Bisulfite Sequencing Research Articles

Reduced expression of FOXE1 in differentiated thyroid cancer, the contribution of CPG methylation, and their clinical relevance

IntroductionForkhead box E1 (FOXE1) is a transcription factor with a crucial role in thyroid morphogenesis and differentiation. Promoter hypermethylation downregulates FOXE1 expression in different tumor types; nevertheless, its expression and relationship with methylation status in differentiated thyroid cancer (DTC) remain unclear.MethodsA total of 33 pairs of matched samples of PTC tumors and non-tumors were included. Tumor cell cultures were treated with either 5-Aza-2′-deoxycytidine demethylating agent or dimethyl sulfoxide (DMSO). A real-time polymerase chain reaction (RT-PCR) and Western blotting were performed to assess FOXE1 expression. The methylation status was quantified using bisulfite sequencing. A luciferase gene assay was used to determine CpG-island functionality. Gene expression and promoter methylation of FOXE1 and FOXE1-regulated genes were also analyzed with data from The Cancer Genome Atlas (TCGA) thyroid samples.ResultsAfter demethylating treatment, increased FOXE1 mRNA was observed concomitantly with reduced promoter methylation of CpGisland2. A negative correlation between mRNA downregulation and an increased methylation level of CpGisland2 was observed in tumors. Diminished protein expression was also detected in some DTC cell lines and in some tumor samples, suggesting the involvement of post-transcriptional regulatory mechanisms. CPGisland2 was proved to be an enhancer. TCGA data analysis showed low FOXE1 mRNA expression in tumors with a negative correlation with methylation status and a positive correlation with the expression of most of its target genes. Reduced FOXE1 expression, accompanied by a high methylation level, was associated with PTC aggressiveness (tall cell variant, advanced extra thyroid extension, T4 American Joint Committee on Cancer (AJCC) classification), age at diagnosis (over 45 years old), and presence of a BRAFV600E mutation.ConclusionFOXE1 mRNA was downregulated in DTC compared with non-tumors, followed by high CpGisland methylation. A coupling of low mRNA expression and high methylation status was related to characteristics of aggressiveness in DTC tumors.

PATH-24. NOVEL MOLECULAR SUBTYPES IDENTIFIED IN INTRACRANIAL GERM CELL TUMORS SHOWED UNIQUE WHOLE-GENOME METHYLATION PROFILE

Abstract BACKGROUND By integrating whole-genome sequencing (WGS) and RNA-seq data, we found that intracranial germ cell tumors (IGCTs) can be divided into three distinct molecular subtypes: immune-hot, MYC/E2F, and SHH, each characterized by a unique profile of genomic abnormalities. To further define these subtypes, we subsequently performed whole-genome bisulfate sequencing (WGBS) on IGCT tumors METHODS Within our prior discovery cohort (n=110), DNA from 84 samples was available for profiling methylation levels using Whole Genome Bisulfite Sequencing (WGBS). Fastq data was aligned to the human reference genome (hg19), with duplicates removed, and DNA methylation was extracted using bismark pipeline (ver0.24.0). After quality control, weight methylation level was calculated for each CpG island as the sum of methylated reads divided by coverage. To identify IGCTs molecular groups, we applied hierarchical consensus k-means clustering to the most variant methylated CpG island (n=1000) across 84 IGCTs tumor tissues. The methylation level of RNA-seq subtypes was visualized to further clarify the molecular subtypes of IGCTs RESULTS Unsupervised clustering revealed that IGCTs can be divided into three groups based on the CpG island methylation levels: hypomethylation group (n=32), moderate methylation group (n=27), and hyper methylation group (n=25). The hypomethylation and moderate methylation group are predominantly observed in germinoma (>90%). In contrast, hypermethylation group comprises the majority of non-germinomatous germ cell tumors. Furthermore, these subgroups derived from methylation analysis are highly consistent with the molecular subtypes previously defined through gene transcription profile analysis. We observed that the majority of MYC/E2F subtype (24/33) belonged to the hypomethylation group, whereas the majority of immune-hot subtype (14/23) fell into the moderate methylation group. The SHH subtype was the most common within the hypermethylation group (18/26) CONCLUSIONS The three molecular IGCT subtypes showed unique methylation profile. These results indicate different mechanisms of tumorigenesis among the subtypes and pave the way for the clinical application of methylation-based subtyping.

