Pattern Of Methylation Changes Research Articles

Dynamics of DNA methylation during osteogenic differentiation of porcine synovial membrane mesenchymal stem cells from two metabolically distinct breeds

ABSTRACT Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell’s metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

Comprehensive analysis of plasma methylome reveals distinct patterns of methylation changes between responders and non-responders to neoadjuvant chemotherapy in breast cancer

The potential of cell-free DNA (cfDNA) methylation profiling in oncology is increasingly recognized for its ability to offer a comprehensive assessment of tumor biology. In this report, we explore the plasma methylome dynamics in patients with early breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT) using an agnostic genome-wide approach with cell-free DNA immunoprecipitation and sequencing (cfMeDIP-seq). Our cohort comprised seven women with triple-positive BC, assessed for cfDNA methylation changes at multiple time points during their treatment course. Genome-wide analysis revealed distinctive patterns of differential methylation in patients achieving pathological complete response (pCR), with no significant epigenomic modifications in patients with residual disease (RD). Gene set enrichment analysis highlighted dynamic variations in functional pathways associated with therapy response, including metabolism, immune response, and response to xenobiotics, with a notable reversibility post-treatment. Principal component analysis of the differentially methylated regions demonstrated a clear distinction between pCR and RD patients, indicating the potential of cfMeDIP-seq for the non-invasive assessment of therapy response. Such findings suggest that cfMeDIP-seq could complement ctDNA offering broader insights into the global modifications induced by chemotherapy. Although future research is needed to validate and expand on our findings, we offer a proof-of-principle of the potential utility of cfMeDIP-seq in the personalized management of BC.

Targeted bisulfite sequencing analysis of candidate genes associated with Alzheimer’s disease.

AbstractBackgroundRecent epigenome‐wide association studies (EWAS) have identified a number of loci in specific genes that show robust and reproducible alterations in DNA methylation in Alzheimer’s disease brain samples. The standard method to assess methylation in EWAS is via microarrays, however, these only target a limited number of methylation sites in each gene. Therefore, further analysis of methylation changes across the entire gene are required to determine the exact extent and pattern of methylation changes in disease. In this study we have performed targeted bisulfite sequencing for candidate genes in the Brains for Dementia Research (BDR) tissue sample resource, which is a highly characterized cohort containing tissue with a high degree of standardized pathological, clinical and administrative data available to allow comparative studies.MethodPrefrontal cortex brain samples from 60 individuals were selected from the BDR cohort and grouped by Braak stage (Control 0‐II; mild cognitive impairment III‐IV; AD V‐VI). The DNA was extracted, before 30 genomic regions of interest, identified from previous AD EWAS, were captured using Agilent Sure Select target baits. The DNA was next‐generation bisulfite sequenced, before the sequence reads were aligned and the methylation status of cytosine residues were called using the Bismark Bisulfite Mapper program. Differentially methylated positions (DMPs) were then analyzed across the three groups.ResultThe degree and extent of methylation within the targeted genomic regions were identified and quantified for each group before being analyzed using linear regression models.ConclusionThis study builds on previous work that identified differential methylation in several genomic regions that were associated with Braak stage. By identifying the exact positions that are subjected to differential methylation this work provides further evidence that dysregulation of methylation is associated with pathological changes in AD prefrontal cortex.

Targeted bisulfite sequencing analysis of candidate genes associated with Alzheimer's disease.

The histopathological changes in Alzheimer's disease (AD), including extensive deposits of amyloid β plaques and neurofibrillary tangles occur many years before the onset of clinical symptoms and epigenetic processes such as DNA methylation may contribute to this delay. Recent epigenome-wide association studies (EWAS) have identified a number of loci in specific genes that show robust and reproducible alterations in DNA methylation changes in AD brain samples. However, the technologies used for these studies only assess a limited number of methylation sites in each gene and further analysis of methylation changes across the entire gene are required to determine the exact extent and pattern of methylation changes in disease. In this study we have performed targeted bisulfite sequencing and RNA sequencing in the Brains for Dementia Research (BDR) tissue sample resource, which is a highly characterised cohort containing tissue with a high degree of standardised pathological, clinical and administrative data available to allow comparative studies. Prefrontal cortex brain samples from 96 individuals were selected from the BDR cohort and grouped by Braak stage (Control 0-II; mild cognitive impairment III-IV; AD V-VI). DNA and RNA was simultaneously extracted before next generation RNA-seq was performed on all 96 samples to analyse the AD transcriptome and data extracted for 30 genomic regions of interest identified from previous EWAS. Concurrently for the DNA, Agilent Sure Select target baits captured the same 30 target genomic regions that were bisulfite sequenced for a subset of 60 samples, allowing analysis of differentially methylated positions (DMPs). The exact location of DMPs within the targeted genomic regions were identified and compared between each group. Similarly, differential expression of mRNA transcripts encoded by these genes were also identified and related to methylation levels. This study builds on previous work that identified differential methylation in several genomic regions that were associated with Braak stage. By identifying the exact positions that are subjected to differential methylation and the potential impact these changes have on gene expression this work provides further evidence that dysregulation of methylation is associated with pathological changes in AD prefrontal cortex.

