Heterogeneity of DNA methylation in multifocal prostate cancer.

PubMed

Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

2015-01-01

Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p < 0.001). Hierarchical cluster analysis revealed a divergent methylation profile of paired PCa foci, while the foci from separate cases with biochemical recurrence showed similar methylation profile and the highest mean levels of DNA methylation. Our findings suggest that PCa tissue is heterogeneous, as between paired foci differences in DNA methylation status were found. Common epigenetic profile of recurrent tumors can be inferred from our data.

The Effect of Metabolic and Bariatric Surgery on DNA Methylation Patterns.

PubMed

Morcillo, Sonsoles; Macías-González, Manuel; Tinahones, Francisco J

2017-08-30

Metabolic and bariatric surgery (MBS) is considered to be the most effective treatment for obesity. Not only due to the significant weight reduction but also because of the many health benefits associated with it. In the last 5 years, several studies have suggested that epigenetic modifications could be involved in the mechanisms underlying the response to bariatric surgery. In this review, we will compile the different studies (2012-2017) concerning the effect of this surgical procedure on DNA methylation patterns (the most studied epigenetic marker) and its association with metabolic improvement. This is an emerging area, and currently, there are not many studies in the literature. The aim is to show what has been done so far and what the future direction in this emerging area might be. Recent findings have shown how metabolic and bariatric surgery modifies the DNA methylation profile of the specific genes associated with the pathophysiology of the disease. The studies were performed in morbidly obese subjects, mainly in women, with the aim of reducing weight and improving the obesity-associated comorbidities. DNA methylation has been measured both in specific tissue and in peripheral blood samples. In general, studies about site-specific DNA methylation have shown a change in the methylation profile after surgery, whereas the studies analyzing global DNA methylation are not so conclusive. Summing up, metabolic and bariatric surgery can modify the DNA methylation profile of different genes and contributes to the metabolic health benefits that are often seen after metabolic and bariatric surgery. Although there are still many issues to be resolved, the capacity to revert the DNA methylation profile of specific sites opens a window for searching for target markers to treat obesity-related comorbidities.

Concordance of DNA methylation profiles between breast core biopsy and surgical excision specimens containing ductal carcinoma in situ (DCIS).

PubMed

Chen, Youdinghuan; Marotti, Jonathan D; Jenson, Erik G; Onega, Tracy L; Johnson, Kevin C; Christensen, Brock C

2017-08-01

The utility and reliability of assessing molecular biomarkers for translational applications on pre-operative core biopsy specimens assume consistency of molecular profiles with larger surgical specimens. Whether DNA methylation in ductal carcinoma in situ (DCIS), measured in core biopsy and surgical specimens are similar, remains unclear. Here, we compared genome-scale DNA methylation measured in matched core biopsy and surgical specimens from DCIS, including specific DNA methylation biomarkers of subsequent invasive cancer. DNA was extracted from guided 2mm cores of formalin fixed paraffin embedded (FFPE) specimens, bisulfite-modified, and measured on the Illumina HumanMethylation450 BeadChip. DNA methylation profiles of core biopsies exhibited high concordance with matched surgical specimens. Within-subject variability in DNA methylation was significantly lower than between-subject variability (all P<2.20E-16). In 641 CpGs whose methylation was related with increased hazard of invasive breast cancer, lower within-subject than between-subject variability was observed in 92.3% of the study participants (P<0.05). Between patient-matched core biopsy and surgical specimens, <0.6% of CpGs measured had changes in median DNA methylation >15%, and a pathway analysis of these CpGs indicated enrichment for genes related with wound healing. Our results indicate that DNA methylation measured in core biopsies are representative of the matched surgical specimens and suggest that DCIS biomarkers measured in core biopsies can inform clinical decision-making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

DNA methylation profile distinguishes clear cell sarcoma of the kidney from other pediatric renal tumors.

PubMed

Ueno, Hitomi; Okita, Hajime; Akimoto, Shingo; Kobayashi, Kenichiro; Nakabayashi, Kazuhiko; Hata, Kenichiro; Fujimoto, Junichiro; Hata, Jun-Ichi; Fukuzawa, Masahiro; Kiyokawa, Nobutaka

2013-01-01

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms' tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing's sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms' tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.

The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana.

PubMed

Wollmann, Heike; Stroud, Hume; Yelagandula, Ramesh; Tarutani, Yoshiaki; Jiang, Danhua; Jing, Li; Jamge, Bhagyshree; Takeuchi, Hidenori; Holec, Sarah; Nie, Xin; Kakutani, Tetsuji; Jacobsen, Steven E; Berger, Frédéric

2017-05-18

Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

PubMed

Nilsson, Emil K; Boström, Adrian E; Mwinyi, Jessica; Schiöth, Helgi B

2016-06-01

Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data.

Changes in Liver Cell DNA Methylation Status in Diabetic Mice Affect Its FT-IR Characteristics

PubMed Central

Vidal, Benedicto de Campos; Ghiraldini, Flávia Gerelli; Mello, Maria Luiza S.

2014-01-01

Background Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. Methodology/Principal Findings The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Conclusions/Significance Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas –CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH3 groups. Other spectral differences were found at 1700–1500 cm−1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice. PMID:25019512

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study

PubMed Central

Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard

2018-01-01

Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

PubMed

Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard

2018-01-01

To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

Early life lead exposure causes gender-specific changes in the DNA methylation profile of DNA extracted from dried blood spots

PubMed Central

Sen, Arko; Heredia, Nicole; Senut, Marie-Claude; Hess, Matthew; Land, Susan; Qu, Wen; Hollacher, Kurt; Dereski, Mary O; Ruden, Douglas M

2015-01-01

Aims In this paper, we tested the hypothesis that early life lead (Pb) exposure associated DNA methylation (5mC) changes are dependent on the sex of the child and can serve as biomarkers for Pb exposure. Methods In this pilot study, we measured the 5mC profiles of DNA extracted from dried blood spots (DBS) in a cohort of 43 children (25 males and 18 females; ages from 3 months to 5 years) from Detroit. Result & Discussion We found that the effect of Pb-exposure on the 5-mC profiles can be separated into three subtypes: affected methylation loci which are conserved irrespective of the sex of the child (conserved); affected methylation loci unique to males (male-specific); and affected methylation loci unique to females (female-specific). PMID:26077427

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PubMed

Elliott, Giles O; Johnson, Ian T; Scarll, Jane; Dainty, Jack; Williams, Elizabeth A; Garg, D; Coupe, Amanda; Bradburn, David M; Mathers, John C; Belshaw, Nigel J

2013-01-01

The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

DNA methylation-based reclassification of olfactory neuroblastoma.

PubMed

Capper, David; Engel, Nils W; Stichel, Damian; Lechner, Matt; Glöss, Stefanie; Schmid, Simone; Koelsche, Christian; Schrimpf, Daniel; Niesen, Judith; Wefers, Annika K; Jones, David T W; Sill, Martin; Weigert, Oliver; Ligon, Keith L; Olar, Adriana; Koch, Arend; Forster, Martin; Moran, Sebastian; Tirado, Oscar M; Sáinz-Japeado, Miguel; Mora, Jaume; Esteller, Manel; Alonso, Javier; Del Muro, Xavier Garcia; Paulus, Werner; Felsberg, Jörg; Reifenberger, Guido; Glatzel, Markus; Frank, Stephan; Monoranu, Camelia M; Lund, Valerie J; von Deimling, Andreas; Pfister, Stefan; Buslei, Rolf; Ribbat-Idel, Julika; Perner, Sven; Gudziol, Volker; Meinhardt, Matthias; Schüller, Ulrich

2018-05-05

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

Quantitative DNA Methylation Profiling in Cancer.

PubMed

Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

2016-01-01

Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

Whole DNA methylome profiling in mice exposed to secondhand smoke.

PubMed

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-11-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.

Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury.

PubMed

Haghighi, Fatemeh; Ge, Yongchao; Chen, Sean; Xin, Yurong; Umali, Michelle U; De Gasperi, Rita; Gama Sosa, Miguel A; Ahlers, Stephen T; Elder, Gregory A

2015-08-15

Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that have been associated with long-term anxiogenic manifestations weeks or months following the initial exposures. Rat frontal cortex eight months post-exposure was used for cell sorting of whole brain tissue into neurons and glia. We interrogated DNA methylation profiles in these cells using Expanded Reduced Representation Bisulfite Sequencing. We obtained data for millions of cytosines, showing distinct methylation profiles for neurons and glia and an increase in global methylation in neuronal versus glial cells (p<10(-7)). We detected DNA methylation perturbations in blast overpressure-exposed animals, compared with sham blast controls, within 458 and 379 genes in neurons and glia, respectively. Differentially methylated neuronal genes showed enrichment in cell death and survival and nervous system development and function, including genes involved in transforming growth factor β and nitric oxide signaling. Functional validation via gene expression analysis of 30 differentially methylated neuronal and glial genes showed a 1.2 fold change in gene expression of the serotonin N-acetyltransferase gene (Aanat) in blast animals (p<0.05). These data provide the first genome-based evidence for changes in DNA methylation induced in response to multiple blast overpressure exposures. In particular, increased methylation and decreased gene expression were observed in the Aanat gene, which is involved in converting serotonin to the circadian hormone melatonin and is implicated in sleep disturbance and depression associated with traumatic brain injury.

DNMT1-interacting RNAs block gene specific DNA methylation

PubMed Central

Di Ruscio, Annalisa; Ebralidze, Alexander K.; Benoukraf, Touati; Amabile, Giovanni; Goff, Loyal A.; Terragni, Joylon; Figueroa, Maria Eugenia; De Figureido Pontes, Lorena Lobo; Alberich-Jorda, Meritxell; Zhang, Pu; Wu, Mengchu; D’Alò, Francesco; Melnick, Ari; Leone, Giuseppe; Ebralidze, Konstantin K.; Pradhan, Sriharsa; Rinn, John L.; Tenen, Daniel G.

2013-01-01

Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease. PMID:24107992

Profile analysis and prediction of tissue-specific CpG island methylation classes

PubMed Central

2009-01-01

Background The computational prediction of DNA methylation has become an important topic in the recent years due to its role in the epigenetic control of normal and cancer-related processes. While previous prediction approaches focused merely on differences between methylated and unmethylated DNA sequences, recent experimental results have shown the presence of much more complex patterns of methylation across tissues and time in the human genome. These patterns are only partially described by a binary model of DNA methylation. In this work we propose a novel approach, based on profile analysis of tissue-specific methylation that uncovers significant differences in the sequences of CpG islands (CGIs) that predispose them to a tissue- specific methylation pattern. Results We defined CGI methylation profiles that separate not only between constitutively methylated and unmethylated CGIs, but also identify CGIs showing a differential degree of methylation across tissues and cell-types or a lack of methylation exclusively in sperm. These profiles are clearly distinguished by a number of CGI attributes including their evolutionary conservation, their significance, as well as the evolutionary evidence of prior methylation. Additionally, we assess profile functionality with respect to the different compartments of protein coding genes and their possible use in the prediction of DNA methylation. Conclusion Our approach provides new insights into the biological features that determine if a CGI has a functional role in the epigenetic control of gene expression and the features associated with CGI methylation susceptibility. Moreover, we show that the ability to predict CGI methylation is based primarily on the quality of the biological information used and the relationships uncovered between different sources of knowledge. The strategy presented here is able to predict, besides the constitutively methylated and unmethylated classes, two more tissue specific methylation classes conserving the accuracy provided by leading binary methylation classification methods. PMID:19383127

Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: a bioinformatics meta-analysis.

PubMed

Brown, William M

2015-12-01

Epigenetics is the study of processes--beyond DNA sequence alteration--producing heritable characteristics. For example, DNA methylation modifies gene expression without altering the nucleotide sequence. A well-studied DNA methylation-based phenomenon is genomic imprinting (ie, genotype-independent parent-of-origin effects). We aimed to elucidate: (1) the effect of exercise on DNA methylation and (2) the role of imprinted genes in skeletal muscle gene networks (ie, gene group functional profiling analyses). Gene ontology (ie, gene product elucidation)/meta-analysis. 26 skeletal muscle and 86 imprinted genes were subjected to g:Profiler ontology analysis. Meta-analysis assessed exercise-associated DNA methylation change. g:Profiler found four muscle gene networks with imprinted loci. Meta-analysis identified 16 articles (387 genes/1580 individuals) associated with exercise. Age, method, sample size, sex and tissue variation could elevate effect size bias. Only skeletal muscle gene networks including imprinted genes were reported. Exercise-associated effect sizes were calculated by gene. Age, method, sample size, sex and tissue variation were moderators. Six imprinted loci (RB1, MEG3, UBE3A, PLAGL1, SGCE, INS) were important for muscle gene networks, while meta-analysis uncovered five exercise-associated imprinted loci (KCNQ1, MEG3, GRB10, L3MBTL1, PLAGL1). DNA methylation decreased with exercise (60% of loci). Exercise-associated DNA methylation change was stronger among older people (ie, age accounted for 30% of the variation). Among older people, genes exhibiting DNA methylation decreases were part of a microRNA-regulated gene network functioning to suppress cancer. Imprinted genes were identified in skeletal muscle gene networks and exercise-associated DNA methylation change. Exercise-associated DNA methylation modification could rewind the 'epigenetic clock' as we age. CRD42014009800. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

EFEMP1 as a novel DNA methylation marker for prostate cancer: array-based DNA methylation and expression profiling.

PubMed

Kim, Yong-June; Yoon, Hyung-Yoon; Kim, Seon-Kyu; Kim, Young-Won; Kim, Eun-Jung; Kim, Isaac Yi; Kim, Wun-Jae

2011-07-01

Abnormal DNA methylation is associated with many human cancers. The aim of the present study was to identify novel methylation markers in prostate cancer (PCa) by microarray analysis and to test whether these markers could discriminate normal and PCa cells. Microarray-based DNA methylation and gene expression profiling was carried out using a panel of PCa cell lines and a control normal prostate cell line. The methylation status of candidate genes in prostate cell lines was confirmed by real-time reverse transcriptase-PCR, bisulfite sequencing analysis, and treatment with a demethylation agent. DNA methylation and gene expression analysis in 203 human prostate specimens, including 106 PCa and 97 benign prostate hyperplasia (BPH), were carried out. Further validation using microarray gene expression data from the Gene Expression Omnibus (GEO) was carried out. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) was identified as a lead candidate methylation marker for PCa. The gene expression level of EFEMP1 was significantly higher in tissue samples from patients with BPH than in those with PCa (P < 0.001). The sensitivity and specificity of EFEMP1 methylation status in discriminating between PCa and BPH reached 95.3% (101 of 106) and 86.6% (84 of 97), respectively. From the GEO data set, we confirmed that the expression level of EFEMP1 was significantly different between PCa and BPH. Genome-wide characterization of DNA methylation profiles enabled the identification of EFEMP1 aberrant methylation patterns in PCa. EFEMP1 might be a useful indicator for the detection of PCa.

The DNA methylation profile of oocytes in mice with hyperinsulinaemia and hyperandrogenism as detected by single-cell level whole genome bisulphite sequencing (SC-WGBS) technology.

PubMed

Li, Qian-Nan; Guo, Lei; Hou, Yi; Ou, Xiang-Hong; Liu, Zhonghua; Sun, Qing-Yuan

2018-06-22

Polycystic ovary syndrome (PCOS), a familial aggregation disease that causes anovulation in women, has well-recognised characteristics, two of which are hyperinsulinaemia and hyperandrogenaemia. To determine whether the DNA methylation status is altered in oocytes by high insulin and androgen levels, we generated a mouse model with hyperinsulinaemia and hyperandrogenaemia by injection of insulin and human chorionic gonadotrophin and investigated DNA methylation changes through single-cell level whole genome bisulphite sequencing. Our results showed that hyperinsulinaemia and hyperandrogenaemia had no significant effects on the global DNA methylation profile and different functional regions of genes, but did alter methylation status of some genes, which were significantly enriched in 17 gene ontology (GO) terms (P<0.05) by GO analysis. Among differently methylated genes, some were related to the occurrence of PCOS. Based on our results, we suggest that hyperinsulinaemia and hyperandrogenaemia may cause changes in some DNA methylation loci in oocytes.

Detecting differential DNA methylation from sequencing of bisulfite converted DNA of diverse species.

PubMed

Huh, Iksoo; Wu, Xin; Park, Taesung; Yi, Soojin V

2017-07-21

DNA methylation is one of the most extensively studied epigenetic modifications of genomic DNA. In recent years, sequencing of bisulfite-converted DNA, particularly via next-generation sequencing technologies, has become a widely popular method to study DNA methylation. This method can be readily applied to a variety of species, dramatically expanding the scope of DNA methylation studies beyond the traditionally studied human and mouse systems. In parallel to the increasing wealth of genomic methylation profiles, many statistical tools have been developed to detect differentially methylated loci (DMLs) or differentially methylated regions (DMRs) between biological conditions. We discuss and summarize several key properties of currently available tools to detect DMLs and DMRs from sequencing of bisulfite-converted DNA. However, the majority of the statistical tools developed for DML/DMR analyses have been validated using only mammalian data sets, and less priority has been placed on the analyses of invertebrate or plant DNA methylation data. We demonstrate that genomic methylation profiles of non-mammalian species are often highly distinct from those of mammalian species using examples of honey bees and humans. We then discuss how such differences in data properties may affect statistical analyses. Based on these differences, we provide three specific recommendations to improve the power and accuracy of DML and DMR analyses of invertebrate data when using currently available statistical tools. These considerations should facilitate systematic and robust analyses of DNA methylation from diverse species, thus advancing our understanding of DNA methylation. © The Author 2017. Published by Oxford University Press.

Genome-wide comparative analysis of DNA methylation between soybean cytoplasmic male-sterile line NJCMS5A and its maintainer NJCMS5B.

PubMed

Li, Yanwei; Ding, Xianlong; Wang, Xuan; He, Tingting; Zhang, Hao; Yang, Longshu; Wang, Tanliu; Chen, Linfeng; Gai, Junyi; Yang, Shouping

2017-08-10

DNA methylation is an important epigenetic modification. It can regulate the expression of many key genes without changing the primary structure of the genomic DNA, and plays a vital role in the growth and development of the organism. The genome-wide DNA methylation profile of the cytoplasmic male sterile (CMS) line in soybean has not been reported so far. In this study, genome-wide comparative analysis of DNA methylation between soybean CMS line NJCMS5A and its maintainer NJCMS5B was conducted by whole-genome bisulfite sequencing. The results showed 3527 differentially methylated regions (DMRs) and 485 differentially methylated genes (DMGs), including 353 high-credible methylated genes, 56 methylated genes coding unknown protein and 76 novel methylated genes with no known function were identified. Among them, 25 DMRs were further validated that the genome-wide DNA methylation data were reliable through bisulfite treatment, and 9 DMRs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. Finally, 8 key DMGs possibly associated with soybean CMS were identified. Genome-wide DNA methylation profile of the soybean CMS line NJCMS5A and its maintainer NJCMS5B was obtained for the first time. Several specific DMGs which participated in pollen and flower development were further identified to be probably associated with soybean CMS. This study will contribute to further understanding of the molecular mechanism behind soybean CMS.

Whole DNA methylome profiling in mice exposed to secondhand smoke

PubMed Central

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-01-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease. PMID:23051858

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

PubMed

Scala, Giovanni; Affinito, Ornella; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Chiariotti, Lorenzo; Cocozza, Sergio

2016-11-25

CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

A DNA methylation map of human cancer at single base-pair resolution

PubMed Central

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-01-01

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities

PubMed Central

Sayols, Sergi; Chianese, Chiara; Giachini, Claudia; Heyn, Holger; Esteller, Manel

2012-01-01

Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research. PMID:23071498

Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer?

PubMed

Karsli-Ceppioglu, Seher; Ngollo, Marjolaine; Adjakly, Mawussi; Dagdemir, Aslihan; Judes, Gaëlle; Lebert, André; Boiteux, Jean-Paul; Penault-LLorca, Frédérique; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

2015-04-01

In prostate cancer, DNA methylation is significantly associated with tumor initiation, progression, and metastasis. Previous studies have suggested that soy phytoestrogens might regulate DNA methylation at individual candidate gene loci and that they play a crucial role as potential therapeutic agents for prostate cancer. The purpose of our study was to examine the modulation effects of phytoestrogens on a genome-wide scale in regards to DNA methylation in prostate cancer. Prostate cancer cell lines DU-145 and LNCaP were treated with 40 μM of genistein and 110 μM of daidzein. DNMT inhibitor 5-azacytidine (2 μM) and the methylating agent budesonide (2 μM) were used to compare their demethylation/methylation effects with phytoestrogens. The regulatory effects of phytoestrogens on DNA methylation were analyzed by using a methyl-DNA immunoprecipitation method coupled with Human DNA Methylation Microarrays (MeDIP-chip). We observed that the methylation profiles of 58 genes were altered by genistein and daidzein treatments in DU-145 and LNCaP prostate cancer cells. In addition, the methylation frequencies of the MAD1L1, TRAF7, KDM4B, and hTERT genes were remarkably modified by genistein treatment. Our results suggest that the modulation effects of phytoestrogens on DNA methylation essentially lead to inhibition of cell growth and induction of apoptosis. Genome-wide methylation profiling reported here suggests that epigenetic regulation mechanisms and, by extension, epigenetics-driven novel therapeutic candidates warrant further consideration in future "omics" studies of prostate cancer.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles.

PubMed

Souren, Nicole Y P; Lutsik, Pavlo; Gasparoni, Gilles; Tierling, Sascha; Gries, Jasmin; Riemenschneider, Matthias; Fryns, Jean-Pierre; Derom, Catherine; Zeegers, Maurice P; Walter, Jörn

2013-05-26

Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes. Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences. Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

PubMed Central

Fernández, Agustín F.; Bayón, Gustavo F.; Urdinguio, Rocío G.; Toraño, Estela G.; García, María G.; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M.; Riancho, José A.; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A.

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. PMID:25271306

Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

PubMed Central

Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

2015-01-01

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID:25552301

DNA modifications in models of alcohol use disorders

PubMed Central

Tulisiak, Christopher T.; Harris, R. Adron; Ponomarev, Igor

2016-01-01

Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol use disorders (AUD). Gene expression is, in part, controlled by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types and clarifying how alcohol influences the methylomes of cell type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder. PMID:27865607

Association of Childhood Chronic Physical Aggression with a DNA Methylation Signature in Adult Human T Cells

PubMed Central

Guillemin, Claire; Vitaro, Frank; Côté, Sylvana M.; Hallett, Michael; Tremblay, Richard E.; Szyf, Moshe

2014-01-01

Background Chronic physical aggression (CPA) is characterized by frequent use of physical aggression from early childhood to adolescence. Observed in approximately 5% of males, CPA is associated with early childhood adverse environments and long-term negative consequences. Alterations in DNA methylation, a covalent modification of DNA that regulates genome function, have been associated with early childhood adversity. Aims To test the hypothesis that a trajectory of chronic physical aggression during childhood is associated with a distinct DNA methylation profile during adulthood. Methods We analyzed genome-wide promoter DNA methylation profiles of T cells from two groups of adult males assessed annually for frequency of physical aggression between 6 and 15 years of age: a group with CPA and a control group. Methylation profiles covering the promoter regions of 20 000 genes and 400 microRNAs were generated using MeDIP followed by hybridization to microarrays. Results In total, 448 distinct gene promoters were differentially methylated in CPA. Functionally, many of these genes have previously been shown to play a role in aggression and were enriched in biological pathways affected by behavior. Their locations in the genome tended to form clusters spanning millions of bases in the genome. Conclusions This study provides evidence of clustered and genome-wide variation in promoter DNA methylation in young adults that associates with a history of chronic physical aggression from 6 to 15 years of age. However, longitudinal studies of methylation during early childhood will be necessary to determine if and how this methylation variation in T cells DNA plays a role in early development of chronic physical aggression. PMID:24691403

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

PubMed

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

2017-04-11

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

PubMed Central

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

2017-01-01

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles. PMID:28398335

Epigenomic profiling of DNA methylation in paired prostate cancer versus adjacent benign tissue

PubMed Central

Geybels, Milan S.; Zhao, Shanshan; Wong, Chao-Jen; Bibikova, Marina; Klotzle, Brandy; Wu, Michael; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

2016-01-01

Background Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites. Methods The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed. Results In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value <0.001), the majority of which were hypermethylated (n = 1,946; 95%). DNA methylation profiles accurately distinguished between PCa and benign tissue samples. Twenty-seven top-ranked hypermethylated CpGs had a mean methylation difference of at least 40% between tissue types, which included 25 CpGs in 17 genes. Furthermore, for ten genes over 50% of promoter region CpGs were hypermethylated in PCa versus benign tissue. The top-ranked differentially methylated genes included three genes that were associated with both promoter hypermethylation and reduced gene expression: SCGB3A1, HIF3A, and AOX1. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. Conclusions This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets. PMID:26383847

Epigenomic profiling of DNA methylation in paired prostate cancer versus adjacent benign tissue.

PubMed

Geybels, Milan S; Zhao, Shanshan; Wong, Chao-Jen; Bibikova, Marina; Klotzle, Brandy; Wu, Michael; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L

2015-12-01

Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites. The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed. In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value < 0.001), the majority of which were hypermethylated (n = 1,946; 95%). DNA methylation profiles accurately distinguished between PCa and benign tissue samples. Twenty-seven top-ranked hypermethylated CpGs had a mean methylation difference of at least 40% between tissue types, which included 25 CpGs in 17 genes. Furthermore, for 10 genes over 50% of promoter region CpGs were hypermethylated in PCa versus benign tissue. The top-ranked differentially methylated genes included three genes that were associated with both promoter hypermethylation and reduced gene expression: SCGB3A1, HIF3A, and AOX1. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets. © 2015 Wiley Periodicals, Inc.

DNA methylation in insects: on the brink of the epigenomic era.

PubMed

Glastad, K M; Hunt, Brendan G; Yi, S V; Goodisman, M A D

2011-10-01

DNA methylation plays an important role in gene regulation in animals. However, the evolution and function of DNA methylation has only recently emerged as the subject of widespread study in insects. In this review we profile the known distribution of DNA methylation systems across insect taxa and synthesize functional inferences from studies of DNA methylation in insects and vertebrates. Unlike vertebrate genomes, which tend to be globally methylated, DNA methylation is primarily targeted to genes in insects. Nevertheless, mounting evidence suggests that a specialized role exists for genic methylation in the regulation of transcription, and possibly mRNA splicing, in both insects and mammals. Investigations in several insect taxa further reveal that DNA methylation is preferentially targeted to ubiquitously expressed genes and may play a key role in the regulation of phenotypic plasticity. We suggest that insects are particularly amenable to advancing our understanding of the biological functions of DNA methylation, because insects are evolutionarily diverse, display several lineage-specific losses of DNA methylation and possess tractable patterns of DNA methylation in moderately sized genomes. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

[Applications of DNA methylation markers in forensic medicine].

PubMed

Zhao, Gui-sen; Yang, Qing-en

2005-02-01

DNA methylation is a post-replication modification that is predominantly found in cytosines of the dinucleotide sequence CpG. Epigenetic information is stored in the distribution of the modified base 5-methylcytosine. DNA methylation profiles represent a more chemically and biologically stable source of molecular diagnostic information than RNA or most proteins. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic. In the past decade, DNA methylation analysis has been revolutionized by two technological advances--bisulphite modification of DNA and methylation-specific polymerase chain reaction (MSP). The methylation pattern of human genome is space-time specific, sex-specific, parent-of-origin specific and disease specific, providing us an alternative way to solve forensic problems.

Forensic DNA methylation profiling from minimal traces: How low can we go?

PubMed

Naue, Jana; Hoefsloot, Huub C J; Kloosterman, Ate D; Verschure, Pernette J

2018-03-01

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg - 1ng) is rather the norm than the exception when analyzing forensic DNA samples. This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications. As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Understanding the relationship between DNA methylation and histone lysine methylation☆

PubMed Central

Rose, Nathan R.; Klose, Robert J.

2014-01-01

DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24560929

Analysis of DNA methylation in FFPE tissues using the MethyLight technology.

PubMed

Dallol, Ashraf; Al-Ali, Waleed; Al-Shaibani, Amina; Al-Mulla, Fahd

2011-01-01

Novel biomarkers are sought after by mining DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Such tissues offer the great advantage of often having complete clinical data (including survival), as well as the tissues are amenable for laser microdissection targeting specific tissue areas. Downstream analysis of such DNA includes mutational screens and methylation profiling. Screening for mutations by sequencing requires a significant amount of DNA for PCR and cycle sequencing. This is self-inhibitory if the gene screened has a large number of exons. Profiling DNA methylation using the MethyLight technology circumvents this problem and allows for the mining of several biomarkers from DNA extracted from a single microscope slide of the tissue of interest. We describe in this chapter a detailed protocol for MethyLight and its use in the determination of CpG Island Methylator Phenotype status in FFPE colorectal cancer samples.

Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

PubMed Central

Marsh, Adam G.; Cottrell, Matthew T.; Goldman, Morton F.

2016-01-01

Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites (p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression. PMID:27853465

Neurobehavior related to epigenetic differences in preterm infants

PubMed Central

Lester, Barry M; Marsit, Carmen J; Giarraputo, James; Hawes, Katheleen; LaGasse, Linda L; Padbury, James F

2015-01-01

Preterm birth is associated with medical problems affecting the neuroendocrine system, altering cortisol levels resulting in negative effects on newborn neurobehavior. Newborn neurobehavior is regulated by DNA methylation of NR3C1 and HSD11B2. Aim: Determine if methylation of HSD11B2 and NR3C1 is associated with neurobehavioral profiles in preterm infants. Patients & methods: Neurobehavior was measured before discharge from the hospital in 67 preterm infants. Cheek swabs were collected for DNA extraction. Results: Infants with the high-risk neurobehavioral profile showed more methylation than infants with the low-risk neurobehavioral profile at CpG3 for NR3C1 and less methylation of CpG3 for HSD11B2. Infants with these profiles were more likely to have increased methylation of NR3C1 and decreased methylation of HSD11B2 at these CpG sites. Conclusion: Preterm birth is associated with epigenetic differences in genes that regulate cortisol levels related to high-risk neurobehavioral profiles. PMID:26585459

DNA methylation patterns of behavior-related gene promoter regions dissect the gray wolf from domestic dog breeds.

PubMed

Banlaki, Zsofia; Cimarelli, Giulia; Viranyi, Zsofia; Kubinyi, Eniko; Sasvari-Szekely, Maria; Ronai, Zsolt

2017-06-01

A growing body of evidence highlights the relationship between epigenetics, especially DNA methylation, and population divergence as well as speciation. However, little is known about how general the phenomenon of epigenetics-wise separation of different populations is, or whether population assignment is, possible based on solely epigenetic marks. In the present study, we compared DNA methylation profiles between four different canine populations: three domestic dog breeds and their ancestor the gray wolf. Altogether, 79 CpG sites constituting the 65 so-called CpG units located in the promoter regions of genes affecting behavioral and temperamental traits (COMT, HTR1A, MAOA, OXTR, SLC6A4, TPH1, WFS1)-regions putatively targeted during domestication and breed selection. Methylation status of buccal cells was assessed using EpiTYPER technology. Significant inter-population methylation differences were found in 52.3% of all CpG units investigated. DNA methylation profile-based hierarchical cluster analysis indicated an unambiguous segregation of wolf from domestic dog. In addition, one of the three dog breeds (Golden Retriever) investigated also formed a separate, autonomous group. The findings support that population segregation is interrelated with shifts in DNA methylation patterns, at least in putative selection target regions, and also imply that epigenetic profiles could provide a sufficient basis for population assignment of individuals.

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

PubMed Central

Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500–2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation. PMID:22802633

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

PubMed Central

2013-01-01

Background Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes. Results Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences. Conclusions Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies. PMID:23706164

MethBank: a database integrating next-generation sequencing single-base-resolution DNA methylation programming data.

PubMed

Zou, Dong; Sun, Shixiang; Li, Rujiao; Liu, Jiang; Zhang, Jing; Zhang, Zhang

2015-01-01

DNA methylation plays crucial roles during embryonic development. Here we present MethBank (http://dnamethylome.org), a DNA methylome programming database that integrates the genome-wide single-base nucleotide methylomes of gametes and early embryos in different model organisms. Unlike extant relevant databases, MethBank incorporates the whole-genome single-base-resolution methylomes of gametes and early embryos at multiple different developmental stages in zebrafish and mouse. MethBank allows users to retrieve methylation levels, differentially methylated regions, CpG islands, gene expression profiles and genetic polymorphisms for a specific gene or genomic region. Moreover, it offers a methylome browser that is capable of visualizing high-resolution DNA methylation profiles as well as other related data in an interactive manner and thus is of great helpfulness for users to investigate methylation patterns and changes of gametes and early embryos at different developmental stages. Ongoing efforts are focused on incorporation of methylomes and related data from other organisms. Together, MethBank features integration and visualization of high-resolution DNA methylation data as well as other related data, enabling identification of potential DNA methylation signatures in different developmental stages and accordingly providing an important resource for the epigenetic and developmental studies. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm

USDA-ARS?s Scientific Manuscript database

Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperms through comparison with three bovine somatic tissues (mammary grand, brain and blood). Large differences between them were observed in the methylation patterns of global CpGs, pericentromeric satellites, p...