Integrative DNA methylome and transcriptome analysis illuminate leaf phenotype alterations in tetraploid citrus

Whole-genome duplication (WGD) in plants triggers profound morphological and physiological changes, with DNA modification being a key epigenetic factor that helps neo-polyploids overcome challenges and gain adaptive advantages. Tetraploids were previously mined from diploid citrus seedlings, showing enhanced environmental adaptability and potential as rootstocks. These tetraploids exhibited increased leaf and cell wall thickness compared to their diploid counterparts. To explore the impact of WGD, transcriptomic and whole-genome bisulfite sequencing (WGBS) were conducted on two pairs of citrus tetraploids and their diploid controls, revealing significant molecular changes. Notably, tetraploid citrus displayed lower CG methylation levels in gene and transposable element (TE) bodies relative to diploids. Differentially methylated genes (DMGs) between tetraploids and diploids were primarily associated with immune stress, organ development, metabolic pathways, and secondary metabolism. In Trifoliate orange (Poncirus trifoliata L. Raf.) and Ziyang Xiangcheng (Citrus junos Sieb. ex Tanaka), only 150 and 58 differentially expressed genes (DEGs) were identified, respectively, with enrichment in critical cellular processes such as cell wall synthesis, plastid development, and pathways modulating chloroplasts and plasma membranes. A total of 70 genes showed both differential methylation and expression, including ACN1, Nac036, and ASMT1, which are involved in stomatal development, leaf morphology, and melatonin synthesis, respectively, offering insight into the regulatory mechanisms of phenotype alterations after polyploidization. These findings reveal the epigenetic modifications in polyploid citrus and highlight the role of polyploidization-induced methylation in driving phenotypic changes.

Methylation Patterns of Diabetes and Obesity Susceptibility Genes in Gestational Diabetes Mellitus: A Cross-Sectional Analysis from Karachi, Pakistan.

Background: Women with gestational diabetes mellitus (GDM) and their offspring have an increased risk of adverse perinatal and long-term health outcomes, which may be attributable to epigenetic modification of diabetes and obesity susceptibility genes. We aimed to investigate the methylation patterns of eight genes in GDM and normoglycemic (NG) mothers, and their respective offspring. Methods: This cross-sectional study, conducted at Aga Khan University from August 2019 to December 2022, recruited pregnant women in the first trimester of gestation from the outpatient obstetrics clinic. Participants were classified as NG or GDM based on the Society of Obstetricians and Gynecologists Pakistan. Venous blood samples were collected from mothers and cord blood from neonates. Peripheral blood mononuclear cells were used for DNA extraction and methylation analysis using methylation-specific PCR. Maternal and neonatal clinical data were recorded. Statistical analysis was performed using R, including binary logistic regression to assess the association between various gene methylation levels and GDM. Results: The study found that GDM mothers had significantly higher fasting blood glucose, 2-hr OGTT, and serum carboxymethyl lysine (CML) levels compared to NG mothers, but no significant differences in neonatal birth weight or serum CML levels. Chemerin methylation was significantly lower in GDM mothers and their babies, while NAMPT, MTNR1B, FNDC5, FAT4, and FTO methylation levels were higher in GDM offspring compared to NG offspring. GDM mothers also had higher methylation levels of brain-derived neurotrophic factor gene (BDNF). Multivariable binary logistic regression identified methylation levels of maternal BDNF and neonatal MTNR1B to be independently associated with GDM. Conclusions: Our study shows a trend of epigenetic modifications in both GDM mothers and their offspring in various genes related to metabolism and inflammation, suggesting an intergenerational transmission of increased risk of developing metabolic disorders. These findings emphasize the need for high throughput studies, early screening, tight glucose control during pregnancy, and postnatal follow-up to mitigate long-term health risks.

