker for multi-cancer early detection. We previously developed SPOT-MAS, a multimodal ctDNA-based assay analyzing methylation and fragmentomic profiles, effective in detecting common cancers (breast, colorectal, liver, lung and gastric). This study extends the analysis to five LSS cancers: endometrial, esophageal, head and neck, ovarian and pancreatic.Methods: SPOT-MAS was applied to profile cfDNA methylation and fragmentomic patterns in 739 healthy individuals and 135 LSS cancer patients.Results: We identified 347 differentially methylated regions and observed genome-wide hypomethylation across all five LSS cancers. Esophageal and head and neck cancers showed an enrichment of short cfDNA fragments (<150bp). Eleven 4-mer end motifs were consistently altered in cfDNA fragments across all LSS cancers. Many significant signatures were consistent with previous observations in common cancers. Notably, SPOT-MAS achieved 96.2% specificity and 74.8% overall sensitivity, with a lower sensitivity of 60.7% in early-stage cancers.Conclusion: This proof-of-concept study demonstrates that SPOT-MAS a non-invasive test trained on five common cancer types, could detect a number of LSS cancer cases, potentially complementing existing screening programs.

N6-Methyladenosine RNA Modification Regulates the Differential Muscle Development in Large White and Ningxiang Pigs.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Growing research indicates that m6A methylation is crucial for a multitude of biological processes. However, research on the m6A modifications in the regulation of porcine muscle growth is lacking. In this study, we identified differentially expressed genes in the neonatal period of muscle development between Large White (LW) and NingXiang (NX) pigs and further reported m6A methylation patterns via MeRIP-seq. We found that m6A modification regulates muscle cell development, myofibrils, cell cycle, and phosphatase regulator activity during the neonatal phase of muscle development. Interestingly, differentially expressed genes in LW and NX pigs were mainly enriched in pathways involved in protein synthesis. Furthermore, we performed a conjoint analysis of MeRIP-seq and RNA-seq data and identified 27 differentially expressed and m6A-modified genes. Notably, a typical muscle-specific envelope transmembrane protein, WFS1, was differentially regulated by m6A modifications in LW and NX pigs. We further revealed that the m6A modification accelerated the degradation of WFS1 in a YTHDF2-dependent manner. Noteworthy, we identified a single nucleotide polymorphism (C21551T) within the last exon of WFS1 that resulted in variable m6A methylation, contributing to the differing WFS1 expression levels observed in LW and NX pigs. Our study conducted a comprehensive analysis of the m6A modification on NX and LW pigs during the neonatal period of muscle development, and elucidated the mechanism by which m6A regulates the differential expression of WFS1 in the two breeds.

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines

BackgroundUveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines.MethodsFour primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared.ResultsIn all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA.ConclusionsThis data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Potential role of heteroplasmic mitochondrial DNA mutations in modulating the subtype-specific adaptation of oral squamous cell carcinoma to cisplatin therapy

Cancer cells are constantly evolving to adapt to environmental changes, particularly during exposure to drug treatment. In this work, we aimed to characterize genetic and epigenetic changes in mitochondrial DNA (mtDNA) that may increase the resistance of oral squamous cell carcinoma (OSCC) to cisplatin. We first derived drug-resistant cells from two human OSCC cell lines, namely SAS and H103, by continual cisplatin treatments for about 4 months. To determine mtDNA changes induced by cisplatin, we performed nanopore sequencing and quantitative polymerase chain reaction analysis of mtDNA extracted from the cells pre- and post-treatment. We also assessed the mitochondrial functions of the cells and their capacity to generate intracellular reactive oxygen species (ROS). We found that in the cisplatin-resistant cells derived from SAS, there was a reduction in mtDNA content and significant enrichment of a m.3910G > C mutation in the MT-ND1 gene. However, such changes were not detected in cisplatin-resistant H103 cells. The cisplatin treatment also altered methylation patterns in both SAS and H103 cells and decreased their sensitivity to ROS-induced cytotoxicity. We suggest that the sequence alterations and epigenetic changes in mtDNA and the reduction in mtDNA content could be key drivers of cisplatin resistance in OSCC. These mtDNA alterations may participate in cellular adaptation that serves as a response to adverse changes in the environment, particularly exposure to cytotoxic agents. Importantly, the observed mtDNA changes may be influenced by the distinct genetic landscapes of various cancer subtypes. Overall, this study reveals significant insights into cisplatin resistance driven by complex mtDNA dynamics, particularly in OSCC. This underscores the need for targeted therapies tailored to the genetic profiles of individual OSCC patients to improve disease prognosis.