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

PubMed

Fernández, Agustín F; Bayón, Gustavo F; Urdinguio, Rocío G; Toraño, Estela G; García, María G; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M; Riancho, José A; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A; Fraga, Mario F

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. © 2015 Fernández et al.; Published by Cold Spring Harbor Laboratory Press.

A collaborative exercise on DNA methylation based body fluid typing.

PubMed

Jung, Sang-Eun; Cho, Sohee; Antunes, Joana; Gomes, Iva; Uchimoto, Mari L; Oh, Yu Na; Di Giacomo, Lisa; Schneider, Peter M; Park, Min Sun; van der Meer, Dieudonne; Williams, Graham; McCord, Bruce; Ahn, Hee-Jung; Choi, Dong Ho; Lee, Yang Han; Lee, Soong Deok; Lee, Hwan Young

2016-10-01

A collaborative exercise on DNA methylation based body fluid identification was conducted by seven laboratories. For this project, a multiplex methylation SNaPshot reaction composed of seven CpG markers was used for the identification of four body fluids, including blood, saliva, semen, and vaginal fluid. A total of 30 specimens were prepared and distributed to participating laboratories after thorough testing. The required experiments included four increasingly complex tasks: (1) CE of a purified single-base extension reaction product, (2) multiplex PCR and multiplex single-base extension reaction of bisulfite-modified DNA, (3) bisulfite conversion of genomic DNA, and (4) extraction of genomic DNA from body fluid samples. In tasks 2, 3 and 4, one or more mixtures were analyzed, and specimens containing both known and unknown body fluid sources were used. Six of the laboratories generated consistent body fluid typing results for specimens of bisulfite-converted DNA and genomic DNA. One laboratory failed to set up appropriate conditions for capillary analysis of reference single-base extension products. In general, variation in the values obtained for DNA methylation analysis between laboratories increased with the complexity of the required experiments. However, all laboratories concurred on the interpretation of the DNA methylation profiles produced. Although the establishment of interpretational guidelines on DNA methylation based body fluid identification has yet to be performed, this study supports the addition of DNA methylation profiling to forensic body fluid typing. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile

PubMed Central

Dehghanizadeh, Somaye; Khoddami, Vahid; Mosbruger, Timothy L.; Hammoud, Sue S.; Edes, Kornelia; Berry, Therese S.; Done, Michelle; Samowitz, Wade S.; DiSario, James A.; Luba, Daniel G.; Burt, Randall W.

2018-01-01

Background Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples. Methods We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps. Results Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no additional major genetic mutations detected. The occurrence of BRAF-V600E was coincident with a unique DNA methylation pattern revealing a set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples. These methylation patterns effectively distinguished sessile serrated polys from adenomatous polyps and did so more effectively than parallel gene expression profiles. Conclusions This study provides an important example of a single oncogenic mutation leading to reproducible global DNA methylation changes. These methylated markers are specific to SSPs and could be of important clinical relevance for the early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing. PMID:29590112

DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

PubMed Central

Liang, Gangning; Weisenberger, Daniel J.

2017-01-01

ABSTRACT DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. PMID:28358281

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status

PubMed Central

Li, Dongming; Palanca, Ana Marie S; Won, So Youn; Gao, Lei; Feng, Ying; Vashisht, Ajay A; Liu, Li; Zhao, Yuanyuan; Liu, Xigang; Wu, Xiuyun; Li, Shaofang; Le, Brandon; Kim, Yun Ju; Yang, Guodong; Li, Shengben; Liu, Jinyuan; Wohlschlegel, James A; Guo, Hongwei; Mo, Beixin; Chen, Xuemei; Law, Julie A

2017-01-01

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing. DOI: http://dx.doi.org/10.7554/eLife.19893.001 PMID:28452714

DNA modifications in models of alcohol use disorders.

PubMed

Tulisiak, Christopher T; Harris, R Adron; Ponomarev, Igor

2017-05-01

Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol-use disorders (AUD). Gene expression is controlled, in part, by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types, thus clarifying how alcohol influences the methylomes of cell-type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder. Copyright © 2016 Elsevier Inc. All rights reserved.

Basic mechanics of DNA methylation and the unique landscape of the DNA methylome in metal-induced carcinogenesis.

PubMed

Brocato, Jason; Costa, Max

2013-07-01

DNA methylation plays an intricate role in the regulation of gene expression and events that compromise the integrity of the methylome may potentially contribute to disease development. DNA methylation is a reversible and regulatory modification that elicits a cascade of events leading to chromatin condensation and gene silencing. In general, normal cells are characterized by gene-specific hypomethylation and global hypermethylation, while cancer cells portray a reverse profile to this norm. The unique methylome displayed in cancer cells is induced after exposure to carcinogenic metals such as nickel, arsenic, cadmium, and chromium (VI). These metals alter the DNA methylation profile by provoking both hyper- and hypo-methylation events. The metal-stimulated deviations to the methylome are possible mechanisms for metal-induced carcinogenesis and may provide potential biomarkers for cancer detection. Development of therapies based on the cancer methylome requires further research including human studies that supply results with larger impact and higher human relevance.

Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

PubMed Central

Long, Hannah K; Sims, David; Heger, Andreas; Blackledge, Neil P; Kutter, Claudia; Wright, Megan L; Grützner, Frank; Odom, Duncan T; Patient, Roger; Ponting, Chris P; Klose, Robert J

2013-01-01

Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI: http://dx.doi.org/10.7554/eLife.00348.001 PMID:23467541

Inferring genome-wide interplay landscape between DNA methylation and transcriptional regulation.

PubMed

Tang, Binhua; Wang, Xin

2015-01-01

DNA methylation and transcriptional regulation play important roles in cancer cell development and differentiation processes. Based on the currently available cell line profiling information from the ENCODE Consortium, we propose a Bayesian inference model to infer and construct genome-wide interaction landscape between DNA methylation and transcriptional regulation, which sheds light on the underlying complex functional mechanisms important within the human cancer and disease context. For the first time, we select all the currently available cell lines (>=20) and transcription factors (>=80) profiling information from the ENCODE Consortium portal. Through the integration of those genome-wide profiling sources, our genome-wide analysis detects multiple functional loci of interest, and indicates that DNA methylation is cell- and region-specific, due to the interplay mechanisms with transcription regulatory activities. We validate our analysis results with the corresponding RNA-sequencing technique for those detected genomic loci. Our results provide novel and meaningful insights for the interplay mechanisms of transcriptional regulation and gene expression for the human cancer and disease studies.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome.

PubMed

Howell, Kate Joanne; Kraiczy, Judith; Nayak, Komal M; Gasparetto, Marco; Ross, Alexander; Lee, Claire; Mak, Tim N; Koo, Bon-Kyoung; Kumar, Nitin; Lawley, Trevor; Sinha, Anupam; Rosenstiel, Philip; Heuschkel, Robert; Stegle, Oliver; Zilbauer, Matthias

2018-02-01

We analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis. We obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn's disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed. We found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents. We identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

DNA methylation profiles of donor nuclei cells and tissues of cloned bovine fetuses.

PubMed

Kremenskoy, Maksym; Kremenska, Yuliya; Suzuki, Masako; Imai, Kei; Takahashi, Seiya; Hashizume, Kazuyoshi; Yagi, Shintaro; Shiota, Kunio

2006-04-01

Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissue-specific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.

First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

PubMed

Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

2013-05-01

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

Integrated genomic classification of melanocytic tumors of the central nervous system using mutation analysis, copy number alterations and DNA methylation profiling.

PubMed

Griewank, Klaus; Koelsche, Christian; van de Nes, Johannes A P; Schrimpf, Daniel; Gessi, Marco; Möller, Inga; Sucker, Antje; Scolyer, Richard A; Buckland, Michael E; Murali, Rajmohan; Pietsch, Torsten; von Deimling, Andreas; Schadendorf, Dirk

2018-06-11

In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria can be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis. Targeted next-generation-sequencing, array-based genome-wide methylation analysis and BAP1 immunohistochemistry was performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, incl. 47 primary tumors of the central nervous system, 16 uveal melanomas. 13 cutaneous melanoma metastasis and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation and copy-number profiles were correlated with clinicopathological features. Combining mutation, copy-number and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group. Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations markers of poor prognosis. Copyright ©2018, American Association for Cancer Research.

Assisted reproductive technology alters deoxyribonucleic acid methylation profiles in bloodspots of newborn infants

USDA-ARS?s Scientific Manuscript database

To evaluate the effect of infertility and intracytoplasmic sperm injection (ICSI) on DNA methylation of offspring. Microarray analysis of DNA methylation in archived neonatal bloodspots of in vitro fertilization (IVF)/ICSI-conceived children compared with controls born to fertile and infertile paren...

Assessment of DNA methylation profiling and copy number variation as indications of clonal relationship in ipsilateral and contralateral breast cancers to distinguish recurrent breast cancer from a second primary tumour.

PubMed

Huang, Katie T; Mikeska, Thomas; Li, Jason; Takano, Elena A; Millar, Ewan K A; Graham, Peter H; Boyle, Samantha E; Campbell, Ian G; Speed, Terence P; Dobrovic, Alexander; Fox, Stephen B

2015-10-09

Patients with breast cancer have an increased risk of developing subsequent breast cancers. It is important to distinguish whether these tumours are de novo or recurrences of the primary tumour in order to guide the appropriate therapy. Our aim was to investigate the use of DNA methylation profiling and array comparative genomic hybridization (aCGH) to determine whether the second tumour is clonally related to the first tumour. Methylation-sensitive high-resolution melting was used to screen promoter methylation in a panel of 13 genes reported as methylated in breast cancer (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, CDH13, RARβ, BRCA1, CDH1, CDKN2A, TP73, and GSTP1) in 29 tumour pairs (16 ipsilateral and 13 contralateral). Using the methylation profile of these genes, we employed a Bayesian and an empirical statistical approach to estimate clonal relationship. Copy number alterations were analysed using aCGH on the same set of tumour pairs. There is a higher probability of the second tumour being recurrent in ipsilateral tumours compared with contralateral tumours (38 % versus 8 %; p <0.05) based on the methylation profile. Using previously reported recurrence rates as Bayesian prior probabilities, we classified 69 % of ipsilateral and 15 % of contralateral tumours as recurrent. The inferred clonal relationship results of the tumour pairs were generally concordant between methylation profiling and aCGH. Our results show that DNA methylation profiling as well as aCGH have potential as diagnostic tools in improving the clinical decisions to differentiate recurrences from a second de novo tumour.

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

USDA-ARS?s Scientific Manuscript database

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

PubMed

Ikegami, Kohta; Iwatani, Misa; Suzuki, Masako; Tachibana, Makoto; Shinkai, Yoichi; Tanaka, Satoshi; Greally, John M; Yagi, Shintaro; Hattori, Naka; Shiota, Kunio

2007-01-01

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

PubMed Central

Harris, R. Alan; Wang, Ting; Coarfa, Cristian; Nagarajan, Raman P.; Hong, Chibo; Downey, Sara L.; Johnson, Brett E.; Fouse, Shaun D.; Delaney, Allen; Zhao, Yongjun; Olshen, Adam; Ballinger, Tracy; Zhou, Xin; Forsberg, Kevin J.; Gu, Junchen; Echipare, Lorigail; O’Geen, Henriette; Lister, Ryan; Pelizzola, Mattia; Xi, Yuanxin; Epstein, Charles B.; Bernstein, Bradley E.; Hawkins, R. David; Ren, Bing; Chung, Wen-Yu; Gu, Hongcang; Bock, Christoph; Gnirke, Andreas; Zhang, Michael Q.; Haussler, David; Ecker, Joseph; Li, Wei; Farnham, Peggy J.; Waterland, Robert A.; Meissner, Alexander; Marra, Marco A.; Hirst, Martin; Milosavljevic, Aleksandar; Costello, Joseph F.

2010-01-01

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. PMID:20852635

DNA methylation profiles of elderly individuals subjected to indentured childhood labor and trauma.

PubMed

Marinova, Zoya; Maercker, Andreas; Küffer, Andreas; Robinson, Mark D; Wojdacz, Tomasz K; Walitza, Susanne; Grünblatt, Edna; Burri, Andrea

2017-02-27

Childhood trauma is associated with increased vulnerability to mental and somatic disorders later in life. Epigenetic modifications such as DNA methylation are one potential mechanism through which such long-lasting impairments/consequences can be explained. The aim of the present study was to investigate whether childhood trauma is associated with long-term DNA methylation alterations in old age. We assessed genome-wide DNA methylation profiles in a cohort of former indentured child laborers ("Verdingkinder") who suffered severe childhood adversities (N = 30; M age = 75.9 years), and compared them to control group with similar demographic characteristics (N = 15, M age = 72.8 years). DNA was isolated from epithelial buccal cells and hybridized to the Illumina Infinium 450 k DNA methylation array, which provides coverage of 485,000 methylation sites. After accounting for batch effects, age, gender and multiple testing, 71 differentially methylated CpG positions were identified between the two groups. They were annotated among others to genes involved in neuronal projections and neuronal development. Some of the identified genes with differential methylation (DLG associated protein 2, mechanistic target of rapamycin) have previously been associated with traumatic stress. The results indicate specific epigenetic alterations in elderly individuals who were subjected to childhood adversities. Psychiatric and somatic comorbidities as well as differences in buccal epithelial cells proportion may contribute to the observed epigenetic differences.

PRKCZ methylation is associated with sunlight exposure in a North American but not a Mediterranean population

USDA-ARS?s Scientific Manuscript database

Sunlight exposure has been shown to alter DNA methylation patterns across several human cell-types, including T-lymphocytes. Since epigenetic changes establish gene expression profiles, changes in DNA methylation induced by sunlight exposure warrant investigation. The purpose of this study was to as...

Gene promoter methylation and DNA repair capacity in monozygotic twins with discordant smoking habits.

PubMed

Ottini, Laura; Rizzolo, Piera; Siniscalchi, Ester; Zijno, Andrea; Silvestri, Valentina; Crebelli, Riccardo; Marcon, Francesca

2015-02-01

The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile. Copyright © 2015 Elsevier B.V. All rights reserved.

Circadian gene methylation profiles are associated with obesity, metabolic disturbances and carbohydrate intake.

PubMed

Ramos-Lopez, Omar; Samblas, Mirian; Milagro, Fermin I; Riezu-Boj, Jose I; Crujeiras, A B; Martinez, J Alfredo; Project, Mena

2018-03-26

The circadian clock regulates the daily rhythms of several physiological and behavioral processes. Disruptions in clock genes have been associated with obesity and related comorbidities. This study aimed to analyze the association of DNA methylation signatures at circadian rhythm pathway genes with body mass index (BMI), metabolic profiles and dietary intakes. DNA methylation profiling was determined by microarray in white blood cells from 474 adults from the Methyl Epigenome Network Association (MENA) project. Kyoto Encyclopedia of Genes and Genomes database was used to identify the genes integrating the circadian rhythm pathway. Network enrichment analyses were performed with the PathDIP platform. Associations between circadian methylation patterns with anthropometric measurements, the metabolic profile, clinical data and dietary intakes were analyzed. DNA methylation patterns of nine CpG sites at six circadian rhythm pathway genes were strongly correlated with BMI (false discovery rates <0.0001). These CpGs encompassed cg09578018 (RORA), cg20406576 (PRKAG2), cg10059324 (PER3), cg01180628 (BHLHE40), cg23871860 (FBXL3), cg16964728 (RORA), cg14129040 (CREB1), cg07012178 (PRKAG2) and cg24061580 (PRKAG2). Interestingly, network enrichment analyses revealed that the six BMI-associated genes statistically contributed to the regulation of the circadian rhythm pathway (p = 1.9E-10). In addition, methylation signatures at cg09578018 (RORA), cg24061580 (PRKAG2), cg01180628 (BHLHE40) and cg10059324 (PER3) also correlated with insulin resistance (p < 0.0001) and mean arterial blood pressure (p < 0.0001). Furthermore, relevant correlations (p < 0.05) between methylation at cg09578018 (RORA) and cg01180628 (BHLHE40) with total energy and carbohydrate intakes were found. This investigation revealed potential associations of DNA methylation profiles at circadian genes with obesity, metabolic disturbances and carbohydrate intake, with potential impact on weight homeostasis.

DNA Methylation Profiles of Selected Pro-Inflammatory Cytokines in Alzheimer Disease.

PubMed

Nicolia, Vincenzina; Cavallaro, Rosaria A; López-González, Irene; Maccarrone, Mauro; Scarpa, Sigfrido; Ferrer, Isidre; Fuso, Andrea

2017-01-01

By means of functional genomics analysis, we recently described the mRNA expression profiles of various genes involved in the neuroinflammatory response in the brains of subjects with late-onset Alzheimer Disease (LOAD). Some of these genes, namely interleukin (IL)-1β and IL-6, showed distinct expression profiles with peak expression during the first stages of the disease and control-like levels at later stages. IL-1β and IL-6 genes are modulated by DNA methylation in different chronic and degenerative diseases; it is also well known that LOAD may have an epigenetic basis. Indeed, we and others have previously reported gene-specific DNA methylation alterations in LOAD and in related animal models. Based on these data, we studied the DNA methylation profiles, at single cytosine resolution, of IL-1β and IL-6 5'-flanking region by bisulphite modification in the cortex of healthy controls and LOAD patients at 2 different disease stages: Braak I-II/A and Braak V-VI/C. Our analysis provides evidence that neuroinflammation in LOAD is associated with (and possibly mediated by) epigenetic modifications. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

[Genome-scale sequence data processing and epigenetic analysis of DNA methylation].

PubMed

Wang, Ting-Zhang; Shan, Gao; Xu, Jian-Hong; Xue, Qing-Zhong

2013-06-01

A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under-stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive sequences, such as transposable elements. In this paper, we introduce the preprocessing steps of DNA methylation data, by which cytosine (C) and guanine (G) in the reference sequence are transferred to thymine (T) and adenine (A), and cytosine in reads is transferred to thymine, respectively. We also comprehensively review the main content of the DNA methylation analysis on the genomic scale: (1) the cytosine methylation under the context of different sequences; (2) the distribution of genomic methylcytosine; (3) DNA methylation context and the preference for the nucleotides; (4) DNA- protein interaction sites of DNA methylation; (5) degree of methylation of cytosine in the different structural elements of genes. DNA methylation analysis technique provides a powerful tool for the epigenome study in human and other species, and genes and environment interaction, and founds the theoretical basis for further development of disease diagnostics and therapeutics in human.

Investigating the epigenetic effects of a prototype smoke-derived carcinogen in human cells.

PubMed

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P; Besaratinia, Ahmad

2010-05-12

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

PubMed Central

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P.; Besaratinia, Ahmad

2010-01-01

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease. PMID:20485678

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

PubMed Central

Furukawa, Ryohei; Hachiya, Tsuyoshi; Ohmomo, Hideki; Shiwa, Yuh; Ono, Kanako; Suzuki, Sadafumi; Satoh, Mamoru; Hitomi, Jiro; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Cytosine methylation at CpG dinucleotides is an epigenetic mechanism that affects the gene expression profiles responsible for the functional differences in various cells and tissues. Although gene expression patterns are dynamically altered in response to various stimuli, the intraindividual dynamics of DNA methylation in human cells are yet to be fully understood. Here, we investigated the extent to which DNA methylation contributes to the dynamics of gene expression by collecting 24 blood samples from two individuals over a period of 3 months. Transcriptome and methylome association analyses revealed that only ~2% of dynamic changes in gene expression could be explained by the intraindividual variation of DNA methylation levels in peripheral blood mononuclear cells and purified monocytes. These results showed that DNA methylation levels remain stable for at least several months, suggesting that disease-associated DNA methylation markers are useful for estimating the risk of disease manifestation. PMID:27192970

DNA methylation profiles distinguish different subtypes of gastroenteropancreatic neuroendocrine tumors.

PubMed

How-Kit, Alexandre; Dejeux, Emelyne; Dousset, Bertrand; Renault, Victor; Baudry, Marion; Terris, Benoit; Tost, Jörg

2015-01-01

Most studies have considered gastroenteropancreatic neuroendocrine tumors (GEP-NETs) as a homogenous group of samples or distinguish only gastrointestinal from pancreatic endocrine tumors. This article investigates if DNA methylation patterns could distinguish subtypes of GEP-NETs. The DNA methylation level of 807 cancer-related genes was investigated in insulinomas, gastrinomas, non-functioning pancreatic endocrine tumors and small intestine endocrine tumors. DNA methylation patterns were found to be tumor type specific for each of the pancreatic tumor subtypes and identified two distinct methylation-based groups in small intestine endocrine tumors. Differences of DNA methylation levels were validated by pyrosequencing for 20 candidate genes and correlated with differences at the transcriptional level for four candidate genes. The heterogeneity of DNA methylation patterns in the different subtypes of gastroenteropancreatic neuroendocrine tumors suggests different underlying pathways and, therefore, these tumors should be considered as distinct entities in molecular and clinical studies.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood.

PubMed

Lee, Hwan Young; Jung, Sang-Eun; Lee, Eun Hee; Yang, Woo Ick; Shin, Kyoung-Jin

2016-09-01

The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed multiplex methylation SNaPshot reaction includes the 4CpG markers of which specificities have been confirmed by multiple studies, it will facilitate confirmatory tests for body fluids that are frequently observed in forensic casework. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice.

PubMed

Schraut, K G; Jakob, S B; Weidner, M T; Schmitt, A G; Scholz, C J; Strekalova, T; El Hajj, N; Eijssen, L M T; Domschke, K; Reif, A; Haaf, T; Ortega, G; Steinbusch, H W M; Lesch, K P; Van den Hove, D L

2014-10-21

The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/- offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt genotype, PS exposure and their interaction.

missMethyl: an R package for analyzing data from Illumina's HumanMethylation450 platform.

PubMed

Phipson, Belinda; Maksimovic, Jovana; Oshlack, Alicia

2016-01-15

DNA methylation is one of the most commonly studied epigenetic modifications due to its role in both disease and development. The Illumina HumanMethylation450 BeadChip is a cost-effective way to profile >450 000 CpGs across the human genome, making it a popular platform for profiling DNA methylation. Here we introduce missMethyl, an R package with a suite of tools for performing normalization, removal of unwanted variation in differential methylation analysis, differential variability testing and gene set analysis for the 450K array. missMethyl is an R package available from the Bioconductor project at www.bioconductor.org. alicia.oshlack@mcri.edu.au Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

DNA methylation-based classification and grading system for meningioma: a multicentre, retrospective analysis.

PubMed

Sahm, Felix; Schrimpf, Daniel; Stichel, Damian; Jones, David T W; Hielscher, Thomas; Schefzyk, Sebastian; Okonechnikov, Konstantin; Koelsche, Christian; Reuss, David E; Capper, David; Sturm, Dominik; Wirsching, Hans-Georg; Berghoff, Anna Sophie; Baumgarten, Peter; Kratz, Annekathrin; Huang, Kristin; Wefers, Annika K; Hovestadt, Volker; Sill, Martin; Ellis, Hayley P; Kurian, Kathreena M; Okuducu, Ali Fuat; Jungk, Christine; Drueschler, Katharina; Schick, Matthias; Bewerunge-Hudler, Melanie; Mawrin, Christian; Seiz-Rosenhagen, Marcel; Ketter, Ralf; Simon, Matthias; Westphal, Manfred; Lamszus, Katrin; Becker, Albert; Koch, Arend; Schittenhelm, Jens; Rushing, Elisabeth J; Collins, V Peter; Brehmer, Stefanie; Chavez, Lukas; Platten, Michael; Hänggi, Daniel; Unterberg, Andreas; Paulus, Werner; Wick, Wolfgang; Pfister, Stefan M; Mittelbronn, Michel; Preusser, Matthias; Herold-Mende, Christel; Weller, Michael; von Deimling, Andreas

2017-05-01

The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. German Cancer Aid, Else Kröner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors.

PubMed

Rechache, Nesrin S; Wang, Yonghong; Stevenson, Holly S; Killian, J Keith; Edelman, Daniel C; Merino, Maria; Zhang, Lisa; Nilubol, Naris; Stratakis, Constantine A; Meltzer, Paul S; Kebebew, Electron

2012-06-01

It is not known whether there are any DNA methylation alterations in adrenocortical tumors. The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes.

PubMed

Matsushita, Junya; Okamura, Kazuyuki; Nakabayashi, Kazuhiko; Suzuki, Takehiro; Horibe, Yu; Kawai, Tomoko; Sakurai, Toshihiro; Yamashita, Satoshi; Higami, Yoshikazu; Ichihara, Gaku; Hata, Kenichiro; Nohara, Keiko

2018-03-22

C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

Basic Mechanics of DNA Methylation and the Unique Landscape of the DNA Methylome in Metal-Induced Carcinogenesis

PubMed Central

Brocato, Jason; Costa, Max

2013-01-01

DNA methylation plays an intricate role in the regulation of gene expression and events that compromise the integrity of the methylome may potentially contribute to disease development. DNA methylation is a reversible and regulatory modification that elicits a cascade of events leading to chromatin condensation and gene silencing. In general, normal cells are characterized by gene-specific hypomethylation and global hypermethylation, while cancer cells portray a reverse profile to this norm. The unique methylome displayed in cancer cells is induced after exposure to carcinogenic metals such as nickel, arsenic, cadmium, and chromium (VI). These metals alter the DNA methylation profile by provoking both hyper- and hypomethylation events. The metal-stimulated deviations to the methylome are possible mechanisms for metal-induced carcinogenesis and may provide potential biomarkers for cancer detection. Development of therapies based on the cancer methylome requires further research including human studies that supply results with larger impact and higher human relevance. PMID:23844698

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress.

PubMed

Wang, Wensheng; Zhao, Xiuqin; Pan, Yajiao; Zhu, Linghua; Fu, Binying; Li, Zhikang

2011-09-20

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development. Copyright © 2011. Published by Elsevier Ltd.

Epigenetic deregulation in chronic lymphocytic leukemia: Clinical and biological impact.

PubMed

Mansouri, Larry; Wierzbinska, Justyna Anna; Plass, Christoph; Rosenquist, Richard

2018-02-07

Deregulated transcriptional control caused by aberrant DNA methylation and/or histone modifications is a hallmark of cancer cells. In chronic lymphocytic leukemia (CLL), the most common adult leukemia, the epigenetic 'landscape' has added a new layer of complexity to our understanding of this clinically and biologically heterogeneous disease. Early studies identified aberrant DNA methylation, often based on single gene promoter analysis with both biological and clinical impact. Subsequent genome-wide profiling studies revealed differential DNA methylation between CLLs and controls and in prognostics subgroups of the disease. From these studies, it became apparent that DNA methylation in regions outside of promoters, such as enhancers, is important for the regulation of coding genes as well as for the regulation of non-coding RNAs. Although DNA methylation profiles are reportedly stable over time and in relation to therapy, a higher epigenetic heterogeneity or 'burden' is seen in more aggressive CLL subgroups, albeit as non-recurrent 'passenger' events. More recently, DNA methylation profiles in CLL analyzed in relation to differentiating normal B-cell populations revealed that the majority of the CLL epigenome reflects the epigenomes present in the cell of origin and that only a small fraction of the epigenetic alterations represents truly CLL-specific changes. Furthermore, CLL patients can be grouped into at least three clinically relevant epigenetic subgroups, potentially originating from different cells at various stages of differentiation and associated with distinct outcomes. In this review, we summarize the current understanding of the DNA methylome in CLL, the role of histone modifying enzymes, highlight insights derived from animal models and attempts made to target epigenetic regulators in CLL along with the future directions of this rapidly advancing field. Copyright © 2018 Elsevier Ltd. All rights reserved.

Global LINE-1 DNA methylation is associated with blood glycaemic and lipid profiles

PubMed Central

Pearce, Mark S; McConnell, James C; Potter, Catherine; Barrett, Laura M; Parker, Louise; Mathers, John C; Relton, Caroline L

2012-01-01

Background Patterns of DNA methylation change with age and these changes are believed to be associated with the development of common complex diseases. The hypothesis that Long Interspersed Nucleotide Element 1 (LINE-1) DNA methylation (an index of global DNA methylation) is associated with biomarkers of metabolic health was investigated in this study. Methods Global LINE-1 DNA methylation was quantified by pyrosequencing in blood-derived DNA samples from 228 individuals, aged 49–51 years, from the Newcastle Thousand Families Study (NTFS). Associations between log-transformed LINE-1 DNA methylation levels and anthropometric and blood biochemical measurements, including triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, fasting glucose and insulin secretion and resistance were examined. Results Linear regression, after adjustment for sex, demonstrated positive associations between log-transformed LINE-1 DNA methylation and fasting glucose {coefficient 2.80 [95% confidence interval (CI) 0.39–5.22]}, total cholesterol [4.76 (95% CI 1.43–8.10)], triglycerides [3.83 (95% CI 1.30–6.37)] and LDL-cholesterol [5.38 (95% CI 2.12–8.64)] concentrations. A negative association was observed between log-transformed LINE-1 methylation and both HDL cholesterol concentration [−1.43 (95% CI −2.38 to −0.48)] and HDL:LDL ratio [−1.06 (95% CI −1.76 to −0.36)]. These coefficients reflect the millimoles per litre change in biochemical measurements per unit increase in log-transformed LINE-1 methylation. Conclusions These novel associations between global LINE-1 DNA methylation and blood glycaemic and lipid profiles highlight a potential role for epigenetic biomarkers as predictors of metabolic disease and may be relevant to future diagnosis, prevention and treatment of this group of disorders. Further work is required to establish the role of confounding and reverse causation in the observed associations. PMID:22422454

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM).

PubMed

Guilleret, Isabelle; Losi, Lorena; Chelbi, Sonia T; Fonda, Sergio; Bougel, Stéphanie; Saponaro, Sara; Gozzi, Gaia; Alberti, Loredana; Braunschweig, Richard; Benhattar, Jean

2016-10-14

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Regional differences in mitochondrial DNA methylation in human post-mortem brain tissue.

PubMed

Devall, Matthew; Smith, Rebecca G; Jeffries, Aaron; Hannon, Eilis; Davies, Matthew N; Schalkwyk, Leonard; Mill, Jonathan; Weedon, Michael; Lunnon, Katie

2017-01-01

DNA methylation is an important epigenetic mechanism involved in gene regulation, with alterations in DNA methylation in the nuclear genome being linked to numerous complex diseases. Mitochondrial DNA methylation is a phenomenon that is receiving ever-increasing interest, particularly in diseases characterized by mitochondrial dysfunction; however, most studies have been limited to the investigation of specific target regions. Analyses spanning the entire mitochondrial genome have been limited, potentially due to the amount of input DNA required. Further, mitochondrial genetic studies have been previously confounded by nuclear-mitochondrial pseudogenes. Methylated DNA Immunoprecipitation Sequencing is a technique widely used to profile DNA methylation across the nuclear genome; however, reads mapped to mitochondrial DNA are often discarded. Here, we have developed an approach to control for nuclear-mitochondrial pseudogenes within Methylated DNA Immunoprecipitation Sequencing data. We highlight the utility of this approach in identifying differences in mitochondrial DNA methylation across regions of the human brain and pre-mortem blood. We were able to correlate mitochondrial DNA methylation patterns between the cortex, cerebellum and blood. We identified 74 nominally significant differentially methylated regions ( p  < 0.05) in the mitochondrial genome, between anatomically separate cortical regions and the cerebellum in matched samples ( N  = 3 matched donors). Further analysis identified eight significant differentially methylated regions between the total cortex and cerebellum after correcting for multiple testing. Using unsupervised hierarchical clustering analysis of the mitochondrial DNA methylome, we were able to identify tissue-specific patterns of mitochondrial DNA methylation between blood, cerebellum and cortex. Our study represents a comprehensive analysis of the mitochondrial methylome using pre-existing Methylated DNA Immunoprecipitation Sequencing data to identify brain region-specific patterns of mitochondrial DNA methylation.