Identification of a DNA methylation signature in whole blood of newborn guinea pigs and human neonates following antenatal betamethasone exposure

Antenatal corticosteroids (ACS) are administered where there is risk of preterm birth to promote fetal lung development and improve perinatal survival. However, treatment may be associated with increased risk of developing neurobehavioural disorders. We have recently identified that ACS results in significant changes to DNA methylation patterns in the newborn and juvenile prefrontal cortex (PFC) of exposed guinea pig offspring. Methylation changes at transcription factor binding sites (TFBS) for PLAGL1, TFAP2C, ZNF263, and SP1 were consistently noted at both post-natal stages, suggesting a long-lasting signature of ACS exposure. In this study, we determined if comparable methylation changes are also present in the newborn blood of ACS-exposed guinea pig offspring, as this would determine whether blood methylation patterns may be used as a peripheral biomarker of changes in the brain. Pregnant guinea pigs were treated with saline or betamethasone (1 mg/kg) on gestational days 50/51. gDNA from whole blood of term-born offspring on post-natal day (PND) 1 was used for reduced representation bisulfite sequencing. Overall, 1677 differentially methylated CpG sites (DMCs) were identified in response to ACS. While no specific DMCs identified in the blood overlapped with those previously reported in the PFC of PND1 offspring, significant differential methylation at TFBSs for PLAGL1, TFAP2C, EGR1, ZNF263, and SP1 was persistently observed. Furthermore, re-examination of our previously reported data of DMCs in human neonatal blood following ACS identified the presence of this same TFBS signature in human infants, suggesting the potential for clinical translation of our epigenomic data.

Blood and Bone-Derived DNA Methylation Ages Predict Mortality After Geriatric Hip Fracture: A Pilot Study.

The purpose of this study was to (1) perform the first analysis of bone-derived DNA methylation, (2) compare DNA methylation clocks derived from bone with those derived from whole blood, and (3) establish a relationship between DNA methylation age and 1-year mortality within the geriatric hip fracture population. Patients ≥65 years old who presented to a Level-I trauma center with a hip fracture were prospectively enrolled from 2020 to 2021. Preoperative whole blood and intraoperative bone samples were collected. Following DNA extraction, RRBS (reduced representation bisulfite sequencing) libraries for methylation clock analysis were prepared. Sequencing data were analyzed using computational algorithms previously described by Horvath et al. to build a regression model of methylation (biological) age for each tissue type. Student t tests were used to analyze differences (Δ) in methylation age versus chronological age. Correlation between blood and bone methylation ages was expressed using the Pearson R coefficient. Blood and bone samples were collected from 47 patients. DNA extraction, sequencing, and methylation analysis were performed on 24 specimens from 12 subjects. Mean age at presentation was 85.4 ± 8.65 years. There was no difference in DNA extraction yield between the blood and bone samples (p = 0.935). The mean follow-up duration was 12.4 ± 4.3 months. The mortality cohort (4 patients, 33%) showed a mean ΔAgeBone of 18.33 ± 6.47 years and mean ΔAgeBlood of 16.93 ± 4.02 years. In comparison, the survival cohort showed a significantly lower mean ΔAgeBone and ΔAgeBlood (7.86 ± 6.7 and 7.31 ± 7.71 years; p = 0.026 and 0.039, respectively). Bone-derived methylation age was strongly correlated with blood-derived methylation age (R = 0.81; p = 0.0016). Bone-derived DNA methylation clocks were found to be both feasible and strongly correlated with those derived from whole blood within a geriatric hip fracture population. Mortality was independently associated with the DNA methylation age, and that age was approximately 17 years greater than chronological age in the mortality cohort. The results of the present study suggest that prevention of advanced DNA methylation may play a key role in decreasing mortality following hip fracture. Prognostic Level I. See Instructions for Authors for a complete description of levels of evidence.

TNF-alpha gene promoter's hypomethylation mediates a pro-inflammatory phenotype in peripheral blood monocytes from apical periodontitis individuals.