The pattern of DNA methylation alteration, and its association with the changes of gene expression and alternative splicing during phosphate starvation in tomato

SUMMARYDNA methylation is changed and associates with gene expression alterations in plant response to phosphate starvation (Pi–), a common stress that impacts plant growth and productivity. However, in the horticultural model species Solanum lycopersicum (tomato), the dynamics of DNA methylation and its relationship with changes in gene transcription and alternative splicing (AS) under Pi– are unknown. Here, we performed integrative methylome and transcriptome analyses of tomato seedlings under Pi‐deficient and ‐sufficient conditions. We found Pi– caused a slight increase in the overall methylation level, with millions of differentially methylated cytosines (DmCs) and a few hundred differentially methylated regions (DMRs). We also identified thousands of differentially expressed (DE) and differential AS (DAS) genes induced by Pi–, and found that DmCs were more abundant in non‐expressed genes than in DE or DAS genes. Moreover, DNA methylation alterations weakly correlated with transcription changes but not with DAS events, and hyper‐CHH‐DMRs overlapping with transposable elements (TEs) were enriched in a subset of Pi starvation response (PSR) genes. We propose that changes in DNA methylation may be associated with the differential expression of some PSR genes, but that most of these changes probably control the expression of nearby TEs, rather than directly affecting the transcription or AS of PSR genes. Besides, the pattern of methylation changes upon Pi– may largely be shaped by TE distributions. Together, our study provides comprehensive insights into the association of DNA methylation with gene transcription and AS under Pi– in tomato and may contribute to unveiling novel roles of epigenetic mechanisms in plant stress response.

Integrated Bioinformatics Analysis of DNA Methylation Biomarkers in Thyroid Cancer Based on TCGA Database.

Previous studies have reported a cluster of aberrant promoter methylation changes associated with silencing of tumor suppressor genes in thyroid cancer (TC), but these results of individual genes are far from enough. In this work, we aimed to investigate the onset and pattern of methylation changes during the progression of TC by informatics analysis. We downloaded the DNA methylation and RNA sequencing datasets from The Cancer Genome Atlas focusing on TC. Abnormally methylated differentially expressed genes (DEGs) were sorted and pathways were analyzed. The KEGG and GO were then used to perform enrichment and functional analysis of identified pathways and genes. Gene-drug interaction network and human protein atlas were applied to obtain feature DNA methylation biomarkers. In total, we identified 2170 methylation-driven DEGs, including 1054 hypermethylatedlow-expression DEGs and 1116 hypomethylated-high-expression DEGs at the screening step. Further analysis screened total of eight feature DNA methylation biomarkers (RXRG, MET, PDGFRA, FCGR3A, VEGFA, CSF1R, FCGR1A and C1QA). Pathway analysis showed that aberrantly methylated DEGs mainly associated with transcriptional misregulation in cancer, MAPK signaling, and intrinsic apoptotic signaling in TC. Taken together, we have identified novel aberrantly methylated genes and pathways linked to TC, which might serve as novel biomarkers for precision diagnosis and disease treatment.

Growing Old Before Their Time: Measuring Aging.

In this issue of Pediatrics , Raffington et al1 describe an association between early childhood disadvantage and a measure of aging based on DNA methylation. Pediatricians will be intrigued by the concept that a genome-based measure can quantify and perhaps partially explain the well-known link between social equality and remote health effects, including obesity, hypertension, and cardiovascular disease, as well as an easily measured early predictor of whether an initiative to mitigate these morbidities is likely to be successful. Because some of the underlying biological concepts may not be familiar, it will be helpful to discuss DNA methylation and “methylation age.” The genetic code is embedded in the DNA sequence of the 4 canonical nucleobases: adenine, guanine, thymine, and cytosine. This sequence specifies the messenger RNA that codes proteins, several types of functionally active non–messenger RNAs, and diverse control and structural regions. DNA bases are also subject to chemical modification. The most relevant modification for this discussion is methylation of some cytosines (addition of CH3) to form 5-methylcytosine. Methylation of cytosine regulates the rate of RNA transcription and therefore regulates which proteins a cell expresses. During development, patterns of methylation change in a tissue-specific and age-dependent manner.2 … Address correspondence to Daniel A. Notterman, MD, MA, FAAP, Department of Molecular Biology, Princeton University, 219 Lewis Thomas Laboratory, Princeton, NJ 08544. E-mail: dan1{at}princeton.edu