Comparison of the Genome-Wide DNA Methylation Profiles between Fast-Growing and Slow-Growing Broilers

PubMed Central

Li, Zhenhui; Zheng, Xuejuan; Jia, Xinzheng; Nie, Qinghua; Zhang, Xiquan

2013-01-01

Introduction Growth traits are important in poultry production, however, little is known for its regulatory mechanism at epigenetic level. Therefore, in this study, we aim to compare DNA methylation profiles between fast- and slow-growing broilers in order to identify candidate genes for chicken growth. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation pattern in high and low tails of Recessive White Rock (WRRh; WRRl) and that of Xinhua Chickens (XHh; XHl) at 7 weeks of age. The results showed that the average methylation density was the lowest in CGIs followed by promoters. Within the gene body, the methylation density of introns was higher than that of UTRs and exons. Moreover, different methylation levels were observed in different repeat types with the highest in LINE/CR1. Methylated CGIs were prominently distributed in the intergenic regions and were enriched in the size ranging 200–300 bp. In total 13,294 methylated genes were found in four samples, including 4,085 differentially methylated genes of WRRh Vs. WRRl, 5,599 of XHh Vs. XHl, 4,204 of WRRh Vs. XHh, as well as 7,301 of WRRl Vs. XHl. Moreover, 132 differentially methylated genes related to growth and metabolism were observed in both inner contrasts (WRRh Vs. WRRl and XHh Vs. XHl), whereas 129 differentially methylated genes related to growth and metabolism were found in both across-breed contrasts (WRRh Vs. XHh and WRRl Vs. XHl). Further analysis showed that overall 75 genes exhibited altered DNA methylation in all four contrasts, which included some well-known growth factors of IGF1R, FGF12, FGF14, FGF18, FGFR2, and FGFR3. In addition, we validate the MeDIP-seq results by bisulfite sequencing in some regions. Conclusions This study revealed the global DNA methylation pattern of chicken muscle, and identified candidate genes that potentially regulate muscle development at 7 weeks of age at methylation level. PMID:23441189

Across-Platform Imputation of DNA Methylation Levels Incorporating Nonlocal Information Using Penalized Functional Regression.

PubMed

Zhang, Guosheng; Huang, Kuan-Chieh; Xu, Zheng; Tzeng, Jung-Ying; Conneely, Karen N; Guan, Weihua; Kang, Jian; Li, Yun

2016-05-01

DNA methylation is a key epigenetic mark involved in both normal development and disease progression. Recent advances in high-throughput technologies have enabled genome-wide profiling of DNA methylation. However, DNA methylation profiling often employs different designs and platforms with varying resolution, which hinders joint analysis of methylation data from multiple platforms. In this study, we propose a penalized functional regression model to impute missing methylation data. By incorporating functional predictors, our model utilizes information from nonlocal probes to improve imputation quality. Here, we compared the performance of our functional model to linear regression and the best single probe surrogate in real data and via simulations. Specifically, we applied different imputation approaches to an acute myeloid leukemia dataset consisting of 194 samples and our method showed higher imputation accuracy, manifested, for example, by a 94% relative increase in information content and up to 86% more CpG sites passing post-imputation filtering. Our simulated association study further demonstrated that our method substantially improves the statistical power to identify trait-associated methylation loci. These findings indicate that the penalized functional regression model is a convenient and valuable imputation tool for methylation data, and it can boost statistical power in downstream epigenome-wide association study (EWAS). © 2016 WILEY PERIODICALS, INC.

Single-cell multimodal profiling reveals cellular epigenetic heterogeneity.

PubMed

Cheow, Lih Feng; Courtois, Elise T; Tan, Yuliana; Viswanathan, Ramya; Xing, Qiaorui; Tan, Rui Zhen; Tan, Daniel S W; Robson, Paul; Loh, Yuin-Han; Quake, Stephen R; Burkholder, William F

2016-10-01

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

CancerLocator: non-invasive cancer diagnosis and tissue-of-origin prediction using methylation profiles of cell-free DNA.

PubMed

Kang, Shuli; Li, Qingjiao; Chen, Quan; Zhou, Yonggang; Park, Stacy; Lee, Gina; Grimes, Brandon; Krysan, Kostyantyn; Yu, Min; Wang, Wei; Alber, Frank; Sun, Fengzhu; Dubinett, Steven M; Li, Wenyuan; Zhou, Xianghong Jasmine

2017-03-24

We propose a probabilistic method, CancerLocator, which exploits the diagnostic potential of cell-free DNA by determining not only the presence but also the location of tumors. CancerLocator simultaneously infers the proportions and the tissue-of-origin of tumor-derived cell-free DNA in a blood sample using genome-wide DNA methylation data. CancerLocator outperforms two established multi-class classification methods on simulations and real data, even with the low proportion of tumor-derived DNA in the cell-free DNA scenarios. CancerLocator also achieves promising results on patient plasma samples with low DNA methylation sequencing coverage.

DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

PubMed Central

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes. PMID:22016810

DNA methylation profiling of embryonic stem cell differentiation into the three germ layers.

PubMed

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy.

PubMed

Martino, David; Joo, Jihoon E; Sexton-Oates, Alexandra; Dang, Thanh; Allen, Katrina; Saffery, Richard; Prescott, Susan

2014-07-01

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450,000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value<0.05, delta β>0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 92 [corrected] allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.

Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling.

PubMed

Hassler, Melanie R; Pulverer, Walter; Lakshminarasimhan, Ranjani; Redl, Elisa; Hacker, Julia; Garland, Gavin D; Merkel, Olaf; Schiefer, Ana-Iris; Simonitsch-Klupp, Ingrid; Kenner, Lukas; Weisenberger, Daniel J; Weinhaeusel, Andreas; Turner, Suzanne D; Egger, Gerda

2016-10-04

Aberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+) and NPM-ALK-negative (ALK-) anaplastic large-cell lymphoma (ALCL). We find that ALK+ and ALK- ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

DNA demethylation activates genes in seed maternal integument development in rice (Oryza sativa L.).

PubMed

Wang, Yifeng; Lin, Haiyan; Tong, Xiaohong; Hou, Yuxuan; Chang, Yuxiao; Zhang, Jian

2017-11-01

DNA methylation is an important epigenetic modification that regulates various plant developmental processes. Rice seed integument determines the seed size. However, the role of DNA methylation in its development remains largely unknown. Here, we report the first dynamic DNA methylomic profiling of rice maternal integument before and after pollination by using a whole-genome bisulfite deep sequencing approach. Analysis of DNA methylation patterns identified 4238 differentially methylated regions underpin 4112 differentially methylated genes, including GW2, DEP1, RGB1 and numerous other regulators participated in maternal integument development. Bisulfite sanger sequencing and qRT-PCR of six differentially methylated genes revealed extensive occurrence of DNA hypomethylation triggered by double fertilization at IAP compared with IBP, suggesting that DNA demethylation might be a key mechanism to activate numerous maternal controlling genes. These results presented here not only greatly expanded the rice methylome dataset, but also shed novel insight into the regulatory roles of DNA methylation in rice seed maternal integument development. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The caste- and sex-specific DNA methylome of the termite Zootermopsis nevadensis

PubMed Central

Glastad, Karl M.; Gokhale, Kaustubh; Liebig, Jürgen; Goodisman, Michael A. D.

2016-01-01

Epigenetic inheritance plays an important role in mediating alternative phenotype in highly social species. In order to gain a greater understanding of epigenetic effects in societies, we investigated DNA methylation in the termite Zootermopsis nevadensis. Termites are the most ancient social insects, and developmentally distinct from highly-studied, hymenopteran social insects. We used replicated bisulfite-sequencing to investigate patterns of DNA methylation in both sexes and among castes of Z. nevadensis. We discovered that Z. nevadensis displayed some of the highest levels of DNA methylation found in insects. We also found strong differences in methylation between castes. Methylated genes tended to be uniformly and highly expressed demonstrating the antiquity of associations between intragenic methylation and gene expression. Differentially methylated genes were more likely to be alternatively spliced than not differentially methylated genes, and possessed considerable enrichment for development-associated functions. We further observed strong overrepresentation of multiple transcription factor binding sites and miRNA profiles associated with differential methylation, providing new insights into the possible function of DNA methylation. Overall, our results show that DNA methylation is widespread and associated with caste differences in termites. More generally, this study provides insights into the function of DNA methylation and the success of insect societies. PMID:27848993

Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes

PubMed Central

Atilano, Shari R.; Malik, Deepika; Chwa, Marilyn; Cáceres-Del-Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin; Kenney, M. Cristina

2015-01-01

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. PMID:25964427

DNA methylation abnormalities in congenital heart disease.

PubMed

Serra-Juhé, Clara; Cuscó, Ivon; Homs, Aïda; Flores, Raquel; Torán, Núria; Pérez-Jurado, Luis A

2015-01-01

Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation

PubMed Central

Gautier, Jean-François; Porcher, Raphaël; Abi Khalil, Charbel; Bellili-Munoz, Naima; Fetita, Lila Sabrina; Travert, Florence; Choukem, Simeon-Pierre; Riveline, Jean-Pierre; Hadjadj, Samy; Larger, Etienne; Boudou, Philippe; Blondeau, Bertrand; Roussel, Ronan; Ferré, Pascal; Ravussin, Eric; Rouzet, François; Marre, Michel

2015-01-01

Background Fetal exposure to hyperglycemia impacts negatively kidney development and function. Objective Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. Design Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. Results Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)—a key enzyme involved in gene expression during early development–was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. Conclusion Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function. PMID:26258530

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation.

PubMed

Gautier, Jean-François; Porcher, Raphaël; Abi Khalil, Charbel; Bellili-Munoz, Naima; Fetita, Lila Sabrina; Travert, Florence; Choukem, Simeon-Pierre; Riveline, Jean-Pierre; Hadjadj, Samy; Larger, Etienne; Boudou, Philippe; Blondeau, Bertrand; Roussel, Ronan; Ferré, Pascal; Ravussin, Eric; Rouzet, François; Marre, Michel

2015-01-01

Fetal exposure to hyperglycemia impacts negatively kidney development and function. Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)--a key enzyme involved in gene expression during early development--was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.

Effects of cytosine methylation on transcription factor binding sites

PubMed Central

2014-01-01

Background DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. Results We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines “traffic lights”. We observed a strong selection against CpG “traffic lights” within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. Conclusions Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. PMID:24669864

QDMR: a quantitative method for identification of differentially methylated regions by entropy

PubMed Central

Zhang, Yan; Liu, Hongbo; Lv, Jie; Xiao, Xue; Zhu, Jiang; Liu, Xiaojuan; Su, Jianzhong; Li, Xia; Wu, Qiong; Wang, Fang; Cui, Ying

2011-01-01

DNA methylation plays critical roles in transcriptional regulation and chromatin remodeling. Differentially methylated regions (DMRs) have important implications for development, aging and diseases. Therefore, genome-wide mapping of DMRs across various temporal and spatial methylomes is important in revealing the impact of epigenetic modifications on heritable phenotypic variation. We present a quantitative approach, quantitative differentially methylated regions (QDMRs), to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy. QDMR was applied to synthetic methylation patterns and methylation profiles detected by methylated DNA immunoprecipitation microarray (MeDIP-chip) in human tissues/cells. This approach can give a reasonable quantitative measure of methylation difference across multiple samples. Then DMR threshold was determined from methylation probability model. Using this threshold, QDMR identified 10 651 tissue DMRs which are related to the genes enriched for cell differentiation, including 4740 DMRs not identified by the method developed by Rakyan et al. QDMR can also measure the sample specificity of each DMR. Finally, the application to methylation profiles detected by reduced representation bisulphite sequencing (RRBS) in mouse showed the platform-free and species-free nature of QDMR. This approach provides an effective tool for the high-throughput identification of potential functional regions involved in epigenetic regulation. PMID:21306990

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

2011-11-04

Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.« less

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

PubMed

Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

2017-10-18

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

DNA Methylation in Osteoarthritis: Current Status and Therapeutic Implications

PubMed Central

Miranda-Duarte, Antonio

2018-01-01

Background: Primary Osteoarthritis (OA) is a multifactorial disease in which genetic factors are strongly associated with its development; however, recently it has been observed that epigenetic modifications are also involved in the pathogenesis of OA. DNA methylation is related to gene silencing, and several studies have investigated its role in the loci of different pathways or molecules associated to OA. Objective: This review is focused on the current status of DNA methylation studies related to OA pathogenesis. Method: A review of the literature was conducted on searching in PUBMED for original papers on DNA methylation in OA. Conclusion: The DNA methylation research of loci related to OA pathogenesis has shown a correlation between methylation and gene repression; however, there are some exceptions to this rule. Recently, the development of genome-wide methylation and genome-wide hydroxymethylation profiles has demonstrated that several genes previously associated with OA can have changes in their methylation status, favoring the development of the disease, and these have even shown the role of other epigenetic markers. PMID:29682093

Potential roles of DNA methylation in the initiation and establishment of replicative senescence revealed by array-based methylome and transcriptome analyses

PubMed Central

Sakaki, Mizuho; Ebihara, Yukiko; Okamura, Kohji; Nakabayashi, Kazuhiko; Igarashi, Arisa; Matsumoto, Kenji; Hata, Kenichiro; Kobayashi, Yoshiro

2017-01-01

Cellular senescence is classified into two groups: replicative and premature senescence. Gene expression and epigenetic changes are reported to differ between these two groups and cell types. Normal human diploid fibroblast TIG-3 cells have often been used in cellular senescence research; however, their epigenetic profiles are still not fully understood. To elucidate how cellular senescence is epigenetically regulated in TIG-3 cells, we analyzed the gene expression and DNA methylation profiles of three types of senescent cells, namely, replicatively senescent, ras-induced senescent (RIS), and non-permissive temperature-induced senescent SVts8 cells, using gene expression and DNA methylation microarrays. The expression of genes involved in the cell cycle and immune response was commonly either down- or up-regulated in the three types of senescent cells, respectively. The altered DNA methylation patterns were observed in replicatively senescent cells, but not in prematurely senescent cells. Interestingly, hypomethylated CpG sites detected on non-CpG island regions (“open sea”) were enriched in immune response-related genes that had non-CpG island promoters. The integrated analysis of gene expression and methylation in replicatively senescent cells demonstrated that differentially expressed 867 genes, including cell cycle- and immune response-related genes, were associated with DNA methylation changes in CpG sites close to the transcription start sites (TSSs). Furthermore, several miRNAs regulated in part through DNA methylation were found to affect the expression of their targeted genes. Taken together, these results indicate that the epigenetic changes of DNA methylation regulate the expression of a certain portion of genes and partly contribute to the introduction and establishment of replicative senescence. PMID:28158250

Computational Micromodel for Epigenetic Mechanisms

PubMed Central

Raghavan, Karthika; Ruskin, Heather J.; Perrin, Dimitri; Goasmat, Francois; Burns, John

2010-01-01

Characterization of the epigenetic profile of humans since the initial breakthrough on the human genome project has strongly established the key role of histone modifications and DNA methylation. These dynamic elements interact to determine the normal level of expression or methylation status of the constituent genes in the genome. Recently, considerable evidence has been put forward to demonstrate that environmental stress implicitly alters epigenetic patterns causing imbalance that can lead to cancer initiation. This chain of consequences has motivated attempts to computationally model the influence of histone modification and DNA methylation in gene expression and investigate their intrinsic interdependency. In this paper, we explore the relation between DNA methylation and transcription and characterize in detail the histone modifications for specific DNA methylation levels using a stochastic approach. PMID:21152421

Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.

PubMed

Guo, Hongshan; Zhu, Ping; Guo, Fan; Li, Xianlong; Wu, Xinglong; Fan, Xiaoying; Wen, Lu; Tang, Fuchou

2015-05-01

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

Epigenetic regulation in gallbladder cancer: Promoter methylation profiling as emergent novel biomarkers.

PubMed

Tekcham, Dinesh Singh; Tiwari, Pramod Kumar

2016-12-01

DNA methylation, once considered to rule the sex determination in Mary Lyon's hypothesis, has now reached the epicenter of human diseases, from monogenic (e.g. Prader Willi syndrome, Angelman syndromes and Beckwith-Wiedemann syndrome) to polygenic diseases, like cancer. Technological developments from gold standard to high throughput technologies have made tremendous advancement to define the epigenetic mechanism of cancer. Gallbladder cancer (GBC) is a fatal health issue affecting mostly the middle-aged women, whose survival rate is very low due to late symptomatic diagnosis. DNA methylation has become one of the key molecular mechanisms in the tumorigenesis of gallbladder. Various molecules have been reported to be epigenetically altered in GBC. In this review, we have discussed the classes of epigenetics, an overview of DNA methylation, technological approaches for its study, profile of methylated genes, their likely roles in GBC, future prospects of biomarker development and other discovery approaches, including therapeutics. © 2016 John Wiley & Sons Australia, Ltd.

Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

PubMed Central

van Dongen, Jenny; Ehli, Erik A.; Slieker, Roderick C.; Bartels, Meike; Weber, Zachary M.; Davies, Gareth E.; Slagboom, P. Eline; Heijmans, Bastiaan T.; Boomsma, Dorret I.

2014-01-01

DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ) twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment). We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19) using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho) was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs), compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins. PMID:24802513

Comprehensive analysis of genome-wide DNA methylation across human polycystic ovary syndrome ovary granulosa cell.

PubMed

Xu, Jiawei; Bao, Xiao; Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

2016-05-10

Polycystic ovary syndrome (PCOS) affects approximately 7% of the reproductive-age women. A growing body of evidence indicated that epigenetic mechanisms contributed to the development of PCOS. The role of DNA modification in human PCOS ovary granulosa cell is still unknown in PCOS progression. Global DNA methylation and hydroxymethylation were detected between PCOS' and controls' granulosa cell. Genome-wide DNA methylation was profiled to investigate the putative function of DNA methylaiton. Selected genes expressions were analyzed between PCOS' and controls' granulosa cell. Our results showed that the granulosa cell global DNA methylation of PCOS patients was significant higher than the controls'. The global DNA hydroxymethylation showed low level and no statistical difference between PCOS and control. 6936 differentially methylated CpG sites were identified between control and PCOS-obesity. 12245 differential methylated CpG sites were detected between control and PCOS-nonobesity group. 5202 methylated CpG sites were significantly differential between PCOS-obesity and PCOS-nonobesity group. Our results showed that DNA methylation not hydroxymethylation altered genome-wide in PCOS granulosa cell. The different methylation genes were enriched in development protein, transcription factor activity, alternative splicing, sequence-specific DNA binding and embryonic morphogenesis. YWHAQ, NCF2, DHRS9 and SCNA were up-regulation in PCOS-obesity patients with no significance different between control and PCOS-nonobesity patients, which may be activated by lower DNA methylaiton. Global and genome-wide DNA methylation alteration may contribute to different genes expression and PCOS clinical pathology.

Early life lipid profile and metabolic programming in very young children.

PubMed

Wijnands, K P J; Obermann-Borst, S A; Steegers-Theunissen, R P M

2015-06-01

Lipid derangements during early postnatal life may induce stable epigenetic changes and alter metabolic programming. We investigated associations between serum lipid profiles in very young children and DNA methylation of tumor necrosis factor-alpha (TNFα) and leptin (LEP). Secondly, we explored if the maternal serum lipid profile modifies DNA methylation in the child. In 120 healthy children at 17 months of age, DNA methylation of TNFα and LEP was measured in DNA derived from whole blood. Linear mixed models were used to calculate exposure-specific differences and associations. Total cholesterol in children was associated with decreased methylation of TNFα (-5.8%, p = 0.036), and HDL-cholesterol was associated with decreased methylation of both TNFα (-6.9%, p = 0.013) and LEP (-3.4%, p = 0.021). Additional adjustment for gestational age at birth, birth weight, sex, breastfeeding and educational level attenuated the effects, TNFα (-6.1%, p = 0.058) and LEP (-3.1%, p = 0.041). In mothers, HDL-cholesterol only was associated with decreased methylation of TNFα in the child (-8.7%, p = 0.001). Our data support the developmental origin of health and disease hypothesis by showing that total cholesterol and HDL-cholesterol levels in very young children are associated with epigenetic metabolic programming, which may affect their vulnerability for developing cardiovascular diseases in later life. Copyright © 2015 Elsevier B.V. All rights reserved.

Prognostic Classifier Based on Genome-Wide DNA Methylation Profiling in Well-Differentiated Thyroid Tumors.

PubMed

Bisarro Dos Reis, Mariana; Barros-Filho, Mateus Camargo; Marchi, Fábio Albuquerque; Beltrami, Caroline Moraes; Kuasne, Hellen; Pinto, Clóvis Antônio Lopes; Ambatipudi, Srikant; Herceg, Zdenko; Kowalski, Luiz Paulo; Rogatto, Silvia Regina

2017-11-01

Even though the majority of well-differentiated thyroid carcinoma (WDTC) is indolent, a number of cases display an aggressive behavior. Cumulative evidence suggests that the deregulation of DNA methylation has the potential to point out molecular markers associated with worse prognosis. To identify a prognostic epigenetic signature in thyroid cancer. Genome-wide DNA methylation assays (450k platform, Illumina) were performed in a cohort of 50 nonneoplastic thyroid tissues (NTs), 17 benign thyroid lesions (BTLs), and 74 thyroid carcinomas (60 papillary, 8 follicular, 2 Hürthle cell, 1 poorly differentiated, and 3 anaplastic). A prognostic classifier for WDTC was developed via diagonal linear discriminant analysis. The results were compared with The Cancer Genome Atlas (TCGA) database. A specific epigenetic profile was detected according to each histological subtype. BTLs and follicular carcinomas showed a greater number of methylated CpG in comparison with NTs, whereas hypomethylation was predominant in papillary and undifferentiated carcinomas. A prognostic classifier based on 21 DNA methylation probes was able to predict poor outcome in patients with WDTC (sensitivity 63%, specificity 92% for internal data; sensitivity 64%, specificity 88% for TCGA data). High-risk score based on the classifier was considered an independent factor of poor outcome (Cox regression, P < 0.001). The methylation profile of thyroid lesions exhibited a specific signature according to the histological subtype. A meaningful algorithm composed of 21 probes was capable of predicting the recurrence in WDTC. Copyright © 2017 Endocrine Society

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls.

PubMed

Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

2010-03-05

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P < or = 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P < or = 0.05). The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.

Establishment and functions of DNA methylation in the germline

PubMed Central

Stewart, Kathleen R; Veselovska, Lenka; Kelsey, Gavin

2016-01-01

Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization. PMID:27659720

Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice

PubMed Central

Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi

2011-01-01

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis. PMID:21455306

Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice.

PubMed

Lempiäinen, Harri; Müller, Arne; Brasa, Sarah; Teo, Soon-Siong; Roloff, Tim-Christoph; Morawiec, Laurent; Zamurovic, Natasa; Vicart, Axel; Funhoff, Enrico; Couttet, Philippe; Schübeler, Dirk; Grenet, Olivier; Marlowe, Jennifer; Moggs, Jonathan; Terranova, Rémi

2011-03-24

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

PubMed

van den Dungen, Myrthe W; Murk, Albertinka J; Kok, Dieuwertje E; Steegenga, Wilma T

2017-04-01

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes. Copyright © 2017 Wageningen University. Published by Elsevier Ltd.. All rights reserved.

A DNA methylation fingerprint of 1628 human samples

PubMed Central

Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

2012-01-01

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state.

PubMed

Khulan, B; Manning, J R; Dunbar, D R; Seckl, J R; Raikkonen, K; Eriksson, J G; Drake, A J

2014-09-23

Early-life stress (ELS) is known to be associated with an increased risk of neuropsychiatric and cardiometabolic disease in later life. One of the potential mechanisms underpinning this is through effects on the epigenome, particularly changes in DNA methylation. Using a well-phenotyped cohort of 83 men from the Helsinki Birth Cohort Study, who experienced ELS in the form of separation from their parents during childhood, and a group of 83 matched controls, we performed a genome-wide analysis of DNA methylation in peripheral blood. We found no differences in DNA methylation between men who were separated from their families and non-separated men; however, we did identify differences in DNA methylation in association with the development of at least mild depressive symptoms over the subsequent 5-10 years. Notably, hypomethylation was identified at a number of genes with roles in brain development and/or function in association with depressive symptoms. Pathway analysis revealed an enrichment of DNA methylation changes in pathways associated with development and morphogenesis, DNA and transcription factor binding and programmed cell death. Our results support the concept that DNA methylation differences may be important in the pathogenesis of psychiatric disease.

Variation of DNA Methylome of Zebrafish Cells under Cold Pressure

PubMed Central

Xu, Qiongqiong; Luo, Juntao; Shi, Yingdi; Li, Xiaoxia; Yan, Xiaonan; Zhang, Junfang

2016-01-01

DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure. PMID:27494266

Tobacco exposure-related alterations in DNA methylation and gene expression in human monocytes: the Multi-Ethnic Study of Atherosclerosis (MESA)

PubMed Central

Reynolds, Lindsay M.; Lohman, Kurt; Pittman, Gary S.; Barr, R. Graham; Chi, Gloria C.; Kaufman, Joel; Wan, Ma; Bell, Douglas A.; Blaha, Michael J.; Rodriguez, Carlos J.; Liu, Yongmei

2017-01-01

ABSTRACT Alterations in DNA methylation and gene expression in blood leukocytes are potential biomarkers of harm and mediators of the deleterious effects of tobacco exposure. However, methodological issues, including the use of self-reported smoking status and mixed cell types have made previously identified alterations in DNA methylation and gene expression difficult to interpret. In this study, we examined associations of tobacco exposure with DNA methylation and gene expression, utilizing a biomarker of tobacco exposure (urine cotinine) and CD14+ purified monocyte samples from 934 participants of the community-based Multi-Ethnic Study of Atherosclerosis (MESA). Urine cotinine levels were measured using an immunoassay. DNA methylation and gene expression were measured with microarrays. Multivariate linear regression was used to test for associations adjusting for age, sex, race/ethnicity, education, and study site. Urine cotinine levels were associated with methylation of 176 CpGs [false discovery rate (FDR)<0.01]. Four CpGs not previously identified by studies of non-purified blood samples nominally replicated (P value<0.05) with plasma cotinine-associated methylation in 128 independent monocyte samples. Urine cotinine levels associated with expression of 12 genes (FDR<0.01), including increased expression of P2RY6 (Beta ± standard error = 0.078 ± 0.008, P = 1.99 × 10−22), a gene previously identified to be involved in the release of pro-inflammatory cytokines. No cotinine-associated (FDR<0.01) methylation profiles significantly (FDR<0.01) correlated with cotinine-associated (FDR<0.01) gene expression profiles. In conclusion, our findings i) identify potential monocyte-specific smoking-associated methylation patterns and ii) suggest that alterations in methylation may not be a main mechanism regulating gene expression in monocytes in response to cigarette smoking. PMID:29166816

The von Hippel-Lindau (VHL) tumor-suppressor gene is down-regulated by selenium deficiency in Caco-2 cells and rat colon mucosa.

PubMed

Uthus, Eric; Begaye, Adrienne; Ross, Sharon; Zeng, Huawei

2011-08-01

To test the hypothesis that selenium affects DNA methylation and hence gene regulation, we employed a methylation array (Panomics) in the human colonic epithelial Caco-2 cell model. The array profiles DNA methylation from promoter regions of 82 human genes. After conditioning cells to repeatedly reduced concentrations of fetal bovine serum, a serum-free culture was established. Se-methylselenocysteine (SeMSC) was added at 0 (deficient Se) or 250 (control Se) nM to cells maintained in DMEM. After 7 days, cells were collected and stored at -80 °C until analysis; experiments were replicated three times. Glutathione peroxidase activity was significantly decreased in cells grown in low SeMSC. Cells grown in 250 nM SeMSC had maximal GPx activity. Genomic DNA from cells grown in the low-SeMSC media and media containing 250 nM SeMSC was incubated with methylation-binding protein followed by isolation of methylated DNA. The methylated DNA was labeled with biotin and hybridized to the methylation array. Thus, genes with promoter methylation will produce a higher chemiluminescence signal than those genes with no promoter methylation. Of the genes profiled, the von Hippel-Lindau (VHL) gene was most different as indicated by quantification following chemiluminescence detection demonstrating that the promoter region of VHL was hypermethylated in cells from the low-SeMSC media. To determine whether promoter methylation affected transcription, we isolated RNA from replicate samples and performed real-time RT PCR. VHL (mRNA) was down-regulated (fold change significantly <1) in cells grown in low SeMSC compared to cells grown in 250 nM SeMSC (control; fold change = 1). We also show that (mRNA) Vhl expression is significantly reduced in mucosa from rats fed a diet deficient in Se. Our results suggest that low Se status affects DNA promoter region methylation and that this can result in down-regulation of the tumor suppressor gene VHL.

Maternal adversities during pregnancy and cord blood oxytocin receptor (OXTR) DNA methylation

PubMed Central

Unternaehrer, Eva; Bolten, Margarete; Nast, Irina; Staehli, Simon; Meyer, Andrea H.; Dempster, Emma; Hellhammer, Dirk H.; Lieb, Roselind

2016-01-01

The aim of this study was to investigate whether maternal adversities and cortisol levels during pregnancy predict cord blood DNA methylation of the oxytocin receptor (OXTR). We collected cord blood of 39 babies born to mothers participating in a cross-sectional study (N = 100) conducted in Basel, Switzerland (2007–10). Mothers completed the Inventory of Life Events (second trimester: T2), the Edinburgh Postnatal Depression Scale (EPDS, third trimester: T3), the Trier Inventory of Chronic Stress (TICS-K, 1–3 weeks postpartum) and provided saliva samples (T2, T3) for maternal cortisol profiles, as computed by the area under the curve with respect to ground (AUCg) or increase (AUCi) for the cortisol awakening response (CAR) and for diurnal cortisol profiles (DAY). OXTR DNA methylation was quantified using Sequenom EpiTYPER. The number of stressful life events (P = 0.032), EPDS score (P = 0.007) and cortisol AUCgs at T2 (CAR: P = 0.020; DAY: P = 0.024) were negatively associated with OXTR DNA methylation. Our findings suggest that distinct prenatal adversities predict decreased DNA methylation in a gene that is relevant for childbirth, maternal behavior and wellbeing of mother and offspring. If a reduced OXTR methylation increases OXTR expression, our findings could suggest an epigenetic adaptation to an adverse early environment. PMID:27107296

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss.

PubMed

Benton, Miles C; Johnstone, Alice; Eccles, David; Harmon, Brennan; Hayes, Mark T; Lea, Rod A; Griffiths, Lyn; Hoffman, Eric P; Stubbs, Richard S; Macartney-Coxson, Donia

2015-01-22

Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3' untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Genome-Wide DNA Methylation Profiling Reveals Epigenetic Adaptation of Stickleback to Marine and Freshwater Conditions.

PubMed

Artemov, Artem V; Mugue, Nikolai S; Rastorguev, Sergey M; Zhenilo, Svetlana; Mazur, Alexander M; Tsygankova, Svetlana V; Boulygina, Eugenia S; Kaplun, Daria; Nedoluzhko, Artem V; Medvedeva, Yulia A; Prokhortchouk, Egor B

2017-09-01

The three-spined stickleback (Gasterosteus aculeatus) represents a convenient model to study microevolution-adaptation to a freshwater environment. Although genetic adaptations to freshwater environments are well-studied, epigenetic adaptations have attracted little attention. In this work, we investigated the role of DNA methylation in the adaptation of the marine stickleback population to freshwater conditions. DNA methylation profiling was performed in marine and freshwater populations of sticklebacks, as well as in marine sticklebacks placed into a freshwater environment and freshwater sticklebacks placed into seawater. We showed that the DNA methylation profile after placing a marine stickleback into fresh water partially converged to that of a freshwater stickleback. For six genes including ATP4A ion pump and NELL1, believed to be involved in skeletal ossification, we demonstrated similar changes in DNA methylation in both evolutionary and short-term adaptation. This suggested that an immediate epigenetic response to freshwater conditions can be maintained in freshwater population. Interestingly, we observed enhanced epigenetic plasticity in freshwater sticklebacks that may serve as a compensatory regulatory mechanism for the lack of genetic variation in the freshwater population. For the first time, we demonstrated that genes encoding ion channels KCND3, CACNA1FB, and ATP4A were differentially methylated between the marine and the freshwater populations. Other genes encoding ion channels were previously reported to be under selection in freshwater populations. Nevertheless, the genes that harbor genetic and epigenetic changes were not the same, suggesting that epigenetic adaptation is a complementary mechanism to selection of genetic variants favorable for freshwater environment. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dynamic integrated analysis of DNA methylation and gene expression profiles in in vivo and in vitro fertilized mouse post-implantation extraembryonic and placental tissues.