Epigenetic regulation of the key inflammatory genes plays a crucial role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges. However, it has not been addressed in apical periodontitis (AP). We aimed to explore the methylation pattern of the TNF-α gene promoter and its association with the inflammatory phenotype of peripheral blood monocytes from individuals with AP and controls. A cross-sectional study was conducted, including otherwise healthy individuals with AP (n = 25) and controls (n = 29). Monocytes were isolated from the volunteer's blood samples using a Ficoll gradient followed by negative immunoselection. RNA and DNA were extracted. The DNA methylation profiles of the TNF-α gene promoter region were analyzed using bisulfite sequencing PCR. The mRNA expression levels of DNA methyltransferases 3a (DNMT3a) and Ten Eleven Translocation enzymes 1(TET1) were assessed by qPCR. A fraction of primary monocytes was also cultured for 24 h, and the supernatant was collected to measure cytokine levels through a Luminex assay. Generalized structural equation models (GSEM) evaluated the association between AP, DNA methylation, and TNF-α protein expression controlled for potential covariates. Models included the effect of the methylation of TNF-α gene promoter as a mediator of the association between AP and TNF-α protein expression levels. Monocytes from AP individuals exhibited a heightened secretion of TNF-α and IL-1β and hypomethylation of the TNF gene promoter (p < .05). AP diagnosis was associated with the TNF-α gene promoter´s hypomethylated profile and enhanced pro-inflammatory cytokine levels, while lower methylation of the gene promoter region and -163 CpG single site mediated TNF-α overexpression (p < .05). DNA hypomethylation at the TNF-α gene mediates a proinflammatory phenotype in monocytes from AP patients, supporting a role in the systemic response.

Epigenetic dynamics of partially methylated domains in human placenta and trophoblast stem cells

BackgroundThe placenta is essential for nutrient exchange and hormone production between the mother and the developing fetus and serves as an invaluable model for epigenetic research. Most epigenetic studies of the human placenta have used whole placentas from term pregnancies and have identified the presence of partially methylated domains (PMDs). However, the origin of these domains, which are typically absent in most somatic cells, remains unclear in the placental context.ResultsUsing whole-genome bisulfite sequencing and analysis of histone H3 modifications, we generated epigenetic profiles of human cytotrophoblasts during the first trimester and at term, as well as human trophoblast stem cells. Our study focused specifically on PMDs. We found that genomic regions likely to form PMDs are resistant to global DNA demethylation during trophectoderm reprogramming, and that PMDs arise through a slow methylation process within condensed chromatin near the nuclear lamina. In addition, we found significant differences in histone H3 modifications between PMDs in cytotrophoblasts and trophoblast stem cells.ConclusionsOur findings suggest that spatiotemporal genomic features shape megabase-scale DNA methylation patterns, including PMDs, in the human placenta and highlight distinct differences in PMDs between human cytotrophoblasts and trophoblast stem cells. These findings advance our understanding of placental biology and provide a basis for further research into human development and related diseases.

BS-clock, advancing epigenetic age prediction with high-resolution DNA methylation bisulfite sequencing data.

DNA methylation patterns provide precise and accurate estimates of biological age due to their robustness and predictable changes associated with aging processes. Although several methylation aging clocks have been developed in recent years, they are primarily designed for DNA methylation array data, which has limited CpG coverage and detection sensitivity compared to bisulfite sequencing data. Here, we present BS-clock, a novel DNA methylation clock for human aging based on bisulfite sequencing data. Using BS-seq data from 529 samples retrieved from four tissues, our BS-clock achieves higher correlations with chronological age in multiple tissue types compared to existing array-based clocks. Our study revealed age-dependent aging rates across different age stages and disease conditions, and overall low cross-tissue prediction capability by applying the model trained on one tissue type to others. In summary, BS-clock overcomes limitations of array-based techniques, offering genome-wide CpG site coverage and more robust and accurate aging quantification. This research paves the way for advanced epigenetic studies of aging and holds promise for developing targeted interventions to promote healthy aging. All analysis codes for reproducing the results of the study are publicly available at https://github.com/hucongcong97/BS-clock. Supplementary data are available at Bioinformatics online.

Downregulation of C1R promotes hepatocellular carcinoma development by activating HIF-1α-regulated glycolysis.

