Significant Methylation Changes Research Articles

Alterations in Skeletal Muscle Insulin Signaling DNA Methylation: A Pilot Randomized Controlled Trial of Olanzapine in Healthy Volunteers.

Antipsychotics are associated with severe metabolic side effects including insulin resistance; however, the mechanisms underlying this side effect are not fully understood. The skeletal muscle plays a critical role in insulin-stimulated glucose uptake, and changes in skeletal muscle DNA methylation by antipsychotics may play a role in the development of insulin resistance. A double-blind, placebo-controlled trial of olanzapine was performed in healthy volunteers. Twelve healthy volunteers were randomized to receive 10 mg/day of olanzapine for 7 days. Participants underwent skeletal muscle biopsies to analyze DNA methylation changes using a candidate gene approach for the insulin signaling pathway. Ninety-seven methylation sites were statistically significant (false discovery rate < 0.05 and beta difference between the groups of ≥10%). Fifty-five sites had increased methylation in the skeletal muscle of olanzapine-treated participants while 42 were decreased. The largest methylation change occurred at a site in the Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (PPARGC1A) gene, which had 52% lower methylation in the olanzapine group. Antipsychotic treatment in healthy volunteers causes significant changes in skeletal muscle DNA methylation in the insulin signaling pathway. Future work will need to expand on these findings with expression analyses.

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

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

Alterations in Blood Methylome as Potential Epigenetic Biomarker in Sporadic Parkinson's Disease.

To characterize DNA methylation (DNAm) differences between sporadic Parkinson's disease (PD) and healthy control (HC) individuals enrolled in the Parkinson's Progression Markers Initiative (PPMI). Using whole blood, we characterized longitudinal differences in DNAm between sporadic PD patients (n = 196) and HCs (n = 86) enrolled in PPMI. RNA sequencing (RNAseq) was used to conduct gene expression analyses for genes mapped to differentially methylated cytosine-guanine sites (CpGs). At the time of patient enrollment, 5,178 CpGs were differentially methylated (2,683 hypermethylated and 2,495 hypomethylated) in PD compared to HC. Of these, 579 CpGs underwent significant methylation changes over 3 years. Several differentially methylated CpGs were found near the cytochrome P450 family 2 subfamily E member 1 (CYP2E1) gene. Additionally, multiple hypermethylated CpGs were associated with the N-myc downregulated gene family member 4 (NDRG4) gene. RNA-Seq analyses showed 75 differentially expressed genes in PD patients compared to controls. An integrative analysis of both differentially methylated sites and differentially expressed genes revealed 20 genes that exhibited hypomethylation concomitant with overexpression. Additionally, 1 gene, cathepsin H (CTSH), displayed hypermethylation that was associated with its decreased expression. We provide initial evidence of alterations in DNAm in blood of PD patients that may serve as potential epigenetic biomarker of disease. To evaluate the significance of these changes throughout the progression of PD, additional profiling at longer intervals and during the prodromal stages of disease will be necessary. ANN NEUROL 2024.

A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Non-concordant epigenetic and transcriptional responses to acute thermal stress in western mosquitofish (Gambusia affinis).

Climate change is intensifying the frequency and severity of extreme temperatures. Understanding the molecular mechanisms underlying the ability to cope with acute thermal stress is key for predicting species' responses to extreme temperature events. While many studies have focused on the individual roles of gene expression, post-transcriptional processes and epigenetic modifications in response to acute thermal stress, the relative contribution of these molecular mechanisms remains unclear. The wide range of thermal limits of western mosquitofish (Gambusia affinis) provides an opportunity to explore this interplay. Here, we quantified changes in gene expression, alternative splicing, DNA methylation and microRNA (miRNA) expression in muscle tissue dissected from mosquitofish immediately after reaching high (CTmax) or low thermal limit (CTmin). Although the numbers of genes showing expression and splicing changes in response to acute temperature stress were small, we found a possibly larger and non-redundant role of splicing compared to gene expression, with more genes being differentially spliced (DSGs) than differentially expressed (DEGs), and little overlap between DSGs and DEGs. We also identified a small proportion of CpGs showing significant methylation change (i.e. differentially methylated cytosines, DMCs) in fish at thermal limits; however, there was no overlap between DEGs and genes annotated with DMCs in both CTmax and CTmin experiments. The weak interplay between epigenetic modifications and gene expression was further supported by our discoveries of no differentially expressed miRNAs. These findings provide novel insights into the relative role of different molecular mechanisms underlying immediate responses to extreme temperatures and demonstrate non-concordant responses of epigenetic and transcriptional mechanisms to acute temperature stress.

Abstract 3496: EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors

Abstract Introduction. Aberrant DNA methylation patterns are frequent in colorectal adenocarcinoma (CRC) tissues. The investigation of this layer of epigenetic regulation holds the promise of a better understanding of tumour progression and the identification of novel biomarker candidates. Purpose. Our goal was to collect and analyse publicly available DNA methylation data of colorectal samples and create a user-friendly tool for exploring and visualizing the CRC methylome. Methods. We used Illumina Infinium Methylation Arrays, which is a popular platform for genome-wide methylation analysis of large cohorts. Gene Expression Omnibus database (GEO) and TCGA-COAD (The Cancer Genome Atlas Colon Adenocarcinoma) datasets including normal colorectal mucosa, adenoma, and adenocarcinoma samples were systematically collected and filtered. Differentially methylated regions were identified using the Kruskal-Wallis test. Genes with significant methylation changes were further analysed using ROC (Receiver Operator Characteristic) analysis. Finally, we established a web application using the shiny R package. Results. We assembled a database including methylation data of 2,586 samples. Adenoma and adenocarcinoma samples were globally hypomethylated. Hypermethylation (p &amp;lt; 0.05, Δβ &amp;gt;= 0.2) in adenocarcinoma samples was predominantly present in regions near proximal promoters. Out of the 74 previously proposed biomarkers examined, 50 had a cross-validated AUC of over 0.8. FDA-approved biomarkers BMP3, NDRG4, and SEPT9 have shown the highest performance in the TSS200 region (cvAUC = 0.8, 0.83, and 0.76, respectively). Genes with the highest performance were ITGA4 (5’UTR, first exon: cvAUC = 0.9), MDFI (5’UTR: cvAUC = 0.9), CNRIP1 (5’UTR, first exon: cvAUC = 0.89). The established web application can visualize methylation at CpG sites and gene regions, and KEGG pathway genes. The platform is available at https://epigenplot.com. Conclusion. We collected and analysed a sizeable database of colorectal tissue data. Gene regions with the best performance include previously proposed and novel diagnostic biomarker candidates. The web platform provides an tool for the efficient exploration of the assembled data. Citation Format: Dalma Müller, Balazs Gyorffy. EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3496.

Abstract 3666: Multi-cancer early detection using metrics of DNA methylation based epigenetic instability

Abstract Cancers present significant changes in DNA methylation, which has proven to be highly useful in cell-free DNA (cfDNA) based cancer detection. Cancer epigenomes are marked by a high degree of intra- and inter-tumor epigenetic variation, indicative of the high level of epigenetic instability. However, the nature of the regions with high epigenetic instability and its utility as biomarker has not been explored. Here we developed a novel methodology and metric to measure the degree of epigenetic perturbation, termed the Epigenetic Instability Index (EII), for multiple cancer screening through cfDNA. Our novel methodology provides a sample specific score of epigenetic perturbation in relation to the expected normal state as opposed to only looking at CpG sites that change between samples. Through machine learning, we have elucidated 269 CpG-island regions which sufficiently capture the epigenetic instability in cancers. We have built classifier models using the EII metrics of these 269 regions and demonstrate that they can efficiently identify breast and lung cancer cases from cfDNA methylation data. Particularly, the models can differentiate even Stage IA of NSCLC with ~75% sensitivity at 95% specificity and early-stage breast cancer at ~68% sensitivity and 95% specificity. The EII metrics perform significantly better than metrics that measure absolute DNA methylation levels. Our studies highlight the potential of measuring epigenetic instability from cfDNA in any given sample in addition to traditional methods of methylation measurement for non-invasive screenings via liquid biopsies. Citation Format: Sara-Jayne Thursby, Zhicheng Jin, Steve B. Baylin, Malcolm Brock, Thomas Pisanic, Hariharan Easwaran. Multi-cancer early detection using metrics of DNA methylation based epigenetic instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3666.

The study of HMOX1 DNA methylation and gene expression and the diagnostic potential of miR-153-3p in preeclampsia.

Background: The objective was to elucidate the potential epigenetic regulatory mechanisminHMOX1 expression in preeclampsia. Materials & methods: HMOX1 promoter DNA methylation was evaluated in the placental tissue and blood of preeclamptic and normotensive pregnant women. HMOX1 and miR-153-3p gene expression were assessed in placental tissue and peripheral blood mononuclear cells (PBMCs). Related microarray datasets in the Gene Expression Omnibus database were also analyzed. Results: In placental tissue, despite HMOX1 expression downregulation, there was no significant change in HMOX1 methylation. In PBMCs, there was no significant alteration in HMOX1 expression, while hypomethylation wasobserved in blood. The miR-153-3p expressionincreased in theplacental tissue and inthePBMCs of preeclampsia. Conclusion: DNA methylation does not affect HMOX1 expression, while miR-153-3p might be a biomarker for preeclampsia.

DNA methylation may partly explain psychotropic drug-induced metabolic side effects: results from a prospective 1-month observational study

BackgroundMetabolic side effects of psychotropic medications are a major drawback to patients’ successful treatment. Using an epigenome-wide approach, we aimed to investigate DNA methylation changes occurring secondary to psychotropic treatment and evaluate associations between 1-month metabolic changes and both baseline and 1-month changes in DNA methylation levels. Seventy-nine patients starting a weight gain inducing psychotropic treatment were selected from the PsyMetab study cohort. Epigenome-wide DNA methylation was measured at baseline and after 1 month of treatment, using the Illumina Methylation EPIC BeadChip.ResultsA global methylation increase was noted after the first month of treatment, which was more pronounced (p < 2.2 × 10–16) in patients whose weight remained stable (< 2.5% weight increase). Epigenome-wide significant methylation changes (p < 9 × 10−8) were observed at 52 loci in the whole cohort. When restricting the analysis to patients who underwent important early weight gain (≥ 5% weight increase), one locus (cg12209987) showed a significant increase in methylation levels (p = 3.8 × 10–8), which was also associated with increased weight gain in the whole cohort (p = 0.004). Epigenome-wide association analyses failed to identify a significant link between metabolic changes and methylation data. Nevertheless, among the strongest associations, a potential causal effect of the baseline methylation level of cg11622362 on glycemia was revealed by a two-sample Mendelian randomization analysis (n = 3841 for instrument-exposure association; n = 314,916 for instrument-outcome association).ConclusionThese findings provide new insights into the mechanisms of psychotropic drug-induced weight gain, revealing important epigenetic alterations upon treatment, some of which may play a mediatory role.

Alteration of DNA methyltransferases by eribulin elicits broad DNA methylation changes with potential therapeutic implications for triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with limited treatment options. Eribulin, a chemotherapeutic drug, induces epigenetic changes in cancer cells, suggesting a unique mechanism of action. Materials & methods: MDA-MB 231 cells were treated with eribulin and paclitaxel, and the samples from 53 patients treated with neoadjuvant eribulin were compared with those from 14 patients who received the standard-of-care treatment using immunohistochemistry. Results: Eribulin treatment caused significant DNA methylation changes in drug-tolerant persister TNBC cells, and it also elicited changes in the expression levels of epigenetic modifiers (DNMT1, TET1, DNMT3A/B) in vitro and in primary TNBC tumors. Conclusion: These findings provide new insights into eribulin's mechanism of action and potential biomarkers for predicting TNBC treatment response.

Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD.

Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.

Epigenetic Profiling of Type 2 Diabetes Mellitus: An Epigenome-Wide Association Study of DNA Methylation in the Korean Genome and Epidemiology Study.

Diabetes is characterized by persistently high blood glucose levels and severe complications and affects millions of people worldwide. In this study, we explored the epigenetic landscape of diabetes using data from the Korean Genome and Epidemiology Study (KoGES), specifically the Ansung-Ansan (AS-AS) cohort. Using epigenome-wide association studies, we investigated DNA methylation patterns in patients with type 2 diabetes mellitus (T2DM) and those with normal glucose regulation. Differential methylation analysis revealed 106 differentially methylated probes (DMPs), with the 10 top DMPs prominently associated with TXNIP, PDK4, NBPF20, ARRDC4, UFM1, PFKFB2, C7orf50, and ABCG1, indicating significant changes in methylation. Correlation analysis highlighted the association between the leading DMPs (e.g., cg19693031 and cg26974062 for TXNIP and cg26823705 for NBPF20) and key glycemic markers (fasting plasma glucose and hemoglobin A1c), confirming their relevance in T2DM. Moreover, we identified 62 significantly differentially methylated regions (DMRs) spanning 61 genes. A DMR associated with PDE1C showed hypermethylation, whereas DMRs associated with DIP2C, FLJ90757, PRSS50, and TDRD9 showed hypomethylation. PDE1C and TDRD9 showed a strong positive correlation between the CpG sites included in each DMR, which have previously been implicated in T2DM-related processes. This study contributes to the understanding of epigenetic modifications in T2DM. These valuable insights can be utilized in identifying potential biomarkers and therapeutic targets for effective management and prevention of diabetes.

Analysis of DNA Methylation Differences during the JIII Formation of Bursaphelenchus xylophilus.

DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pinewood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives at low temperatures through third-stage dispersal juvenile, making it a major pathogen for pines in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal juvenile, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal juvenile and three other propagative juvenile stages of PWN. Our findings revealed that the average methylation rate of cytosine in the samples ranged from 0.89% to 0.99%. Moreover, we observed significant DNA methylation changes in the third-stage dispersal juvenile and the second-stage propagative juvenile of PWN, including differentially methylated cytosine (DMCs, n = 435) and regions (DMRs, n = 72). In the joint analysis of methylation-associated transcription, we observed that 23 genes exhibited overlap between differentially methylated regions and differential gene expression during the formation of the third-stage dispersal juvenile of PWN. Further functional analysis of these genes revealed enrichment in processes related to lipid metabolism and fatty acid synthesis. These findings emphasize the significance of DNA methylation in the development of third-stage dispersal juvenile of PWN, as it regulates transcription to enhance the probability of rapid expansion in PWN.

Epigenetic Profiling of PTPN11 Mutant JMML Hematopoietic Stem and Progenitor Cells Reveals an Aberrant Histone Landscape.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

High-throughput Sequencing Reveals N6-methyladenosine-modified LncRNAs as Potential Biomarkers in Mice with Liver Fibrosis.

N6-methyladenosine (m6A) is the most frequent internal modification in eukaryotic RNA. Long noncoding RNAs (lncRNAs) are a new type of noncoding regulatory molecule with multiple cellular functions. Both are closely related to the occurrence and development of liver fibrosis (LF). However, the role of m6A-methylated lncRNAs in the progression of LF remains largely unknown. In this study, HE and Masson staining were used to observe pathological changes in the liver, m6A-modified RNA immunoprecipitation sequencing (m6A-seq) was performed to systematically evaluate the m6A modification level of lncRNAs in LF mice, meRIP-qPCR and RT-qPCR were used to detect the m6A methylation level and RNA expression level of the target lncRNAs. A total of 415 m6A peaks were detected in 313 lncRNAs in liver fibrosis tissues. There were 98 significantly different m6A peaks in LF, which were located on 84 lncRNAs, of which 45.2% of the lncRNA length was between 200-400 bp. At the same time, the first three chromosomes of these methylated lncRNAs were chromosomes 7, 5 and 1. RNA sequencing identified 154 differentially expressed lncRNAs in LF. The joint analysis of m6A-seq and RNA-seq found that there were three lncRNAs with significant changes in m6A methylation and RNA expression levels: lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586. Subsequently, the verification results showed that the m6A methylation levels of lncRNA H19 and lncRNA Gm17586 were significantly increased, while that of lncRNA Gm16023 was significantly decreased, and the RNA expression of three lncRNAs was significantly decreased. Through the establishment of a lncRNA-miRNA-mRNA regulatory network, the possible regulatory relationships of lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586 in LF were revealed. This study revealed the unique m6A methylation pattern of lncRNAs in LF mice, suggesting that the m6A methylation modification of lncRNAs is related to the occurrence and development of LF.

Long-term exposure to ambient fine particulate components and leukocyte epigenome-wide DNA Methylation in older men: the Normative Aging Study

BackgroundEpigenome-wide association studies of ambient fine particulate matter (PM2.5) have been reported. However, few have examined PM2.5 components (PMCs) and sources or included repeated measures. The lack of high-resolution exposure measurements is the key limitation. We hypothesized that significant changes in DNA methylation might vary by PMCs and the sources.MethodsWe predicted the annual average of 14 PMCs using novel high-resolution exposure models across the contiguous U.S., between 2000–2018. The resolution was 50 m × 50 m in the Greater Boston Area. We also identified PM2.5 sources using positive matrix factorization. We repeatedly collected blood samples and measured leukocyte DNAm with the Illumina HumanMethylation450K BeadChip in the Normative Aging Study. We then used median regression with subject-specific intercepts to estimate the associations between long-term (one-year) exposure to PMCs / PM2.5 sources and DNA methylation at individual cytosine-phosphate-guanine CpG sites. Significant probes were identified by the number of independent degrees of freedom approach, using the number of principal components explaining > 95% of the variation of the DNA methylation data. We also performed regional and pathway analyses to identify significant regions and pathways.ResultsWe included 669 men with 1,178 visits between 2000–2013. The subjects had a mean age of 75 years. The identified probes, regions, and pathways varied by PMCs and their sources. For example, iron was associated with 6 probes and 6 regions, whereas nitrate was associated with 15 probes and 3 regions. The identified pathways from biomass burning, coal burning, and heavy fuel oil combustion sources were associated with cancer, inflammation, and cardiovascular diseases, whereas there were no pathways associated with all traffic.ConclusionsOur findings showed that the effects of PM2.5 on DNAm varied by its PMCs and sources.

Long-Term Use of Amoxicillin Is Associated with Changes in Gene Expression and DNA Methylation in Patients with Low Back Pain and Modic Changes.

Long-term antibiotics are prescribed for a variety of medical conditions, recently including low back pain with Modic changes. The molecular impact of such treatment is unknown. We conducted longitudinal transcriptome and epigenome analyses in patients (n = 100) receiving amoxicillin treatment or placebo for 100 days in the Antibiotics in Modic Changes (AIM) study. Gene expression and DNA methylation were investigated at a genome-wide level at screening, after 100 days of treatment, and at one-year follow-up. We identified intra-individual longitudinal changes in gene expression and DNA methylation in patients receiving amoxicillin, while few changes were observed in patients receiving placebo. After 100 days of amoxicillin treatment, 28 genes were significantly differentially expressed, including the downregulation of 19 immunoglobulin genes. At one-year follow-up, the expression levels were still not completely restored. The significant changes in DNA methylation (n = 4548 CpGs) were mainly increased methylation levels between 100 days and one-year follow-up. Hence, the effects on gene expression occurred predominantly during treatment, while the effects on DNA methylation occurred after treatment. In conclusion, unrecognized side effects of long-term amoxicillin treatment were revealed, as alterations were observed in both gene expression and DNA methylation that lasted long after the end of treatment.

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs).Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

NALCN is a potential biomarker and therapeutic target in human cancers.

Background: Sodium leak channel non-selective (NALCN), known as a voltage-independent Na+ channel, is increasingly considered to play vital roles in tumorigenesis and metastasis of human cancers. However, no comprehensive pan-cancer analysis of NALCN has been conducted. Our study aims to explore the potential diagnostic, prognostic and therapeutic value of NALCN in human cancers. Methods: Through comprehensive application of datasets from Human Protein Atlas (HPA), The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), Enhanced Version of Tumor Immune Estimation Resource (TIMER2.0), Tumor and Immune System Interaction Database (TISIDB), The University of Alabama at Birmingham Cancer data analysis Portal (UALCAN), cBioPortal, GeneMANIA and Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) databases, we explored the potential roles of NALCN in different cancers. The differential expression, prognostic implications, pathological stages and grades, molecular and immune subtypes, diagnostic accuracy, tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR) genes, immune checkpoint genes, chemokine genes, major histocompatibility complex (MHC)-related genes, tumor-infiltrating immune cells (TIICs), promoter methylation, mutations, copy number alteration (CNA), and functional enrichment related to NALCN were analyzed. Results: Most cancers lowly expressed NALCN. Upregulated NALCN expression was associated with poor or better prognosis in different cancers. Moreover, NALCN was correlated with clinicopathological features in multiple cancers. NALCN showed high diagnostic accuracy in 5 caner types. NALCN is highly linked with immune-related biomarkers, immune-related genes and TIICs. Significant methylation changes and genetic alteration of NALCN can be observed in many cancers. Enrichment analysis showed that NALCN is closely related to multiple tumor-related signaling pathways. Conclusion: Our study revealed the vital involvement of NALCN in cancer. NALCN can be used as a prognostic biomarker for immune infiltration and clinical outcomes, and has potential diagnostic and therapeutic implications.

Critical role of transcriptome-wide m6A methylation in the aqueous humor of patients with pseudoexfoliation glaucoma

N6-methyladenosine (m6A) modification is one of the most common types of methylation modifications in eukaryotic mRNA. However, its role in the pathogenesis of pseudoexfoliation glaucoma (PXG) has not yet been reported. To enhance understanding in this regard, we assessed the m6A methylome in the aqueous humor of patients with PXG. MeRIP-Seq and RNA-Seq analyses were performed to compare the m6A methylomes and gene expression profiles of the aqueous humor of patients with PXG with those of patients with age-related cataract (ARC). Colorimetric m6A quantification was performed to detect global m6A levels. Quantitative reverse transcription PCR confirmed the expression of m6A-related enzymes and mRNAs in both groups. Results showed significantly higher aqueous humor m6A levels in the PXG group than in the ARC group. Five m6A-related enzymes, including METTL3, YTHDC2, HNRNPA2B1, HNRNPC, and LRPPRC, were significantly up-regulated in PXG specimens. We also observed 9728 m6A-modified peaks related to 6126 gene transcripts in the PXG group, with more than 250 genes containing one m6A peak (hypomethylated or hypermethylated). The distribution of the m6A peaks was enriched in coding sequences and 3′-untranslated regions for both groups. GGAC motif structures were also significantly enriched. Bioinformatics analysis further revealed that m6A plays a critical role in extracellular matrix formation and histone deacetylation. Additionally, MMP14, ADAMTSL1, FN1, and HDAC1 showed significant changes in m6A methylation and mRNA expression in the PXG group. Therefore, m6A methylation may regulate extracellular matrix composition in PXG and METTL3 may be a pivotal regulator of this process. In the future, it would be necessary to investigate MMP14, ADAMTSL1, FN1, and HDAC1, which are potential target genes.