PubMed

Tan, Kun; Zhang, Zhenni; Miao, Kai; Yu, Yong; Sui, Linlin; Tian, Jianhui; An, Lei

2016-07-01

How does in vitro fertilization (IVF) alter promoter DNA methylation patterns and its subsequent effects on gene expression profiles during placentation in mice? IVF-induced alterations in promoter DNA methylation might have functional consequences in a number of biological processes and functions during IVF placentation, including actin cytoskeleton organization, hematopoiesis, vasculogenesis, energy metabolism and nutrient transport. During post-implantation embryonic development, both embryonic and extraembryonic tissues undergo de novo DNA methylation, thereby establishing a global DNA methylation pattern, and influencing gene expression profiles. Embryonic and placental tissues of IVF conceptuses can have aberrant morphology and functions, resulting in adverse pregnancy outcomes such as pregnancy loss, low birthweight, and long-term health effects. To date, the IVF-induced global profiling of DNA methylation alterations, and their functional consequences on aberrant gene expression profiles in IVF placentas have not been systematically studied. Institute for Cancer Research mice (6 week-old females and 8-9 week-old males) were used to generate in vivo fertilization (IVO) and IVF blastocysts. After either IVO and development (IVO group as control) or in vitro fertilization and culture (IVF group), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Extraembryonic (ectoplacental cone and extraembryonic ectoderm) and placental tissues from both groups were sampled at embryonic day (E) 7.5 (IVO, n = 822; IVF, n = 795) and E10.5 (IVO, n = 324; IVF, n = 278), respectively. The collected extraembryonic (E7.5) and placental tissues (E10.5) were then used for high-throughput RNA sequencing (RNA-seq) and methylated DNA immunoprecipitation sequencing (MeDIP-seq). The main dysfunctions indicated by bioinformatic analyses were further validated using molecular detection, and morphometric and phenotypic analyses. Dynamic functional profiling of high-throughput data, together with molecular detection, and morphometric and phenotypic analyses, showed that differentially expressed genes dysregulated by DNA methylation were functionally involved in: (i) actin cytoskeleton disorganization in IVF extraembryonic tissues, which may impair allantois or chorion formation, and chorioallantoic fusion; (ii) disturbed hematopoiesis and vasculogenesis, which may lead to abnormal placenta labyrinth formation and thereby impairing nutrition transport in IVF placentas; (iii) dysregulated energy and amino acid metabolism, which may cause placental dysfunctions, leading to delayed embryonic development or even lethality; (iv) disrupted genetic information processing, which can further influence gene transcriptional and translational processes. Findings in mouse placental tissues may not be fully representative of human placentas. Further studies are necessary to confirm these findings and determine their clinical significance. Our study is the first to provide the genome-wide analysis of gene expression dysregulation caused by DNA methylation during IVF placentation. Systematic understanding of the molecular mechanisms implicated in IVF placentation can be useful for the improvement of existing assisted conception systems to prevent these IVF-associated safety concerns. This work was supported by grants from the National Natural Science Foundation of China (No. 31472092), and the National High-Tech R&D Program (Nos. 2011|AA100303, 2013AA102506). There was no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Integrative DNA methylome analysis of pan-cancer biomarkers in cancer discordant monozygotic twin-pairs.

PubMed

Roos, Leonie; van Dongen, Jenny; Bell, Christopher G; Burri, Andrea; Deloukas, Panos; Boomsma, Dorret I; Spector, Tim D; Bell, Jordana T

2016-01-01

A key focus in cancer research is the discovery of biomarkers that accurately diagnose early lesions in non-invasive tissues. Several studies have identified malignancy-associated DNA methylation changes in blood, yet no general cancer biomarker has been identified to date. Here, we explore the potential of blood DNA methylation as a biomarker of pan-cancer (cancer of multiple different origins) in 41 female cancer discordant monozygotic (MZ) twin-pairs sampled before or after diagnosis using the Illumina HumanMethylation450 BeadChip. We analysed epigenome-wide DNA methylation profiles in 41 cancer discordant MZ twin-pairs with affected individuals diagnosed with tumours at different single primary sites: the breast, cervix, colon, endometrium, thyroid gland, skin (melanoma), ovary, and pancreas. No significant global differences in whole blood DNA methylation profiles were observed. Epigenome-wide analyses identified one novel pan-cancer differentially methylated position at false discovery rate (FDR) threshold of 10 % (cg02444695, P = 1.8 × 10(-7)) in an intergenic region 70 kb upstream of the SASH1 tumour suppressor gene, and three suggestive signals in COL11A2, AXL, and LINC00340. Replication of the four top-ranked signals in an independent sample of nine cancer-discordant MZ twin-pairs showed a similar direction of association at COL11A2, AXL, and LINC00340, and significantly greater methylation discordance at AXL compared to 480 healthy concordant MZ twin-pairs. The effects at cg02444695 (near SASH1), COL11A2, and LINC00340 were the most promising in biomarker potential because the DNA methylation differences were found to pre-exist in samples obtained prior to diagnosis and were limited to a 5-year period before diagnosis. Gene expression follow-up at the top-ranked signals in 283 healthy individuals showed correlation between blood methylation and gene expression in lymphoblastoid cell lines at PRL, and in the skin tissue at AXL. A significant enrichment of differential DNA methylation was observed in enhancer regions (P = 0.03). We identified DNA methylation signatures in blood associated with pan-cancer, at or near SASH1, COL11A2, AXL, and LINC00340. Three of these signals were present up to 5 years prior to cancer diagnosis, highlighting the potential clinical utility of whole blood DNA methylation analysis in cancer surveillance.

Expression and methylation of BDNF in the human brain in schizophrenia.

PubMed

Cheah, Sern-Yih; McLeay, Robert; Wockner, Leesa F; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

2017-08-01

To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood.

PubMed

Huang, R C; Garratt, E S; Pan, H; Wu, Y; Davis, E A; Barton, S J; Burdge, G C; Godfrey, K M; Holbrook, J D; Lillycrop, K A

2015-01-01

Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value < 0.05). The top pathways enriched among the DMCpGs included developmental processes, immune system regulation, regulation of cell signaling, and small GTPase-mediated signal transduction. The associations between the methylation of selected DMCpGs with childhood obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P < 0.01), while obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 - 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 - 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy

PubMed Central

Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

2016-01-01

It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring’s Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18–22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11–13 and weeks 18–22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy. PMID:27509522

Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy.

PubMed

Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

2016-08-06

It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring's Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18-22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11-13 and weeks 18-22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy.

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

PubMed

Ashktorab, Hassan; Daremipouran, M; Goel, Ajay; Varma, Sudhir; Leavitt, R; Sun, Xueguang; Brim, Hassan

2014-04-01

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject's colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands-in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)-were significantly hypermethylated in tumor vs. normal tissues (P<0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network-the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.

Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

PubMed

Kuan, Pei Fen; Chiang, Derek Y

2012-09-01

DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. © 2012, The International Biometric Society.

Validation study of genes with hypermethylated promoter regions associated with prostate cancer recurrence

PubMed Central

Stott-Miller, Marni; Zhao, Shanshan; Wright, Jonathan L.; Kolb, Suzanne; Bibikova, Marina; Klotzle, Brandy; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

2014-01-01

Background One challenge in prostate cancer (PCa) is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in PCa, but few studies of DNA methylation in relation to features of more aggressive tumors or PCa recurrence have been completed. Methods We used the Infinium® HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized PCa who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the SEER registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared to those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined. Results During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared to those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46x10−6), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2×10−4). DNA methylation profiles did not differ by recurrence status for the other genes. Conclusions These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and PCa recurrence. Impact Tumor DNA methylation profiling may help distinguish PCa patients at higher risk for disease recurrence. PMID:24718283

DNA methylation-based variation between human populations.

PubMed

Kader, Farzeen; Ghai, Meenu

2017-02-01

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

Epigenetic dysregulation of key developmental genes in radiation-induced rat mammary carcinomas.

PubMed

Daino, Kazuhiro; Nishimura, Mayumi; Imaoka, Tatsuhiko; Takabatake, Masaru; Morioka, Takamitsu; Nishimura, Yukiko; Shimada, Yoshiya; Kakinuma, Shizuko

2018-02-13

With the increase in the number of long-term cancer survivors worldwide, there is a growing concern about the risk of secondary cancers induced by radiotherapy. Epigenetic modifications of genes associated with carcinogenesis are attractive targets for the prevention of cancer owing to their reversible nature. To identify genes with possible changes in functionally relevant DNA methylation patterns in mammary carcinomas induced by radiation exposure, we performed microarray-based global DNA methylation and expression profiling in γ-ray-induced rat mammary carcinomas and normal mammary glands. The gene expression profiling identified dysregulation of developmentally related genes, including the downstream targets of polycomb repressive complex 2 (PRC2) and overexpression of enhancer of zeste homolog 2, a component of PRC2, in the carcinomas. By integrating expression and DNA methylation profiles, we identified ten hypermethylated and three hypomethylated genes that possibly act as tumor-suppressor genes and oncogenes dysregulated by aberrant DNA methylation; half of these genes encode developmental transcription factors. Bisulfite sequencing and quantitative PCR confirmed the dysregulation of the polycomb-regulated developmentally related transcription-factor genes Dmrt2, Hoxa7, Foxb1, Sox17, Lhx8, Gata3 and Runx1. Silencing of Hoxa7 was further verified by immunohistochemistry. These results suggest that, in radiation-induced mammary gland carcinomas, PRC2-mediated aberrant DNA methylation leads to dysregulation of developmentally related transcription-factor genes. Our findings provide clues to molecular mechanisms linking epigenetic regulation and radiation-induced breast carcinogenesis and underscore the potential of such epigenetic mechanisms as targets for cancer prevention. © 2018 UICC.

TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours.

PubMed

Maleva Kostovska, Ivana; Jakimovska, Milena; Popovska-Jankovic, Katerina; Kubelka-Sabit, Katerina; Karagjozov, Mitko; Plaseska-Karanfilska, Dijana

2018-03-09

Tumours presenting BRCAness profile behave more aggressively and are more invasive as a consequence of their complex genetic and epigenetic alterations, caused by impaired fidelity of the DNA repair processes. Methylation of promoter CpG islands represents an alternative mechanism to inactivate DNA repair and tumour suppressor genes. In our study, we analyzed the frequency of methylation changes of 24 tumour suppressor genes and explored their association with BRCAness profile. BRCA1ness profile and aberrant methylation were studied in 233 fresh frozen breast tumour tissues by Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation Specific (MS)-MLPA methods, respectively. Our analyses revealed that 12.4% of the breast cancer (BC) patients had tumours with a BRCA1ness profile. TIMP3 showed significantly higher (p = 5.8х10 -5 ) methylation frequency in tumours with BRCA1ness, while methylation of APC, GSTP1 and RASSF1 promoters was negatively associated with BRCA1ness (р = 0.0017, р = 0.007 and р = 0.046, respectively). TIMP3 methylation was also associated with triple negative (TN) BC. Furthermore, TN tumours showing BRCA1ness showed stronger association with TIMP3 methylation (p = 0.0008) in comparison to TN tumours without BRCA1ness (p = 0.009). In conclusion, we confirmed that TIMP3 methylation is a marker for TN tumours and furthermore we showed for the first time that TIMP3 promoter methylation is an epigenetic marker of BRCA1ness tumours.

DNA methylation of ESR-1 and N-33 in colorectal mucosa of patients with ulcerative colitis (UC).

PubMed

Arasaradnam, Ramesh P; Khoo, Kevin; Bradburn, Mike; Mathers, John C; Kelly, Seamus B

2010-07-01

Epigenetic marking such as DNA methylation influence gene transcription and chromosomal stability and may also be affected by environmental exposures. Few studies exist on alteration in DNA methylation profiles (genomic and gene specific methylation) in patients with Ulcerative Colitis (UC) and no studies exist that assess its relationship with lifestyle exposures. The methylation level of both ESR-1 and N-33 genes were significantly higher in UC subjects compared with controls (7.9% vs. 5.9%; p = 0.015 and 66% vs. 9.3%; p < 0.001 respectively). There was no detectable difference in global DNA methylation between patients with UC and age and sex matched controls. No associations between indices of DNA methylation and anthropometric measures or smoking patterns were detected. To assess genomic methylation and promoter methylation of the ESR-1 (oestrogen receptor-1) and N-33 (tumor suppressor candidate-3) genes in the macroscopically normal mucosa of UC patients as well as to investigate effects of anthropometric and lifestyle exposures on DNA methylation. Sixty eight subjects were recruited (24 UC and 44 age and sex matched controls). Colorectal mucosal biopsies were obtained and DNA was extracted. Genomic DNA methylation was quantified using the tritium-labelled cytosine extension assay (3[H] dCTP) while gene specific methylation was quantified using the COBRA method. For the first time, we have shown increased methylation in the promoter regions of the putative tumor suppressor gene N-33 in macroscopically normal mucosa of patients with UC. In addition, we have confirmed that methylation of ESR-1 promoter is higher in UC patients compared with age and sex matched controls. These findings suggest that inactivation through methylation of the putative tumor suppressor genes N-33 and ESR-1 may not be associated with colorectal carcinogenesis in UC.

Identification of body fluid-specific DNA methylation markers for use in forensic science.

PubMed

Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Links between DNA methylation and nucleosome occupancy in the human genome.

PubMed

Collings, Clayton K; Anderson, John N

2017-01-01

DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Altered DNA Methylation Patterns Associated With Clinically Relevant Increases in PTSD Symptoms and PTSD Symptom Profiles in Military Personnel.

PubMed

Martin, Christiana; Cho, Young-Eun; Kim, Hyungsuk; Yun, Sijung; Kanefsky, Rebekah; Lee, Hyunhwa; Mysliwiec, Vincent; Cashion, Ann; Gill, Jessica

2018-05-01

Military personnel experience posttraumatic stress disorder (PTSD), which is associated with differential DNA methylation across the whole genome. However, the relationship between these DNA methylation patterns and clinically relevant increases in PTSD severity is not yet clearly understood. The purpose of this study was to identify differences in DNA methylation associated with PTSD symptoms and investigate DNA methylation changes related to increases in the severity of PTSD in military personnel. In this pilot study, a cross-sectional comparison was made between military personnel with PTSD (n = 8) and combat-matched controls without PTSD (n = 6). Symptom measures were obtained, and genome-wide DNA methylation was measured using methylated DNA immunoprecipitation (MeDIP-seq) from whole blood samples at baseline and 3 months later. A longitudinal comparison measured DNA methylation changes in military personnel with clinically relevant increases in PTSD symptoms between time points (PTSD onset) and compared methylation patterns to controls with no clinical changes in PTSD. In military personnel with elevated PTSD symptoms 3 months following baseline, 119 genes exhibited reduced methylation and 8 genes exhibited increased methylation. Genes with reduced methylation in the PTSD-onset group relate to the canonical pathways of netrin signaling, Wnt/Ca + pathway, and axonal guidance signaling. These gene pathways relate to neurological disorders, and the current findings suggest that these epigenetic changes potentially relate to PTSD symptomology. This study provides some novel insights into the role of epigenetic changes in PTSD symptoms and the progression of PTSD symptoms in military personnel.

Novel epigenetic changes unveiled by monozygotic twins discordant for smoking habits.

PubMed

Allione, Alessandra; Marcon, Francesca; Fiorito, Giovanni; Guarrera, Simonetta; Siniscalchi, Ester; Zijno, Andrea; Crebelli, Riccardo; Matullo, Giuseppe

2015-01-01

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.

Gestational Diabetes Alters Offspring DNA Methylation Profiles in Human and Rat: Identification of Key Pathways Involved in Endocrine System Disorders, Insulin Signaling, Diabetes Signaling, and ILK Signaling.

PubMed

Petropoulos, Sophie; Guillemin, Claire; Ergaz, Zivanit; Dimov, Sergiy; Suderman, Matthew; Weinstein-Fudim, Liza; Ornoy, Asher; Szyf, Moshe

2015-06-01

Gestational diabetes is associated with risk for metabolic disease later in life. Using a cross-species approach in rat and humans, we examined the hypothesis that gestational diabetes during pregnancy triggers changes in the methylome of the offspring that might be mediating these risks. We show in a gestation diabetes rat model, the Cohen diabetic rat, that gestational diabetes triggers wide alterations in DNA methylation in the placenta in both candidate diabetes genes and genome-wide promoters, thus providing evidence for a causal relationship between diabetes during pregnancy and DNA methylation alterations. There is a significant overlap between differentially methylated genes in the placenta and the liver of the rat offspring. Several genes differentially methylated in rat placenta exposed to maternal diabetes are also differentially methylated in the human placenta of offspring exposed to gestational diabetes in utero. DNA methylation changes inversely correlate with changes in expression. The changes in DNA methylation affect known functional gene pathways involved in endocrine function, metabolism, and insulin responses. These data provide support to the hypothesis that early-life exposures and their effects on metabolic disease are mediated by DNA methylation changes. This has important diagnostic and therapeutic implications.

Genomic methylation and transcriptomic profiling provides insights into heading depression in inbred Brassica rapa L. ssp. pekinensis.

PubMed

Liu, Yan; Xu, Cui; Tang, Xuebing; Pei, Surui; Jin, Di; Guo, Minghao; Yang, Meng; Zhang, Yaowei

2018-07-30

Inbreeding depression is the reduction in fitness observed in inbred populations. In plants, it leads to disease, weaker resistance to adverse environmental conditions, inhibition of growth, and decrease of yield. To elucidate molecular mechanisms behind inbreeding depression, we compared global DNA methylation and transcriptome profiles of a normal and a highly inbred heading degenerated variety of the Chinese cabbage (Brassica rapa L. ssp. pekinensis). DNA methylation was reduced in inbred plants, suggesting a change in the epigenetic landscape. Transcriptome analysis by RNA-Seq revealed that genes in auxin-response and synthesis pathways were differentially expressed in the inbreeding depression lines. Interestingly, methylation levels of some of those genes were also changed. Furthermore, endogenous IAA content was decreased in inbred plants, in agreement with expression and methylation data. Chemical inhibition of auxin also replicated the degenerated phenotype in normal plants, while exogenous IAA application had no effect in inbred depression plants, suggesting a more complex mechanism. These data indicate DNA methylation-regulated auxin pathways play a role in establishing inbred depression phenotypes in plants. Our findings reveal new insights into inbreeding depression and leafy head development in Chinese cabbage. Copyright © 2018 Elsevier B.V. All rights reserved.

Maternal adversities during pregnancy and cord blood oxytocin receptor (OXTR) DNA methylation.

PubMed

Unternaehrer, Eva; Bolten, Margarete; Nast, Irina; Staehli, Simon; Meyer, Andrea H; Dempster, Emma; Hellhammer, Dirk H; Lieb, Roselind; Meinlschmidt, Gunther

2016-09-01

The aim of this study was to investigate whether maternal adversities and cortisol levels during pregnancy predict cord blood DNA methylation of the oxytocin receptor (OXTR). We collected cord blood of 39 babies born to mothers participating in a cross-sectional study (N = 100) conducted in Basel, Switzerland (2007-10). Mothers completed the Inventory of Life Events (second trimester: T2), the Edinburgh Postnatal Depression Scale (EPDS, third trimester: T3), the Trier Inventory of Chronic Stress (TICS-K, 1-3 weeks postpartum) and provided saliva samples (T2, T3) for maternal cortisol profiles, as computed by the area under the curve with respect to ground (AUCg) or increase (AUCi) for the cortisol awakening response (CAR) and for diurnal cortisol profiles (DAY). OXTR DNA methylation was quantified using Sequenom EpiTYPER. The number of stressful life events (P = 0.032), EPDS score (P = 0.007) and cortisol AUCgs at T2 (CAR: P = 0.020; DAY: P = 0.024) were negatively associated with OXTR DNA methylation. Our findings suggest that distinct prenatal adversities predict decreased DNA methylation in a gene that is relevant for childbirth, maternal behavior and wellbeing of mother and offspring. If a reduced OXTR methylation increases OXTR expression, our findings could suggest an epigenetic adaptation to an adverse early environment. © The Author (2016). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Exploratory analysis of ERCC2 DNA methylation in survival among pediatric medulloblastoma patients.

PubMed

Banfield, Emilyn; Brown, Austin L; Peckham, Erin C; Rednam, Surya P; Murray, Jeffrey; Okcu, M Fatih; Mitchell, Laura E; Chintagumpala, Murali M; Lau, Ching C; Scheurer, Michael E; Lupo, Philip J

2016-10-01

Medulloblastoma is the most frequent malignant pediatric brain tumor. While survival rates have improved due to multimodal treatment including cisplatin-based chemotherapy, there are few prognostic factors for adverse treatment outcomes. Notably, genes involved in the nucleotide excision repair pathway, including ERCC2, have been implicated in cisplatin sensitivity in other cancers. Therefore, this study evaluated the role of ERCC2 DNA methylation profiles on pediatric medulloblastoma survival. The study population included 71 medulloblastoma patients (age <18years at diagnosis) and recruited from Texas Children's Cancer Center between 2004 and 2009. DNA methylation profiles were generated from peripheral blood samples using the Illumina Infinium Human Methylation 450 Beadchip. Sixteen ERCC2-associated CpG sites were evaluated in this analysis. Multivariable regression models were used to determine the adjusted association between DNA methylation and survival. Cox regression and Kaplan-Meier curves were used to compare 5-year overall survival between hyper- and hypo-methylation at each CpG site. In total, 12.7% (n=9) of the patient population died within five years of diagnosis. In our population, methylation of the cg02257300 probe (Hazard Ratio=9.33; 95% Confidence Interval: 1.17-74.64) was associated with death (log-rank p=0.01). This association remained suggestive after correcting for multiple comparisons (FDR p<0.2). No other ERCC2-associated CpG site was associated with survival in this population of pediatric medulloblastoma patients. These findings provide the first evidence that DNA methylation within the promoter region of the ERCC2 gene may be associated with survival in pediatric medulloblastoma. If confirmed in future studies, this information may lead to improved risk stratification or promote the development of novel, targeted therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fatty acid synthase methylation levels in adipose tissue: effects of an obesogenic diet and phenolic compounds

USDA-ARS?s Scientific Manuscript database

DNA methylation is an epigenetic mechanism that can inhibit gene transcription. The aim of this study was to assess changes induced by an obesogenic diet in the methylation profile of genes involved in adipose tissue triacylglycerol metabolism, and to determine whether this methylation pattern can b...

Genome-wide DNA methylation profiles and their replationship with mRNA and the microRNA transcriptome in bovine muscle tissue (Bos Taurine)

USDA-ARS?s Scientific Manuscript database

DNA methylation is a key epigenetic modification in mammals, having essential and important roles in muscle development. We sample longissimus thoracis tissues from a well-known elite native breed of Chinese Qinchuan cattle living within comparable environments at fetal and adult stages, using methy...

DNA methylation profiling reveals the presence of population-specific signatures correlating with phenotypic characteristics.

PubMed

Giri, Anil K; Bharadwaj, Soham; Banerjee, Priyanka; Chakraborty, Shraddha; Parekatt, Vaisak; Rajashekar, Donaka; Tomar, Abhishek; Ravindran, Aarthi; Basu, Analabha; Tandon, Nikhil; Bharadwaj, Dwaipayan

2017-06-01

Phenotypic characteristics are known to vary substantially among different ethnicities around the globe. These variations are mediated by number of stochastic events and cannot be attributed to genetic architecture alone. DNA methylation is a well-established mechanism that sculpts our epigenome influencing phenotypic variation including disease manifestation. Since DNA methylation is an important determinant for health issues of a population, it demands a thorough investigation of the natural differences in genome wide DNA methylation patterns across different ethnic groups. This study is based on comparative analyses of methylome from five different ethnicities with major focus on Indian subjects. The current study uses hierarchical clustering approaches, principal component analysis and locus specific differential methylation analysis on Illumina 450K methylation data to compare methylome of different ethnic subjects. Our data indicates that the variations in DNA methylation patterns of Indians are less among themselves compared to other global population. It empirically correlated with dietary, cultural and demographical divergences across different ethnic groups. Our work further suggests that Indians included in this study, despite their genetic similarity with the Caucasian population, are in close proximity with Japanese in terms of their methylation signatures.

Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort.

PubMed

Chen, Zhuo; Miao, Feng; Paterson, Andrew D; Lachin, John M; Zhang, Lingxiao; Schones, Dustin E; Wu, Xiwei; Wang, Jinhui; Tompkins, Joshua D; Genuth, Saul; Braffett, Barbara H; Riggs, Arthur D; Natarajan, Rama

2016-05-24

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort

PubMed Central

Chen, Zhuo; Miao, Feng; Paterson, Andrew D.; Lachin, John M.; Zhang, Lingxiao; Schones, Dustin E.; Wu, Xiwei; Wang, Jinhui; Tompkins, Joshua D.; Genuth, Saul; Braffett, Barbara H.; Riggs, Arthur D.; Natarajan, Rama

2016-01-01

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16–17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory. PMID:27162351

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

PubMed Central

Xu, Yaomin; Hu, Bo; Choi, Ae-Jin; Gopalan, Banu; Lee, Byron H.; Kalady, Matthew F.; Church, James M.; Ting, Angela H.

2012-01-01

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes. PMID:21990380

Lnc2Meth: a manually curated database of regulatory relationships between long non-coding RNAs and DNA methylation associated with human disease

PubMed Central

Zhi, Hui; Li, Xin; Wang, Peng; Gao, Yue; Gao, Baoqing; Zhou, Dianshuang; Zhang, Yan; Guo, Maoni; Yue, Ming; Shen, Weitao

2018-01-01

Abstract Lnc2Meth (http://www.bio-bigdata.com/Lnc2Meth/), an interactive resource to identify regulatory relationships between human long non-coding RNAs (lncRNAs) and DNA methylation, is not only a manually curated collection and annotation of experimentally supported lncRNAs-DNA methylation associations but also a platform that effectively integrates tools for calculating and identifying the differentially methylated lncRNAs and protein-coding genes (PCGs) in diverse human diseases. The resource provides: (i) advanced search possibilities, e.g. retrieval of the database by searching the lncRNA symbol of interest, DNA methylation patterns, regulatory mechanisms and disease types; (ii) abundant computationally calculated DNA methylation array profiles for the lncRNAs and PCGs; (iii) the prognostic values for each hit transcript calculated from the patients clinical data; (iv) a genome browser to display the DNA methylation landscape of the lncRNA transcripts for a specific type of disease; (v) tools to re-annotate probes to lncRNA loci and identify the differential methylation patterns for lncRNAs and PCGs with user-supplied external datasets; (vi) an R package (LncDM) to complete the differentially methylated lncRNAs identification and visualization with local computers. Lnc2Meth provides a timely and valuable resource that can be applied to significantly expand our understanding of the regulatory relationships between lncRNAs and DNA methylation in various human diseases. PMID:29069510

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Decreased expression level of BER genes in Alzheimer's disease patients is not derivative of their DNA methylation status.

PubMed

Sliwinska, Agnieszka; Sitarek, Przemysław; Toma, Monika; Czarny, Piotr; Synowiec, Ewelina; Krupa, Renata; Wigner, Paulina; Bialek, Katarzyna; Kwiatkowski, Dominik; Korycinska, Anna; Majsterek, Ireneusz; Szemraj, Janusz; Galecki, Piotr; Sliwinski, Tomasz

2017-10-03

Neurodegeneration in Alzheimer's disease can be caused by accumulation of oxidative DNA damage resulting from altered expression of genes involved in the base excision repair system (BER). Promoter methylation can affect the profile of BER genes expression. Decreased expression of BER genes was observed in the brains of AD patients. The aim of our study was to compare the expression and methylation profiles of six genes coding for proteins involved in BER, namely: hOGG1, APE1, MUTYH, NEIL1, PARP1 and XRCC1, in the peripheral blood cells of AD patients and healthy volunteers. The study consisted of 100 persons diagnosed with Alzheimer's disease according to DSM-IV criteria, and 110 healthy volunteers. DNA and total RNA were isolated from venous blood cells. Promoter methylation profiles were obtained by High Resolution Melting (HRM) analysis of bisulfide converted DNA samples. Real-time PCR with TaqMan probes was employed for gene expression analysis. APE1, hOGG1, MUTYH, PARP1 and NEIL1 were significantly (p<0.001) down-regulated in the lymphocytes of AD patients, as compared to healthy volunteers. Expression of XRCC1 didn't differ significantly between both groups. We did not find any differences in the methylation pattern of any of the investigated BER genes. The methylation status of promoters is not associated with downregulation of BER genes. Our results show that downregulation of BER genes detected in peripheral blood samples could reflect the changes occurring in the brain of patients with AD, and may be a useful biomarker of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA Methylation and Hydroxymethylation Profile of CD34+-Enriched Cell Products Intended for Autologous CD34+ Cell Transplantation.

PubMed

Rozman, Jasmina-Ziva; Pohar Perme, Maja; Jez, Mojca; Malicev, Elvira; Krasna, Metka; Vrtovec, Bojan; Rozman, Primoz

2017-09-01

Epigenetic dysregulation has been shown to limit functional capacity of aging hematopoietic stem cells, which may contribute to impaired outcome of hematopoietic stem cell-based therapies. The aim of our study was to gain better insight into the epigenetic profile of CD34 + -enriched cell products intended for autologous CD34 + cell transplantation in patients with cardiomyopathy. We found global DNA methylation content significantly higher in immunoselected CD34 + cells compared to leukocytes in leukapheresis products (2.33 ± 1.03% vs. 1.84 ± 0.86%, p = 0.04). Global DNA hydroxymethylation content did not differ between CD34 + cells and leukocytes (p = 0.30). By measuring methylation levels of 94 stem cell transcription factors on a ready-to-use array, we identified 15 factors in which average promoter methylation was significantly different between leukocytes and CD34 + cells. The difference was highest for HOXC12 (58.18 ± 6.47% vs. 13.34 ± 24.18%, p = 0.0009) and NR2F2 (51.65 ± 25.89% vs. 7.66 ± 21.43%, p = 0.0045) genes. Our findings suggest that global DNA methylation and hydroxymethylation patterns as well as target methylation profile of selected genes in CD34 + -enriched cell products do not differ significantly compared to leukapheresis products and, thus, can tell us little about the functional capacity and regenerative properties of CD34 + cells. Future studies should examine other CD34 + cell graft characteristics, which may serve as prognostic tools for autologous CD34 + cell transplantation.

Identification of DNA methylation changes associated with disease progression in subchondral bone with site-matched cartilage in knee osteoarthritis.

PubMed

Zhang, Yanfei; Fukui, Naoshi; Yahata, Mitsunori; Katsuragawa, Yozo; Tashiro, Toshiyuki; Ikegawa, Shiro; Lee, Ming Ta Michael

2016-09-30

Subchondral bone plays a key role in the development of osteoarthritis, however, epigenetics of subchondral bone has not been extensively studied. In this study, we examined the genome-wide DNA methylation profiles of subchondral bone from three regions on tibial plateau representing disease progression using HumanMethylation450 BeadChip to identify progression associated DNA methylation alterations. Significant differential methylated probes (DMPs) and differential methylated genes (DMGs) were identified in the intermediate and late stages and during the transition from intermediate to late stage of OA in the subchondral bone. Over half of the DMPs were hyper-methylated. Genes associated with OA and bone remodeling were identified. DMGs were enriched in morphogenesis and development of skeletal system, and HOX transcription factors. Comparison of DMGs identified in subchondral bone and site-matched cartilage indicated that DNA methylation changes occurred earlier in subchondral bone and identified different methylation patterns at the late stage of OA. However, shared DMPs, DMGs and common pathways that implicated the tissue reparation were also identified. Methylation is one key mechanism to regulate the crosstalk between cartilage and subchondral bone.

Identification of DNA methylation changes associated with disease progression in subchondral bone with site-matched cartilage in knee osteoarthritis

PubMed Central

Zhang, Yanfei; Fukui, Naoshi; Yahata, Mitsunori; Katsuragawa, Yozo; Tashiro, Toshiyuki; Ikegawa, Shiro; Lee, Ming Ta Michael

2016-01-01

Subchondral bone plays a key role in the development of osteoarthritis, however, epigenetics of subchondral bone has not been extensively studied. In this study, we examined the genome-wide DNA methylation profiles of subchondral bone from three regions on tibial plateau representing disease progression using HumanMethylation450 BeadChip to identify progression associated DNA methylation alterations. Significant differential methylated probes (DMPs) and differential methylated genes (DMGs) were identified in the intermediate and late stages and during the transition from intermediate to late stage of OA in the subchondral bone. Over half of the DMPs were hyper-methylated. Genes associated with OA and bone remodeling were identified. DMGs were enriched in morphogenesis and development of skeletal system, and HOX transcription factors. Comparison of DMGs identified in subchondral bone and site-matched cartilage indicated that DNA methylation changes occurred earlier in subchondral bone and identified different methylation patterns at the late stage of OA. However, shared DMPs, DMGs and common pathways that implicated the tissue reparation were also identified. Methylation is one key mechanism to regulate the crosstalk between cartilage and subchondral bone. PMID:27686527

Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Xu, Rui-Hua; Wei, Wei; Krawczyk, Michal; Wang, Wenqiu; Luo, Huiyan; Flagg, Ken; Yi, Shaohua; Shi, William; Quan, Qingli; Li, Kang; Zheng, Lianghong; Zhang, Heng; Caughey, Bennett A.; Zhao, Qi; Hou, Jiayi; Zhang, Runze; Xu, Yanxin; Cai, Huimin; Li, Gen; Hou, Rui; Zhong, Zheng; Lin, Danni; Fu, Xin; Zhu, Jie; Duan, Yaou; Yu, Meixing; Ying, Binwu; Zhang, Wengeng; Wang, Juan; Zhang, Edward; Zhang, Charlotte; Li, Oulan; Guo, Rongping; Carter, Hannah; Zhu, Jian-Kang; Hao, Xiaoke; Zhang, Kang

2017-11-01

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive `liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

Differential DNA methylation and lymphocyte proportions in a Costa Rican high longevity region.