C1R has been identified to have a distinct function in cutaneous squamous cell carcinoma that goes beyond its role in the complement system. However, it is currently unknown whether C1R is involved in the progression of hepatocellular carcinoma (HCC). HCC tissues were used to examine C1R expression in relation to clinical and pathological factors. Malignant characteristics of HCC cells were assessed through in vitro and in vivo experiments. The mechanism underlying the role of C1R in HCC was explored through RNA-seq, methylation-specific PCR, immuno-precipitation, and dual-luciferase reporter assays. This study found that the expression of C1R decreased as the malignancy of HCC increased and was associated with poor prognosis. C1R promoter was highly methylated through DNMT1 and DNMT3a, resulting in a decrease in C1R expression. Downregulation of C1R expression resulted in heightened malignant characteristics of HCC cells through the activation of HIF-1α-mediated glycolysis. Additionally, decreased C1R expression was found to promote xenograft tumor formation. We found that C-reactive protein (CRP) binds to C1R, and the free CRP activates the NF-κB signaling pathway, which in turn boosts the expression of HIF-1α. This increase in HIF-1α leads to higher glycolysis levels, ultimately promoting aggressive behavior in HCC. Methylation of the C1R promoter region results in the downregulation of C1R expression in HCC. C1R inhibits aggressive behavior in HCC in vitro and in vivo by inhibiting HIF-1α-regulated glycolysis. These findings indicate that C1R acts as a tumor suppressor gene during HCC progression, opening up new possibilities for innovative therapeutic approaches.

Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells

Butyric acid as a short-chain fatty acid (SCFA) is one of the key microbial metabolites of ruminants. Numerous studies indicate that butyrate is crucial in muscle growth and development, and plays an important molecular regulatory role mainly by inhibiting histone deacetylation. DNA methylation, a major epigenetic modification, is involved in cell differentiation. Butyrate, in addition to its role in acetylation modifications, can alter the DNA methylation status of cells. However, the impact of butyrate on the DNA methylation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. In this study, we developed a differentiation model of SMSCs and employed RNA sequencing (RNA-seq) alongside whole genome bisulfite sequencing (WGBS) to explore the effects of butyrate treatment on DNA methylation status and its relationship with gene expression. Treatment of SMSCs with sodium butyrate (NaB) at 1.0 mM for 2 days significantly inhibited the expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A) at the mRNA and protein levels while promoting the expression of demethylases (TET1, TET2, TET3) at mRNA levels. WGBS identified 4292 differentially methylated regions (DMRs), comprising 2294 hypermethylated and 1998 hypomethylated regions. These DMRs were significantly enriched in the MAPK, cAMP, and Wnt signaling pathways, all of which are implicated in myogenesis and development. Combining RNA-seq and WGBS data revealed a total of 130 overlapping genes, including MDFIC, CREBBP, DMD, LTBP2 and KLF4. These genes are predominantly involved in regulating the FoxO, MAPK, PI3K-Akt, and Wnt signaling pathways. This study provides new insights into the effects of butyrate-mediated DNA methylation on SMSC development and enhances our understanding of butyrate as an epigenetic modifier beyond its role in acetylation.

Induction of FAM46C expression mediated by DNMT3A downregulation is involved in early-onset preeclampsia through gene body methylation

BackgroundAberrant methylation of genomic DNA has been found in preeclamptic placentas, which is characterized by elevated DNA methylation and hypermethylation of gene body regions, but the underlying mechanism is not yet fully understood. MethodsGlobal DNA methylation was assessed through ELISA and HPLC. The methylation sites were detected using the Illumina Human Methylation 450 K Microarray. The methylation level of FAM46C promoter and gene body was detected through the bisulfite sequencing. RNA-seq was utilized to investigate the mechanism by which DNMT3A and FAM46C mediate the migration and invasion of trophoblast cells. ResultsWe discovered that DNMT3A knockdown led to elevated levels of gene body methylation and FAM46C transcription. FAM46C downregulation completely rescued the suppressive effects caused by DNMT3A knockdown on the migration and invasion of trophoblast cells. Mechanistically, DNMT3A reduction led to an increase in the enrichment of DNMT3B and DNMT1 in the gene body region of FAM46C. The results of transcriptome sequencing showed that DNMT3A and FAM46C regulate the adhesion of trophoblast cells. Elevated expression of FAM46C and increased methylation levels within its gene body region were observed in extravillous trophoblast cells of early-onset preeclamptic placentas. ConclusionsDNMT3A-mediated aberrant FAM46C gene body methylation is relevant to the development of early-onset preeclampsia.