Decoding the Methylation Patterns of ABC Transporters in Colorectal Cancer

Colorectal cancer (CRC) is a significant global health concern, posing a major threat to morbidity and mortality rates. The molecular mechanisms that drive CRC, particularly the DNA methylation patterns in ATP-binding cassette (ABC) genes, have attracted considerable attention because of their potential roles in CRC drug resistance, initiation, and progression. This study aimed to investigate the methylation patterns of ABC genes in CRC using a high-throughput microarray technology. Microarray methylation data from CRC and adjacent normal tissues were subjected to preprocessing, differential methylation analysis, and correlation analysis using the MethSurv tool for a detailed study of methylation patterns. The study revealed global hypomethylation of 43 ABC transporters and two pseudogenes in CRC compared to normal tissues. Receiver operating characteristic curve analysis identified 369 CpG sites as potential biomarkers for CRC diagnosis, with area under the curve values ranging from acceptable to outstanding. Survival analysis demonstrated the correlation between DNA methylation of individual CpG sites and patient survival probability in colon and rectal adenocarcinoma datasets from The Cancer Genome Atlas. The study provides insights into the epigenetic landscape of ABC genes in CRC, highlighting their potential roles in drug resistance, disease initiation, and progression. The findings offer opportunities for developing innovative therapeutic approaches and improving patient outcomes in the fight against CRC. Further research is needed to validate these results and investigate the functional implications of ABC gene methylation in CRC pathogenesis and treatment response.

The Diversity of Methylation Patterns in Serous Borderline Ovarian Tumors and Serous Ovarian Carcinomas.

Background: Changes in DNA methylation patterns are a pivotal mechanism of carcinogenesis. In some tumors, aberrant methylation precedes genetic changes, while gene expression may be more frequently modified due to methylation alterations than by mutations. Methods: Herein, 128 serous ovarian tumors were analyzed, including borderline ovarian tumors (BOTS) with (BOT.V600E) and without (BOT) the BRAF V600E mutation, low-grade (lg), and high-grade (hg) ovarian cancers (OvCa). The methylome of the samples was profiled with Infinium MethylationEPIC microarrays. Results: The biggest number of differentially methylated (DM) CpGs and regions (DMRs) was found between lgOvCa and hgOvCa. By contrast, the BOT.V600E tumors had the lowest number of DM CpGs and DMRs compared to all other groups and, in relation to BOT, their genome was strongly downmethylated. Remarkably, the ten most significant DMRs, discriminating BOT from lgOvCa, encompassed the MHC region on chromosome 6. We also identified hundreds of DMRs, being of potential use as predictive biomarkers in BOTS and hgOvCa. DMRs with the best discriminative capabilities overlapped the following genes: BAIAP3, IL34, WNT10A, NEU1, SLC44A4, and HMOX1, TCN2, PES1, RP1-56J10.8, ABR, NCAM1, RP11-629G13.1, AC006372.4, NPTXR in BOTS and hgOvCa, respectively. Conclusions: The global genome-wide hypomethylation positively correlates with the increasing aggressiveness of ovarian tumors. We also assume that the immune system may play a pivotal role in the transition from BOTS to lgOvCa. Given that the BOT.V600E tumors had the lowest number of DM CpGs and DMRs compared to all other groups, when methylome is considered, such tumors might be placed in-between BOT and OvCa.