PubMed

McEwen, Lisa M; Morin, Alexander M; Edgar, Rachel D; MacIsaac, Julia L; Jones, Meaghan J; Dow, William H; Rosero-Bixby, Luis; Kobor, Michael S; Rehkopf, David H

2017-01-01

The Nicoya Peninsula in Costa Rica has one of the highest old-age life expectancies in the world, but the underlying biological mechanisms of this longevity are not well understood. As DNA methylation is hypothesized to be a component of biological aging, we focused on this malleable epigenetic mark to determine its association with current residence in Nicoya versus elsewhere in Costa Rica. Examining a population's unique DNA methylation pattern allows us to differentiate hallmarks of longevity from individual stochastic variation. These differences may be characteristic of a combination of social, biological, and environmental contexts. In a cross-sectional subsample of the Costa Rican Longevity and Healthy Aging Study, we compared whole blood DNA methylation profiles of residents from Nicoya ( n  = 48) and non-Nicoya (other Costa Rican regions, n  = 47) using the Infinium HumanMethylation450 microarray. We observed a number of differences that may be markers of delayed aging, such as bioinformatically derived differential CD8+ T cell proportions. Additionally, both site- and region-specific analyses revealed DNA methylation patterns unique to Nicoyans. We also observed lower overall variability in DNA methylation in the Nicoyan population, another hallmark of younger biological age. Nicoyans represent an interesting group of individuals who may possess unique immune cell proportions as well as distinct differences in their epigenome, at the level of DNA methylation.

Accounting for cell lineage and sex effects in the identification of cell-specific DNA methylation using a Bayesian model selection algorithm.

PubMed

White, Nicole; Benton, Miles; Kennedy, Daniel; Fox, Andrew; Griffiths, Lyn; Lea, Rodney; Mengersen, Kerrie

2017-01-01

Cell- and sex-specific differences in DNA methylation are major sources of epigenetic variation in whole blood. Heterogeneity attributable to cell type has motivated the identification of cell-specific methylation at the CpG level, however statistical methods for this purpose have been limited to pairwise comparisons between cell types or between the cell type of interest and whole blood. We developed a Bayesian model selection algorithm for the identification of cell-specific methylation profiles that incorporates knowledge of shared cell lineage and allows for the identification of differential methylation profiles in one or more cell types simultaneously. Under the proposed methodology, sex-specific differences in methylation by cell type are also assessed. Using publicly available, cell-sorted methylation data, we show that 51.3% of female CpG markers and 61.4% of male CpG markers identified were associated with differential methylation in more than one cell type. The impact of cell lineage on differential methylation was also highlighted. An evaluation of sex-specific differences revealed differences in CD56+NK methylation, within both single and multi- cell dependent methylation patterns. Our findings demonstrate the need to account for cell lineage in studies of differential methylation and associated sex effects.

Genome-wide analysis of DNA methylation in five tissues of sika deer (Cervus nippon).

PubMed

Yang, Chun; Zhang, Yan; Liu, Wenyuan; Lu, Xiao; Li, Chunyi

2018-03-01

DNA methylation plays an important role in regulating gene expression during tissue development and differentiation in eukaryotes. In contrast to domestic animals, epigenetic studies have been seldom conducted in wild animals. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of sika deer using the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique. Overall, a total of 104,131 fragments were amplified including 41,951 methylated fragments using 32 pairs of selected primers. The average incidence of DNA methylation was approximately 38.18% in muscle, 40.32% in heart, 41.86% in liver, 41.20% in lung, and 41.68% in kidney, respectively. Also, the significant differences of the DNA methylation levels were found between the different tissue types (P<0.05), which indicates that the differences of genome-wide DNA methylation levels may be related to gene expression during tissue development and differentiation. In addition, 37 tissue-specific differentially methylated regions (T-DMRs) were identified and recovered by MSAP in five tissues, and were further confirmed by Southern blot analysis. Our study presents the first look at the T-DMRs in sika deer and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in sika deer. Copyright © 2017. Published by Elsevier B.V.

Genetic Perturbation of the Maize Methylome[W

PubMed Central

Li, Qing; Hermanson, Peter J.; Zaunbrecher, Virginia M.; Song, Jawon; Wendt, Jennifer; Rosenbaum, Heidi; Madzima, Thelma F.; Sloan, Amy E.; Huang, Ji; Burgess, Daniel L.; Richmond, Todd A.; McGinnis, Karen M.; Meeley, Robert B.; Danilevskaya, Olga N.; Vaughn, Matthew W.; Kaeppler, Shawn M.; Jeddeloh, Jeffrey A.

2014-01-01

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana. PMID:25527708

The Influence of Metabolic Syndrome and Sex on the DNA Methylome in Schizophrenia

PubMed Central

Lines, Brittany N.

2018-01-01

Introduction The mechanism by which metabolic syndrome occurs in schizophrenia is not completely known; however, previous work suggests that changes in DNA methylation may be involved which is further influenced by sex. Within this study, the DNA methylome was profiled to identify altered methylation associated with metabolic syndrome in a schizophrenia population on atypical antipsychotics. Methods Peripheral blood from schizophrenia subjects was utilized for DNA methylation analyses. Discovery analyses (n = 96) were performed using an epigenome-wide analysis on the Illumina HumanMethylation450K BeadChip based on metabolic syndrome diagnosis. A secondary discovery analysis was conducted based on sex. The top hits from the discovery analyses were assessed in an additional validation set (n = 166) using site-specific methylation pyrosequencing. Results A significant increase in CDH22 gene methylation in subjects with metabolic syndrome was identified in the overall sample. Additionally, differential methylation was found within the MAP3K13 gene in females and the CCDC8 gene within males. Significant differences in methylation were again observed for the CDH22 and MAP3K13 genes, but not CCDC8, in the validation sample set. Conclusions This study provides preliminary evidence that DNA methylation may be associated with metabolic syndrome and sex in schizophrenia. PMID:29850476

Integrating Colon Cancer Microarray Data: Associating Locus-Specific Methylation Groups to Gene Expression-Based Classifications.

PubMed

Barat, Ana; Ruskin, Heather J; Byrne, Annette T; Prehn, Jochen H M

2015-11-23

Recently, considerable attention has been paid to gene expression-based classifications of colorectal cancers (CRC) and their association with patient prognosis. In addition to changes in gene expression, abnormal DNA-methylation is known to play an important role in cancer onset and development, and colon cancer is no exception to this rule. Large-scale technologies, such as methylation microarray assays and specific sequencing of methylated DNA, have been used to determine whole genome profiles of CpG island methylation in tissue samples. In this article, publicly available microarray-based gene expression and methylation data sets are used to characterize expression subtypes with respect to locus-specific methylation. A major objective was to determine whether integration of these data types improves previously characterized subtypes, or provides evidence for additional subtypes. We used unsupervised clustering techniques to determine methylation-based subgroups, which are subsequently annotated with three published expression-based classifications, comprising from three to six subtypes. Our results showed that, while methylation profiles provide a further basis for segregation of certain (Inflammatory and Goblet-like) finer-grained expression-based subtypes, they also suggest that other finer-grained subtypes are not distinctive and can be considered as a single subtype.

Integrating Colon Cancer Microarray Data: Associating Locus-Specific Methylation Groups to Gene Expression-Based Classifications

PubMed Central

Barat, Ana; Ruskin, Heather J.; Byrne, Annette T.; Prehn, Jochen H. M.

2015-01-01

Recently, considerable attention has been paid to gene expression-based classifications of colorectal cancers (CRC) and their association with patient prognosis. In addition to changes in gene expression, abnormal DNA-methylation is known to play an important role in cancer onset and development, and colon cancer is no exception to this rule. Large-scale technologies, such as methylation microarray assays and specific sequencing of methylated DNA, have been used to determine whole genome profiles of CpG island methylation in tissue samples. In this article, publicly available microarray-based gene expression and methylation data sets are used to characterize expression subtypes with respect to locus-specific methylation. A major objective was to determine whether integration of these data types improves previously characterized subtypes, or provides evidence for additional subtypes. We used unsupervised clustering techniques to determine methylation-based subgroups, which are subsequently annotated with three published expression-based classifications, comprising from three to six subtypes. Our results showed that, while methylation profiles provide a further basis for segregation of certain (Inflammatory and Goblet-like) finer-grained expression-based subtypes, they also suggest that other finer-grained subtypes are not distinctive and can be considered as a single subtype. PMID:27600244

Identification and validation of seven new loci showing differential DNA methylation related to serum lipid profile: an epigenome-wide approach. The REGICOR study

USDA-ARS?s Scientific Manuscript database

Lipid traits (total, low-density and high-density lipoprotein cholesterol, and triglycerides) are risk factors for cardiovascular disease. DNA methylation is not only an inherited but also modifiable epigenetic mark that has been related to cardiovascular risk factors. Our aim was to identify loci s...

Genome-wide DNA methylomes from discrete developmental stages reveal the predominance of non-CpG methylation in Tribolium castaneum.

PubMed

Song, Xiaowen; Huang, Fei; Liu, Juanjuan; Li, Chengjun; Gao, Shanshan; Wu, Wei; Zhai, Mengfan; Yu, Xiaojuan; Xiong, Wenfeng; Xie, Jia; Li, Bin

2017-10-01

Cytosine DNA methylation is a vital epigenetic regulator of eukaryotic development. Whether this epigenetic modification occurs in Tribolium castaneum has been controversial, its distribution pattern and functions have not been established. Here, using bisulphite sequencing (BS-Seq), we confirmed the existence of DNA methylation and described the methylation profiles of the four life stages of T. castaneum. In the T. castaneum genome, both symmetrical CpG and non-CpG methylcytosines were observed. Symmetrical CpG methylation, which was catalysed by DNMT1 and occupied a small part in T. castaneum methylome, was primarily enriched in gene bodies and was positively correlated with gene expression levels. Asymmetrical non-CpG methylation, which was predominant in the methylome, was strongly concentrated in intergenic regions and introns but absent from exons. Gene body methylation was negatively correlated with gene expression levels. The distribution pattern and functions of this type of methylation were similar only to the methylome of Drosophila melanogaster, which further supports the existence of a novel methyltransferase in the two species responsible for this type of methylation. This first life-cycle methylome of T. castaneum reveals a novel and unique methylation pattern, which will contribute to the further understanding of the variety and functions of DNA methylation in eukaryotes. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Grandmaternal stress during pregnancy and DNA methylation of the third generation: an epigenome-wide association study

PubMed Central

Serpeloni, F; Radtke, K; de Assis, S G; Henning, F; Nätt, D; Elbert, T

2017-01-01

Stress during pregnancy may impact subsequent generations, which is demonstrated by an increased susceptibility to childhood and adulthood health problems in the children and grandchildren. Although the importance of the prenatal environment is well reported with regards to future physical and emotional outcomes, little is known about the molecular mechanisms that mediate the long-term consequences of early stress across generations. Recent studies have identified DNA methylation as a possible mediator of the impact of prenatal stress in the offspring. Whether psychosocial stress during pregnancy also affects DNA methylation of the grandchildren is still not known. In the present study we examined the multigenerational hypothesis, that is, grandmaternal exposure to psychosocial stress during pregnancy affecting DNA methylation of the grandchildren. We determined the genome-wide DNA methylation profile in 121 children (65 females and 56 males) and tested for associations with exposure to grandmaternal interpersonal violence during pregnancy. We observed methylation variations of five CpG sites significantly (FDR<0.05) associated with the grandmother’s report of exposure to violence while pregnant with the mothers of the children. The results revealed differential methylation of genes previously shown to be involved in circulatory system processes (FDR<0.05). This study provides support for DNA methylation as a biological mechanism involved in the transmission of stress across generations and motivates further investigations to examine prenatal-dependent DNA methylation as a potential biomarker for health problems. PMID:28809857

Grandmaternal stress during pregnancy and DNA methylation of the third generation: an epigenome-wide association study.

PubMed

Serpeloni, F; Radtke, K; de Assis, S G; Henning, F; Nätt, D; Elbert, T

2017-08-15

Stress during pregnancy may impact subsequent generations, which is demonstrated by an increased susceptibility to childhood and adulthood health problems in the children and grandchildren. Although the importance of the prenatal environment is well reported with regards to future physical and emotional outcomes, little is known about the molecular mechanisms that mediate the long-term consequences of early stress across generations. Recent studies have identified DNA methylation as a possible mediator of the impact of prenatal stress in the offspring. Whether psychosocial stress during pregnancy also affects DNA methylation of the grandchildren is still not known. In the present study we examined the multigenerational hypothesis, that is, grandmaternal exposure to psychosocial stress during pregnancy affecting DNA methylation of the grandchildren. We determined the genome-wide DNA methylation profile in 121 children (65 females and 56 males) and tested for associations with exposure to grandmaternal interpersonal violence during pregnancy. We observed methylation variations of five CpG sites significantly (FDR<0.05) associated with the grandmother's report of exposure to violence while pregnant with the mothers of the children. The results revealed differential methylation of genes previously shown to be involved in circulatory system processes (FDR<0.05). This study provides support for DNA methylation as a biological mechanism involved in the transmission of stress across generations and motivates further investigations to examine prenatal-dependent DNA methylation as a potential biomarker for health problems.

Characterization of the Cartilage DNA Methylome in Knee and Hip Osteoarthritis

PubMed Central

Rushton, Michael D; Reynard, Louise N; Barter, Matt J; Refaie, Ramsay; Rankin, Kenneth S; Young, David A; Loughlin, John

2014-01-01

Objective The aim of this study was to characterize the genome-wide DNA methylation profile of chondrocytes from knee and hip cartilage obtained from patients with osteoarthritis (OA) and hip cartilage obtained from patients with femoral neck fracture, providing the first comparison of DNA methylation between OA and non-OA hip cartilage, and between OA hip and OA knee cartilage. Methods The study was performed using the Illumina Infinium HumanMethylation450 BeadChip array, which allows the annotation of ∼480,000 CpG sites. Genome-wide methylation was assessed in chondrocyte DNA extracted from 23 hip OA patients, 73 knee OA patients, and 21 healthy hip control patients with femoral neck fracture. Results Analysis revealed that chondrocytes from the hip cartilage of OA patients and healthy controls have unique methylation profiles, with 5,322 differentially methylated loci (DMLs) identified between the 2 groups. In addition, a comparison between hip and knee OA chondrocytes revealed 5,547 DMLs between the 2 groups, including DMLs in several genes known to be involved in the pathogenesis of OA. Hip OA samples were found to cluster into 2 groups. A total of 15,239 DMLs were identified between the 2 clusters, with an enrichment of genes involved in inflammation and immunity. Similarly, we confirmed a previous report of knee OA samples that also clustered into 2 groups. Conclusion We demonstrated that global DNA methylation using a high-density array can be a powerful tool in the characterization of OA at the molecular level. Identification of pathways enriched in DMLs between OA and OA-free cartilage highlight potential etiologic mechanisms that are involved in the initiation and/or progression of the disease and that could be therapeutically targeted. PMID:24838673

Genome-wide analysis of salinity-stress induced DNA methylation alterations in cotton (Gossypium hirsutum L.) using the Me-DIP sequencing technology.

PubMed

Lu, X K; Shu, N; Wang, J J; Chen, X G; Wang, D L; Wang, S; Fan, W L; Guo, X N; Guo, L X; Ye, W W

2017-06-29

Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.

DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

PubMed

Oda, Masaaki; Kumaki, Yuichi; Shigeta, Masaki; Jakt, Lars Martin; Matsuoka, Chisa; Yamagiwa, Akiko; Niwa, Hitoshi; Okano, Masaki

2013-06-01

DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

Lack of Evidence for Green Tea Polyphenols as DNA Methylation Inhibitors in Murine Prostate

PubMed Central

Morey Kinney, Shannon R.; Zhang, Wa; Pascual, Marien; Greally, John M.; Gillard, Bryan M.; Karasik, Ellen; Foster, Barbara A.; Karpf, Adam R.

2009-01-01

Green tea polyphenols (GTPs) have been reported to inhibit DNA methylation in cultured cells. Here we tested whether oral consumption of GTPs affects normal or cancer specific DNA methylation in vivo, using mice. Wildtype (WT) and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice were administered 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HELP assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate. PMID:19934341

Genome-wide DNA methylomes from discrete developmental stages reveal the predominance of non-CpG methylation in Tribolium castaneum

PubMed Central

Song, Xiaowen; Huang, Fei; Liu, Juanjuan; Li, Chengjun; Gao, Shanshan; Wu, Wei; Zhai, Mengfan; Yu, Xiaojuan; Xiong, Wenfeng; Xie, Jia

2017-01-01

Abstract Cytosine DNA methylation is a vital epigenetic regulator of eukaryotic development. Whether this epigenetic modification occurs in Tribolium castaneum has been controversial, its distribution pattern and functions have not been established. Here, using bisulphite sequencing (BS-Seq), we confirmed the existence of DNA methylation and described the methylation profiles of the four life stages of T. castaneum. In the T. castaneum genome, both symmetrical CpG and non-CpG methylcytosines were observed. Symmetrical CpG methylation, which was catalysed by DNMT1 and occupied a small part in T. castaneum methylome, was primarily enriched in gene bodies and was positively correlated with gene expression levels. Asymmetrical non-CpG methylation, which was predominant in the methylome, was strongly concentrated in intergenic regions and introns but absent from exons. Gene body methylation was negatively correlated with gene expression levels. The distribution pattern and functions of this type of methylation were similar only to the methylome of Drosophila melanogaster, which further supports the existence of a novel methyltransferase in the two species responsible for this type of methylation. This first life-cycle methylome of T. castaneum reveals a novel and unique methylation pattern, which will contribute to the further understanding of the variety and functions of DNA methylation in eukaryotes. PMID:28449092

Effects of As2O3 on DNA methylation, genomic instability, and LTR retrotransposon polymorphism in Zea mays.

PubMed

Erturk, Filiz Aygun; Aydin, Murat; Sigmaz, Burcu; Taspinar, M Sinan; Arslan, Esra; Agar, Guleray; Yagci, Semra

2015-12-01

Arsenic is a well-known toxic substance on the living organisms. However, limited efforts have been made to study its DNA methylation, genomic instability, and long terminal repeat (LTR) retrotransposon polymorphism causing properties in different crops. In the present study, effects of As2O3 (arsenic trioxide) on LTR retrotransposon polymorphism and DNA methylation as well as DNA damage in Zea mays seedlings were investigated. The results showed that all of arsenic doses caused a decreasing genomic template stability (GTS) and an increasing Random Amplified Polymorphic DNAs (RAPDs) profile changes (DNA damage). In addition, increasing DNA methylation and LTR retrotransposon polymorphism characterized a model to explain the epigenetically changes in the gene expression were also found. The results of this experiment have clearly shown that arsenic has epigenetic effect as well as its genotoxic effect. Especially, the increasing of polymorphism of some LTR retrotransposon under arsenic stress may be a part of the defense system against the stress.

Lnc2Meth: a manually curated database of regulatory relationships between long non-coding RNAs and DNA methylation associated with human disease.

PubMed

Zhi, Hui; Li, Xin; Wang, Peng; Gao, Yue; Gao, Baoqing; Zhou, Dianshuang; Zhang, Yan; Guo, Maoni; Yue, Ming; Shen, Weitao; Ning, Shangwei; Jin, Lianhong; Li, Xia

2018-01-04

Lnc2Meth (http://www.bio-bigdata.com/Lnc2Meth/), an interactive resource to identify regulatory relationships between human long non-coding RNAs (lncRNAs) and DNA methylation, is not only a manually curated collection and annotation of experimentally supported lncRNAs-DNA methylation associations but also a platform that effectively integrates tools for calculating and identifying the differentially methylated lncRNAs and protein-coding genes (PCGs) in diverse human diseases. The resource provides: (i) advanced search possibilities, e.g. retrieval of the database by searching the lncRNA symbol of interest, DNA methylation patterns, regulatory mechanisms and disease types; (ii) abundant computationally calculated DNA methylation array profiles for the lncRNAs and PCGs; (iii) the prognostic values for each hit transcript calculated from the patients clinical data; (iv) a genome browser to display the DNA methylation landscape of the lncRNA transcripts for a specific type of disease; (v) tools to re-annotate probes to lncRNA loci and identify the differential methylation patterns for lncRNAs and PCGs with user-supplied external datasets; (vi) an R package (LncDM) to complete the differentially methylated lncRNAs identification and visualization with local computers. Lnc2Meth provides a timely and valuable resource that can be applied to significantly expand our understanding of the regulatory relationships between lncRNAs and DNA methylation in various human diseases. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

PubMed Central

Aberg, Karolina A.; Xie, Lin Y.; Nerella, Srilaxmi; Copeland, William E.; Costello, E. Jane; van den Oord, Edwin J.C.G.

2013-01-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach. PMID:23644822

High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot.

PubMed

Aberg, Karolina A; Xie, Lin Y; Nerella, Srilaxmi; Copeland, William E; Costello, E Jane; van den Oord, Edwin J C G

2013-05-01

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.

Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

PubMed

Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

2016-09-01

DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

Methylation of the oxytocin receptor gene in clinically depressed patients compared to controls: The role of OXTR rs53576 genotype.

PubMed

Reiner, I; Van IJzendoorn, M H; Bakermans-Kranenburg, M J; Bleich, S; Beutel, M; Frieling, H

2015-06-01

The emerging field of epigenetics provides a biological basis for gene-environment interactions relevant to depression. We focus on DNA methylation of exon 1 and 2 of the oxytocin receptor gene (OXTR) promoter. The research aims of the current study were to compare OXTR DNA methylation of depressed patients with healthy control subjects and to investigate possible influences of the OXTR rs53576 genotype. The sample of the present study consisted of 43 clinically depressed women recruited from a psychosomatic inpatient unit and 42 healthy, female control subjects - mean age 30 years (SD = 9). DNA methylation profiles of the OXTR gene were assessed from leukocyte DNA by means of bisulfite sequencing. Depressed female patients had decreased OXTR exon 1 DNA methylation compared to non-depressed women. The association between depression and methylation level was moderated by OXTR rs53576 genotype. Exon 2 methylation was associated with OXTR rs53576 genotype but not with depression. Our findings suggest exon-specific methylation mechanisms. Exon 1 methylation appears to be associated with depressive phenotypes whereas exon 2 methylation is influenced by genotype. Previously reported divergent associations between OXTR genotype and depression might be explained by varying exon 1 methylation. In order to further understand the etiology of depression, research on the interplay between genotype, environmental influences and exon-specific methylation patterns is needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

An integrated genomics analysis of epigenetic subtypes in human breast tumors links DNA methylation patterns to chromatin states in normal mammary cells.

PubMed

Holm, Karolina; Staaf, Johan; Lauss, Martin; Aine, Mattias; Lindgren, David; Bendahl, Pär-Ola; Vallon-Christersson, Johan; Barkardottir, Rosa Bjork; Höglund, Mattias; Borg, Åke; Jönsson, Göran; Ringnér, Markus

2016-02-29

Aberrant DNA methylation is frequently observed in breast cancer. However, the relationship between methylation patterns and the heterogeneity of breast cancer has not been comprehensively characterized. Whole-genome DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChip arrays was performed on 188 human breast tumors. Unsupervised bootstrap consensus clustering was performed to identify DNA methylation epigenetic subgroups (epitypes). The Cancer Genome Atlas data, including methylation profiles of 669 human breast tumors, was used for validation. The identified epitypes were characterized by integration with publicly available genome-wide data, including gene expression levels, DNA copy numbers, whole-exome sequencing data, and chromatin states. We identified seven breast cancer epitypes. One epitype was distinctly associated with basal-like tumors and with BRCA1 mutations, one epitype contained a subset of ERBB2-amplified tumors characterized by multiple additional amplifications and the most complex genomes, and one epitype displayed a methylation profile similar to normal epithelial cells. Luminal tumors were stratified into the remaining four epitypes, with differences in promoter hypermethylation, global hypomethylation, proliferative rates, and genomic instability. Specific hyper- and hypomethylation across the basal-like epitype was rare. However, we observed that the candidate genomic instability drivers BRCA1 and HORMAD1 displayed aberrant methylation linked to gene expression levels in some basal-like tumors. Hypomethylation in luminal tumors was associated with DNA repeats and subtelomeric regions. We observed two dominant patterns of aberrant methylation in breast cancer. One pattern, constitutively methylated in both basal-like and luminal breast cancer, was linked to genes with promoters in a Polycomb-repressed state in normal epithelial cells and displayed no correlation with gene expression levels. The second pattern correlated with gene expression levels and was associated with methylation in luminal tumors and genes with active promoters in normal epithelial cells. Our results suggest that hypermethylation patterns across basal-like breast cancer may have limited influence on tumor progression and instead reflect the repressed chromatin state of the tissue of origin. On the contrary, hypermethylation patterns specific to luminal breast cancer influence gene expression, may contribute to tumor progression, and may present an actionable epigenetic alteration in a subset of luminal breast cancers.

[Comparative analysis of methylation profiles in tissues of oral leukoplakia and oral squamous cell carcinoma].

PubMed

Fu, J; Su, Y; Liu, Y; Zhang, X Y

2018-04-09

Objective: To compare the methylation profiles in tissues of oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC) with healthy tissues of oral mucosa, in order to identify the role of DNA methylation played in tumorigenesis. Methods: DNA samples extracted from tissues of 4 healthy oral mucosa, 4 OSCC and 4 OLK collected from patients of the Department of Oral Medicine, Capital Medical University School of Stomatology were examined and compared using Methylation 450 Bead Chip. The genes associated with differentially methylated CpG sites were selected for gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. Results: Multiple differentially methylated CpG sites were identified by using the above mentioned assay. Hypermethylation constitutes 86.18% (23 290/27 025) of methylation changes in OLK and hypomethylation accounts for 13.82% (3 734/27 025) of methylation changes. Both hypermethylated and hypomethylated CpG sites were markedly increased in OSCC tissue compared with OLK tissue. The majority of differentially methylated CpG sites were located outside CpG islands, with approximately one-fourth in CpG shores flanking the islands, which were considered highly important for gene regulation and tumorigenesis. Pathway analysis revealed that differentially methylated CpG sites in both OLK and OSCC patients shared the same pathway enrichments, most of which were correlated with carcinogenesis and cancer progression (e.g., DNA repair, cell cycle, and apoptosis). Conclusions: In the present study, methylation-associated alterations affect almost all pathways in the cellular network in both OLK and OSCC. OLK and OSCC shared similar methylation changes whether in pathways or genes, indicating that epigenetically they might have the same molecular basis for disease progression.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm

PubMed Central

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P

2018-01-01

Abstract Background Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Results Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Conclusions Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility. PMID:29635292

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

PubMed

Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

2015-08-01

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm.

PubMed

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P; Liu, George E

2018-05-01

Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility.

Altered intragenic DNA methylation of HOOK2 gene in adipose tissue from individuals with obesity and type 2 diabetes

PubMed Central

Fernández-Bayón, Gustavo; Morales-Sánchez, Paula; Sanz, Lourdes; Turienzo, Estrella; González, Juan José; Martinez-Faedo, Ceferino; Suarez-Gutiérrez, Lorena; Ares, Jessica; Díaz-Naya, Lucia; Martin-Nieto, Alicia; Fernández-Morera, Juan L.; Fraga, Mario F.

2017-01-01

Aims/Hypothesis Failure in glucose response to insulin is a common pathology associated with obesity. In this study, we analyzed the genome wide DNA methylation profile of visceral adipose tissue (VAT) samples in a population of individuals with obesity and assessed whether differential methylation profiles are associated with the presence of type 2 diabetes (T2D). Methods More than 485,000 CpG genome sites from VAT samples from women with obesity undergoing gastric bypass (n = 18), and classified as suffering from type 2 diabetes (T2D) or not (no type 2 diabetes, NT2D), were analyzed using DNA methylation arrays. Results We found significant differential methylation between T2D and NT2D samples in 24 CpGs that map with sixteen genes, one of which, HOOK2, demonstrated a significant correlation between differentially hypermethylated regions on the gene body and the presence of type 2 diabetes. This was validated by pyrosequencing in a population of 91 samples from both males and females with obesity. Furthermore, when these results were analyzed by gender, female T2D samples were found hypermethylated at the cg04657146-region and the cg 11738485-region of HOOK2 gene, whilst, interestingly, male samples were found hypomethylated in this latter region. Conclusion The differential methylation profile of the HOOK2 gene in individuals with T2D and obesity might be related to the attendant T2D, but further studies are required to identify the potential role of HOOK2 gene in T2D disease. The finding of gender differences in T2D methylation of HOOK2 also warrants further investigation. PMID:29228058

CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

PubMed

Kava, Hieronimus W; Murray, Vincent

2016-10-01

This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-wide analysis of DNA methylation variations caused by chronic glucolipotoxicity in beta-cells.

PubMed

Hu, Y; Xu, X-H; He, K; Zhang, L-L; Wang, S-K; Pan, Y-Q; He, B-S; Feng, T-T; Mao, X-M

2014-02-01

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium.We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively.We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells.We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma.

PubMed

Zhang, Wei; Yan, Wei; You, Gan; Bao, Zhaoshi; Wang, Yongzhi; Liu, Yanwei; You, Yongping; Jiang, Tao

2013-01-01

To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP- primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ⩾18months) and 20 short-term survivors (STS; overall survival ⩽9months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP- samples. Methylation levels of 11 CpG loci (10genes) were statistically significantly lower, and 43 CpG loci (40genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP- samples (P<0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP- samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP- primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP- primary GBMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Metabolic Imbalance Associated with Methylation Dysregulation and Oxidative Damage in Children with Autism

ERIC Educational Resources Information Center

Melnyk, Stepan; Fuchs, George J.; Schulz, Eldon; Lopez, Maya; Kahler, Stephen G.; Fussell, Jill J.; Bellando, Jayne; Pavliv, Oleksandra; Rose, Shannon; Seidel, Lisa; Gaylor, David W.; James, S. Jill

2012-01-01

Oxidative stress and abnormal DNA methylation have been implicated in the pathophysiology of autism. We investigated the dynamics of an integrated metabolic pathway essential for cellular antioxidant and methylation capacity in 68 children with autism, 54 age-matched control children and 40 unaffected siblings. The metabolic profile of unaffected…

TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells

PubMed Central

Yamamoto, Eiichiro; Harada, Taku; Aoki, Hironori; Maruyama, Reo; Toyota, Mutsumi; Sasaki, Yasushi; Sugai, Tamotsu; Tokino, Takashi; Nakase, Hiroshi

2016-01-01

Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2’-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2’-deoxycytidine in CRC cells. PMID:27977763

DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).

PubMed

Canovas, Sebastian; Ross, Pablo J; Kelsey, Gavin; Coy, Pilar

2017-11-01

DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children. © 2017 WILEY Periodicals, Inc.

Human Endometrial DNA Methylome Is Cycle-Dependent and Is Associated With Gene Expression Regulation

PubMed Central

Houshdaran, Sahar; Zelenko, Zara; Irwin, Juan C.

2014-01-01

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders. PMID:24877562

Evaluation of massively parallel sequencing for forensic DNA methylation profiling.

PubMed

Richards, Rebecca; Patel, Jayshree; Stevenson, Kate; Harbison, SallyAnn

2018-05-11

Epigenetics is an emerging area of interest in forensic science. DNA methylation, a type of epigenetic modification, can be applied to chronological age estimation, identical twin differentiation and body fluid identification. However, there is not yet an agreed, established methodology for targeted detection and analysis of DNA methylation markers in forensic research. Recently a massively parallel sequencing-based approach has been suggested. The use of massively parallel sequencing is well established in clinical epigenetics and is emerging as a new technology in the forensic field. This review investigates the potential benefits, limitations and considerations of this technique for the analysis of DNA methylation in a forensic context. The importance of a robust protocol, regardless of the methodology used, that minimises potential sources of bias is highlighted. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers

PubMed Central

Begue, Gwénaëlle; Raue, Ulrika; Jemiolo, Bozena

2017-01-01

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men. PMID:28057818

DNA methylation in schizophrenia in different patient-derived cell types.