Comprehensive evaluation of the impact of whole-genome bisulfite sequencing (WGBS) on the fragmentomic characteristics of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA) is non-randomly fragmented in human body fluids. Analyzing such fragmentation patterns of cfDNA holds great promise for liquid biopsy. Whole-genome bisulfite sequencing (WGBS) is widely used for cfDNA methylation profiling. However, its applicability for studying fragmentomic characteristics remains largely unexplored. MethodsWe performed paired WGBS and whole-genome sequencing (WGS) on 66 peripheral plasma samples from 58 pregnant women. Then, we systematically compared the fragmentation patterns of cell-free nuclear DNA and mitochondrial DNA (mtDNA) sequenced from these two approaches. Additionally, we evaluated the extent of the size shortening in fetal-derived cfDNA and estimated the fetal DNA fraction in maternal plasma using both sequencing methods. ResultsCompared to WGS samples, WGBS samples demonstrated a significantly lower genome coverage and higher GC content in cfDNA. They also showed a significant decrease in the size of cell-free nuclear DNA, along with alterations in the end motif pattern that were specifically associated with CpG and “CC” sites. While there was a slight shift in the inferred nucleosome footprint from cfDNA coverages in WGBS samples, the cfDNA coverage patterns in CTCF and TSS regions remained highly consistent between these two sequencing methods. Both methods accurately reflected gene expression levels through their TSS coverages. Additionally, WGBS samples exhibited an increased abundance and longer length of mtDNA in plasma. Furthermore, we observed the size shortening of fetal cfDNA in plasma consistently, with a highly correlated fetal DNA fraction inferred by cfDNA coverage between WGBS and WGS samples (r = 0.996). However, the estimated fetal cfDNA fraction in WGBS samples was approximately 7 % lower than in WGS samples. ConclusionsWe confirmed that WGBS can introduce artificial breakages to cfDNA, leading to altered fragmentomic patterns in both nuclear and mitochondrial DNA. However, WGBS cfDNA remains suitable for analyzing certain cfDNA fragmentomic characteristics, such as coverage in genome regulation regions and the essential characteristics of fetal DNA in maternal plasma.

Prenatal exposures and cell type proportions are main drivers of FKBP5 DNA methylation in maltreated and non-maltreated children

DNA methylation in peripheral tissues may be a relevant biomarker of risk for developing mental disorders after exposure to early life adversity. Genes involved in HPA axis regulation, such as FKBP5, might play a key role. In this study, we aimed to identify the main drivers of salivary FKBP5 methylation in a cohort of 162 maltreated and non-maltreated children aged 3-5 years at two measurement timepoints. We combined data from a targeted bisulfite sequencing approach for fine-mapping 49 CpGs in regulatory regions of FKBP5 and epigenetic scores for exposure to alcohol, cigarette smoke, and glucocorticoids derived from the EPIC microarray.Most variability of methylation in the FKBP5 locus was explained by estimated cell type proportions as well as epigenetic exposure scores, most prominently the glucocorticoid exposure score. While not surviving correction for multiple testing, we replicate previously reported associations of FKBP5 methylation with CM. We also detected synergistic effects of both rs1360780 genotype and the glucocorticoid exposure score on FKBP5 hypomethylation. These effects were identified in the 3’TAD, a distal regulatory of FKBP5 which is not extensively covered in Illumina arrays, emphasizing the need for fine mapping approaches. Additionally, the epigenetic glucocorticoid exposure score was associated with childhood maltreatment, maternal mental disorders, and pregnancy complications, thereby highlighting the role of glucocorticoid signaling in the epigenetic consequences of early adversity.These results underscore the need to assess cell type heterogeneity in targeted assessments of DNA methylation and show the impact of exposures beyond just childhood maltreatment such as glucocorticoid exposure.

Binding of PtoRAP2.12 to demethylated and accessible chromatin regions in the PtoGntK promoter stimulates growth of poplar.