NRN1 genetic variability and methylation changes as biomarkers for cognitive remediation therapy response in schizophrenia

Cognitive remediation therapy (CRT) demonstrates potential in enhancing cognitive function in schizophrenia (SZ), though the identification of molecular biomarkers remains challenging. The Neuritin-1 gene (NRN1) emerges as a promising candidate gene due to its association with SZ, cognitive performance and response to neurotherapeutic treatments. We aimed to investigate whether NRN1 genetic variability and methylation changes following CRT are related to cognitive improvements.Twenty-five SZ patients were randomly assigned to CRT or treatment-as-usual (TAU) groups, with cognitive function and NRN1 methylation assessed pre- and post-intervention using the MATRICS Consensus Cognitive Battery and EpiTYPER. Besides, eleven NRN1 polymorphisms were genotyped. Methylation changes (Δm = post - pre) were analyzed via sparse Partial Least Square Discriminant Analysis (sPLS-DA) to identify latent components (LCs) distinguishing CRT from TAU. To further explore methylation patterns of these LCs, CpG units were grouped into two subsets, yielding Δm means for those with increased and decreased methylation. Cognitive changes (Δcog = post - pre) were used to identify CRT improvers (CRT-I, Δcog ≥ 1), and the association between methylation changes and cognitive improvements post-therapy was also tested.We identified two LCs that differentiated CRT from TAU with a classification error rate of 0.28. The main component, LC1, included 25 CpG units. The subsets of CpG units with increased and decreased post-therapy methylation differed significantly between the two treatment arms, suggesting that differences were not merely data-driven but reflected meaningful biological variation. Additionally, CpG units linked to therapy were also associated with cognitive improvement, with LC1 and the subset of CpG units showing increased methylation post-therapy distinguishing CRT-I from the rest of the patients across multiple cognitive domains. Furthermore, the effect of LC1 on speed processing improvement after CRT was enhanced by considering the NRN1-rs9405890 polymorphism. Notably, these CpG units, particularly those with increased methylation after CRT, overlapped with key gene regulatory elements.Our model, integrating genetics and epigenetics, boosts the understanding of CRT response variability and highlights this multi-level approach as a promising strategy for identifying potential NRN1-related biomarkers of CRT effects, though further studies with larger samples are needed.

MethyLasso: a segmentation approach to analyze DNA methylation patterns and identify differentially methylated regions from whole-genome datasets.

DNA methylation is an epigenetic mark involved in the regulation of gene expression, and patterns of DNA methylation anticorrelate with chromatin accessibility and transcription factor binding. DNA methylation can be profiled at the single cytosine resolution in the whole genome and has been performed in many cell types and conditions. Computational approaches are then essential to study DNA methylation patterns in a single condition or capture dynamic changes of DNA methylation levels across conditions. Toward this goal, we developed MethyLasso, a new approach to segment DNA methylation data. We use it as an all-in-one tool to perform the identification of low-methylated regions, unmethylated regions, DNA methylation valleys and partially methylated domains in a single condition as well as differentially methylated regions between two conditions. We performed a rigorous benchmarking comparing existing approaches by evaluating the agreement of the regions across tools, their number, size, level of DNA methylation, boundaries, cytosine-guanine content and coverage using several real datasets as well as the sensitivity and precision of the approaches using simulated data and show that MethyLasso performs best overall. MethyLasso is freely available at https://github.com/bardetlab/methylasso.

Methylation Clocks Do Not Predict Age or Alzheimer's Disease Risk Across Genetically Admixed Individuals.

Epigenetic clocks that quantify rates of aging from DNA methylation patterns across the genome have emerged as a potential biomarker for risk of age-related diseases, like Alzheimer's disease (AD), and environmental and social stressors. However, methylation clocks have not been validated in genetically diverse cohorts. Here we evaluate a set of methylation clocks in 621 AD patients and matched controls from African American, Hispanic, and white cohorts. The clocks are less accurate at predicting age in genetically admixed individuals, especially those with substantial African ancestry, than in the white cohort. The clocks also do not consistently identify age acceleration in admixed AD cases compared to controls. Methylation QTL (meQTL) commonly influence CpGs in clocks, and these meQTL have significantly higher frequencies in African genetic ancestries. Our results demonstrate that methylation clocks often fail to predict age and AD risk beyond their training populations and suggest avenues for improving their portability.