PubMed

Vitale, Alejandra M; Matigian, Nicholas A; Cristino, Alexandre S; Nones, Katia; Ravishankar, Sugandha; Bellette, Bernadette; Fan, Yongjun; Wood, Stephen A; Wolvetang, Ernst; Mackay-Sim, Alan

2017-01-01

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a "ground state" upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

Genome-scale case-control analysis of CD4+ T-cell DNA methylation in juvenile idiopathic arthritis reveals potential targets involved in disease.

PubMed

Ellis, Justine A; Munro, Jane E; Chavez, Raul A; Gordon, Lavinia; Joo, Jihoon E; Akikusa, Jonathan D; Allen, Roger C; Ponsonby, Anne-Louise; Craig, Jeffrey M; Saffery, Richard

2012-11-13

Juvenile Idiopathic Arthritis (JIA) is a complex autoimmune rheumatic disease of largely unknown cause. Evidence is growing that epigenetic variation, particularly DNA methylation, is associated with autoimmune disease. However, nothing is currently known about the potential role of aberrant DNA methylation in JIA. As a first step to addressing this knowledge gap, we have profiled DNA methylation in purified CD4+ T cells from JIA subjects and controls. Genomic DNA was isolated from peripheral blood CD4+ T cells from 14 oligoarticular and polyarticular JIA cases with active disease, and healthy age- and sex-matched controls. Genome-scale methylation analysis was carried out using the Illumina Infinium HumanMethylation27 BeadChip. Methylation data at >25,000 CpGs was compared in a case-control study design. Methylation levels were significantly different (FDR adjusted p<0.1) at 145 loci. Removal of four samples exposed to methotrexate had a striking impact on the outcome of the analysis, reducing the number of differentially methylated loci to 11. The methotrexate-naive analysis identified reduced methylation at the gene encoding the pro-inflammatory cytokine IL32, which was subsequently replicated using a second analysis platform and a second set of case-control pairs. Our data suggests that differential T cell DNA methylation may be a feature of JIA, and that reduced methylation at IL32 is associated with this disease. Further work in larger prospective and longitudinal sample collections is required to confirm these findings, assess whether the identified differences are causal or consequential of disease, and further investigate the epigenetic modifying properties of therapeutic regimens.

DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma

PubMed Central

Anwar, Sumadi Lukman; Lehmann, Ulrich

2014-01-01

Epigenetic alterations have been identified as a major characteristic in human cancers. Advances in the field of epigenetics have contributed significantly in refining our knowledge of molecular mechanisms underlying malignant transformation. DNA methylation and microRNA expression are epigenetic mechanisms that are widely altered in human cancers including hepatocellular carcinoma (HCC), the third leading cause of cancer related mortality worldwide. Both DNA methylation and microRNA expression patterns are regulated in developmental stage specific-, cell type specific- and tissue-specific manner. The aberrations are inferred in the maintenance of cancer stem cells and in clonal cell evolution during carcinogenesis. The availability of genome-wide technologies for DNA methylation and microRNA profiling has revolutionized the field of epigenetics and led to the discovery of a number of epigenetically silenced microRNAs in cancerous cells and primary tissues. Dysregulation of these microRNAs affects several key signalling pathways in hepatocarcinogenesis suggesting that modulation of DNA methylation and/or microRNA expression can serve as new therapeutic targets for HCC. Accumulative evidence shows that aberrant DNA methylation of certain microRNA genes is an event specifically found in HCC which correlates with unfavorable outcomes. Therefore, it can potentially serve as a biomarker for detection as well as for prognosis, monitoring and predicting therapeutic responses in HCC. PMID:24976726

Correspondence of DNA Methylation Between Blood and Brain Tissue and Its Application to Schizophrenia Research.

PubMed

Walton, Esther; Hass, Johanna; Liu, Jingyu; Roffman, Joshua L; Bernardoni, Fabio; Roessner, Veit; Kirsch, Matthias; Schackert, Gabriele; Calhoun, Vince; Ehrlich, Stefan

2016-03-01

Given the difficulty of procuring human brain tissue, a key question in molecular psychiatry concerns the extent to which epigenetic signatures measured in more accessible tissues such as blood can serve as a surrogate marker for the brain. Here, we aimed (1) to investigate the blood-brain correspondence of DNA methylation using a within-subject design and (2) to identify changes in DNA methylation of brain-related biological pathways in schizophrenia.We obtained paired blood and temporal lobe biopsy samples simultaneously from 12 epilepsy patients during neurosurgical treatment. Using the Infinium 450K methylation array we calculated similarity of blood and brain DNA methylation for each individual separately. We applied our findings by performing gene set enrichment analyses (GSEA) of peripheral blood DNA methylation data (Infinium 27K) of 111 schizophrenia patients and 122 healthy controls and included only Cytosine-phosphate-Guanine (CpG) sites that were significantly correlated across tissues.Only 7.9% of CpG sites showed a statistically significant, large correlation between blood and brain tissue, a proportion that although small was significantly greater than predicted by chance. GSEA analysis of schizophrenia data revealed altered methylation profiles in pathways related to precursor metabolites and signaling peptides.Our findings indicate that most DNA methylation markers in peripheral blood do not reliably predict brain DNA methylation status. However, a subset of peripheral data may proxy methylation status of brain tissue. Restricting the analysis to these markers can identify meaningful epigenetic differences in schizophrenia and potentially other brain disorders. © The Author 2015. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny

PubMed Central

Marttila, Saara; Kananen, Laura; Jylhävä, Juulia; Nevalainen, Tapio; Hervonen, Antti; Jylhä, Marja; Hurme, Mikko

2015-01-01

The heritability of lifespan is 20-30%, but only a few genes associated with longevity have been identified. To explain this discrepancy, the inheritance of epigenetic features, such as DNA methylation, have been proposed to contribute to the heritability of lifespan. We investigated whether parental lifespan is associated with DNA methylation profile in nonagenarians. A regression model, adjusted for differences in blood cell proportions, identified 659 CpG sites where the level of methylation was associated with paternal lifespan. However, no association was observed between maternal lifespan and DNA methylation. The 659 CpG sites associated with paternal lifespan were enriched outside of CpG islands and were located in genes associated with development and morphogenesis, as well as cell signaling. The largest difference in the level of methylation between the progeny of the shortest-lived and longest-lived fathers was identified for CpG sites mapping to CXXC5. In addition, the level of methylation in three Notch-genes (NOTCH1, NOTCH3 and NOTCH4) was also associated with paternal lifespan. There are implications for the inheritance of acquired traits via epigenetic mechanisms in mammals. Here we describe DNA methylation features that are associated with paternal lifespan, and we speculate that the identified CpG sites may represent intergenerational epigenetic inheritance. PMID:26436701

Sex differences in DNA methylation of the cord blood are related to sex-bias psychiatric diseases

NASA Astrophysics Data System (ADS)

Maschietto, Mariana; Bastos, Laura Caroline; Tahira, Ana Carolina; Bastos, Elen Pereira; Euclydes, Veronica Luiza Vale; Brentani, Alexandra; Fink, Günther; de Baumont, Angelica; Felipe-Silva, Aloísio; Francisco, Rossana Pulcineli Vieira; Gouveia, Gisele; Grisi, Sandra Josefina Ferraz Ellero; Escobar, Ana Maria Ulhoa; Moreira-Filho, Carlos Alberto; Polanczyk, Guilherme Vanoni; Miguel, Euripedes Constantino; Brentani, Helena

2017-03-01

Sex differences in the prevalence of psychiatric disorders are well documented, with exposure to stress during gestation differentially impacting females and males. We explored sex-specific DNA methylation in the cord blood of 39 females and 32 males born at term and with appropriate weight at birth regarding their potential connection to psychiatric outcomes. Mothers were interviewed to gather information about environmental factors (gestational exposure) that could interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. Excluding XYS probes, there were 2,332 differentially methylated CpG sites (DMSs) between sexes, which were enriched within brain modules of co-methylated CpGs during brain development and also differentially methylated in the brains of boys and girls. Genes associated with the DMSs were enriched for neurodevelopmental disorders, particularly for CpG sites found differentially methylated in brain tissue between patients with schizophrenia and controls. Moreover, the DMS had an overlap of 890 (38%) CpG sites with a cohort submitted to toxic exposition during gestation. This study supports the evidences that sex differences in DNA methylation of autosomes act as a primary driver of sex differences that are found in psychiatric outcomes.

Sex differences in DNA methylation of the cord blood are related to sex-bias psychiatric diseases

PubMed Central

Maschietto, Mariana; Bastos, Laura Caroline; Tahira, Ana Carolina; Bastos, Elen Pereira; Euclydes, Veronica Luiza Vale; Brentani, Alexandra; Fink, Günther; de Baumont, Angelica; Felipe-Silva, Aloísio; Francisco, Rossana Pulcineli Vieira; Gouveia, Gisele; Grisi, Sandra Josefina Ferraz Ellero; Escobar, Ana Maria Ulhoa; Moreira-Filho, Carlos Alberto; Polanczyk, Guilherme Vanoni; Miguel, Euripedes Constantino; Brentani, Helena

2017-01-01

Sex differences in the prevalence of psychiatric disorders are well documented, with exposure to stress during gestation differentially impacting females and males. We explored sex-specific DNA methylation in the cord blood of 39 females and 32 males born at term and with appropriate weight at birth regarding their potential connection to psychiatric outcomes. Mothers were interviewed to gather information about environmental factors (gestational exposure) that could interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. Excluding XYS probes, there were 2,332 differentially methylated CpG sites (DMSs) between sexes, which were enriched within brain modules of co-methylated CpGs during brain development and also differentially methylated in the brains of boys and girls. Genes associated with the DMSs were enriched for neurodevelopmental disorders, particularly for CpG sites found differentially methylated in brain tissue between patients with schizophrenia and controls. Moreover, the DMS had an overlap of 890 (38%) CpG sites with a cohort submitted to toxic exposition during gestation. This study supports the evidences that sex differences in DNA methylation of autosomes act as a primary driver of sex differences that are found in psychiatric outcomes. PMID:28303968

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats.

PubMed

Amaral, Cátia Lira Do; Bueno, Rafaela de Barros E Lima; Burim, Regislaine Valéria; Queiroz, Regina Helena Costa; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

2011-05-18

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation. 2011 Elsevier B.V. All rights reserved.

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

PubMed

Kuo, Lu-Ting; Lu, Hsueh-Yi; Lee, Chien-Chang; Tsai, Jui-Chang; Lai, Hong-Shiee; Tseng, Ham-Min; Kuo, Meng-Fai; Tu, Yong-Kwang

2016-08-01

Aberrant methylation has been associated with transcriptional inactivation of tumor-related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan-Meier survival curve analysis demonstrated longer duration of progression-free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression-free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression-free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Epigenomics of Development in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Steve; Freitag, Michael; Mockler, Todd

2013-01-10

We conducted research to determine the role of epigenetic modifications during tree development using poplar (Populus trichocarpa), a model woody feedstock species. Using methylated DNA immunoprecipitation (MeDIP) or chromatin immunoprecipitation (ChIP), followed by high-throughput sequencing, we are analyzed DNA and histone methylation patterns in the P. trichocarpa genome in relation to four biological processes: bud dormancy and release, mature organ maintenance, in vitro organogenesis, and methylation suppression. Our project is now completed. We have 1) produced 22 transgenic events for a gene involved in DNA methylation suppression and studied its phenotypic consequences; 2) completed sequencing of methylated DNA from elevenmore » target tissues in wildtype P. trichocarpa; 3) updated our customized poplar genome browser using the open-source software tools (2.13) and (V2.2) of the P. trichocarpa genome; 4) produced summary data for genome methylation in P. trichocarpa, including distribution of methylation across chromosomes and in and around genes; 5) employed bioinformatic and statistical methods to analyze differences in methylation patterns among tissue types; and 6) used bisulfite sequencing of selected target genes to confirm bioinformatics and sequencing results, and gain a higher-resolution view of methylation at selected genes 7) compared methylation patterns to expression using available microarray data. Our main findings of biological significance are the identification of extensive regions of the genome that display developmental variation in DNA methylation; highly distinctive gene-associated methylation profiles in reproductive tissues, particularly male catkins; a strong whole genome/all tissue inverse association of methylation at gene bodies and promoters with gene expression; a lack of evidence that tissue specificity of gene expression is associated with gene methylation; and evidence that genome methylation is a significant impediment to tissue dedifferentiation and redifferentiation in vitro.« less

Genomewide analysis indicates that queen larvae have lower methylation levels in the honey bee ( Apis mellifera)

NASA Astrophysics Data System (ADS)

Shi, Yuan Yuan; Yan, Wei Yu; Huang, Zachary Y.; Wang, Zi Long; Wu, Xiao Bo; Zeng, Zhi Jiang

2013-02-01

The honey bee is a social insect characterized by caste differentiation, by which a young larva can develop into either a queen or a worker. Despite possessing the same genome, queen and workers display marked differences in reproductive capacity, physiology, and behavior. Recent studies have shown that DNA methylation plays important roles in caste differentiation. To further explore the roles of DNA methylation in this process, we analyzed DNA methylome profiles of both queen larvae (QL) and worker larvae (WL) of different ages (2, 4, and 6 day old), by using methylated DNA immunoprecipitation-sequencing (meDIP-seq) technique. The global DNA methylation levels varied between the larvae of two castes. DNA methylation increased from 2-day- to 4-day-old QL and then decreased in 6-day-old larvae. In WL, methylation levels increased with age. The methylcytosines in both larvae were enriched in introns, followed by coding sequence (CDS) regions, CpG islands, 2 kbp downstream and upstream of genes, and 5' and 3' untranslated regions (UTRs). The number of differentially methylated genes (DMGs) in 2-, 4-, and 6-day-old QL and WL was 725, 3,013, and 5,049, respectively. Compared to 4- and 6-day-old WL, a large number of genes in QL were downmethylated, which were involved in many processes including development, reproduction, and metabolic regulation. In addition, some DMGs were concerned with caste differentiation.

Extensive epigenetic reprogramming during the life cycle of Marchantia polymorpha.

PubMed

Schmid, Marc W; Giraldo-Fonseca, Alejandro; Rövekamp, Moritz; Smetanin, Dmitry; Bowman, John L; Grossniklaus, Ueli

2018-01-25

In plants, the existence and possible role of epigenetic reprogramming has been questioned because of the occurrence of stably inherited epialleles. Evidence suggests that epigenetic reprogramming does occur during land plant reproduction, but there is little consensus on the generality and extent of epigenetic reprogramming in plants. We studied DNA methylation dynamics during the life cycle of the liverwort Marchantia polymorpha. We isolated thalli and meristems from male and female gametophytes, archegonia, antherozoids, as well as sporophytes at early and late developmental stages, and compared their DNA methylation profiles. Of all cytosines tested for differential DNA methylation, 42% vary significantly in their methylation pattern throughout the life cycle. However, the differences are limited to few comparisons between specific stages of the life cycle and suggest four major epigenetic states specific to sporophytes, vegetative gametophytes, antherozoids, and archegonia. Further analyses indicated clear differences in the mechanisms underlying reprogramming in the gametophytic and sporophytic generations, which are paralleled by differences in the expression of genes involved in DNA methylation. Differentially methylated cytosines with a gain in methylation in antherozoids and archegonia are enriched in the CG and CHG contexts, as well as in gene bodies and gene flanking regions. In contrast, gain of DNA methylation during sporophyte development is mostly limited to the CHH context, LTR retrotransposons, DNA transposons, and repeats. We conclude that epigenetic reprogramming occurs at least twice during the life cycle of M. polymorpha and that the underlying mechanisms are likely different between the two events.

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

PubMed

McInnes, Tyler; Zou, Donghui; Rao, Dasari S; Munro, Francesca M; Phillips, Vicky L; McCall, John L; Black, Michael A; Reeve, Anthony E; Guilford, Parry J

2017-03-28

Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.

Genome-wide DNA Methylation Profiling of CpG Islands in Hypospadias

PubMed Central

Choudhry, Shweta; Deshpande, Archana; Qiao, Liang; Beckman, Kenneth; Sen, Saunak; Baskin, Laurence S.

2013-01-01

Purpose Hypospadias is one of the most frequent genital malformations in the male newborn, and results from abnormal penile and urethral development. The etiology of hypospadias remains largely unknown despite intensive investigations. Fetal androgens have a crucial role in genital differentiation. Recent studies have suggested that molecular mechanisms that underlie the effects of androgens on the fetus may involve disruption of epigenetic programming of gene expression during development. We assessed whether epigenetic modification of DNA methylation is associated with hypospadias in a case-control study of 12 hypospadias and 8 control subjects. Materials and Methods Genome-wide DNA methylation profiling was performed on the study subjects using the Illumina Infinium® HumanMethylation450 Bead-Chip, which enables the direct investigation of methylation status of more than 485,000 individual CpG sites throughout the genome. The methylation level at each CpG site was compared between cases and controls using the t test and logistic regression. Results We identified 14 CpG sites that were associated with hypospadias with p <0.00001. These CpG sites were in or near the SCARB1, MYBPH, SORBS1, LAMA4, HOXD11, MYO1D, EGFL7, C10orf41, LMAN1L and SULF1 genes. Two CpG sites in SCARB1 and MYBPH genes remained statistically significant after correction for multiple testing (p = 2.61×10−09, pcorrected = 0.008; p = 3.06×10−08, pcorrected = 0.02, respectively). Conclusions To our knowledge this is the first study to investigate hypospadias using a unique and novel epigenetic approach. Our findings suggest DNA methylation patterns are useful in identifying new genes such as SCARB1 and MYBPH that may be involved in the etiology of hypospadias. PMID:22906644

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring.

PubMed

Asimes, AnnaDorothea; Torcaso, Audrey; Pinceti, Elena; Kim, Chun K; Zeleznik-Le, Nancy J; Pak, Toni R

2017-05-01

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring. Copyright © 2016 Elsevier Inc. All rights reserved.

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring

PubMed Central

Asimes, AnnaDorothea; Torcaso, Audrey; Pinceti, Elena; Kim, Chun K; Zeleznik-Le, Nancy J.; Pak, Toni R.

2016-01-01

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the last 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring. PMID:27817987

DNA Methylation of the Aryl Hydrocarbon Receptor Repressor Associations with Cigarette Smoking and Subclinical Atherosclerosis

PubMed Central

Reynolds, Lindsay M.; Wan, Ma; Ding, Jingzhong; Taylor, Jackson R.; Mstat, Kurt Lohman; Su, Dan; Bennett, Brian D.; Porter, Devin K.; Gimple, Ryan; Pittman, Gary S.; Wang, Xuting; Howard, Timothy D.; Siscovick, David; Psaty, Bruce M.; Shea, Steven; Burke, Gregory L.; Jacobs, David R.; Rich, Stephen S.; Hixson, James E.; Stein, James H.; Stunnenberg, Hendrik; Barr, R. Graham; Kaufman, Joel D.; Post, Wendy S.; Hoeschele, Ina; Herrington, David M.; Bell, Douglas A.; Liu, Yongmei

2015-01-01

Background Tobacco smoke contains numerous agonists of the aryl-hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the aryl-hydrocarbon receptor repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. Methods and Results DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1,256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (p = 6.1×10−134) with smoking status (current vs. never). Novel associations between cg05575921 methylation and carotid plaque scores (p = 3.1×10−10) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known CVD risk factors. This association replicated in an independent cohort using hepatic DNA (n = 141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer), and had methylation correlated with AHRR mRNA profiles (p = 1.4×10−17) obtained from RNA sequencing conducted on a subset (n = 373) of the samples. Conclusions These findings suggest AHRR methylation may be functionally related to AHRR expression in monocytes, and represents a potential biomarker of subclinical atherosclerosis in smokers. PMID:26307030

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

PubMed

Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

2017-02-28

DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation

PubMed Central

Chan, D.; McGraw, S.; Klein, K.; Wallock, L.M.; Konermann, C.; Plass, C.; Chan, P.; Robaire, B.; Jacob, R.A.; Greenwood, C.M.T.; Trasler, J.M.

2017-01-01

STUDY QUESTION Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996–1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare. PMID:27994001

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

PubMed

Chan, D; McGraw, S; Klein, K; Wallock, L M; Konermann, C; Plass, C; Chan, P; Robaire, B; Jacob, R A; Greenwood, C M T; Trasler, J M

2017-02-01

Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

PubMed

Ogino, S; Cantor, M; Kawasaki, T; Brahmandam, M; Kirkner, G J; Weisenberger, D J; Campan, M; Laird, P W; Loda, M; Fuchs, C S

2006-07-01

The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, p<10(-5)) and non-CIMP MSS tumours (6.6%, p<10(-4)), respectively). CIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

Methylation at CPT1A locus is associated with lipoprotein subfraction profiles

USDA-ARS?s Scientific Manuscript database

Lipoprotein subfractions help discriminate cardiometabolic disease risk. Genetic loci validated as associating with lipoprotein measures do not account for a large proportion of the individual variation in lipoprotein measures. We hypothesized that DNA methylation levels across the genome contribute...

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

PubMed Central

2011-01-01

Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease. PMID:22098709

Methylation and expression profiles of MGMT gene in thymic epithelial tumors.

PubMed

Mokhtar, Mohamed; Kondo, Kazuya; Namura, Toshiaki; Ali, Abdellah H K; Fujita, Yui; Takai, Chikako; Takizawa, Hiromitsu; Nakagawa, Yasushi; Toba, Hiroaki; Kajiura, Koichiro; Yoshida, Mitsuteru; Kawakami, Gyokei; Sakiyama, Shoji; Tangoku, Akira

2014-02-01

A key challenge in diagnosis and treatment of thymic epithelial tumors (TET) is in improving our understanding of the genetic and epigenetic changes of these relatively rare tumors. Methylation specific PCR (MSP) and immunohistochemistry were applied to 66 TET to profile the methylation status of DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) and its protein expression in TET to clarify the association between MGMT status and clinicopathological features, response to chemotherapy and overall survival. MGMT methylation was significantly more frequent in thymic carcinoma than in thymoma (17/23, 74% versus 13/44, 29%; P<0.001). Loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma (20/23, 87% versus 10/44, 23%; P<0.0001). There is a significant correlation between of MGMT methylation and loss of its protein expression (P<0.0003). MGMT methylation and loss of expression were significantly more frequent in advanced thymic epithelial tumors (III/IV) than in early tumors (I/II). MGMT methylation plays a soul role in development of TET, especially in thymic carcinoma. Therefore, translation of our results from basic molecular research to clinical practice may have important implication for considering MGMT methylation as a marker and a target of future therapies in TET. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Genetic predisposition to high anxiety- and depression-like behavior coincides with diminished DNA methylation in the adult rat amygdala

PubMed Central

McCoy, Chelsea R.; Jackson, Nateka L.; Day, Jeremy; Clinton, Sarah M.

2016-01-01

Understanding biological mechanisms that shape vulnerability to emotional dysfunction is critical for elucidating the neurobiology of psychiatric illnesses like anxiety and depression. To elucidate molecular and epigenetic alterations in the brain that contribute to individual differences in emotionality, our laboratory utilized a rodent model of temperamental differences. Rats bred for low response to novelty (Low Responders, LRs) are inhibited in novel situations and display high anxiety, helplessness, and diminished sociability compared to High Novelty Responder (HR) rats. Our current transcriptome profiling experiment identified widespread gene expression differences in the amygdala of adult HR/LR rats; we hypothesize that HR/LR gene expression and downstream behavioral differences stem from distinct epigenetic (specifically DNA methylation) patterning in the HR/LR brain. Although we found similar levels of DNA methyltransferase proteins in the adult HR/LR amygdala, next-generation sequencing analysis of the methylome revealed 793 differentially methylated genomic sites between the groups. Most of the differentially methylated sites were hypermethylated in HR versus LR, so we next tested the hypothesis that enhancing DNA methylation in LRs would improve their anxiety/depression-like phenotype. We found that increasing DNA methylation in LRs (via increased dietary methyl donor content) improved their anxiety-like behavior and decreased their typically high levels of Forced Swim Test (FST) immobility; however, dietary methyl donor depletion exacerbated LRs’ high FST immobility. These data are generally consistent with findings in depressed patients showing that treatment with DNA methylation-promoting agents improves depressive symptoms, and highlight epigenetic mechanisms that may contribute to individual differences in risk for emotional dysfunction. PMID:27965039

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

PubMed Central

Colacino, Justin A.; Arthur, Anna E.; Dolinoy, Dana C.; Sartor, Maureen A.; Duffy, Sonia A.; Chepeha, Douglas B.; Bradford, Carol R.; Walline, Heather M.; McHugh, Jonathan B.; D'Silva, Nisha; Carey, Thomas E.; Wolf, Gregory T.; Taylor, Jeremy M.G.; Peterson, Karen E.; Rozek, Laura S.

2012-01-01

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression. PMID:22722388

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

PubMed

Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA.

PubMed

Aberg, Karolina A; Chan, Robin F; Shabalin, Andrey A; Zhao, Min; Turecki, Gustavo; Staunstrup, Nicklas Heine; Starnawska, Anna; Mors, Ole; Xie, Lin Y; van den Oord, Edwin Jcg

2017-09-01

We recently showed that, after optimization, our methyl-CpG binding domain sequencing (MBD-seq) application approximates the methylome-wide coverage obtained with whole-genome bisulfite sequencing (WGB-seq), but at a cost that enables adequately powered large-scale association studies. A prior drawback of MBD-seq is the relatively large amount of genomic DNA (ideally >1 µg) required to obtain high-quality data. Biomaterials are typically expensive to collect, provide a finite amount of DNA, and may simply not yield sufficient starting material. The ability to use low amounts of DNA will increase the breadth and number of studies that can be conducted. Therefore, we further optimized the enrichment step. With this low starting material protocol, MBD-seq performed equally well, or better, than the protocol requiring ample starting material (>1 µg). Using only 15 ng of DNA as input, there is minimal loss in data quality, achieving 93% of the coverage of WGB-seq (with standard amounts of input DNA) at similar false/positive rates. Furthermore, across a large number of genomic features, the MBD-seq methylation profiles closely tracked those observed for WGB-seq with even slightly larger effect sizes. This suggests that MBD-seq provides similar information about the methylome and classifies methylation status somewhat more accurately. Performance decreases with <15 ng DNA as starting material but, even with as little as 5 ng, MBD-seq still achieves 90% of the coverage of WGB-seq with comparable genome-wide methylation profiles. Thus, the proposed protocol is an attractive option for adequately powered and cost-effective methylome-wide investigations using (very) low amounts of DNA.

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

PubMed

Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

2014-12-01

Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

Identification and validation of seven new loci showing differential DNA methylation related to serum lipid profile: an epigenome-wide approach. The REGICOR study.

PubMed

Sayols-Baixeras, S; Subirana, I; Lluis-Ganella, C; Civeira, F; Roquer, J; Do, A N; Absher, D; Cenarro, A; Muñoz, D; Soriano-Tárraga, C; Jiménez-Conde, J; Ordovas, J M; Senti, M; Aslibekyan, S; Marrugat, J; Arnett, D K; Elosua, R

2016-10-15

Lipid traits (total, low-density and high-density lipoprotein cholesterol, and triglycerides) are risk factors for cardiovascular disease. DNA methylation is not only an inherited but also modifiable epigenetic mark that has been related to cardiovascular risk factors. Our aim was to identify loci showing differential DNA methylation related to serum lipid levels. Blood DNA methylation was assessed using the Illumina Human Methylation 450 BeadChip. A two-stage epigenome-wide association study was performed, with a discovery sample in the REGICOR study (n = 645) and validation in the Framingham Offspring Study (n = 2,542). Fourteen CpG sites located in nine genes (SREBF1, SREBF2, PHOSPHO1, SYNGAP1, ABCG1, CPT1A, MYLIP, TXNIP and SLC7A11) and 2 intergenic regions showed differential methylation in association with lipid traits. Six of these genes and 1 intergenic region were new discoveries showing differential methylation related to total cholesterol (SREBF2), HDL-cholesterol (PHOSPHO1, SYNGAP1 and an intergenic region in chromosome 2) and triglycerides (MYLIP, TXNIP and SLC7A11). These CpGs explained 0.7%, 9.5% and 18.9% of the variability of total cholesterol, HDL cholesterol and triglycerides in the Framingham Offspring Study, respectively. The expression of the genes SREBF2 and SREBF1 was inversely associated with methylation of their corresponding CpGs (P-value = 0.0042 and 0.0045, respectively) in participants of the GOLDN study (n = 98). In turn, SREBF1 expression was directly associated with HDL cholesterol (P-value = 0.0429). Genetic variants in SREBF1, PHOSPHO1, ABCG1 and CPT1A were also associated with lipid profile. Further research is warranted to functionally validate these new loci and assess the causality of new and established associations between these differentially methylated loci and lipid metabolism.

Global epigenetic profiling identifies methylation subgroups associated with recurrence-free survival in meningioma

PubMed Central

Olar, Adriana; Wani, Khalida M; Wilson, Charmaine D; Zadeh, Gelareh; DeMonte, Franco; Jones, David TW; Pfister, Stefan M; Sulman, Erik P; Aldape, Kenneth D

2017-01-01

Meningioma is the most common primary brain tumor and carries a substantial risk of local recurrence. Methylation profiles of meningioma and their clinical implications are not well understood. We hypothesized that aggressive meningiomas have unique DNA methylation patterns that could be used to better stratify patient management. Samples (n=140) were profiled using the Illumina HumanMethylation450 BeadChip. Unsupervised modeling on a training set (n=89) identified 2 molecular methylation subgroups of meningioma (MM) with significantly different recurrence free survival (RFS) times between the groups: a prognostically unfavorable subgroup (MM-UNFAV) and a prognostically favorable subgroup (MM-FAV). This finding was validated in the remaining 51 samples and led to a baseline meningioma methylation classifier (bMMC) defined by 283 CpG loci (283-bMMC). To further optimize a recurrence predictor, probes subsumed within the baseline classifier were subject to additional modeling using a similar training/validation approach, leading to a 64-CpG loci meningioma methylation predictor (64-MMP). After adjustment for relevant clinical variables [WHO grade, mitotic index, Simpson grade, sex, location, and copy number aberrations (CNA)] multivariable analyses for RFS showed that the baseline methylation classifier was not significant (p=0.0793). The methylation predictor however was significantly associated with tumor recurrence (p<0.0001). CNA were extracted from the 450k intensity profiles. Tumor samples in the MM-UNFAV subgroup showed an overall higher proportion of CNAs compared to the MM-FAV subgroup tumors and the CNAs were complex in nature. CNAs in the MM-UNFAV subgroup included recurrent losses of 1p, 6q, 14q and 18q, and gain of 1q, all of which were previously identified as indicators of poor outcome. In conclusion, our analyses demonstrate robust DNA methylation signatures in meningioma that correlate with CNAs and stratify patients by recurrence risk. PMID:28130639

Characterization of the cartilage DNA methylome in knee and hip osteoarthritis.

PubMed

Rushton, Michael D; Reynard, Louise N; Barter, Matt J; Refaie, Ramsay; Rankin, Kenneth S; Young, David A; Loughlin, John

2014-09-01

The aim of this study was to characterize the genome-wide DNA methylation profile of chondrocytes from knee and hip cartilage obtained from patients with osteoarthritis (OA) and hip cartilage obtained from patients with femoral neck fracture, providing the first comparison of DNA methylation between OA and non-OA hip cartilage, and between OA hip and OA knee cartilage. The study was performed using the Illumina Infinium HumanMethylation450 BeadChip array, which allows the annotation of ∼480,000 CpG sites. Genome-wide methylation was assessed in chondrocyte DNA extracted from 23 hip OA patients, 73 knee OA patients, and 21 healthy hip control patients with femoral neck fracture. Analysis revealed that chondrocytes from the hip cartilage of OA patients and healthy controls have unique methylation profiles, with 5,322 differentially methylated loci (DMLs) identified between the 2 groups. In addition, a comparison between hip and knee OA chondrocytes revealed 5,547 DMLs between the 2 groups, including DMLs in several genes known to be involved in the pathogenesis of OA. Hip OA samples were found to cluster into 2 groups. A total of 15,239 DMLs were identified between the 2 clusters, with an enrichment of genes involved in inflammation and immunity. Similarly, we confirmed a previous report of knee OA samples that also clustered into 2 groups. We demonstrated that global DNA methylation using a high-density array can be a powerful tool in the characterization of OA at the molecular level. Identification of pathways enriched in DMLs between OA and OA-free cartilage highlight potential etiologic mechanisms that are involved in the initiation and/or progression of the disease and that could be therapeutically targeted. © 2014 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

DNA methylation changes at infertility genes in newborn twins conceived by in vitro fertilisation.

PubMed

Castillo-Fernandez, Juan E; Loke, Yuk Jing; Bass-Stringer, Sebastian; Gao, Fei; Xia, Yudong; Wu, Honglong; Lu, Hanlin; Liu, Yuan; Wang, Jun; Spector, Tim D; Saffery, Richard; Craig, Jeffrey M; Bell, Jordana T

2017-03-24

The association of in vitro fertilisation (IVF) and DNA methylation has been studied predominantly at regulatory regions of imprinted genes and at just thousands of the ~28 million CpG sites in the human genome. We investigated the links between IVF and DNA methylation patterns in whole cord blood cells (n = 98) and cord blood mononuclear cells (n = 82) from newborn twins using genome-wide methylated DNA immunoprecipitation coupled with deep sequencing. At a false discovery rate (FDR) of 5%, we identified one significant whole blood DNA methylation change linked to conception via IVF, which was located ~3 kb upstream of TNP1, a gene previously linked to male infertility. The 46 most strongly associated signals (FDR of 25%) included a second region in a gene also previously linked to infertility, C9orf3, suggesting that our findings may in part capture the effect of parental subfertility. Using twin modelling, we observed that individual-specific environmental factors appear to be the main overall contributors of methylation variability at the FDR 25% IVF-associated differentially methylated regions, although evidence for methylation heritability was also obtained at several of these regions. We replicated previous findings of differential methylation associated with IVF at the H19/IGF2 region in cord blood mononuclear cells, and we validated the signal at C9orf3 in monozygotic twins. We also explored the impact of intracytoplasmic sperm injection on the FDR 25% signals for potential effects specific to male or female infertility factors. To our knowledge, this is the most comprehensive study of DNA methylation profiles at birth and IVF conception to date, and our results show evidence for epigenetic modifications that may in part reflect parental subfertility.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

PubMed

Alghamian, Yaman; Abou Alchamat, Ghalia; Murad, Hossam; Madania, Ammar

2017-09-01

DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

PubMed

Zackay, Arie; Steinhoff, Christine

2010-12-15

Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

PubMed Central

2010-01-01

Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

Regulation of DNA methylation on EEF1D and RPL8 expression in cattle.

PubMed

Liu, Xuan; Yang, Jie; Zhang, Qin; Jiang, Li

2017-10-01

Dynamic changes to the epigenome play a critical role in a variety of biology processes and complex traits. Many important candidate genes have been identified through our previous genome wide association study (GWAS) on milk production traits in dairy cattle. However, the underlying mechanism of candidate genes have not yet been clearly understood. In this study, we analyzed the methylation variation of the candidate genes, EEF1D and RPL8, which were identified to be strongly associated with milk production traits in dairy cattle in our previous studies, and its effect on protein and mRNA expression. We compared DNA methylation profiles and gene expression levels of EEF1D and RPL8 in five different tissues (heart, liver, mammary gland, ovary and muscle) of three cows. Both genes showed the highest expression level in mammary gland. For RPL8, there was no difference in the DNA methylation pattern in the five tissues, suggesting no effect of DNA methylation on gene expression. For EEF1D, the DNA methylation levels of its first CpG island differed in the five tissues and were negatively correlated with the gene expression levels. To further investigate the function of DNA methylation on the expression of EEF1D, we collected blood samples of three cows at early stage of lactation and in dry period and analyzed its expression and the methylation status of the first CpG island in blood. As a result, the mRNA expression of EEF1D in the dry period was higher than that at the early stage of lactation, while the DNA methylation level in the dry period was lower than that at the early stage of lactation. Our result suggests that the DNA methylation of EEF1D plays an important role in the spatial and temporal regulation of its expression and possibly have an effect on the milk production traits.

Impact of collection season and storage of semen on methylation activity in swine placental and fetal tissues derived from summer or winter breedings

USDA-ARS?s Scientific Manuscript database

DNA methylation patterns in extra-embryonic tissues have been linked to irregular fetal growth and early pregnancy loss. The objective of the current study was to evaluate methylation profiles of placental and fetal tissue collected from pregnancies derived using cooled-extended (ExT) or cryopreserv...

Long-term outdoor air pollution and DNA methylation in circulating monocytes: results from the Multi-Ethnic Study of Atherosclerosis (MESA).

PubMed

Chi, Gloria C; Liu, Yongmei; MacDonald, James W; Barr, R Graham; Donohue, Kathleen M; Hensley, Mark D; Hou, Lifang; McCall, Charles E; Reynolds, Lindsay M; Siscovick, David S; Kaufman, Joel D

2016-12-01

DNA methylation may mediate effects of air pollution on cardiovascular disease. The association between long-term air pollution exposure and DNA methylation in monocytes, which are central to atherosclerosis, has not been studied. We investigated the association between long-term ambient air pollution exposure and DNA methylation (candidate sites and global) in monocytes of adults (aged ≥55). One-year average ambient fine particulate matter (PM 2.5 ) and oxides of nitrogen (NO X ) concentrations were predicted at participants' (n = 1,207) addresses using spatiotemporal models. We assessed DNA methylation in circulating monocytes at 1) 2,713 CpG sites associated with mRNA expression of nearby genes and 2) probes mapping to Alu and LINE-1 repetitive elements (surrogates for global DNA methylation) using Illumina's Infinium HumanMethylation450 BeadChip. We used linear regression models adjusted for demographics, smoking, physical activity, socioeconomic status, methyl-nutrients, and technical variables. For significant air pollution-associated methylation sites, we also assessed the association between expression of gene transcripts previously associated with these CpG sites and air pollution. At a false discovery rate of 0.05, five candidate CpGs (cg20455854, cg07855639, cg07598385, cg17360854, and cg23599683) had methylation significantly associated with PM 2.5 and none were associated with NO X . Cg20455854 had the smallest p-value for the association with PM 2.5 (p = 2.77 × 10 -5 ). mRNA expression profiles of genes near three of the PM 2.5 -associated CpGs (ANKHD1, LGALS2, and ANKRD11) were also significantly associated with PM 2.5 exposure. Alu and LINE-1 methylation were not associated with long-term air pollution exposure. We observed novel associations between long-term ambient air pollution exposure and site-specific DNA methylation, but not global DNA methylation, in purified monocytes of a multi-ethnic adult population. Epigenetic markers may provide insights into mechanisms underlying environmental factors in complex diseases like atherosclerosis.

Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis.

PubMed

Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

2016-07-14

Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis.

Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis

PubMed Central

Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

2016-01-01

Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis. PMID:27411928

Human active X-specific DNA methylation events showing stability across time and tissues

PubMed Central

Joo, Jihoon Eric; Novakovic, Boris; Cruickshank, Mark; Doyle, Lex W; Craig, Jeffrey M; Saffery, Richard

2014-01-01

The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork' of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena. PMID:24713664

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

PubMed

Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R

2015-10-15

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes. © 2015 Yelina et al.; Published by Cold Spring Harbor Laboratory Press.

A Low Glycaemic Index Diet in Pregnancy Induces DNA Methylation Variation in Blood of Newborns: Results from the ROLO Randomised Controlled Trial.

PubMed

Geraghty, Aisling A; Sexton-Oates, Alexandra; O'Brien, Eileen C; Alberdi, Goiuri; Fransquet, Peter; Saffery, Richard; McAuliffe, Fionnuala M

2018-04-06

The epigenetic profile of the developing fetus is sensitive to environmental influence. Maternal diet has been shown to influence DNA methylation patterns in offspring, but research in humans is limited. We investigated the impact of a low glycaemic index dietary intervention during pregnancy on offspring DNA methylation patterns using a genome-wide methylation approach. Sixty neonates were selected from the ROLO (Randomised cOntrol trial of LOw glycaemic index diet to prevent macrosomia) study: 30 neonates from the low glycaemic index intervention arm and 30 from the control, whose mothers received no specific dietary advice. DNA methylation was investigated in 771,484 CpG sites in free DNA from cord blood serum. Principal component analysis and linear regression were carried out comparing the intervention and control groups. Gene clustering and pathway analysis were also explored. Widespread variation was identified in the newborns exposed to the dietary intervention, accounting for 11% of the total level of DNA methylation variation within the dataset. No association was found with maternal early-pregnancy body mass index (BMI), infant sex, or birthweight. Pathway analysis identified common influences of the intervention on gene clusters plausibly linked to pathways targeted by the intervention, including cardiac and immune functioning. Analysis in 60 additional samples from the ROLO study failed to replicate the original findings. Using a modest-sized discovery sample, we identified preliminary evidence of differential methylation in progeny of mothers exposed to a dietary intervention during pregnancy.

DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons.

PubMed

Fish, Trevor J; Benninghoff, Abby D

2017-11-01

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

PubMed

Ichimura, Norihisa; Shinjo, Keiko; An, Byonggu; Shimizu, Yasuhiro; Yamao, Kenji; Ohka, Fumiharu; Katsushima, Keisuke; Hatanaka, Akira; Tojo, Masayuki; Yamamoto, Eiichiro; Suzuki, Hiromu; Ueda, Minoru; Kondo, Yutaka

2015-08-01

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. ©2015 American Association for Cancer Research.

DNA methylation profiling using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)

PubMed Central

Suzuki, Masako; Greally, John M.

2010-01-01

The HELP assay is a technique that allows genome-wide analysis of cytosine methylation. Here we describe the assay, its relative strengths and weaknesses, and the transition of the assay from a microarray to massively-parallel sequencing-based foundation. PMID:20434563

Genome-wide methylomic and transcriptomic analyses identify subtype-specific epigenetic signatures commonly dysregulated in glioma stem cells and glioblastoma.

PubMed

Pangeni, Rajendra P; Zhang, Zhou; Alvarez, Angel A; Wan, Xuechao; Sastry, Namratha; Lu, Songjian; Shi, Taiping; Huang, Tianzhi; Lei, Charles X; James, C David; Kessler, John A; Brennan, Cameron W; Nakano, Ichiro; Lu, Xinghua; Hu, Bo; Zhang, Wei; Cheng, Shi-Yuan

2018-06-21

Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.

Longitudinal analyses of the DNA methylome in deployed military servicemen identify susceptibility loci for post-traumatic stress disorder.

PubMed

Rutten, B P F; Vermetten, E; Vinkers, C H; Ursini, G; Daskalakis, N P; Pishva, E; de Nijs, L; Houtepen, L C; Eijssen, L; Jaffe, A E; Kenis, G; Viechtbauer, W; van den Hove, D; Schraut, K G; Lesch, K-P; Kleinman, J E; Hyde, T M; Weinberger, D R; Schalkwyk, L; Lunnon, K; Mill, J; Cohen, H; Yehuda, R; Baker, D G; Maihofer, A X; Nievergelt, C M; Geuze, E; Boks, M P M

2018-05-01

In order to determine the impact of the epigenetic response to traumatic stress on post-traumatic stress disorder (PTSD), this study examined longitudinal changes of genome-wide blood DNA methylation profiles in relation to the development of PTSD symptoms in two prospective military cohorts (one discovery and one replication data set). In the first cohort consisting of male Dutch military servicemen (n=93), the emergence of PTSD symptoms over a deployment period to a combat zone was significantly associated with alterations in DNA methylation levels at 17 genomic positions and 12 genomic regions. Evidence for mediation of the relation between combat trauma and PTSD symptoms by longitudinal changes in DNA methylation was observed at several positions and regions. Bioinformatic analyses of the reported associations identified significant enrichment in several pathways relevant for symptoms of PTSD. Targeted analyses of the significant findings from the discovery sample in an independent prospective cohort of male US marines (n=98) replicated the observed relation between decreases in DNA methylation levels and PTSD symptoms at genomic regions in ZFP57, RNF39 and HIST1H2APS2. Together, our study pinpoints three novel genomic regions where longitudinal decreases in DNA methylation across the period of exposure to combat trauma marks susceptibility for PTSD.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2013-09-01

accomplishments include creation of relevant plant lines, development of in vitro assays, and profiling of mRNA expression in null mutants. 15. SUBJECT TERMS...DNA methylation, UHRF1, VIM1, ubiquitination, epigenetics, chromatin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...Molecular Basis of Human Disease ,” which covered several weeks’ worth of material specifically related to the molecular and epigenetic basis of cancer

An information-theoretic approach to the modeling and analysis of whole-genome bisulfite sequencing data.

PubMed

Jenkinson, Garrett; Abante, Jordi; Feinberg, Andrew P; Goutsias, John

2018-03-07

DNA methylation is a stable form of epigenetic memory used by cells to control gene expression. Whole genome bisulfite sequencing (WGBS) has emerged as a gold-standard experimental technique for studying DNA methylation by producing high resolution genome-wide methylation profiles. Statistical modeling and analysis is employed to computationally extract and quantify information from these profiles in an effort to identify regions of the genome that demonstrate crucial or aberrant epigenetic behavior. However, the performance of most currently available methods for methylation analysis is hampered by their inability to directly account for statistical dependencies between neighboring methylation sites, thus ignoring significant information available in WGBS reads. We present a powerful information-theoretic approach for genome-wide modeling and analysis of WGBS data based on the 1D Ising model of statistical physics. This approach takes into account correlations in methylation by utilizing a joint probability model that encapsulates all information available in WGBS methylation reads and produces accurate results even when applied on single WGBS samples with low coverage. Using the Shannon entropy, our approach provides a rigorous quantification of methylation stochasticity in individual WGBS samples genome-wide. Furthermore, it utilizes the Jensen-Shannon distance to evaluate differences in methylation distributions between a test and a reference sample. Differential performance assessment using simulated and real human lung normal/cancer data demonstrate a clear superiority of our approach over DSS, a recently proposed method for WGBS data analysis. Critically, these results demonstrate that marginal methods become statistically invalid when correlations are present in the data. This contribution demonstrates clear benefits and the necessity of modeling joint probability distributions of methylation using the 1D Ising model of statistical physics and of quantifying methylation stochasticity using concepts from information theory. By employing this methodology, substantial improvement of DNA methylation analysis can be achieved by effectively taking into account the massive amount of statistical information available in WGBS data, which is largely ignored by existing methods.

Peripheral DNA methylation, cognitive decline and brain aging: pilot findings from the Whitehall II imaging study.

PubMed

Chouliaras, Leonidas; Pishva, Ehsan; Haapakoski, Rita; Zsoldos, Eniko; Mahmood, Abda; Filippini, Nicola; Burrage, Joe; Mill, Jonathan; Kivimäki, Mika; Lunnon, Katie; Ebmeier, Klaus P

2018-05-01

The present study investigated the link between peripheral DNA methylation (DNAm), cognitive impairment and brain aging. We tested the association between blood genome-wide DNAm profiles using the Illumina 450K arrays, cognitive dysfunction and brain MRI measures in selected participants of the Whitehall II imaging sub-study. Eight differentially methylated regions were associated with cognitive impairment. Accelerated aging based on the Hannum epigenetic clock was associated with mean diffusivity and global fractional anisotropy. We also identified modules of co-methylated loci associated with white matter hyperintensities. These co-methylation modules were enriched among pathways relevant to β-amyloid processing and glutamatergic signaling. Our data support the notion that blood DNAm changes may have utility as a biomarker for cognitive dysfunction and brain aging.

Long-term alterations to DNA methylation as a biomarker of prenatal alcohol exposure: From mouse models to human children with fetal alcohol spectrum disorders.

PubMed

Laufer, Benjamin I; Chater-Diehl, Eric J; Kapalanga, Joachim; Singh, Shiva M

2017-05-01

Rodent models of Fetal Alcohol Spectrum Disorders (FASD) have revealed that prenatal alcohol exposure (PAE) results in differential DNA cytosine methylation in the developing brain. The resulting genome-wide methylation changes are enriched in genes with neurodevelopmental functions. The profile of differential methylation is dynamic and present in some form for life. The methylation changes are transmitted across subsequent mitotic divisions, where they are maintained and further modified over time. More recent follow up has identified a profile of the differential methylation in the buccal swabs of young children born with FASD. While distinct from the profile observed in brain tissue from rodent models, there are similarities. These include changes in genes belonging to a number of neurodevelopmental and behavioral pathways. Specifically, there is increased methylation at the clustered protocadherin genes and deregulation of genomically imprinted genes, even though no single gene is affected in all patients studied to date. These novel results suggest further development of a methylation based strategy could enable early and accurate diagnostics and therapeutics, which have remained a challenge in FASD research. There are two aspects of this challenge that must be addressed in the immediate future: First, the long-term differential methylomics observed in rodent models must be functionally confirmed. Second, the similarities in differential methylation must be further established in humans at a methylomic level and overcome a number of technical limitations. While a cure for FASD is challenging, there is an opportunity for the development of early diagnostics and attenuations towards a higher quality of life. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

PubMed Central

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P.; Zhou, Feng C.

2009-01-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development. PMID:20009564

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation.

PubMed

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P; Zhou, Feng C

2009-10-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p<0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development.

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

PubMed

Okamoto, Yasuyuki; Shinjo, Keiko; Shimizu, Yasuhiro; Sano, Tsuyoshi; Yamao, Kenji; Gao, Wentao; Fujii, Makiko; Osada, Hirotaka; Sekido, Yoshitaka; Murakami, Shuko; Tanaka, Yasuhito; Joh, Takashi; Sato, Shinya; Takahashi, Satoru; Wakita, Takaji; Zhu, Jingde; Issa, Jean-Pierre J; Kondo, Yutaka

2014-02-01

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Global DNA Methylation Patterns Can Play a Role in Defining Terroir in Grapevine (Vitis vinifera cv. Shiraz)

PubMed Central

Xie, Huahan; Konate, Moumouni; Sai, Na; Tesfamicael, Kiflu G.; Cavagnaro, Timothy; Gilliham, Matthew; Breen, James; Metcalfe, Andrew; Stephen, John R.; De Bei, Roberta; Collins, Cassandra; Lopez, Carlos M. R.

2017-01-01

Understanding how grapevines perceive and adapt to different environments will provide us with an insight into how to better manage crop quality. Mounting evidence suggests that epigenetic mechanisms are a key interface between the environment and the genotype that ultimately affect the plant’s phenotype. Moreover, it is now widely accepted that epigenetic mechanisms are a source of useful variability during crop varietal selection that could affect crop performance. While the contribution of DNA methylation to plant performance has been extensively studied in other major crops, very little work has been done in grapevine. To study the genetic and epigenetic diversity across 22 vineyards planted with the cultivar Shiraz in six wine sub-regions of the Barossa, South Australia. Methylation sensitive amplified polymorphisms (MSAPs) were used to obtain global patterns of DNA methylation. The observed epigenetic profiles showed a high level of differentiation that grouped vineyards by their area of provenance despite the low genetic differentiation between vineyards and sub-regions. Pairwise epigenetic distances between vineyards indicate that the main contributor (23–24%) to the detected variability is associated to the distribution of the vineyards on the N–S axis. Analysis of the methylation profiles of vineyards pruned with the same system increased the positive correlation observed between geographic distance and epigenetic distance suggesting that pruning system affects inter-vineyard epigenetic differentiation. Finally, methylation sensitive genotyping by sequencing identified 3,598 differentially methylated genes in grapevine leaves that were assigned to 1,144 unique gene ontology terms of which 8.6% were associated with response to environmental stimulus. Our results suggest that DNA methylation differences between vineyards and sub-regions within The Barossa are influenced both by the geographic location and, to a lesser extent, by pruning system. Finally, we discuss how epigenetic variability can be used as a tool to understand and potentially modulate terroir in grapevine. PMID:29163587

Targeted and genome-scale methylomics reveals gene body signatures in human cell lines

PubMed Central

Ball, Madeleine Price; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily; Park, In-Hyun; Xie, Bin; Daley, George Q.; Church, George M.

2012-01-01

Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions. PMID:19329998

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors

PubMed Central

Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D.; Ambrosone, Christine B.; Higgins, Michael J.; Sucheston-Campbell, Lara E.

2017-01-01

Background DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. Results For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Materials and Methods Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. Conclusions FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues. PMID:28118602

Genome-wide expression and methylation profiling in the aged rodent brain due to early-life Pb exposure and its relevance to aging.

PubMed

Dosunmu, Remi; Alashwal, Hany; Zawia, Nasser H

2012-06-01

In this study, we assessed global gene expression patterns in adolescent mice exposed to lead (Pb) as infants and their aged siblings to identify reprogrammed genes. Global expression on postnatal day 20 and 700 was analyzed and genes that were down- and up-regulated (≥2 fold) were identified, clustered and analyzed for their relationship to DNA methylation. About 150 genes were differentially expressed in old age. In normal aging, we observed an up-regulation of genes related to the immune response, metal-binding, metabolism and transcription/transduction coupling. Prior exposure to Pb revealed a repression in these genes suggesting that disturbances in developmental stages of the brain compromise the ability to defend against age-related stressors, thus promoting the neurodegenerative process. Overexpression and repression of genes corresponded with their DNA methylation profile. Published by Elsevier Ireland Ltd.

Gut microbiota as an epigenetic regulator: pilot study based on whole-genome methylation analysis.

PubMed

Kumar, Himanshu; Lund, Riikka; Laiho, Asta; Lundelin, Krista; Ley, Ruth E; Isolauri, Erika; Salminen, Seppo

2014-12-16

The core human gut microbiota contributes to the developmental origin of diseases by modifying metabolic pathways. To evaluate the predominant microbiota as an epigenetic modifier, we classified 8 pregnant women into two groups based on their dominant microbiota, i.e., Bacteroidetes, Firmicutes, and Proteobacteria. Deep sequencing of DNA methylomes revealed a clear association between bacterial predominance and epigenetic profiles. The genes with differentially methylated promoters in the group in which Firmicutes was dominant were linked to risk of disease, predominantly to cardiovascular disease and specifically to lipid metabolism, obesity, and the inflammatory response. This is one of the first studies that highlights the association of the predominant bacterial phyla in the gut with methylation patterns. Further longitudinal and in-depth studies targeting individual microbial species or metabolites are recommended to give us a deeper insight into the molecular mechanism of such epigenetic modifications. Epigenetics encompasses genomic modifications that are due to environmental factors and do not affect the nucleotide sequence. The gut microbiota has an important role in human metabolism and could be a significant environmental factor affecting our epigenome. To investigate the association of gut microbiota with epigenetic changes, we assessed pregnant women and selected the participants based on their predominant gut microbiota for a study on their postpartum methylation profile. Intriguingly, we found that blood DNA methylation patterns were associated with gut microbiota profiles. The gut microbiota profiles, with either Firmicutes or Bacteroidetes as a dominant group, correlated with differential methylation status of gene promoters functionally associated with cardiovascular diseases. Furthermore, differential methylation of gene promoters linked to lipid metabolism and obesity was observed. For the first time, we report here a position of the predominant gut microbiota in epigenetic profiling, suggesting one potential mechanism in obesity with comorbidities, if proven in further in-depth studies. Copyright © 2014 Kumar et al.

A new approach to epigenome-wide discovery of non-invasive methylation biomarkers for colorectal cancer screening in circulating cell-free DNA using pooled samples.

PubMed

Gallardo-Gómez, María; Moran, Sebastian; Páez de la Cadena, María; Martínez-Zorzano, Vicenta Soledad; Rodríguez-Berrocal, Francisco Javier; Rodríguez-Girondo, Mar; Esteller, Manel; Cubiella, Joaquín; Bujanda, Luis; Castells, Antoni; Balaguer, Francesc; Jover, Rodrigo; De Chiara, Loretta

2018-01-01

Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

DNA methylation in human papillomavirus-infected cervical cells is elevated in high-grade squamous intraepithelial lesions and cancer.

PubMed

Kim, Mi-Kyung; Lee, In-Ho; Lee, Ki-Heon; Lee, Yoo Kyung; So, Kyeong A; Hong, Sung Ran; Hwang, Chang-Sun; Kee, Mee-Kyung; Rhee, Jee Eun; Kang, Chun; Hur, Soo Young; Park, Jong Sup; Kim, Tae-Jin

2016-03-01

DNA methylation has been shown to be a potential biomarker for early cancer detection. The aim of this study was to evaluate DNA methylation profiles according to liquid-based Pap (LBP) test results and to assess their diagnostic value in a Korean population. A total of 205 patients with various Papanicolaou test results were enrolled to this study (negative, 26; atypical squamous cells of undetermined significance, 39; low grade squamous intraepithelial lesion, 44; high grade squamous intraepithelial lesion (HSIL), 48; and cancer, 48). DNA methylation analysis of four genes, ADCYAP1, PAX1, MAL, and CADM1, was performed on residual cervical cells from LBP samples using a quantitative bisulfite pyrosequencing method. To evaluate the diagnostic performance of the four methylated genes for cancer detection, receiver operating characteristic (ROC) curves were drawn. Sensitivities and specificities were also tested at cutoffs determined from the ROC curves. Cervical cancer cells showed dramatically increased methylation levels for the four genes analyzed. ADCYAP1 and PAX1 also trended toward elevated methylation levels in HSIL samples, although the levels were much lower than those in cancer cells. The sensitivities of methylated ADCYAP1, PAX1, MAL, and CADM1 for the detection of cancer were 79.2%, 75.0%, 70.8%, and 52.1%, and the specificities were 92.0%, 94.0%, 94.7%, and 94.0%, respectively. Methylated ADCYAP1 and PAX1 demonstrated relatively better discriminatory ability than did methylated MAL and CADM1 (area under the curves 0.911 and 0.916 vs. 0.854 and 0.756, respectively). DNA methylation status, especially in the ADCYAP1 and PAX1 genes, showed relatively good specificity, ranging from 90% to 94%. The possible additive and complementary roles of DNA methylation testing with respect to conventional cervical cancer screening programs will need to be validated in prospective population-based studies.

DNA methylation in human papillomavirus-infected cervical cells is elevated in high-grade squamous intraepithelial lesions and cancer

PubMed Central

Lee, Ki-Heon; So, Kyeong A; Hong, Sung Ran; Hwang, Chang-Sun; Kee, Mee-Kyung; Rhee, Jee Eun; Kang, Chun; Hur, Soo Young; Park, Jong Sup

2016-01-01

Objective DNA methylation has been shown to be a potential biomarker for early cancer detection. The aim of this study was to evaluate DNA methylation profiles according to liquid-based Pap (LBP) test results and to assess their diagnostic value in a Korean population. Methods A total of 205 patients with various Papanicolaou test results were enrolled to this study (negative, 26; atypical squamous cells of undetermined significance, 39; low grade squamous intraepithelial lesion, 44; high grade squamous intraepithelial lesion (HSIL), 48; and cancer, 48). DNA methylation analysis of four genes, ADCYAP1, PAX1, MAL, and CADM1, was performed on residual cervical cells from LBP samples using a quantitative bisulfite pyrosequencing method. To evaluate the diagnostic performance of the four methylated genes for cancer detection, receiver operating characteristic (ROC) curves were drawn. Sensitivities and specificities were also tested at cutoffs determined from the ROC curves. Results Cervical cancer cells showed dramatically increased methylation levels for the four genes analyzed. ADCYAP1 and PAX1 also trended toward elevated methylation levels in HSIL samples, although the levels were much lower than those in cancer cells. The sensitivities of methylated ADCYAP1, PAX1, MAL, and CADM1 for the detection of cancer were 79.2%, 75.0%, 70.8%, and 52.1%, and the specificities were 92.0%, 94.0%, 94.7%, and 94.0%, respectively. Methylated ADCYAP1 and PAX1 demonstrated relatively better discriminatory ability than did methylated MAL and CADM1 (area under the curves 0.911 and 0.916 vs. 0.854 and 0.756, respectively). Conclusion DNA methylation status, especially in the ADCYAP1 and PAX1 genes, showed relatively good specificity, ranging from 90% to 94%. The possible additive and complementary roles of DNA methylation testing with respect to conventional cervical cancer screening programs will need to be validated in prospective population-based studies. PMID:26768780

Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

PubMed Central

Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

2010-01-01

Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

PubMed

Taspinar, Mahmut Sinan; Aydin, Murat; Sigmaz, Burcu; Yildirim, Nalan; Agar, Guleray

2017-10-01

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

PubMed

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

DNA methylation profiling of phyllodes and fibroadenoma tumours of the breast.

PubMed

Huang, Katie T; Dobrovic, Alexander; Yan, Max; Karim, Rooshdiya Z; Lee, C Soon; Lakhani, Sunil R; Fox, Stephen B

2010-11-01

Phyllodes tumours and cellular fibroadenomas are both fibroepithelial tumours of the breast. Phyllodes tumours, unlike fibroadenomas, have the ability to recur and metastasise. Although these lesions can be distinguished by their stromal cellularity, mitotic index, presence or absence of stromal overgrowth and cellular atypia, there is overlap and not infrequently a definitive diagnosis cannot be made, particularly on biopsy. We sought to evaluate whether DNA promoter methylation profiling using selected genes known to be methylated in cancer would allow us to learn more about the biology of these tumours, and whether it could identify methylation markers that could differentiate phyllodes tumours from fibroadenomas and/or distinguish phyllodes tumours of different grades. Methylation-sensitive high resolution melting (MS-HRM) was used to screen promoter DNA methylation changes in 86 phyllodes tumours (15 benign, 28 borderline, 43 malignant) and 26 fibroadenomas. A panel of 11 genes (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, RARβ, CDKN2A, CDH1, TP73 and MLH1) was tested. Methylation status was correlated with histology and with clinicopathological parameters. Five of the gene promoters showed some methylation in a proportion of phyllodes tumours; RASSF1A, 45.3%; TWIST1, 10.7%; APC, 4.1%; WIF1, 2.9% and MGMT, 1.3%. Only two genes showed any methylation in fibroadenomas usually at background levels; RASSF1A, 53.8% and MGMT, 8.3%. No CDKN2A methylation was observed in either tumour type, contrary to previous reports. Overall, the methylation patterns differed little from that which might be seen in normal cells. However, significant levels of methylation of RASSF1A (24.4%) and TWIST1 (7.1%) was observed in some phyllodes tumours. Elevated RASSF1A and/or TWIST1 methylation was significantly associated with phyllodes tumours compared with fibroadenomas (P = 0.02), TWIST1 methylation correlated with increasing malignancy in phyllodes tumours (P < 0.001). In conclusion, assessment of methylation of RASSF1A and TWIST1 may aid in the diagnosis of phyllodes tumours. The absence of frequent methylation in fibroadenomas supports a non-neoplastic origin.

Epigenetic repression of HOXB cluster in oral cancer cell lines.

PubMed

Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

2014-08-01

Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA methylation pattern of apoptosis-related genes in ameloblastoma.

PubMed

Costa, Sfs; Pereira, N B; Pereira, Kma; Campos, K; de Castro, W H; Diniz, M G; Gomes, C C; Gomez, R S

2017-09-01

DNA methylation is an important mechanism of gene control expression, and it has been poorly addressed in odontogenic tumours. On this basis, we aimed to assess the methylation pattern of 22 apoptosis-related genes in solid ameloblastomas. Ameloblastoma fresh samples (n = 10) and dental follicles (n = 8) were included in the study. The percentage fraction of methylated and unmethylated DNA promoter of 22 apoptosis-related genes was determined using enzymatic restriction digestion and quantitative real-time PCR (qPCR) array. The relative expressions of the genes that showed the most discrepant methylation profile between tumours and controls were analysed by reverse-transcription quantitative PCR (RT-qPCR). Lower methylation percentages of TNFRSF25 (47.2%) and BCL2L11 (33.2%) were observed in ameloblastomas compared with dental follicles (79.3% and 59.5%, respectively). The RT-qPCR analysis showed increased expression of BCL2L11 in ameloblastomas compared with dental follicles, in agreement with the methylation analysis results, while there was no difference between the expression levels of TNFRSF25 between both groups. On the basis of our results, the transcription of the apoptosis-related gene BCL2L11 is possibly regulated by promoter DNA methylation in ameloblastoma. The biological significance of this finding in ameloblastoma pathobiology remains to be clarified. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Integrative analysis of DNA methylation and gene expression data identifies EPAS1 as a key regulator of COPD.

PubMed

Yoo, Seungyeul; Takikawa, Sachiko; Geraghty, Patrick; Argmann, Carmen; Campbell, Joshua; Lin, Luan; Huang, Tao; Tu, Zhidong; Foronjy, Robert F; Feronjy, Robert; Spira, Avrum; Schadt, Eric E; Powell, Charles A; Zhu, Jun

2015-01-01

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a 'causal' role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.

Epigenetic Alterations in Density Selected Human Spermatozoa for Assisted Reproduction.

PubMed

Yu, Bolan; Zhou, Hua; Liu, Min; Zheng, Ting; Jiang, Lu; Zhao, Mei; Xu, Xiaoxie; Huang, Zhaofeng

2015-01-01

Epidemiological evidence indicates that assisted reproductive technologies (ART) may be associated with several epigenetic diseases such as Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Selection of sperm by density-gradients in ART has improved DNA integrity and sperm quality; however, epigenetic alterations associated with this approach are largely unknown. In the present study, we investigated DNA methylation and histone retention profiles in raw sperm and selected sperm derived from the same individual and separated by using density-gradients. Results from a study group consisting of 93 males demonstrated that both global DNA methylation and histone retention levels decreased in density selected sperm. Compared to unselected raw sperm, histone transition rates decreased by an average of 27.2% in selected sperm, and the global methylation rate was 3.8% in unselected sperm and 3.3% in the selected sperm. DNA methylation and histone retention location profiling analyses suggested that these alterations displayed specific location patterns in the human genome. Changes in the pattern of hypomethylation largely occurred in transcriptional factor gene families such as HOX, FOX, and GATA. Histone retention increased in 67 genes, whereas it was significantly clustered in neural development-related gene families, particularly the olfactory sensor gene family. Although a causative relationship could not be established, the results of the present study suggest the possibility that sperm with good density also possess unique epigenetic profiles, particularly for genes involved in neural and olfactory development. As increasing evidence demonstrates that epigenetics plays a key role in embryonic development and offspring growth characteristics, the specific epigenetic alterations we observed in selected sperm may influence the transcriptional process and neural development in embryos.