Genome-wide methylation profiling identified novel differentially hypermethylated biomarker MPPED2 in colorectal cancer

BackgroundEpigenetic alternation is a common contributing factor to neoplastic transformation. Although previous studies have reported a cluster of aberrant promoter methylation changes associated with silencing of tumor suppressor genes, little is known concerning their sequential DNA methylation changes during the carcinogenetic process. The aim of the present study was to address a genome-wide search for identifying potentially important methylated changes and investigate the onset and pattern of methylation changes during the progression of colorectal neoplasia.MethodsA three-phase design was employed in this study. In the screening phase, DNA methylation profile of 12 pairs of colorectal cancer (CRC) and adjacent normal tissues was analyzed by using the Illumina MethylationEPIC BeadChip. Significant CpG sites were selected based on a cross-validation analysis from The Cancer Genome Atlas (TCGA) database. Methylation levels of candidate CpGs were assessed using pyrosequencing in the training dataset (tumor lesions and adjacent normal tissues from 46 CRCs) and the validation dataset (tumor lesions and paired normal tissues from 13 hyperplastic polyps, 129 adenomas, and 256 CRCs). A linear mixed-effects model was used to examine the incremental changes of DNA methylation during the progression of colorectal neoplasia.ResultsThe comparisons between normal and tumor samples in the screening phase revealed an extensive CRC-specific methylomic pattern with 174,006 (21%) methylated CpG sites, of which 22,232 (13%) were hyermethylated and 151,774 (87%) were hypomethylated. Hypermethylation mostly occurred in CpG islands with an overlap of gene promoters, while hypomethylation tended to be mapped far away from functional regions. Further cross validation analysis from TCGA dataset confirmed 265 hypermethylated promoters coupling with downregulated gene expression. Among which, hypermethylated changes in MEEPD2 promoter was successfully replicated in both training and validation phase. Significant hypermethylation appeared since precursor lesions with an extensive modification in CRCs. The linear mixed-effects modeling analysis found that a cumulative pattern of MPPED2 methylation changes from normal mucosa to hyperplastic polyp to adenoma, and to carcinoma (P < 0.001).ConclusionsOur findings indicate that epigenetic alterations of MPPED2 promoter region appear sequentially during the colorectal neoplastic progression. It might be able to serve as a promising biomarker for early diagnosis and stage surveillance of colorectal tumorigenesis.

Abstract WP551: Senescence-Induced Methylation Changes in Global DNA and Atherosclerosis-Related Genes in Human Vascular Endothelial Cells

Background: Endothelial senescence has been known as a pathophysiological alteration related to atherosclerosis development. We investigated whether methylation changes in global DNA and atherosclerosis-related genes are developed in in-vitro senescence of human vascular endothelial cells. Methods: We induced in-vitro replicative senescence by subculturing 5 human umbilical vein endothelial cell (HUVEC) lines from three to fifteen passages. Using DNA extracted from the 5th, 10th, and 15th passages of the 5 HUVECs, we evaluated LINE1 methylation for global methylation status as well as promoter methylation status for 5 genes ( AIRE1, ALOX12, FANK1, NETO1 , and SERHL2 ) profiled from carotid endarterectomy plaques for gene-specific methylation evaluations. Differences in LINE1 methylations and promoter methylations of the 5 genes were evaluated using pyrosequencing and compared among the 5th, 10th, and 15th passages of the 5 HUVECs using analysis of variance (ANOVA) tests. Results: LINE1 global methylation subsequently decreased as passages were advanced from 5 to 15 (p&lt;0.001). On gene-specific promoter methylation analysis, ALOX12 (p&lt;0.001) and SERHL2 (p&lt;0.001) showed significant passage-dependent increase of promoter methylation. AIRE1 and NETO1 also showed an increase of promoter methylation as passages increased, but without statistical significance. FANK1 exhibited an increase-decrease pattern of methylation changes showing an increase of methylation at passage 10 compared to passage 5, but showing a decrease in passage 15 (p=0.005). Conclusion: After replicative senescence for vascular endothelial cells, global methylation subsequently decreased. However, atherosclerosis-related genes showed either a increase or an increase-decrease of promoter methylations. In particular, the increase of ALOX12 methylation showed as a possible epigenetic marker related to vascular endothelial cell senescence.

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease

BackgroundMany genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. MethodsUsing bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively.ResultsWe found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn’s disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly.ConclusionsOur results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.

Combinatorial identification of DNA methylation patterns over age in the human brain.

BackgroundDNA methylation plays a key role in developmental processes, which is reflected in changing methylation patterns at specific CpG sites over the lifetime of an individual. The underlying mechanisms are complex and possibly affect multiple genes or entire pathways.ResultsWe applied a multivariate approach to identify combinations of CpG sites that undergo modifications when transitioning between developmental stages. Monte Carlo feature selection produced a list of ranked and statistically significant CpG sites, while rule-based models allowed for identifying particular methylation changes in these sites.Our rule-based classifier reports combinations of CpG sites, together with changes in their methylation status in the form of easy-to-read IF-THEN rules, which allows for identification of the genes associated with the underlying sites.ConclusionWe utilized machine learning and statistical methods to discretize decision class (age) values to get a general pattern of methylation changes over the lifespan. The CpG sites present in the significant rules were annotated to genes involved in brain formation, general development, as well as genes linked to cancer and Alzheimer’s disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1259-3) contains supplementary material, which is available to authorized users.

Abstract 3449: Epigenomics identifies conserved DNA methylation events associated with medulloblastoma development in humans and mice

Abstract Medulloblastoma, an invasive embryonal tumor of the cerebellum, is the most common malignant brain tumor of childhood. Genome-wide technologies have recently shown that gene-specific changes in DNA methylation status are a significant feature of medulloblastoma development. Mouse models of medulloblastoma may provide important systems in which to study the impact of these DNA methylation changes, however the epigenetic basis of these models has not been widely investigated. We therefore adopted a cross-species approach to identify epigenetic events of relevance in human and mouse tumors, and investigate their impact on medulloblastoma development. Using Illumina Goldengate DNA methylation arrays, we first identified a series of tumor-specific DNA methylation changes (measured relative to the normal cerebellum) which are strongly associated with activation of the Sonic hedgehog (SHH) signalling pathway, which occurs in approximately 25% of human primary tumors. From these, 12 candidate CpG sites, which lay within regions orthologous to the mouse genome, were selected for further investigation, alongside RASSF1A and COL1A2 CpG sites, which show frequent methylation in human primary tumors. The methylation status of these CpG sites was confirmed to be representative of their surrounding CpG islands in human primary tumors and control cerebellar samples, using bisulfite sequencing approaches. We next assessed whether the DNA methylation status of orthologous regions was altered in tumors from four independent mouse models driven by Shh pathway activation (Ptc1+/- Ink4c-/-; Smo/Smo; Sufu+/- p53-/-; p53-/- Ink4c-/-), compared to strain-matched control mouse cerebella. Tissue-specific methylation patterns in the normal cerebellum were conserved between mouse and human, for 10/14 of the candidate regions investigated. Three regions which showed hypermethylation in human medulloblastomas (DSC2, RASSF1A and TAL1) also showed evidence of hypermethylation in mouse tumors and five regions showed evidence of hypomethylation in both human and mouse tumors (CYP2E1, MMP9, SPDEF, TGFB1, and VAV1). CYP2E1 and VAV1 showed the most similar frequencies and patterns of methylation changes in mouse and human tumors, while other changes were either less frequent (RASSF1A), affected fewer CpG sites (TAL1, TGFB1, SPDEF) or had different tissue-specific methylation patterns (DSC2, MMP9) in mice. Ongoing work investigates the relationship of these methylation changes to gene expression and tumorigenesis. Assessment of the conservation of epigenetic changes between human tumors and disease relevant mouse models may help highlight those alterations most important in driving tumorigenesis, and identify candidate genes to take forward for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3449. doi:10.1158/1538-7445.AM2011-3449

Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression

Background and aimsAlthough aberrant methylation of key genes in the progression of colorectal neoplasia has been reported, no model-based analysis of the incremental changes through the intermediate adenoma stage has...

Methylation analysis of 79 patients with growth restriction reveals novel patterns of methylation change at imprinted loci

This study was an investigation of 79 patients referred to the Wessex Regional Genetics Laboratory with suspected Russell-Silver Syndrome or unexplained short stature/intra uterine growth restriction, warranting genetic investigation. Methylation status was analysed at target sequences within eleven imprinted loci (PLAGL1, IGF2R, PEG10, MEST1, GRB10, KCNQ1OT1, H19, IGF2P0, DLK1, PEG3, NESPAS). Thirty seven percent (37%) (29 of 79) of samples were shown to have a methylation abnormality. The commonest finding was a loss of methylation at H19 (23 of 29), as previously reported in Russell-Silver Syndrome. In addition, four of these patients had methylation anomalies at other loci, of whom two showed hypomethylation of multiple imprinted loci, and two showed a complete gain of methylation at IGF2R. This latter finding was also present in five other patients who did not have demonstrable changes at H19. In total, 7 of 79 patients showed a gain of methylation at IGF2R and this was significantly different from a normal control population of 267 individuals (P=0.002). This study in patients with growth restriction shows the importance of widening the epigenetic investigation to include multiple imprinted loci and highlights potential involvement of the IGF2R locus.

Epigenetic diagnostics of cancer — the application of DNA methylation markers

In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers.

Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.