Genome-wide DNA Methylation Changes Research Articles

Genome-wide DNA methylation changes in human spermatogenesis

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

DNA methylation: an emerging paradigm of gene regulation under drought stress in plants.

Drought is an enormous threat to global crop production. In order to ensure food security for the burgeoning population, we must develop drought tolerant crop varieties. This necessitates the identification of drought-responsive genes and understanding the mechanisms involved in their regulation. DNA methylation is a widely studied mechanism of epigenetic regulation of gene expression, which is known to play vital role in conferring tolerance to various biotic and abiotic stress factors. The recent advances in next-generation sequencing (NGS) technologies, has allowed unprecedented access to genome-wide methylation marks, with single base resolution. The most important roles of DNA methylation have been studied in terms of gene body methylation (gbM), which is associated with regulation of both transcript abundance and its stability. The availability of mutants for the various genes encoding enzymes involved in methylation of DNA has allowed ascertainment of the biological significance of methylation. Even though a vast number of reports have emerged in the recent past, where both genome-wide methylation landscape and locus specific changes in DNA methylation have been studied, a conclusive picture with regards to the biological role of DNA methylation is still lacking. Compounding this, is the lack of sufficient evidence supporting the heritability of these epigenetic changes. Amongst the various epigenetic variations, the DNA methylation changes are observed to be the most stable. This review describes the drought-induced changes in DNA methylation identified across different plant species. We also briefly describe the stress memory contributed by these changes. The identification of heritable, drought-induced methylation marks would broaden the scope of crop improvement in the future.

A role for DNA methylation in bumblebee morphogenesis hints at female-specific developmental erasure.

Epigenetic mechanisms, such as DNA methylation, are crucial factors in animal development. In some mammals, almost all DNA methylation is erased during embryo development and re-established in a sex- and cell-specific manner. This erasure and re-establishment is thought to primarily be a vertebrate-specific trait. Insects are particularly interesting in terms of development as many species often undergo remarkable morphological changes en route to maturity, that is, morphogenesis. However, little is known about the role of epigenetic mechanisms in this process across species. We have used whole-genome bisulfite sequencing to track genome-wide DNA methylation changes through the development of an economically and environmentally important pollinator species, the bumblebee Bombus terrestris (Hymenoptera:Apidae Linnaeus). We find overall levels of DNA methylation vary throughout development, and we find developmentally relevant differentially methylated genes throughout. Intriguingly, we have identified a depletion of DNA methylation in ovaries/eggs and an enrichment of highly methylated genes in sperm. We suggest this could represent a sex-specific DNA methylation erasure event. To our knowledge, this is the first suggestion of possible developmental DNA methylation erasure in an insect species. This study lays the required groundwork for functional experimental work to determine if there is a causal nature to the DNA methylation differences identified. Additionally, the application of single-cell methylation sequencing to this system will enable more accurate identification of if or when DNA methylation is erased during development.

Resistance and aerobic training increases genome-wide DNA methylation in women with polycystic ovary syndrome

ABSTRACT Physical activity is a first-line treatment for polycystic ovary syndrome (PCOS). Resistance or aerobic exercise improves metabolic complications, reproductive outcomes, and quality of life in PCOS. DNA methylation reprogramming during exercise may be the major modifier behind these changes. We sought to evaluate genome-wide DNA methylation changes after supervised resistance and aerobic exercise in women with PCOS. Exercises were performed in 56 women with PCOS (resistance, n = 30; aerobic, n = 26), for 16 weeks (wks), three times per week, in 50-minute to one-hour sessions. Anthropometric indices and hormonal and metabolic parameters were measured before and after training. Genome-wide leukocyte DNA methylation was analysed by Infinium Human MethylationEPIC 850K BeadChip microarrays (Illumina). Both resistance and aerobic exercise improved anthropometric indices, metabolic dysfunction, and hyperandrogenism in PCOS after the training programme, but no differences were observed between the two exercises. Resistance and aerobic exercise increased genome-wide DNA methylation, although resistance changed every category in the CpG island context (islands, shores, shelve, and open sea), whereas aerobic exercise altered CpG shores and the open sea. Using a stringent FDR (>40), 6 significantly differentially methylated regions (DMRs) were observed in the resistance exercise cohort and 14 DRMs in the aerobic cohort, all of which were hypermethylated. The increase in genome-wide DNA methylation may be related to the metabolic and hormonal changes observed in PCOS after resistance and aerobic exercise. Since the mammalian genome is hypermethylated globally to prevent genomic instability and ageing, resistance and aerobic exercise may promote health and longevity through environmentally induced epigenetic changes.

Maternal exposure to E 551 during pregnancy leads to genome-wide DNA methylation changes and metabolic disorders in the livers of pregnant mice and their fetuses

The widespread use of nanoparticles in the food industry has raised concerns regarding their potential adverse effects on human health, particularly in vulnerable populations, including pregnant mothers and fetuses. However, studies evaluating the reproductive and developmental toxicity of food-grade nanomaterials are limited. This study investigated the potential risks of prenatal dietary exposure to food-grade silica nanoparticles (E 551) on maternal health and fetal growth using conventional toxicological and epigenetic methods. The results showed that prenatal exposure to a high-dose of E 551 induces fetal resorption. Moreover, E 551 significantly accumulates in maternal and fetal livers, triggering a hepatic inflammatory response. At the epigenetic level, global DNA methylation is markedly altered in the maternal and fetal livers. Genome-wide DNA methylation sequencing revealed affected mCG, mCHG, and mCHH methylation landscapes. Subsequent bioinformatic analysis of the differentially methylated genes suggests that E 551 poses a risk of inducing metabolic disorders in maternal and fetal livers. This is further evidenced by impaired glucose tolerance in pregnant mice and altered expression of key metabolism-related genes and proteins in maternal and fetal livers. Collectively, the results of this study highlighted the importance of epigenetics in characterizing the potential toxicity of maternal exposure to food-grade nanomaterials during pregnancy.

Genome-wide DNA methylation changes upon DOT1L inhibition in hormone-responsive breast cancer cells

Genome-wide DNA methylation changes upon DOT1L inhibition in hormone-responsive breast cancer cells

Co-Analysis of Genome-Wide DNA Methylation and Gene Expression Suggests Novel Regulatory Mechanism for RUNX1 in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by recurring chromosomal abnormalities that encode oncogenic fusion proteins. Approximately 10% of AML cases are associated with a chromosome 16 inversion [inv(16)(p13q22)] or translocation t(16;16)(p13q22). These chromosomal rearrangements results in the formation of the fusion oncogene CBFB-MYH11 and ultimately the fusion protein CBFβ-SMMHC. Although CBFβ-SMMHC was initially considered a dominant negative repressor of RUNX1, we have recently shown that it works together with RUNX1 to activate gene expression through direct target gene binding. RUNX1 is known to regulate gene expression at both transcriptional and epigenetic levels. Aberrant DNA methylation patterns have been reported across different cancer types including AML. Recent studies have shown that DNA methylation negatively influences RUNX1 DNA binding. We hypothesize that changes to DNA methylation and consequently dysregulated RUNX1 DNA binding contribute to the pathogenesis of inv(16) AML. In this study, we examined the epigenetic and transcriptomic landscapes of CBFB-MYH11 knockin mice and assessed the contribution of the resulting fusion protein CBFβ-SMMHC to RUNX1 DNA target binding. To assess the effects of DNA methylation on RUNX1 DNA binding in vitro, we initially performed fluorescence polarization and electrophoretic mobility shift assays using unmethylated and methylated RUNX1 DNA target sequences. RUNX1, RUNX1/CBFβ and RUNX1/CBFβ-SMMHC showed significantly lower binding affinity for methylated DNA compared to unmethylated DNA probes. To determine genome-wide DNA methylation changes, we performed enzymatic methyl sequencing on sorted Lin -Sca1 -c-Kit + bone marrow cells from control and pre-leukemic inv(16) mice. Inv(16) mice showed a significant genome-wide DNA hypermethylation compared to controls (13,478 hyper- vs 705 hypomethylated regions; q-value < 0.05), with 14% of the differentially methylated regions (DMRs) lying in promoters. Genes associated with these DMRs were enriched for gene ontology (GO) processes related to hematopoiesis, such as myeloid cell homeostasis and erythrocyte differentiation, and DNA replication, such as mitotic DNA replication initiation, among others. In addition, several RUNX1 target genes showed promoter hypermethylation, such as Klf1 or the thrombopoietin receptor Mpl. To understand the impact of aberrant DNA methylation on gene expression, we performed RNA-seq using the same cell population. Gene expression changes were observed in inv(16) mice compared to controls, with 741 genes being upregulated and 278 downregulated from a total of 1019 differentially expressed genes (DEG; absolute fold-change ≥ 2, q-value < 0.05). Enriched GO processes identified from the DEGs were associated with immune responses, such as response to interferon-alpha (and beta) and leukocyte activation involved in inflammatory response. We then intersected the DMR (focusing on promoters) and DEG lists to identify genes whose expression might be directly influenced by DNA methylation changes in inv(16) leukemia. Hypermethylation of promoters was associated with 102 genes, with 67% of them showing downregulation of gene expression in inv(16) mice, whereas hypomethylation was associated with 18 genes with 94% showing upregulation of gene expression. These results show that in inv(16) mice, promoter hypermethylation is associated with downregulation of gene expression, and hypomethylation with increased expression. We also confirmed that several RUNX1 target genes showing promoter hypermethylation also had reduced gene expression in inv(16) mice. The initial analysis suggests that CBFβ-SMMHC expression significantly affects the methylation landscape in mice, with important RUNX1 target genes exhibiting promoter hypermethylation and subsequent downregulation of gene expression. We are currently performing chromatin immunocleavage sequencing using the same cell population to determine RUNX1 binding in vivo. Overall, this study explores a novel regulatory mechanism of RUNX1 function through DNA methylation, and ultimately a new role for RUNX1 in the pathogenesis of inv(16) AML.

DNA methylation profiles in individuals with rare, atypical 7q11.23 CNVs correlate with GTF2I and GTF2IRD1 copy number

Williams-Beuren syndrome (WBS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders caused by deletion and duplication of a 1.5 Mb region that includes at least five genes with a known role in epigenetic regulation. We have shown that CNV of this chromosome segment causes dose-dependent, genome-wide changes in DNA methylation, but the specific genes driving these changes are unknown. We measured genome-wide whole blood DNA methylation in six participants with atypical CNV of 7q11.23 (three with deletions and three with duplications) using the Illumina HumanMethylation450k array and compared their profiles with those from groups of individuals with classic WBS or classic Dup7 and with typically developing (TD) controls. Across the top 1000 most variable positions we found that only the atypical rearrangements that changed the copy number of GTF2IRD1 and/or GTF2I (coding for the TFII-IRD1 and TFII-I proteins) clustered with their respective syndromic cohorts. This finding was supported by results from hierarchical clustering across a selection of differentially methylated CpGs, in addition to pyrosequencing validation. These findings suggest that CNV of the GTF2I genes at the telomeric end of the 7q11.23 interval is a key contributor to the large changes in DNA methylation that are seen in blood DNA from our WBS and Dup7 cohorts, compared to TD controls. Our findings suggest that members of the TFII-I protein family are involved in epigenetic processes that alter DNA methylation on a genome-wide level.

Whole-genome DNA methylation and gene expression profiling in the livers of mice with nonalcoholic steatohepatitis

AimsNon-alcoholic fatty liver disease (NAFLD) has emerged as one of the major causes of liver-related morbidity and mortality. It ranges simple steatosis to non-alcoholic steatohepatitis (NASH). Previous studies have shown that epigenetic factors, such as DNA methylation, can contribute to the development and progression of simple steatosis. However, the profiling of whole-genome DNA methylation remains poorly characterized in NASH. Main methodsIn this study, we established a mouse model of diet-induced NASH, by maintaining male mice on a high-fructose-high-cholesterol diet (HFHC), to generate hepatic steatosis, inflammation and injury. We profiled hepatic gene expression by RNA-Sequencing and locus-specific 5-methylcytosine level, using Whole Genome Bisulfite Sequencing (WGBS). Key findingsWe identified >1000 differentially methylated regions in NASH versus control group, indicating that NASH diet could modulate the liver methylome. Furthermore, integrated analysis of methylome and transcriptome identified certain key methylated genes and pathways, which may be involved in steroid metabolism and inflammation response. The liver methylation levels of key genes especially Tgfb, Msn, Iqgap1, Cyba, Fcgr1 decreased, and their consequent increased expression may lead to NASH development. SignificanceWe found that HFHC diet-induced NASH could induces genome-wide differential DNA methylation changes. Thus, we proposed that DNA methylation profiles of genomes may be a useful signature of gene transcription and may play an important role in the development of NASH. We also screened and validated the changes of key genes, which may provide new perspectives for the mechanistic study of NASH in future.

A Glimpse into the Genome-wide DNA Methylation Changes in 6-hydroxydopamine-induced In Vitro Model of Parkinson's Disease.

A cell-based model of Parkinson's disease (PD) is a well-established in vitro experimental prototype to investigate the disease mechanism and therapeutic approach for a potential anti-PD drug. The SH-SY5Y human neuroblastoma cells and 6-OHDA combo is one of the many neurotoxininduced neuronal cell models employed in numerous neuroscience-related research for discovering neuroprotective drug compounds. Emerging studies have reported a significant correlation between PD and epigenetic alterations, particularly DNA methylation. However, the DNA methylation changes of PD-related CpG sites on the 6-OHDA-induced toxicity on human neuronal cells have not yet been reported. We performed a genome-wide association study (GWAS) using Infinium Epic beadchip array surveying 850000 CpG sites in differentiated human neuroblastoma cells exposed to 6-OHDA. We identified 236 differentially methylated probes (DMPs) or 163 differentially methylated regions (DMRs) in 6-OHDA treated differentiated neuroblastoma cells than the untreated reference group with p<0.01, Δbeta cut-off of 0.1. Among 236 DMPs, hypermethylated DMPs are 110 (47%), whereas 126 (53%) are hypomethylated. Our bioinformatic analysis revealed 3 DMRs that are significantly hypermethylated and associated with neurological disorders, namely AKT1, ITPR1 and GNG7. This preliminary study demonstrates the methylation status of PD-related CpGs in the 6-OHDA-induced toxicity in the differentiated neuroblastoma cells model.

Developmental stress does not induce genome-wide DNA methylation changes in wild great tit (Parus major) nestlings.

The environment experienced during early life is a crucial factor in the life of many organisms. This early life environment has been shown to have profound effects on morphology, physiology and fitness. However, the molecular mechanisms that mediate these effects are largely unknown, even though they are essential for our understanding of the processes that induce phenotypic variation in natural populations. DNA methylation is an epigenetic mechanism that has been suggested to explain such environmentally induced phenotypic changes early in life. To investigate whether DNA methylation changes are associated with experimentally induced early developmental effects, we cross-fostered great tit (Parus major) nestlings and manipulated their brood sizes in a natural study population. We assessed experimental brood size effects on pre-fledging biometry and behaviour. We linked this to genome-wide DNA methylation levels of CpG sites in erythrocyte DNA, using 122 individuals and an improved epiGBS2 laboratory protocol. Brood enlargement caused developmental stress and negatively affected nestling condition, predominantly during the second half of the breeding season, when conditions are harsher. Brood enlargement, however, affected nestling DNA methylation in only one CpG site and only if the hatch date was taken into account. In conclusion, this study shows that nutritional stress in enlarged broods does not associate with direct effects on genome-wide DNA methylation. Future studies should assess whether genome-wide DNA methylation variation may arise later in life as a consequence of phenotypic changes during early development.

Dietary intake of household cadmium-contaminated rice caused genome-wide DNA methylation changes on gene/hubs related to metabolic disorders and cancers

Cadmium (Cd) contamination in food has raised broad concerns in food safety and human health. The toxicity of Cd to animals/humans have been widely reported, yet little is known about the health risk of dietary Cd intake at the epigenetic level. Here, we investigated the effect of a household Cd-contaminated rice (Cd-rice) on genome-wide DNA methylation (DNAm) changes in the model mouse. Feeding Cd-rice increased kidney Cd and urinary Cd concentrations compared with the Control rice (low-Cd rice), whereas supplementation of ethylenediamine tetraacetic acid iron sodium salt (NaFeEDTA) in the diet significantly increased urinary Cd and consequently decreased kidney Cd concentrations. Genome-wide DNAm sequencing revealed that dietary Cd-rice exposure caused the differentially methylated sites (DMSs), which were mainly located in the promoter (32.5%), downstream (32.5%), and intron (26.1%) regions of genes. Notably, Cd-rice exposure induced hypermethylation at the promoter sites of genes Caspase-8 and interleukin-1β (Il-1β), and consequently, their expressions were down-regulated. The two genes are critical in apoptosis and inflammation, respectively. In contrast, Cd-rice induced hypomethylation of the gene midline 1 (Mid1), which is vital to neurodevelopment. Furthermore, ‘pathways in cancer’ was significantly enriched as the leading canonical pathway. Supplementation of NaFeEDTA partly alleviated the toxic symptoms and DNAm alternations induced by Cd-rice exposure. These results highlight the broad effects of elevated dietary Cd intake on the level of DNAm, providing epigenetic evidence on the specific endpoints of health risks induced by Cd-rice exposure.

Abstract 5434: Machine-learning-based epigenetic detection of early-stage lung cancers using the EpiCheck liquid biopsy platform

Abstract Alterations in DNA methylation are one of the earliest, most common signatures of cancer development, making them ideal biomarkers for early detection. Methylation profiling of plasma cell-free DNA (cfDNA) has significant potential to expand the use of liquid biopsies to cancer screening. The EpiCheck platform combines methylation-sensitive restriction endonuclease (MSRE) digestion with whole genome sequencing to comprehensively map genome-wide DNA methylation changes with high fidelity. In this proof-of-concept study, MSRE-NGS was used to interrogate a liquid biopsy atlas focused on early-stage lung cancers to i) identify informative NGS methylation biomarkers in plasma, and ii) develop a machine learning method to discriminate between cancer and high-risk, non-cancer controls. The EpiCheck lung cancer atlas was constructed using biospecimens from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks, and from a prospective multi-center study (NCT04968548). Plasma samples were collected from 93 high-risk primary lung cancer patients (62% stage I) and 90 high-risk individuals without cancer. Extracted DNA was digested with methylation-sensitive endonucleases and sequenced at an average depth of 600x. Methylation levels of ~6 million genomic loci were rank ordered using Student's t-test. Gene set enrichment analysis (GSEA) was performed on the top ranking 1000 differentially methylated loci. A logistic regression classifier with Lasso regularization was trained on 100,000 loci, and performance was examined by mean AUC using 5-fold cross-validation. A total of 23,130 loci exhibited significant differential methylation patterns between cancers and controls (p&amp;lt;0.01, FDR corrected). Of these, 20,159 and 2,971 lung cancer loci were hypermethylated and hypomethylated, respectively. Biological characterization using GSEA identified enrichments in transcriptional regulation and developmental control. In particular, loci were enriched for Polycomb Repressive Complex regulated genes, suggesting a possible connection to abnormal epigenetic regulation via histone modification in lung cancer. Construction of a machine learning logistic regression model based on the five training folds utilized 224 loci on average, and achieved a mean cross-validation AUC of 0.93 when distinguishing plasma cancer cases vs controls. Our findings demonstrate that the MSRE-NGS EpiCheck platform identified putative biomarkers within the plasma methylome for detecting early-stage lung cancer. A machine learning model trained on methylation targets performed with high accuracy in discriminating lung cancer patients from high-risk healthy individuals. Additional studies are required for defining the strength of the approach and for validating its use in non-invasive lung cancer screening. Citation Format: Dvir Netanely, Stephen Lam, Anna McGuire, Stephen Deppen, Eric Grogan, Fabien Maldonado, Michael Gieske, Joseph Seaman, Kimberly Rieger-Christ, Satish Kalanjeri, Luis Herrera, Nichole Tanner, Garrett B. Sherwood, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Revital Knirsh, Ofir Shliefer, Keren Manor, Radha Duttagupta, Aharona Shuali, Peter J. Mazzone, Gerard A. Silvestri, Catherine A. Schnabel, Danny Frumkin, Adam Wasserstrom. Machine-learning-based epigenetic detection of early-stage lung cancers using the EpiCheck liquid biopsy platform. [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 5434.

Epigenomic variability is associated with age-specific naïve CD4 T cell response to activation in infants and adolescents.

Childhood is a critical period of immune development. During this time, naïve CD4 (nCD4) T cells undergo programmed cell differentiation, mediated by epigenetic changes, in response to external stimuli leading to a baseline homeostatic state that may determine lifelong disease risk. However, the ontogeny of epigenetic signatures associated with CD4 T cell activation during key developmental periods are yet to be described. We investigated genome-wide DNA methylation (DNAm) changes associated with nCD4 T activation following 72 h culture in media+anti-CD3/CD28 beads in healthy infants (aged 12 months, n=18) and adolescents (aged 10-15 years, n=15). We integrated these data with transcriptomic and cytokine profiling from the same samples. nCD4 T cells from both age groups show similar extensive epigenetic reprogramming following activation, with the majority of genes involved in the T cell receptor signaling pathway associated with differential methylation. Additionally, we identified differentially methylated probes showing age-specific responses, that is, responses in only infants or adolescents, including within a cluster of T cell receptor (TCR) genes. These encoded several TCR alpha joining (TRAJ), and TCR alpha variable (TRAV) genes. Cytokine data analysis following stimulation revealed enhanced release of IFN-γ, IL-2 and IL-10, in nCD4 T cells from adolescents compared with infants. Overlapping differential methylation and cytokine responses identified four probes potentially underpinning these age-specific responses. We show that DNAm in nCD4T cells in response to activation is dynamic in infancy and adolescence, with additional evidence for age-specific effects potentially driving variation in cytokine responses between these ages.

Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway.

DNA methylation, including aberrant hypomethylation and hypermethylation, plays a significant role in atherosclerosis (AS); therefore, targeting the unbalanced methylation in AS is a potential treatment strategy. Gualou-xiebai herb pair (GXHP), a classic herb combination, have been used for the treatment of atherosclerotic-associated diseases in traditional Chinese medicine. However, the effects and underlying mechanism of GXHP on AS remain nebulous. In this study, the CCK-8 method was applied to determine the non-toxic treatment concentrations for GXHP. The formation of foam cells played a critical role in AS, so the foam cells model was established after RAW264.7 cells were treated with ox-LDL. The contents of total cholesterol (TC) and free cholesterol (FC) were determined by Gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was used to check the expressions of inflammatory factors including IL-1β, TNF-α, and VCAM-1. Methyl-capture sequencing (MC-seq) and RNA-seq were applied to observe the changes in genome-wide DNA methylation and gene expression, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze differentially methylated genes (DMGs) and differentially expressed genes (DEGs). The targeted signaling pathway was selected and verified using western blotting (WB). The results showed that the lipids and inflammatory factors in foam cells significantly increased. GXHP significantly reduced the expression of TC, FC, and inflammatory factors. MC-seq and RNA-seq showed that GXHP not only corrected the aberrant DNA hypermethylation, but also DNA hypomethylation, thus restored the aberrant DEGs in foam cells induced by ox-LDL. GXHP treatment may target the PI3K-Akt signaling pathway. GXHP reduced the protein levels of phosphorylated(p)-PI3K and p-AKT in foam cells. Our data suggest that treatment with GXHP showed protective effects against AS through the inhibition of DNA methylation mediated PI3K-AKT signaling pathway, suggesting GXHP as a novel methylation-based agent.

Transcriptome and methylome dynamics in the gills of large yellow croaker (Larimichthys crocea) during low-salinity adaption

DNA methylation is a critical epigenetic modification that dynamically regulates gene expression in organisms facing abiotic stress. However, few studies have comprehensively examined the role of DNA methylation in marine fish during environmental adaptation. Therefore, this study explored the methylome dynamics and DNA methylation regulation mechanisms in large yellow croaker (Larimichthys crocea) during low-salinity adaption. The methylation level in the gills was notably raised in the S-group (5‰ salinity) compared to C-group (25‰ salinity). A total of 109 differentially methylated promoter target genes and 581 differentially expressed genes were identified via whole-genome bisulfite sequencing (WGBS) and RNA-seq of gills in the two salinity groups, respectively. Moreover, 23 hypo-methylated/up-regulated differentially methylated genes (DMGs) and 28 hyper-methylated/down-regulated DMGs were identified through integrative analysis, which were mainly enriched in signal transduction, ion exchange, energy metabolism, and cytoskeleton system and other biological processes. Collectively, our findings suggested that low-salinity stress can induce adaptive genome-wide DNA methylation changes, which can in turn affect the transcription of genes in large yellow croaker during low-salinity adaptation. Therefore, our findings provide new insights into the regulatory mechanisms of marine fish in response to rapid environmental changes.

Short-term exposure to fine particulate matter and genome-wide DNA methylation in chronic obstructive pulmonary disease: A panel study conducted in Beijing, China.

Fine particulate matter (PM2.5) is a crucial risk factor for chronic obstructive pulmonary disease (COPD). However, the mechanisms whereby PM2.5 contribute to COPD risk have not been fully elucidated. Accumulating evidence suggests that epigenetics, including DNA methylation, play an important role in this process; however, the association between PM2.5 exposure and genome-wide DNA methylation in patients with COPD has not been studied. To evaluate the association of personal exposure to PM2.5 and genome-wide DNA methylation changes in the peripheral blood of patients with COPD. A panel study was conducted in Beijing, China. We repeatedly measured and collected personal PM2.5 data for 72 h. Genome-wide DNA-methylation of peripheral blood was analyzed using the Illumina Infinium Human Methylation BeadChip (850 k). A linear-mixed effect model was used to identify the differentially methylated probe (DMP) associated with PM2.5. Finally, we performed a functional enrichment analysis of the DMPs that were significantly associated with PM2.5. A total of 24 COPD patients were enrolled and 48 repeated DNA methylation measurements were associated in this study. When the false discovery rate was < 0.05, 19 DMPs were significantly associated with PM2.5 and were annotated to corresponding genes. Functional enrichment analysis of these genes showed that they were related to the response to toxic substances, regulation of tumor necrosis factor superfamily cytokine production, regulation of photosensitivity 3-kinase signaling, and other pathways. This study provided evidence for a significant relationship between personal PM2.5 exposure and DNA methylation in patients with COPD. Our research also revealed a new biological pathway explaining the adverse effects of PM2.5 exposure on COPD risk.

Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells.

Epithelial-mesenchymal-transition (EMT) is an epigenetic-based mechanism contributing to the acquired treatment resistance against receptor tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR)-mutations. Delineating the exact epigenetic and gene-expression alterations in EMT-associated EGFR TKI-resistance (EMT-E-TKI-R) is vital for improved diagnosis and treatment of NSCLC patients. We characterized genome-wide changes in mRNA-expression, DNA-methylation and the histone-modification H3K36me3 in EGFR-mutated NSCLC HCC827 cells in result of acquired EMT-E-TKI-R. CRISPR/Cas9 was used to functional examine key findings from the omics analyses. Acquired EMT-E-TKI-R was analyzed with three omics approaches. RNA-sequencing identified 2,233 and 1,972 up- and down-regulated genes, respectively, and among these were established EMT-markers. DNA-methylation EPIC array analyses identified 14,163 and 7,999 hyper- and hypo-methylated, respectively, differential methylated positions of which several were present in EMT-markers. Finally, H3K36me3 chromatin immunoprecipitation (ChIP)-sequencing detected 2,873 and 3,836 genes with enrichment and depletion, respectively, and among these were established EMT-markers. Correlation analyses showed that EMT-E-TKI-R mRNA-expression changes correlated better with H3K36me3 changes than with DNA-methylation changes. Moreover, the omics data supported the involvement of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 signaling axis for acquired EMT-E-TKI-R. CRISPR/Cas9-mediated analyses corroborated the importance of ZEB1 in acquired EMT-E-TKI-R, MIR200C and MIR141 to be in an EMT-E-TKI-R-associated auto-regulatory loop with ZEB1, and FGFR1 to mediate cell survival in EMT-E-TKI-R. The current study describes the synchronous genome-wide changes in mRNA-expression, DNA-methylation, and H3K36me3 in NSCLC EMT-E-TKI-R. The omics approaches revealed potential novel diagnostic markers and treatment targets. Besides, the study consolidates the functional impact of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1-signaling axis in NSCLC EMT-E-TKI-R.

Genome-wide DNA methylation profile of human dental pulp stem cells during odontogenic differentiation

ObjectivesHuman dental pulp stem cells (hDPSCs) is essential for dentin formation and regeneration, emerging evidence revealed that epigenetic regulation plays vital roles in odontogenic differentiation of hDPSCs. The purpose of this study was to explore the genome-wide DNA methylation changes during odontogenic differentiation of hDPSCs. DesignhDPSCs were isolated from young healthy premolars and reduced representation bisulfite sequencing (RRBS) was taken to detect the genome-wide DNA methylation profile of hDPSCs during odontogenic differentiation. Genome-wide DNA methylation map, differentially methylated region (DMR) analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. ResultsWe found a totally different DNA methylation patterns during the odontogenic differentiation of hDPSCs. A total of 9309 differentially methylated genes (DMG) were identified. Bio-information analysis revealed that calcium signaling pathway, pathways in cancer, and HTLV–I infection signaling pathways may play potential roles in odontogenic differentiation of hDPSCs. NOTCH1, WNT7B, and AXIN2 proteins were related with calcium signaling pathway. ConclusionsThis study revealed a comprehensive analysis of global DNA methylation profiles in odontogeinc differentiation of hDPSCs and provided several possible underlying signaling pathways and candidate genes that may regulate the odontogenic differentiation of hDPSCs.

Exploration of genome-wide DNA methylation profiles in night shift workers

ABSTRACT The past decades, studies indicated that night shift work is associated with adverse health effects, however, molecular mechanisms underlying these effects are poorly understood. A few previous studies have hypothesized a role for DNA-methylation (DNAm) in this relationship. We performed a cross-sectional epigenome-wide association study, to investigate if night shift work is associated with genome-wide DNAm changes and DNAm-based biological age acceleration, based on previously developed so-called ‘epigenetic clocks.’ Short term (2–6 years) and intermediate term (10–16 years) night shift workers, along with age and sex matched dayworkers (non-shift workers) were selected from the Lifelines Cohort Study. For genome-wide methylation analysis the Infinium Methylation EPIC array (Ilumina) was used. Linear regression analyses were used to detect differences in methylation at individual CpG-sites associated with night shift work. Pathway analysis was performed based on KEGG pathways and predictions of age acceleration in night shift workers were performed based on four previously developed epigenetic age calculators. Only in women, differences in methylation at individual CpG-sites were observed between night shift workers and non-shift workers. Most of these differentially methylated positions (DMPs) were observed in intermediate term night shift workers. Pathway analysis shows involvement of pathways related to circadian rhythm and cellular senescence. Increased age acceleration was observed only in short-term night shift workers (men and women). This might be indicative of adaptation to night shift work or a so-called healthy worker effect. In conclusion, these results show that DNA methylation changes are associated with night shift work, specifically in women.