DNA methylation is an essential epigenetic modification for gene regulation in plant growth and development. However, the precise mechanisms of DNA methylation remain poorly understood, especially in woody plants. We employed whole-genome bisulfite sequencing (WGBS), assays for transposase-accessible chromatin using sequencing (ATAC-seq), and RNA-Seq to investigate epigenetic regulatory relationships in Populus tomentosa treated with DNA methylation inhibitor 5-azacitidine. Expression-quantitative trait methylation analysis (eQTM), epigenome-wide association study (EWAS), and joint linkage-linkage disequilibrium mapping were used to explore the epigenetic regulatory genes, and using CRISPR/Cas9 to identify the role of candidate genes. Plant developmental abnormalities occurred when DNA methylation levels were substantially reduced. DNA methylation regulated 112 expressed genes via chromatin accessibility, of which 61 genes were significantly influenced by DNA methylation variation at the population level. One DNA methylation-regulated gene, PtoGntK, was located in a major quantitative trait locus (QTL) for poplar growth. Overexpression and CRISPR/Cas9 of PtoGntK revealed it affected poplar height and stem diameter. The PtoRAP2.12 was found to bind to the demethylated accessible region in the PtoGntK promoter, thereby promoting growth in poplar. This study identified key genes with epigenetic regulation for plant growth and provides insights into epigenetic regulation mechanisms in woody plants.

Enhancing the differential diagnosis of small pulmonary nodules: a comprehensive model integrating plasma methylation, protein biomarkers, and LDCT imaging features

BackgroundAccurate differentiation between malignant and benign pulmonary nodules, especially those measuring 5–10 mm in diameter, continues to pose a significant diagnostic challenge. This study introduces a novel, precise approach by integrating circulating cell-free DNA (cfDNA) methylation patterns, protein profiling, and computed tomography (CT) imaging features to enhance the classification of pulmonary nodules.MethodsBlood samples were collected from 419 participants diagnosed with pulmonary nodules ranging from 5 to 30 mm in size, before any disease-altering procedures such as treatment or surgical intervention. High-throughput bisulfite sequencing was used to conduct DNA methylation profiling, while protein profiling was performed utilizing the Olink proximity extension assay. The dataset was divided into a training set and an independent test set. The training set included 162 matched cases of benign and malignant nodules, balanced for sex and age. In contrast, the test set consisted of 46 benign and 49 malignant nodules. By effectively integrating both molecular (DNA methylation and protein profiling) and CT imaging parameters, a sophisticated deep learning-based classifier was developed to accurately distinguish between benign and malignant pulmonary nodules.ResultsOur results demonstrate that the integrated model is both accurate and robust in distinguishing between benign and malignant pulmonary nodules. It achieved an AUC score 0.925 (sensitivity = 83.7%, specificity = 82.6%) in classifying test set. The performance of the integrated model was significantly higher than that of individual methylation (AUC = 0.799, P = 0.004), protein (AUC = 0.846, P = 0.009), and imaging models (AUC = 0.866, P = 0.01). Importantly, the integrated model achieved a higher AUC of 0.951 (sensitivity = 83.9%, specificity = 89.7%) in 5–10 mm small nodules. These results collectively confirm the accuracy and robustness of our model in detecting malignant nodules from benign ones.ConclusionsOur study presents a promising noninvasive approach to distinguish the malignancy of pulmonary nodules using multiple molecular and imaging features, which has the potential to assist in clinical decision-making.Trial registration: This study was registered on ClinicalTrials.gov on 01/01/2020 (NCT05432128). https://classic.clinicaltrials.gov/ct2/show/NCT05432128.

Epigenetic insights into creep-feeding: methylation profiling of Longissimus thoracis muscle at weaning in crossbred cattle