Distinct methylome profile of cfDNA in AMI patients reveals significant alteration in cAMP signaling pathway genes regulating cardiac muscle contraction

BackgroundThe role of epigenetics in cardiovascular diseases has paved the way for innovative therapeutic approaches. Investigating epigenetic changes using cell-free DNA (cfDNA) holds substantial promise beyond mere diagnostics, especially for heart-related conditions like acute myocardial infarction (AMI), where obtaining tissue samples is a challenge. This study explores the methylation patterns of cfDNA in AMI patients and compares them with genomic DNA (gDNA) from the same individuals, aiming to evaluate the effectiveness of cfDNA as a valuable resource for studying heart-related diseases.MethodologyWe generated global methylome profiles of cfDNA and gDNA from 25 AMI patients using EM-Seq. Tissue deconvolution analysis was performed to estimate tissue specificity based on the methylation patterns. Differentially methylated loci were identified and explored to understand AMI pathophysiology.ResultsComparative analysis of cfDNA and gDNA methylation patterns in AMI patients reveals cfDNA holds more significance than gDNA. Principal component analysis revealed distinct clusters for cfDNA and gDNA, indicating distinct methylome profiles. cfDNA originated from multiple sources, predominantly from neutrophils (~ 75%) and about 10% from the left atrium, highlighting cardiac-specific changes. In contrast, immune cells are the major source of gDNA, indicative of inflammatory responses. Gene set enrichment analysis (GSEA) associates cfDNA methylation patterns with pathways related to cardiac muscle contraction, inflammation, hypoxia, and lipid metabolism. The affected genes include G protein-coupled receptors (GHSR, FFAR2, HTR1A, and VIPR2) that are part of the cAMP signaling pathway.ConclusionEpigenetic changes in cfDNA are more specific to cardiac tissue compared to those in gDNA, providing better insights into the molecular mechanisms involved in AMI. Genes that are differentially methylated in cfDNA and regulate core pathways, such as cAMP signaling, could be targeted for clinical applications, including the development of effective biomarkers and therapeutic targets.

Maternal e-cigarette exposure alters DNA methylome, site-specific CpG and CH methylation, and transcriptomic signatures in the neonatal brain

Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development, potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis. Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure, underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life.

Genome-wide methylation profiling reveals extracellular vesicle DNA as an ex vivo surrogate of cancer cell-derived DNA

Extracellular vesicle-derived DNA (evDNA) encapsulates the complete genome and mutational status of cells; however, whether cancer cell-derived evDNA mirrors the epigenetic features of parental genomic DNA remains uncertain. This study aimed to assess and compare the DNA methylation patterns of evDNA from cancer cell lines and primary cancer tissues with those of the nuclear genomic DNA. We isolated evDNA secreted by two cancer cell lines (HCT116 and MDA-MB-231) from various subcellular compartments, including the nucleus and cytoplasm. Additionally, we obtained evDNA and nuclear DNA (nDNA) from the primary cancer tissues of colon cancer patients. We conducted a comprehensive genome-wide DNA methylation analysis using the Infinium Methylation EPIC BeadChip, examining > 850,000 CpG sites. Remarkable similarities were observed between evDNA and nDNA methylation patterns in cancer cell lines and patients. This concordance extended to clinical cancer tissue samples, showcasing the potential utility of evDNA methylation patterns in deducing cellular origin within heterogeneous populations through methylation-based deconvolution. The observed concordance underscores the potential of evDNA as a noninvasive surrogate marker for discerning tissue origin, particularly in cancer tissues, offering a promising future for cancer diagnostics. This finding enhances our understanding of cellular origins and would help develop innovative diagnostic and therapeutic strategies for cancer.

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

Effects of Differentially Methylated CpG Sites in Enhancer and Promoter Regions on the Chromatin Structures of Target LncRNAs in Breast Cancer.

Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer-promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation's role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis.