Global DNA Methylation Analysis Research Articles

Reprogramming of DNA methylation and changes of gene expression in grafted Hevea brasiliensis.

Rubber tree (Hevea brasiliensis) is reproduced by bud grafting for commercial planting, but significant intraclonal variations exist in bud-grafted clones. DNA methylation changes related to grafting may be partly responsible for intraclonal variations. In the current study, whole-genome DNA methylation profiles of grafted rubber tree plants (GPs) and their donor plants (DPs) were evaluated by whole-genome bisulfite sequencing. Data showed that DNA methylation was downregulated and DNA methylations in CG, CHG, and CHH sequences were reprogrammed in GPs, suggesting that grafting induced the reprogramming of DNA methylation. A total of 5,939 differentially methylated genes (DMGs) were identified by comparing fractional methylation levels between GPs and DPs. Transcriptional analysis revealed that there were 9,798 differentially expressed genes (DEGs) in the DP and GP comparison. A total of 1,698 overlapping genes between DEGs and DMGs were identified. These overlapping genes were markedly enriched in the metabolic pathway and biosynthesis of secondary metabolites by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Global DNA methylation and transcriptional analyses revealed that reprogramming of DNA methylation is correlated with gene expression in grafted rubber trees. The study provides a whole-genome methylome of rubber trees and an insight into the molecular mechanisms underlying the intraclonal variations existing in the commercial planting of grafted rubber trees.

Global DNA Methylation analysis of imatinib resistant and sensitive K562 cells

OBJECTIVE: Chronic myeloid leukemia (CML) is a hematological disease which is known by the presence of Philadelphia chromosome (Ph+). BCR-ABL protein is expressed by Ph+ chromosome, represents constant increased tyrosine kinase activity. Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) which is utilized as a first line treatment in CML. Emergence of IMA resistance at some point of therapy leads to treatment failure. DNA methylation is considered to be the most stable epigenetic change and several studies have shown that epigenetic changes may play a role in drug resistance. We investigated the global methylation profile of IMA-sensitive K562S, IMA-resistant K562R and IMA-resistant and adherent K562R (K562R-adh) cells to determine whether epigenetic reprogramming is involved in the resistance to IMA and the change in phenotype due to this resistance. MATERIAL AND METHODS: In this study, morphologically distinct, IMA-sensitive K562S and 5µM IMA-resistant K562R and K562R-adh in-vitro CML cell models were used to analyze the global DNA methylation profile. After DNA was isolated from the cells, global 5mC DNA methylation profiles were investigated by ELISA using equal amounts of DNA. RESULTS: Compared to K562S, the global methylation of K562R showed an increase in DNA methylation profile, but this increase in methylation was not statistically significant. Whereas, a slight hypermethylation was observed in the DNA of the K562R-adh vs K562S and K562R-adh vs K562R which is statistically significant. We observed slight hypermethylation in IMA-resistant cells lines versus to the IMA-sensitive cell line. CONCLUSION: Our observed differences in 5methyl-Cytosine on CpG islands (5mC) in K562S versus K562R and K562R-adh cell lines suggest that the DNA methylation alteration in resistant cells may partly contributed in phenotype switching.

Reinforcement of repressive marks in the chicken primordial germ cell epigenetic signature: divergence from basal state resetting in mammals

BackgroundIn mammals, primordial germ cells (PGCs), the embryonic precursors of the germline, arise from embryonic or extra-embryonic cells upon induction by the surrounding tissues during gastrulation, according to mechanisms which are elucidated in mice but remain controversial in primates. They undergo genome-wide epigenetic reprogramming, consisting of extensive DNA demethylation and histone post-translational modification (PTM) changes, toward a basal, euchromatinized state. In contrast, chicken PGCs are specified by preformation before gastrulation based on maternally-inherited factors. They can be isolated from the bloodstream during their migration to the genital ridges. Our prior research highlighted differences in the global epigenetic profile of cultured chicken PGCs compared with chicken somatic cells and mammalian PGCs. This study investigates the acquisition and evolution of this profile during development.ResultsQuantitative analysis of global DNA methylation and histone PTMs, including their distribution, during key stages of chicken early development revealed divergent PGC epigenetic changes compared with mammals. Unlike mammalian PGCs, chicken PGCs do not undergo genome-wide DNA demethylation or exhibit a decrease in histone H3 lysine 9 dimethylation. However, chicken PGCs show 5‑hydroxymethylcytosine loss, macroH2A redistribution, and chromatin decompaction, mirroring mammalian processes. Chicken PGCs initiate their epigenetic signature during migration, progressively accumulating high global levels of H3K9me3, with preferential enrichment in inactive genome regions. Despite apparent global chromatin decompaction, abundant heterochromatin marks, including repressive histone PTMs, HP1 variants, and DNA methylation, persists in chicken PGCs, contrasting with mammalian PGCs.ConclusionsChicken PGCs’ epigenetic signature does not align with the basal chromatin state observed in mammals, suggesting a departure from extensive epigenetic reprogramming. Despite disparities in early PGC development, the persistence of several epigenetic features shared with mammals implies their involvement in chromatin-regulated germ cell properties, with the distinctive elevation of chicken-specific H3K9me3 potentially participating in these processes.

Poor control of oxidative stress index is associated with increased DNA methylation in retinopathy patients of a sample from babylonian population

Abstract Background: Retinopathy is one of almost insidious advanced neuron-vascular complications of diabetes, oneof the most common complications that lead to permanent visual impairment in the middle-aged population. Objectives: The study investigated the association between poor oxidative stress index and DNA methylation. Materials and Methods: The study included 120 participants, of whom 40 were healthy, 40 patients with retinopathy, and 40 non-retinopathy diabetes patients (type 2 diabetes mellitus (T2DM)). The total antioxidant, capacity (TAC) evaluated using Cupric ion reducing antioxidant capacity method. Reactive oxygen species (ROS) was measured using Erel method. Global DNA methylation has measured employing 5 mC% estimating using MethylFlash™ Methylated DNA Quantification Kit. Results: Current investigation indicated significant (P ≤ 0.05) accrue in TAC, ROSs, and oxidative stress index (OSI) in diabetic compared with controls. In addition, a significant (P ≤ 0.05) increase of ROSs and OSI were recorded in retinopathy compared with T2DM patients was reported. The analysis of global DNA methylation revealed that diabetic patients have a significantly (P ≤ 0.05) more 5 mC% as compared with healthy subjects. Also, the present study showed a significant rise in methylation in retinopathy compared as with T2DM patients. Regarding the correlation analysis, the current study conducted significantly (P < 0.05) positive association between DNA methylation and each of the ROSs and OSI. Also, a significant positive correlation was reported between DNA methylation and ROSs and OSI in retinopathy and T2DM. Conclusion: Global DNA methylation has an increased level in patients and could be engaged with emergence and progress of retinopathy in diabetic with poor control of oxidative stress and increased ROSs.

Comparative computational analysis to distinguish mesenchymal stem cells from fibroblasts.

Mesenchymal stem cells (MSCs) are considered to be the most promising stem cell type for cell-based therapies in regenerative medicine. Based on their potential to home to diseased body sites following a therapeutically application, these cells could (i) differentiate then into organ-specific cell types to locally restore injured cells or, most prominently, (ii) foster tissue regeneration including immune modulations more indirectly by secretion of protective growth factors and cytokines. As tissue-resident stem cells of mesenchymal origin, these cells are morphologically and even molecularly- at least concerning the classical marker genes- indistinguishable from similar lineage cells, particularly fibroblasts. Here we used microarray-based gene expression and global DNA methylation analyses as well as accompanying computational tools in order to specify differences between MSCs and fibroblasts, to further unravel potential identity genes and to highlight MSC signaling pathways with regard to their trophic and immunosuppressive action. We identified 1352 differentially expressed genes, of which in the MSCs there is a strong signature for e.g., KRAS signaling, known to play essential role in stemness maintenance, regulation of coagulation and complement being decisive for resolving inflammatory processes, as well as of wound healing particularly important for their regenerative capacity. Genes upregulated in fibroblasts addressed predominately transcription and biosynthetic processes and mapped morphological features of the tissue. Concerning the cellular identity, we specified the already known HOX code for MSCs, established a potential HOX code for fibroblasts, and linked certain HOX genes to functional cell-type-specific properties. Accompanied methylation profiles revealed numerous regions, especially in HOX genes, being differentially methylated, which might provide additional biomarker potential. Conclusively, transcriptomic together with epigenetic signatures can be successfully be used for the definition (cellular identity) of MSCs versus fibroblasts as well as for the determination of the superior functional properties of MSCs, such as their immunomodulatory potential.

Artificial Intelligence‐Assisted Label‐Free Spectroscopic Quantification of Global DNA Cytosine Methylation in a Miniature Plasmonic Pickering Emulsion

AbstractEpigenetic DNA methylations are early and frequently observed events in a diversity of diseases such as cancer. Despite the considerable clinical values for cancer liquid biopsy, quantitative analysis of DNA methylations remains a major challenge due to the lack of rapid, sensitive detection techniques. Here, an artificial intelligence‐assisted label‐free surface‐enhanced Raman spectroscopy (SERS) (iMeSERS) biosensor is reported for simultaneous quantification of C5‐methylcytosine (5mC) level and methylation ratio in DNA samples. This method utilizes the plasmonic Pickering emulsions as the biosensing platform for label‐free SERS detection, formed upon the addition of a sub‐microliter DNA sample to the hydrophobic Au nanostar‐containing n‐decane. Distinct spectral signatures of monophosphates of canonical deoxyribonucleotides (dNMPs) and the common methylation modification 5‐methyl‐2′‐deoxycytidine (d5mCMP) are identified and distinguished by the iMeSERS biosensor. The deep learning algorithms trained with SERS signatures of dNMPs and d5mCMP are then applied to the quantitative analysis of global DNA methylation. The exceptional capability of the deep learning‐driven approach is demonstrated for simultaneous quantification of the methylation ratio and level using a sub‐microliter volume of DNA samples. This work shows the power of label‐free SERS techniques combined with deep learning algorithms for quantitative analysis of epigenetic DNA modifications with great promises for clinical diagnosis.

Global endometrial DNA methylation analysis reveals insights into mQTL regulation and associated endometriosis disease risk and endometrial function

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Herein, we characterize variation in DNA methylation (DNAm) and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genotype data and methylation in endometrial samples from 984 deeply-phenotyped participants. We estimate that 15.4% of the variation in endometriosis is captured by DNAm and identify significant differences in DNAm profiles associated with stage III/IV endometriosis, endometriosis sub-phenotypes and menstrual cycle phase, including opening of the window for embryo implantation. Menstrual cycle phase was a major source of DNAm variation suggesting cellular and hormonally-driven changes across the cycle can regulate genes and pathways responsible for endometrial physiology and function. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTLs including 51 associated with risk of endometriosis, highlighting candidate genes contributing to disease risk. Our work provides functional evidence for epigenetic targets contributing to endometriosis risk and pathogenesis. Data generated serve as a valuable resource for understanding tissue-specific effects of methylation on endometrial biology in health and disease.

Cancer cells same as zombies reprogram normal cells via the secreted microenvironment.

The cancer microenvironment plays a crucial role in promoting metastasis and malignancy even in normal cells. In the present study, the effect of acidic and conditioned media of cancer cells (MDA-MB-231), separately and in combination, was studied for the first time on the cell death mechanisms and DNA methylation of normal fibroblasts (NIH/3T3). Cell survival of conditioned media was rescued by the addition of acidic media to conditioned media, as shown by the results. Cell metabolic activity is deviated in a direction other than the Krebs cycle by acidic media The mitochondrial metabolic activity of all groups was enhanced over time, except for acidic media. Unlike the highest amount of ROS in conditioned media, its level decreased to the level of acidic media in the combination group. Furthermore, cells were deviated towards autophagy, rather than apoptosis, by the addition of acidic media to the conditioned media, unlike the conditioned media. Global DNA methylation analysis revealed significantly higher DNA hypomethylation in acidic media than in normal and combination media. Not only were cells treated with conditioned media rescued by acidic media, but also DNA hypomethylation and apoptosis in the combination group were decreased through epigenetic modifications. The acidic and conditioned media produced by cancer cells can remotely activate malignant signaling pathways, much like zombies, which can cause metabolic and epigenetic changes in normal cells.

Methylome and transcriptome analysis of flowering branches building of Citrus plants induced by drought stress

Citrus plants exhibit positive floral response under water stress conditions, however, the mechanistic understanding of floral induction remains largely unexplored in water deficit. In this study, DNA methylomic and transcriptomic analyses were integrated to explore the flowering bud formation as well as branches building after light drought stress. While comparing with the conventional watering group (CK), the light drought group treated with five months (LD) showed a significant increase in the flowering branches, whereas an apparent decrease in vegetative branches. Global DNA methylation analysis showed that the LD Group acquired DNA methylation in more than 70,090 genomic regions and lost DNA methylation in about 18,421 genomic regions compared with normal watering group, this indicates that water deficiency leads to a global increase in the expression of DNA methylation in citrus. In the same time, we verified that the increase of DNA methylation level in LD group was correlated with the decrease of DNA demethylase related gene expression.Interestingly, in transcription analysis, it was found that the promoting flower genes of the LD group did not increase but decreased similarly with repressing genes, which is contrary to the intended result. Thus, we thought the lower expression of suppressors FLC and BFT were the key influencing factor to stimulate the flowering branches formation after LD treatment. Moreover, there was a strong negative correlation between the genes expression level and methylation level of the flowering induction/flower development genes. In general, we thought high global DNA methylation level induced by water deficit regulate the flowering branches building by reducing FLC and BFT genes expression.

The Effect of Nanomaterials on DNA Methylation: A Review.

DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to a cytosine residue in CpG dinucleotides, which are particularly abundant in gene promoter regions. Several studies have highlighted the role that modifications of DNA methylation may have on the adverse health effects caused by exposure to environmental toxicants. One group of xenobiotics that is increasingly present in our daily lives are nanomaterials, whose unique physicochemical properties make them interesting for a large number of industrial and biomedical applications. Their widespread use has raised concerns about human exposure, and several toxicological studies have been performed, although the studies focusing on nanomaterials' effect on DNA methylation are still limited. The aim of this review is to investigate the possible impact of nanomaterials on DNA methylation. From the 70 studies found eligible for data analysis, the majority were in vitro, with about half using cell models related to the lungs. Among the in vivo studies, several animal models were used, but most were mice models. Only two studies were performed on human exposed populations. Global DNA methylation analyses was the most frequently applied approach. Although no trend towards hypo- or hyper-methylation could be observed, the importance of this epigenetic mechanism in the molecular response to nanomaterials is evident. Furthermore, methylation analysis of target genes and, particularly, the application of comprehensive DNA methylation analysis techniques, such as genome-wide sequencing, allowed identifying differentially methylated genes after nanomaterial exposure and affected molecular pathways, contributing to the understanding of their possible adverse health effects.

Epigenetic Modifications Related to Potato Skin Russeting.

Potato tuber skin is a protective corky tissue consisting of suberized phellem cells. Smooth-skinned varieties are characterized by a clean, shiny appearance compared to the darker hue of russeted potatoes. The rough skin of russeted cultivars is a desired, genetically inherited characteristic; however, unwanted russeting of smooth-skinned cultivars often occurs under suboptimal growth conditions. The involvement of epigenetic modifiers in regulating the smooth skin russeting disorder was tested. We used smooth-skin commercial cultivars with and without the russeting disorder and three lines from a breeding population segregating for russeting. Anatomically, the russet skin showed similar characteristics, whether the cause was environmentally triggered or genetically determined. The old outer layers of the corky phellem remain attached to the newly formed phellem layers instead of being sloughed off. Global DNA methylation analysis indicated a significant reduction in the percentage of 5-methylcytosine in mature vs. immature skin and russet vs. smooth skin. This was true for both the smooth-skin commercial cultivars and the russeted lines. The expression level of selected DNA methyltransferases was reduced in accordance. DNA demethylase expression did not change between the skin types and age. Hence, the reduced DNA methylation in mature and russet skin is more likely to be achieved through passive DNA demethylation and loss of methyltransferase activity.

Sex-specific variations in global DNA methylation levels with age: a population-based exploratory study from North India.

Purpose: Aging is one of the most important risk factors for a number of human diseases. Epigenetic alterations, including changes in DNA methylation patterns, have been reported to be one of the hallmarks of aging. Being a malleable process, the role of site-specific DNA methylation in aging is being extensively investigated; however, much less attention has been given to alterations in global DNA methylation with aging at the population level. The present study aims to explore overall and sex-specific variations in global DNA methylation patterns with age. Methods: A total of 1,127 adult individuals (792 females) aged 30-75years belonging to Haryana, North India, were recruited. Socio-demographic data was collected using a pretested interview schedule. Global DNA methylation analysis, of peripheral blood leucocyte (PBL) DNA, was performed using the ELISA-based colorimetric technique. Results: Though the overall correlation analysis revealed a weak inverse trend between global DNA methylation and age, the adjusted regression model showed no significant association between global DNA methylation and age. In age-stratified analysis, global DNA methylation levels were found to be fairly stable until 60years of age, followed by a decline in the above-60 age group. Further, no significant difference in DNA patterns methylation pattern was observed between males and females. Conclusion: Overall, the study suggests a lack of association between global DNA methylation and age, especially until 60years of age, and a similar DNA methylation pattern between males and females with respect to age.

Analysis of global DNA methylation and epigenetic modifiers (DNMTs and HDACs) in human foetal endothelium exposed to gestational and type 2 diabetes

ABSTRACT Foetuses exposed to maternal gestational diabetes (GDM) and type 2 diabetes (T2D) have an increased risk of adverse perinatal outcomes. Epigenetic mechanisms, including DNA methylation and histone modifications, may act as mediators of persistent metabolic memory in endothelial cells (ECs) exposed to hyperglycaemia, even after glucose normalization. Therefore, we investigated alterations in global DNA methylation and epigenetic modifier expression (DNMT1, DNMT3a, DNMT3b, HDAC1, and HDAC2) in human umbilical vein ECs (HUVECs) from the umbilical cords of mothers with GDM (n = 8) and T2D (n = 3) compared to that of healthy mothers (n = 6). Global DNA alteration was measured using a 5-methylation cytosine colorimetric assay, followed by quantitative real-time polymerase chain reaction to measure DNA methyltransferase and histone acetylase transcript expression. We revealed that DNA hypermethylation occurs in both GDM- and T2D-HUVECs compared to that in Control-HUVECs. Furthermore, there was a significant increase in HDAC2 mRNA levels in GDM-HUVECs and increase in DNMT3b mRNA levels in T2D-HUVECs. Overall, our results suggest that GDM and T2D are associated with global DNA hypermethylation in foetal endothelial cells under normoglycemic conditions and the aberrant mRNA expression of HDAC2 and DNMT3b could play a role in this dysregulation.

Molecular Subtyping and Survival Analysis of Osteosarcoma Reveals Prognostic Biomarkers and Key Canonical Pathways.

Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes, MUC6, MUC12, and MUC4, were found to be highly mutated in the OS patients. Results revealed the enrichment of molecular pathways including Wnt signaling, Calcium signaling, and PI3K-Akt signaling in the OS tumors. Survival analyses showed that the expression levels of several genes such as RAMP1, CRIP1, CORT, CHST13, and DDX60L, miRNAs and lncRNAs were associated with survival of OS patients. Molecular subtyping using Cluster-Of-Clusters Analysis (COCA) for mRNA, lncRNA, and miRNA expression; DNA methylation; and mutation data from the TARGET dataset revealed two distinct molecular subtypes, each with a distinctive gene expression profile. Between the two subtypes, three upregulated genes, POP4, HEY1, CERKL, and seven downregulated genes, CEACAM1, ABLIM1, LTBP2, ISLR, LRRC32, PTPRF, and GPX3, associated with OS metastasis were found to be differentially regulated. Thus, the molecular subtyping results provide a strong basis for classification of OS patients that could be used to develop better prognostic treatment strategies.

Abstract 5998: Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma

Abstract Cancer cells recruit and rewire normal cells in their microenvironment to support and protect them by creating a pro-tumorigenic tumor microenvironment (TME). We lack an overarching view of how, despite being genomically stable, stromal cells in the tumor microenvironment are heterogeneously reprogrammed across time and space to promote the evolution of aggressive disease. Recent work by us and others has shown that fibroblasts in the tumor microenvironment are transcriptionally rewired to become protumorigenic cancer associated fibroblasts (CAFs). Transcriptional regulation plays a major role in this reprogramming, but the extent to which epigenetic modifications of DNA also contribute to the rewiring of CAF transcription is not clear. Here we address this question by dissecting the epigenetic landscape of breast CAFs. We applied a sensitive method of whole genome bisulfide sequencing on a model of triple-negative breast cancer in mice to evaluate the methylome profile of CAFs compared to normal mammary fibroblasts (NMFs). we found that fibroblasts undergo massive DNA methylation changes as they transition into CAFs in mice. These changes inversely correlated with transcriptional changes between CAFs and NMFs. We characterized potential regulators of this process, and tested their expression in CAFs in human breast cancer patients, to confirm relevance of our findings to human disease. Our findings suggest that epigenetic alterations contribute to the transcriptional rewiring of fibroblasts to CAFs. This work presents a comprehensive map of DNA-methylation in CAFs, and reveals a previously unknown facet of the dynamic plasticity of the stroma. Citation Format: Coral Halperin, Joschka Hey, Dieter Weichenhan, Yaniv Stein, Shimrit Mayer, Pavlo Lustik, Christoph Plass, Ruth Scherz-Shouval. Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5998.

Global DNA methylation, DNA methyltransferase and stress-related gene expression in ovine oocytes and embryos after exposure to metabolic stressors.

DNA methylation, considered the most prominent epigenetic mark was important for the gene regulation in embryonic development. The present study aimed at evaluating the effects of metabolic stressors [Non-esterified fatty acid (NEFA), β-hydroxy-butyric acid (BHB), ammonia and urea] exposure during the in vitro ovine oocyte maturation, global DNA methylation, DNA methyltransferase and stress-related gene expression. Colorimetric analysis of global DNA methylation and the expression of the DNA methyltransferase genes (DNMT1, DNMT3A, and DNMT3B) were assessed in the matured oocytes, 2-cell embryos and blastocysts produced in vitro from oocytes exposed with the metabolic stressors during 24 h of the in vitro maturation (IVM). Further, the mRNA expression of the stress-related genes (SOD1, SOD2) in the matured oocytes, 2-cell embryos and blastocysts produced was assessed. Significant difference in global DNA methylation levels between all the treatments tested was observed when compared with control in oocytes, two-cell embryos and blastocysts. Elevated concentration of metabolic stressors resulted in increased expressions of several stress-related genes, i.e., SOD1, SOD2 and in mRNA expression of DNA methyltransferase genes. The present study is the first to report that the DNA methylation was sensitive to the effects of the metabolic stressors in ovine oocytes/embryos. The aberrant expressions of genes during oocyte development targeted in the present study can provide evidence for the early embryo developmental arrest and blastocysts quality. These results highlighted the sensitivity of the early embryogenesis and more precisely of the reprogramming period to metabolites challenges, in a realistic situation of elevated concentration of metabolic stressors.

Multi-omics analysis reveals spatiotemporal regulation and function of heteromorphic leaves in Populus.

Despite the high economic and ecological importance of forests, our knowledge of the adaptive evolution of leaf traits remains very limited. Euphrates poplar (Populus euphratica), which has high tolerance to arid environment, has evolved four heteromorphic leaf forms, including narrow (linear and lanceolate) and broad (ovate and broad-ovate) leaves on different crowns. Here, we revealed the significant functional divergence of four P. euphratica heteromorphic leaves at physiological and cytological levels. Through global analysis of transcriptome and DNA methylation across tree and leaf developmental stages, we revealed that gene expression and DNA epigenetics differentially regulated key processes involving development and functional adaptation of heteromorphic leaves, such as hormone signaling pathways, cell division, and photosynthesis. Combined analysis of gene expression, methylation, ATAC-seq, and Hi-C-seq revealed longer interaction of 3D genome, hypomethylation, and open chromatin state upregulates IAA-related genes (such as PIN-FORMED1 and ANGUSTIFOLIA3) and promotes the occurrence of broad leaves while narrow leaves were associated with highly concentrated heterochromatin, hypermethylation, and upregulated abscisic acid pathway genes (such as Pyrabactin Resistance1-like10). Therefore, development of P. euphratica heteromorphic leaves along with functional divergence was regulated by differentially expressed genes, DNA methylation, chromatin accessibility, and 3D genome remodeling to adapt to the arid desert. This study advances our understanding of differential regulation on development and functional divergence of heteromorphic leaves in P. euphratica at the multi-omics level and provides a valuable resource for investigating the adaptive evolution of heteromorphic leaves in Populus.

Unveiling the identities of null cell tumours: Epigenomics corroborate subtle histological cues in pituitary neuroendocrine tumour/adenoma classification.

Pituitary neuroendocrine tumour (PitNET)/adenoma classification is based on cell lineage and requires immunopositivity for adenohypophysial hormones and/or transcription factors (TFs) SF1, TPIT, or PIT1. PitNET/adenomas lacking lineage affiliation are termed "null cell" tumours (NCTs). NCT diagnosis may be afflicted by methodological limitations and inconsistent diagnostic approaches. Previous studies have questioned the existence of true NCTs. In this study, we explore the epigenomic identities of PitNET/adenomas lacking clear TF-immunopositivity. 74 hormone-negative PitNET/adenomas were immunostained and scored for SF1, TPIT, and PIT1 expression. All tumours were classified as gonadotroph, corticotroph, PIT1-positive or "null cell". NCTs were subjected to global DNA methylation analysis. Epigenomic profiles of NCTs were compared to reference tumours using UMAP plotting and methylation-based classification. TF-immunostaining revealed definite lineage identity in 59 of 74 (79.7%) hormone-negative PitNET/adenomas. Of the remaining 15 NCTs, 13 demonstrated minimal and inconclusive nuclear SF1- or TPIT-expression (5 and 8, respectively). Two NCTs were entirely immunonegative. UMAP plotting and methylation-based classification demonstrated that the epigenomes of NCTs with minimal SF1- or TPIT-expression, were adequately affiliated with gonadotroph or corticotroph lineages, respectively. The two immunonegative NCTs were located near the corticotroph PitNET/adenomas via UMAP, while the methylation classifier could not match these two cases to predefined tumour classes. Epigenomic analyses substantiate lineage identification based on minimal TF-immunopositivity in PitNET/adenomas. This strategy dramatically decreases the incidence of NCTs and further challenges the legitimacy of NCTs as a distinct PitNET/adenoma subtype. Our study may be useful for guiding diagnostic efforts and future considerations of PitNET/adenoma classification.

Evaluation of the Effect of Delivery Mode on Methylation Changes in the Global DNA and the PTEN Gene

Objective: This study aimed to investigate the association between global DNA and PTEN gene methylation changes and the type of delivery. Material and Methods: The study included a total of 129 pregnant women at the >37th week of pregnancy who had a cesarean (CS) (n=62) or vaginal delivery (VD) (n=67) and their infant's cord blood (n=129). The 5-mC DNA ELISA method performed global DNA methylation analyses, while PTEN gene methylation change analyses were conducted using the methylation-specific polymerase chain reaction method. Results: The global DNA methylation changes in the women in the CS (Group 1) and VD (Group 2) groups and their infants were found to be CS 9.15±4.82, VD 10.6±5.09 (p=0.09); CS 11.3±6.78, and VD 11.7±5.69 (p=0.75), respectively. Based on age, when the global DNA methylation changes were examined in the pregnant women under and over 30, and their infants, the differences in both groups were statistically significant (p=0.05, p=0.015). The PTEN gene methylation rates of the pregnant women in Group 1 (22.5%) were higher than in Group 2 (13.4%). The PTEN gene methylation rates of the infants in Group 1 (17.7%) were higher than those in Group 2 (14.9%). Conclusion: In this study, a significant increase was determined in mothers and infants regarding their global DNA methylation based on age. Our study is also the first study showing gene-specific methylation changes in the PTEN gene. Our findings are believed to pioneer new studies and positively contribute to scientific research.

Investigation of PM2.5-induced carcinogenic effects through mediation of ErbB family based on DNA methylation and transcriptomics analysis by a lung-mimicking microfluidic platform

Fine particle (PM2.5, less than 2.5 micrometers in diameter) is regarded as a harmful carcinogen. However, the molecular mechanisms of the carcinogenic effects of ambient fine particles have not been fully elucidated, and therapeutic options to address this major public health challenge are lacking. Here, we present global gene-specific DNA methylation and transcriptomic (RNA-Seq) analyses after HBE cells were exposed to fine particles on a portable, small, and all-in-one organ-level lung-mimicking air-liquid interface exposure (MALIE) microfluidic platform. A series of cancer-related signal transduction pathways were activated. ErbB1, ErbB2, and ErbB3 gene expression altered by fine particle exposure was the result of changes in the cellular DNA methylome. The protein expression of ErbB family was inhibited by drugs and could regulate downstream Grb2/Raf pathway and Akt/MDM2 pathway. All of the above results indicated that ErbB family may be promising drug targets for air pollution-related diseases and that inhibitor drugs can be used as therapeutic options to treat these diseases.