IntroductionThis study investigated the impact of creep-feeding supplementation on the genome methylation of the Longissimus thoracis (LT) muscle in crossbred beef cattle (Bos taurus × Bos indicus).MethodsThe experiment involved 48 uncastrated F1 Angus-Nellore males (half-siblings), which were divided into two groups: NCF – no creep-feeding (n = 24) and CF – creep-feeding (n = 24). After weaning at 210 days, all animals were feedlot finished for 180 days under identical conditions. LT muscle biopsies were collected at weaning for genomic DNA methylation analysis by reduced representation bisulfite sequencing (RRBS).Results and discussionThe groups differed significantly (CF &amp;gt; NCF: p &amp;lt; 0.05) to weaning weight (243.57±5.70 vs. 228.92±5.07kg), backfat thickness (12.96±0.86 vs. 10.61±0.42mm), LT muscle marbling score (366.11±12.39 vs. 321.50±13.65), and LT intramuscular fat content (5.80±0.23 vs. 4.95±0.20%). The weights at the beginning of the experiment and at slaughter (390 days) did not differ significantly. Mean methylation levels were higher in CF with 0.18% more CpG, 0.04% CHG, and 0.03% CHH. We identified 974 regions with differential methylation (DMRs: &amp;gt; 25% and q &amp;lt; 0.05), which overlapped with 241 differentially methylated genes (DMGs). Among these genes, 108 were hypermethylated and 133 were hypomethylated in CF group. Notably, 39 of these DMGs were previously identified as differentially expressed genes (DEGs: log2 fold change [0.5]) in the same animal groups. Over-representation analysis highlighted epigenetic regulations related to muscle growth, PPAR signaling, adipogenesis, insulin response, and lipid metabolism. Key DMGs/DEGs included: ACAA1, SORBS1, SMAD3, TRIM63, PRKCA, DNMT3A, RUNX1, NRG3, and SLC2A8. These epigenetic changes improved the performance of supplemented animals up to weaning and enhanced meat quality traits, particularly higher intramuscular fat. The results provided insights into the intricate interplay between nutrition, epigenetics, gene expression and phenotypes in beef cattle production.

Analysis of population heterogeneity in CHO cells by genome-wide DNA methylation analysis and by multi-modal single-cell sequencing

CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.

Investigating the prognostic utility of GSTP1 promoter methylation in prostate cancer

AbstractObjectivesWe aim to determine the prognostic significance of glutathione S‐transferase Pi 1 DNA methylation (mGSTP1) in two independent prostate cancer cohorts with long‐term clinical follow‐up data.Subjects/Patients and MethodsWe first re‐examined a published, in‐house whole genome bisulphite sequencing (WGBS) prostate cancer dataset, derived from radical prostatectomy (RP) tissue (n = 15) with median follow‐up 19.5 years, to confirm and visualise the association between mGSTP1 and patient mortality. To validate prognostic significance, we used a quantitative methylation‐specific head‐loop (MS‐HL) assay to measure mGSTP1 levels in a larger, independent cohort (n = 186), and performed univariable and multivariable Cox survival analysis.ResultsRe‐analysis of WGBS data showed a significant increase in mGSTP1 in RP samples from patients with lethal versus non‐lethal disease. Subsequent analysis in the larger cohort using the MS‐HL assay confirmed that mGSTP1 was detectable in 97% of RP samples, validating the diagnostic potential of mGSTP1. Univariable Cox survival analysis revealed a significant association between mGSTP1 levels and biochemical recurrence and metastatic relapse free survival, with a near‐significant association with prostate cancer specific mortality. Notably, multivariable Cox models demonstrated that mGSTP1 did not add independent prognostic value beyond standard clinicopathological features.ConclusionOur study supports the importance of DNA methylation as a tissue‐based prostate tumour biomarker. GSTP1 methylation is well established as a diagnostic marker, and in this study, we find that GSTP1 methylation levels are also associated with disease prognosis. Further research is required into the clinical utility of prognostic methylation markers and their functional role in disease progression.

A novel N4,N4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily

Ribosome structure and activity are challenged at high temperatures, often demanding modifications to ribosomal RNAs (rRNAs) to retain translation fidelity. LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome identified an RNA modification, N4,N4-dimethylcytidine (m42C), at the universally conserved C918 in the 16S rRNA helix 31 loop. Here, we characterize and structurally resolve a class of RNA methyltransferase that generates m42C whose function is critical for hyperthermophilic growth. m42C is synthesized by the activity of a unique family of RNA methyltransferase containing a Rossman-fold that targets only intact ribosomes. The phylogenetic distribution of the newly identified m42C synthase family implies that m42C is biologically relevant in each domain. Resistance of m42C to bisulfite-driven deamination suggests that efforts to capture m5C profiles via bisulfite sequencing are also capturing m42C.