Epigenetic Alterations in Density Selected Human Spermatozoa for Assisted Reproduction

PubMed Central

Yu, Bolan; Zhou, Hua; Liu, Min; Zheng, Ting; Jiang, Lu; Zhao, Mei; Xu, Xiaoxie; Huang, Zhaofeng

2015-01-01

Epidemiological evidence indicates that assisted reproductive technologies (ART) may be associated with several epigenetic diseases such as Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Selection of sperm by density-gradients in ART has improved DNA integrity and sperm quality; however, epigenetic alterations associated with this approach are largely unknown. In the present study, we investigated DNA methylation and histone retention profiles in raw sperm and selected sperm derived from the same individual and separated by using density-gradients. Results from a study group consisting of 93 males demonstrated that both global DNA methylation and histone retention levels decreased in density selected sperm. Compared to unselected raw sperm, histone transition rates decreased by an average of 27.2% in selected sperm, and the global methylation rate was 3.8% in unselected sperm and 3.3% in the selected sperm. DNA methylation and histone retention location profiling analyses suggested that these alterations displayed specific location patterns in the human genome. Changes in the pattern of hypomethylation largely occurred in transcriptional factor gene families such as HOX, FOX, and GATA. Histone retention increased in 67 genes, whereas it was significantly clustered in neural development-related gene families, particularly the olfactory sensor gene family. Although a causative relationship could not be established, the results of the present study suggest the possibility that sperm with good density also possess unique epigenetic profiles, particularly for genes involved in neural and olfactory development. As increasing evidence demonstrates that epigenetics plays a key role in embryonic development and offspring growth characteristics, the specific epigenetic alterations we observed in selected sperm may influence the transcriptional process and neural development in embryos. PMID:26709917

Authentication of forensic DNA samples.

PubMed

Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

2010-02-01

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

Proteomic and Epigenetic Analysis of Rice after Seed Spaceflight and Ground-Base Ion Radiations

NASA Astrophysics Data System (ADS)

Wang, Wei; Sun, Yeqing; Peng, Yuming; Zhao, Qian; Wen, Bin; Yang, Jun

Highly ionizing radiation (HZE) in space is considered as main factor causing biological effects to plant seeds. In previous work, we compared the proteomic profiles of rice plants growing after seed spaceflights to ground controls by two-dimensional difference gel electrophoresis (2-D DIGE) with mass spectrometry and found that the protein expression profiles were changed and differentially expressed proteins participated in most of the biological processes of rice. To further evaluate the dosage effects of space radiation and compare between low- and high-dose ion effects, we carried out three independent ground-base ionizing radiation experiments with different cumulative doses (low-dose range: 2~1000mGy, high-dose range: 2000~20000mGy) to rice seeds and performed proteomic analysis of seedlings. We found that protein expression profiles showed obvious boundaries between low- and high-dose radiation groups. Rates of differentially expressed proteins presented a dose-dependent effect, it reached the highest value at 2000mGy dosage point in all three radiation experiments coincidently; while proteins responded to low-dose radiations preferred to change their expressions at the minimum dosage (2mGy). Proteins participating in rice biological processes also responded differently between low- and high-dose radiations: proteins involved in energy metabolism and photosynthesis tended to be regulated after low-dose radiations while stress responding, protein folding and cell redox homeostasis related proteins preferred to change their expressions after high-dose radiations. By comparing the proteomic profiles between ground-base radiations and spaceflights, it was worth noting that ground-base low-dose ion radiation effects shared similar biological effects as space environment. In addition, we discovered that protein nucleoside diphosphate kinase 1 (NDPK1) showed obvious increased regulation after spaceflights and ion radiations. NDPK1 catalyzes nucleotide metabolism and is reported to be involved in DNA repair process. Its expression sensitivity and specificity were confirmed by RT-PCR and western blot analysis, indicating its potential to be used as space radiation biomarker. Space radiations might induce epigenetic effects on rice plants, especially changes of DNA methylation. Early results suggested that there were correlations between DNA methylation polymorphic and genomic mutation rates. In addition, the 5-methylcytosine located in coding gene’s promoter and exon regions could regulate gene expressions thus influence protein expressions. So whether there is correlation between genome DNA methylation changes and protein expression profile alterations caused by space radiation is worth for further investigation. Therefore we used the same rice samples treated by carbon ion radiation with different doses (0, 10, 20,100, 200, 1000, 2000, 5000, 20000mGy) and applied methylation sensitive amplification polymorphism (MSAP) for scanning genome DNA methylation changes. Interestingly, DNA methylation polymorphism rates also presented a dose-dependent effect and showed the same changing trend as rates of differentially expressed proteins. Whether there are correlations between epigenetic and proteomic effects of space radiation is worth for further investigation.

Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort.

PubMed

Soubry, Adelheid; Schildkraut, Joellen M; Murtha, Amy; Wang, Frances; Huang, Zhiqing; Bernal, Autumn; Kurtzberg, Joanne; Jirtle, Randy L; Murphy, Susan K; Hoyo, Cathrine

2013-02-06

Data from epidemiological and animal model studies suggest that nutrition during pregnancy may affect the health status of subsequent generations. These transgenerational effects are now being explained by disruptions at the level of the epigenetic machinery. Besides in vitro environmental exposures, the possible impact on the reprogramming of methylation profiles at imprinted genes at a much earlier time point, such as during spermatogenesis or oogenesis, has not previously been considered. In this study, our aim was to determine associations between preconceptional obesity and DNA methylation profiles in the offspring, particularly at the differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene. We examined DNA from umbilical cord blood leukocytes from 79 newborns, born between July 2005 and November 2006 at Duke University Hospital, Durham, NC. Their mothers participated in the Newborn Epigenetics Study (NEST) during pregnancy. Parental characteristics were obtained via standardized questionnaires and medical records. DNA methylation patterns at two DMRs were analyzed by bisulfite pyrosequencing; one DMR upstream of IGF2 (IGF2 DMR), and one DMR upstream of the neighboring H19 gene (H19 DMR). Multiple regression models were used to determine potential associations between the offspring's DNA methylation patterns and parental obesity before conception. Obesity was defined as body mass index (BMI) ≥30 kg/m². Hypomethylation at the IGF2 DMR was associated with paternal obesity. Even after adjusting for several maternal and newborn characteristics, we observed a persistent inverse association between DNA methylation in the offspring and paternal obesity (β-coefficient was -5.28, P = 0.003). At the H19 DMR, no significant associations were detected between methylation patterns and paternal obesity. Our data suggest an increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers, but a larger study is warranted to further explore the potential effects of maternal obesity or lifestyle on the offspring's epigenome. While our small sample size is limited, our data indicate a preconceptional impact of paternal obesity on the reprogramming of imprint marks during spermatogenesis. Given the biological importance of imprinting fidelity, our study provides evidence for transgenerational effects of paternal obesity that may influence the offspring's future health status.

Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort

PubMed Central

2013-01-01

Background Data from epidemiological and animal model studies suggest that nutrition during pregnancy may affect the health status of subsequent generations. These transgenerational effects are now being explained by disruptions at the level of the epigenetic machinery. Besides in vitro environmental exposures, the possible impact on the reprogramming of methylation profiles at imprinted genes at a much earlier time point, such as during spermatogenesis or oogenesis, has not previously been considered. In this study, our aim was to determine associations between preconceptional obesity and DNA methylation profiles in the offspring, particularly at the differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene. Methods We examined DNA from umbilical cord blood leukocytes from 79 newborns, born between July 2005 and November 2006 at Duke University Hospital, Durham, NC. Their mothers participated in the Newborn Epigenetics Study (NEST) during pregnancy. Parental characteristics were obtained via standardized questionnaires and medical records. DNA methylation patterns at two DMRs were analyzed by bisulfite pyrosequencing; one DMR upstream of IGF2 (IGF2 DMR), and one DMR upstream of the neighboring H19 gene (H19 DMR). Multiple regression models were used to determine potential associations between the offspring's DNA methylation patterns and parental obesity before conception. Obesity was defined as body mass index (BMI) ≥30 kg/m2. Results Hypomethylation at the IGF2 DMR was associated with paternal obesity. Even after adjusting for several maternal and newborn characteristics, we observed a persistent inverse association between DNA methylation in the offspring and paternal obesity (β-coefficient was -5.28, P = 0.003). At the H19 DMR, no significant associations were detected between methylation patterns and paternal obesity. Our data suggest an increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers, but a larger study is warranted to further explore the potential effects of maternal obesity or lifestyle on the offspring's epigenome. Conclusions While our small sample size is limited, our data indicate a preconceptional impact of paternal obesity on the reprogramming of imprint marks during spermatogenesis. Given the biological importance of imprinting fidelity, our study provides evidence for transgenerational effects of paternal obesity that may influence the offspring's future health status. PMID:23388414

DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing.

PubMed

Xiang, Yuqian; Zhang, Junyu; Li, Qiaoli; Zhou, Xinyao; Wang, Teng; Xu, Mingqing; Xia, Shihui; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi

2014-09-01

Utilizing epigenetic (DNA methylation) differences to differentiate between maternal peripheral blood (PBL) and fetal (placental) DNA has been a promising strategy for non-invasive prenatal testing (NIPT). However, the differentially methylated regions (DMRs) have yet to be fully ascertained. In the present study, we performed genome-wide comparative methylome analysis between maternal PBL and placental DNA from pregnancies of first trimester by methylated DNA immunoprecipitation-sequencing (MeDIP-Seq) and Infinium HumanMethylation450 BeadChip assays. A total of 36 931 DMRs and 45 804 differentially methylated sites (DMSs) covering the whole genome, exclusive of the Y chromosome, were identified via MeDIP-Seq and Infinium 450k array, respectively, of which 3759 sites in 2188 regions were confirmed by both methods. Not only did we find the previously reported potential fetal DNA markers in our identified DMRs/DMSs but also we verified fully the identified DMRs/DMSs in the validation round by MassARRAY EpiTYPER. The screened potential fetal DNA markers may be used for NIPT on aneuploidies and other chromosomal diseases, such as cri du chat syndrome and velo-cardio-facial syndrome. In addition, these potential markers may have application in the early diagnosis of placental dysfunction, such as pre-eclampsia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia.

PubMed

Rani, Lata; Mathur, Nitin; Gupta, Ritu; Gogia, Ajay; Kaur, Gurvinder; Dhanjal, Jaspreet Kaur; Sundar, Durai; Kumar, Lalit; Sharma, Atul

2017-01-01

In chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients. The integration of DNA methylation profile ( n  = 14) with the gene expression profile ( n  = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients ( n  = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1 , PMEPA1 , SOX7 , SPRY1 , CDK6 , TBX2 , and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup ( p  < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p  = 0.005) or PAX9 (RR = 1.87, p  = 0.001). High expression of CRY1 (HR: 3.53, p  < 0.001) or PAX9 (HR: 3.14, p  < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172-9.272, p  = 0.016) was also predictive of shorter overall survival in CLL. The DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Cell subpopulation deconvolution reveals breast cancer heterogeneity based on DNA methylation signature.

PubMed

Wen, Yanhua; Wei, Yanjun; Zhang, Shumei; Li, Song; Liu, Hongbo; Wang, Fang; Zhao, Yue; Zhang, Dongwei; Zhang, Yan

2017-05-01

Tumour heterogeneity describes the coexistence of divergent tumour cell clones within tumours, which is often caused by underlying epigenetic changes. DNA methylation is commonly regarded as a significant regulator that differs across cells and tissues. In this study, we comprehensively reviewed research progress on estimating of tumour heterogeneity. Bioinformatics-based analysis of DNA methylation has revealed the evolutionary relationships between breast cancer cell lines and tissues. Further analysis of the DNA methylation profiles in 33 breast cancer-related cell lines identified cell line-specific methylation patterns. Next, we reviewed the computational methods in inferring clonal evolution of tumours from different perspectives and then proposed a deconvolution strategy for modelling cell subclonal populations dynamics in breast cancer tissues based on DNA methylation. Further analysis of simulated cancer tissues and real cell lines revealed that this approach exhibits satisfactory performance and relative stability in estimating the composition and proportions of cellular subpopulations. The application of this strategy to breast cancer individuals of the Cancer Genome Atlas's identified different cellular subpopulations with distinct molecular phenotypes. Moreover, the current and potential future applications of this deconvolution strategy to clinical breast cancer research are discussed, and emphasis was placed on the DNA methylation-based recognition of intra-tumour heterogeneity. The wide use of these methods for estimating heterogeneity to further clinical cohorts will improve our understanding of neoplastic progression and the design of therapeutic interventions for treating breast cancer and other malignancies. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

DNA Methylation and Hydroxymethylation Levels in Relation to Two Weight Loss Strategies: Energy-Restricted Diet or Bariatric Surgery.

PubMed

Nicoletti, Carolina Ferreira; Nonino, Carla Barbosa; de Oliveira, Bruno Affonso Parenti; Pinhel, Marcela Augusta de Souza; Mansego, Maria Luisa; Milagro, Fermin Ignacio; Zulet, Maria Angeles; Martinez, José Alfredo

2016-03-01

Weight loss can be influenced by genetic factors and epigenetic mechanisms that participate in the regulation of body weight. This study aimed to investigate whether the weight loss induced by two different obesity treatments (energy restriction or bariatric surgery) may affect global DNA methylation (LINE-1) and hydroxymethylation profile, as well as the methylation patterns in inflammatory genes. This study encompassed women from three differents groups: 1. control group (n = 9), normal weight individuals; 2. energy restriction group (n = 22), obese patients following an energy-restricted Mediterranean-based dietary treatment (RESMENA); and 3. bariatric surgery group (n = 14), obese patients underwent a hypocaloric diet followed by bariatric surgery. Anthropometric measurements and 12-h fasting blood samples were collected before the interventions and after 6 months. Lipid and glucose biomarkers, global hydroxymethylation (by ELISA), LINE-1, SERPINE-1, and IL-6 (by MS-HRM) methylation levels were assessed in all participants. Baseline LINE-1 methylation was associated with serum glucose levels whereas baseline hydroxymethylation was associated with BMI, waist circumference, total cholesterol, and triglycerides. LINE-1 and SERPINE-1 methylation levels did not change after weight loss, whereas IL-6 methylation increased after energy restriction and decreased in the bariatric surgery group. An association between SERPINE-1 methylation and weight loss responses was found. Global DNA methylation and hydroxymethylation might be biomarkers for obesity and associated comorbidities. Depending on the obesity treatment (diet or surgery), the DNA methylation patterns behave differently. Baseline SERPINE-1 methylation may be a predictor of weight loss values after bariatric surgery.

[Epigenetic heredity (deoxyribonucleic acid methylation): Clinical context in neurodegenerative disorders and ATXN2 gene].

PubMed

Laffita-Mesa, José Miguel; Bauer, Peter

2014-10-21

Epigenetics is the group of changes in the phenotype which are related with the process independently of the primary DNA sequence. These changes are intimately related with changes in the gene expression level and its profile across the body. These are mediated by histone tail modifications, DNA methylation, micro-RNAs, with chromatin remodeling remaining as the foundation of epigenetic changes. DNA methylation involves the covalent addition of methyl group to cytosine of the DNA, which is mediated by methyltransferases enzymes. DNA methylation regulates gene expression by repressing transcription, while de-methylation activates gene transcription. Several human diseases are related with the epigenetic process: cancer, Alzheimer disease, stroke, Parkinson disease, and diabetes. We present here the basis of epigenetic inheritance and show the pathogenic mechanisms relating epigenetics in human diseases, specifically with regard to neurodegeneration. We discuss current concepts aimed at understanding the contribution of epigenetics to human neurodegenerative diseases. We also discuss recent findings obtained in our and other centers regarding the ATXN2 gene that causes spinocerebellar ataxia 2 and amyotrophic lateral sclerosis. Epigenetics play a pivotal role in the pathogenesis of human diseases and in several neurodegenerative disorders, and this knowledge will illuminate the pathways in the diagnostic and therapeutic field, which ultimately will be translated into the clinic context of neurodegenerative diseases. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation.

PubMed

Teschendorff, Andrew E; Jones, Allison; Fiegl, Heidi; Sargent, Alexandra; Zhuang, Joanna J; Kitchener, Henry C; Widschwendter, Martin

2012-03-27

Recently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation. We analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions. We observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively). We demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application. The ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821.

Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation

PubMed Central

2012-01-01

Background Recently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation. Methods We analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions. Results We observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively). Conclusions We demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application. Trial registration The ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821. PMID:22453031

Epigenetic profiling of ADHD symptoms trajectories: a prospective, methylome-wide study.

PubMed

Walton, E; Pingault, J-B; Cecil, C A M; Gaunt, T R; Relton, C L; Mill, J; Barker, E D

2017-02-01

Attention-deficit/hyperactivity disorder (ADHD) is a prevalent developmental disorder, associated with a range of long-term impairments. Variation in DNA methylation, an epigenetic mechanism, is implicated in both neurobiological functioning and psychiatric health. However, the potential role of DNA methylation in ADHD symptoms is currently unclear. In this study, we examined data from the Avon Longitudinal Study of Parents and Children (ALSPAC)-specifically the subsample forming the Accessible Resource for Integrated Epigenomics Studies (ARIES)-that includes (1) peripheral measures of DNA methylation (Illumina 450k) at birth (n=817, 49% male) and age 7 (n=892, 50% male) and (2) trajectories of ADHD symptoms (7-15 years). We first employed a genome-wide analysis to test whether DNA methylation at birth associates with later ADHD trajectories; and then followed up at age 7 to investigate the stability of associations across early childhood. We found that DNA methylation at birth differentiated ADHD trajectories across multiple genomic locations, including probes annotated to SKI (involved in neural tube development), ZNF544 (previously implicated in ADHD), ST3GAL3 (linked to intellectual disability) and PEX2 (related to perixosomal processes). None of these probes maintained an association with ADHD trajectories at age 7. Findings lend novel insights into the epigenetic landscape of ADHD symptoms, highlighting the potential importance of DNA methylation variation in genes related to neurodevelopmental and peroxisomal processes that play a key role in the maturation and stability of cortical circuits.

Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men-A pilot study.

PubMed

Gunes, S; Agarwal, A; Henkel, R; Mahmutoglu, A M; Sharma, R; Esteves, S C; Aljowair, A; Emirzeoglu, D; Alkhani, A; Pelegrini, L; Joumah, A; Sabanegh, E

2018-04-01

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation. © 2017 Blackwell Verlag GmbH.

Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

PubMed

Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

2016-01-01

Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation.

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

PubMed Central

Gim, Jeong-An; Hong, Chang Pyo; Kim, Dae-Soo; Moon, Jae-Woo; Choi, Yuri; Eo, Jungwoo; Kwon, Yun-Jeong; Lee, Ja-Rang; Jung, Yi-Deun; Bae, Jin-Han; Choi, Bong-Hwan; Ko, Junsu; Song, Sanghoon; Ahn, Kung; Ha, Hong-Seok; Yang, Young Mok; Lee, Hak-Kyo; Park, Kyung-Do; Do, Kyoung-Tag; Han, Kyudong; Yi, Joo Mi; Cha, Hee-Jae; Ayarpadikannan, Selvam; Cho, Byung-Wook; Bhak, Jong; Kim, Heui-Soo

2015-01-01

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits. PMID:25666347

Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing

PubMed Central

Crampton, Mollee; Sripathi, Venkateswara R.; Hossain, Khwaja; Kalavacharla, Venu

2016-01-01

Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar (“Sierra”) using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer

PubMed Central

Rager, Julia E.; Miller, Sloane; Tulenko, Samantha E.; Smeester, Lisa; Ray, Paul D.; Yosim, Andrew; Currier, Jenna M.; Ishida, María C.; González-Horta, Maria del Carmen; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Gutiérrez-Torres, Daniela S.; Drobná, Zuzana; Del Razo, Luz M.; García-Vargas, Gonzalo G.; Kim, William Y.; Zhou, Yi-Hui; Wright, Fred A.; Stýblo, Miroslav; Fry, Rebecca C.

2016-01-01

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to a myriad of adverse health effects, including cancer of the bladder. The present study set out to identify DNA methylation patterns associated with iAs and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic (As)-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total As (tAs) and As species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 As-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the As- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer. PMID:26039340

Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

PubMed

Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

2015-01-01

CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

Epigenomics and bolting tolerance in sugar beet genotypes.

PubMed

Hébrard, Claire; Peterson, Daniel G; Willems, Glenda; Delaunay, Alain; Jesson, Béline; Lefèbvre, Marc; Barnes, Steve; Maury, Stéphane

2016-01-01

In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

GFRA3 promoter methylation may be associated with decreased postoperative survival in gastric cancer.

PubMed

Eftang, Lars Lohne; Klajic, Jovana; Kristensen, Vessela N; Tost, Jörg; Esbensen, Qin Ying; Blom, Gustav Peter; Bukholm, Ida Rashida Khan; Bukholm, Geir

2016-03-16

A large number of epigenetic alterations has been found to be implicated in the etiology of gastric cancer. We have studied the DNA methylation status of 27 500 gene promoter regions in 24 gastric adenocarcinomas from a Norwegian cohort, and aimed at identifying the hypermethylated regions. We have compared our findings to the gene expression in the same tissue, and linked our results to prognosis and survival. Biopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 24 patients following surgical resection of the tumor. Genome-wide DNA methylation profiling of the tumor and matched non-cancerous mucosa was performed. The results were compared to whole transcriptome cDNA microarray analysis of the same material. Most of the gene promoter regions in both types of tissue showed a low degree of methylation, however there was a small, but significant hypermethylation of the tumors. Hierarchical clustering showed separate grouping of the tumor and normal tissue. Hypermethylation of the promoter region of the GFRA3 gene showed a strong correlation to post-operative survival and several of the clinicopathological parameters, however no difference was found between the two main histological types of gastric cancer. There was only a modest correlation between the DNA methylation status and gene expression. The different DNA methylation clusters of the tumors and normal tissue indicate that aberrant DNA methylation is a distinct feature of gastric cancer, although there is little difference in the overall, and low, methylation levels between the two tissue types. The GFRA3 promoter region showed marked hypermethylation in almost all tumors, and its correlation with survival and other clinicopathological parameters may have important prognostic significance.

Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol

PubMed Central

Medina-Aguilar, Rubiceli; Pérez-Plasencia, Carlos; Marchat, Laurence A.; Gariglio, Patricio; García Mena, Jaime; Rodríguez Cuevas, Sergio; Ruíz-García, Erika; Astudillo-de la Vega, Horacio; Hernández Juárez, Jennifer; Flores-Pérez, Ali; López-Camarillo, César

2016-01-01

Aberrant DNA methylation is a frequent epigenetic alteration in cancer cells that has emerged as a pivotal mechanism for tumorigenesis. Accordingly, novel therapies targeting the epigenome are being explored with the aim to restore normal DNA methylation patterns on oncogenes and tumor suppressor genes. A limited number of studies indicate that dietary compound resveratrol modulates DNA methylation of several cancer-related genes; however a complete view of changes in methylome by resveratrol has not been reported yet. In this study we performed a genome-wide survey of DNA methylation signatures in triple negative breast cancer cells exposed to resveratrol. Our data showed that resveratrol treatment for 24 h and 48 h decreased gene promoter hypermethylation and increased DNA hypomethylation. Of 2476 hypermethylated genes in control cells, 1,459 and 1,547 were differentially hypomethylated after 24 h and 48 h, respectively. Remarkably, resveratrol did not induce widespread non-specific DNA hyper- or hypomethylation as changes in methylation were found in only 12.5% of 27,728 CpG loci. Moreover, resveratrol restores the hypomethylated and hypermethylated status of key tumor suppressor genes and oncogenes, respectively. Importantly, the integrative analysis of methylome and transcriptome profiles in response to resveratrol showed that methylation alterations were concordant with changes in mRNA expression. Our findings reveal for the first time the impact of resveratrol on the methylome of breast cancer cells and identify novel potential targets for epigenetic therapy. We propose that resveratrol may be considered as a dietary epidrug as it may exert its anti-tumor activities by modifying the methylation status of cancer -related genes which deserves further in vivo characterization. PMID:27355345

A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties.

PubMed

Pan, Gaofeng; Jiang, Limin; Tang, Jijun; Guo, Fei

2018-02-08

DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods-especially machine learning methods-have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k -gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria-area under the receiver operating characteristic curve (AUC), Matthew's correlation coefficient (MCC), accuracy (ACC), sensitivity (SN), and specificity-are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA.

PubMed

Tang, Qiuqiong; Holland-Letz, Tim; Slynko, Alla; Cuk, Katarina; Marme, Frederik; Schott, Sarah; Heil, Jörg; Qu, Bin; Golatta, Michael; Bewerunge-Hudler, Melanie; Sutter, Christian; Surowy, Harald; Wappenschmidt, Barbara; Schmutzler, Rita; Hoth, Markus; Bugert, Peter; Bartram, Claus R; Sohn, Christof; Schneeweiss, Andreas; Yang, Rongxi; Burwinkel, Barbara

2016-09-27

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

PubMed

Altobelli, Gioia; Bogdarina, Irina G; Stupka, Elia; Clark, Adrian J L; Langley-Evans, Simon

2013-01-01

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Microarray-based DNA methylation study of Ewing's sarcoma of the bone.

PubMed

Park, Hye-Rim; Jung, Woon-Won; Kim, Hyun-Sook; Park, Yong-Koo

2014-10-01

Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing's sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing's sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing's sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing's sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10 , OSM , APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing's sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing's sarcoma.

Microarray-based DNA methylation study of Ewing’s sarcoma of the bone

PubMed Central

PARK, HYE-RIM; JUNG, WOON-WON; KIM, HYUN-SOOK; PARK, YONG-KOO

2014-01-01

Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing’s sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing’s sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing’s sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing’s sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10, OSM, APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing’s sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing’s sarcoma. PMID:25202378

Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice

PubMed Central

Inoue, Kota; Fukuda, Kei; Sasaki, Hiroyuki

2017-01-01

Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both mutant spermatocytes, suggesting that retrotransposon silencing is important for the maintenance of not only genomic integrity but also transcriptomic integrity. PMID:28749988

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naïve embryonic stem cells.

PubMed

Coluccio, Andrea; Ecco, Gabriela; Duc, Julien; Offner, Sandra; Turelli, Priscilla; Trono, Didier

2018-02-26

The KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation. Here, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others. Our results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation.

PubMed

Duncan, Christopher G; Barwick, Benjamin G; Jin, Genglin; Rago, Carlo; Kapoor-Vazirani, Priya; Powell, Doris R; Chi, Jen-Tsan; Bigner, Darell D; Vertino, Paula M; Yan, Hai

2012-12-01

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

Integrated data analysis reveals potential drivers and pathways disrupted by DNA methylation in papillary thyroid carcinomas.

PubMed

Beltrami, Caroline Moraes; Dos Reis, Mariana Bisarro; Barros-Filho, Mateus Camargo; Marchi, Fabio Albuquerque; Kuasne, Hellen; Pinto, Clóvis Antônio Lopes; Ambatipudi, Srikant; Herceg, Zdenko; Kowalski, Luiz Paulo; Rogatto, Silvia Regina

2017-01-01

Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related. The comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. BRAF mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with BRAF wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in BRAF V600E-positive tumors. The methylation and expression pattern of six selected genes ( ERBB3 , FGF1 , FGFR2 , GABRB2 , HMGA2 , and RDH5 ) were confirmed as altered by pyrosequencing and RT-qPCR. DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring BRAF V600E. In addition to the promoter region, gene body and 3'UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.

Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

PubMed Central

Matsuyama, Tomoki; Kimura, Makoto T.; Koike, Kuniaki; Abe, Tomoko; Nakano, Takeshi; Asami, Tadao; Ebisuzaki, Toshikazu; Held, William A.; Yoshida, Shigeo; Nagase, Hiroki

2003-01-01

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development. PMID:12888509

Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource.

PubMed

Nanan, Kyster K; Ocheltree, Cody; Sturgill, David; Mandler, Mariana D; Prigge, Maria; Varma, Garima; Oberdoerffer, Shalini

2017-12-15

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Effects of methyl substitution on DNA binding enthalpies of enantiopure Ru(phenanthroline)2dipyridophenazine2+ complexes.

PubMed

Mårtensson, Anna K F; Lincoln, Per

2018-04-25

Isothermal titration calorimetry (ITC) has been utilized to investigate the effect of methyl substituents on the intercalating dppz ligand of the enantiomers of the parent complex Ru(phen)2dppz2+ (phen = 1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) on DNA binding thermodynamics. The methylated complexes (10-methyl-dppz and 11,12-dimethyl-dppz) have large, concentration-dependent, positive heats of dilution, and a strong endothermic background is also apparent in the ITC-profiles from titration of methylated complexes into poly(dAdT)2, which make direct comparison between complexes difficult. By augmenting a simple cooperative binding model with one equilibrium for complex self-aggregation in solution and one equilibrium for complex aggregation on saturated DNA, it was possible to find an excellent global fit to the experimental data with DNA affinity parameters restricted to be equal for all Δ-enantiomers as well as for all Λ-enantiomers. In general, enthalpic differences, compared to the unsubstituted complex, were small and less than 4 kJ mol-1, except for the heat of intercalation of Δ-10-methyl-dppz (-11,6 kJ mol-1) and Λ-11,12-dimethyl-dppz (+4.3 kJ mol-1).

Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor.

PubMed

Haller, Florian; Zhang, Jitao David; Moskalev, Evgeny A; Braun, Alexander; Otto, Claudia; Geddert, Helene; Riazalhosseini, Yasser; Ward, Aoife; Balwierz, Aleksandra; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, B Michael; Agaimy, Abbas; Fletcher, Jonathan A; Hoheisel, Jörg; Hartmann, Arndt; Werner, Martin; Wiemann, Stefan; Sahin, Ozgür

2015-03-01

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category. © 2014 UICC.

Self-reported smoking, serum cotinine, and blood DNA methylation.

PubMed

Zhang, Yan; Florath, Ines; Saum, Kai-Uwe; Brenner, Hermann

2016-04-01

Epigenome-wide profiling of DNA methylation pattern with respect to tobacco smoking has given rise to a new measure of smoking exposure. We investigated the relationships of methylation markers with both cotinine, an established marker of internal smoking exposure, and self-reported smoking. Blood DNA methylation levels across the genome and serum cotinine were measured in 1000 older adults aged 50-75 years. Epigenome-wide scans were performed to identify methylation markers associated with cotinine. The inter-dose-response relationships between the number of cigarettes smoked per day, cotinine concentration, and DNA methylation were modeled by restricted cubic spline regression. Of 61 CpGs that passed the genome-wide significance threshold (p<1.13×10(-7)), 40 CpGs in 25 chromosomal regions were successfully replicated, showing 0.2-3% demethylation per 10ng/ml increases in cotinine. The strongest associations were observed for several loci at AHRR, F2RL3, 2q37.1, 6p21.33, and GFI1 that were previously identified to be related to self-reported smoking. One locus at RAB34 was newly discovered. Both cotinine and methylation markers exhibited non-linear relationships with the number of cigarettes smoked per day, where the highest rates of increase in cotinine and decreases in methylation were observed at low smoking intensity (1-15 cigarettes/day) and plateaued at high smoking intensity (>15-20 cigarettes/day). A clear linear relationship was observed between cotinine concentration and methylation level. Both cotinine and methylation markers showed similar accuracy in distinguishing current from never smoker, but only methylation markers distinguished former from never smoker with high accuracy. Our study corroborates and expands the list of smoking-associated DNA methylation markers. Methylation levels were linearly related to cotinine concentration and provided accurate measures for both current and past smoking exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA Hypomethylation Affects Cancer-Related Biological Functions and Genes Relevant in Neuroblastoma Pathogenesis

PubMed Central

Mayol, Gemma; Martín-Subero, José I.; Ríos, José; Queiros, Ana; Kulis, Marta; Suñol, Mariona; Esteller, Manel; Gómez, Soledad; Garcia, Idoia; de Torres, Carmen; Rodríguez, Eva; Galván, Patricia; Mora, Jaume; Lavarino, Cinzia

2012-01-01

Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3′ untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation. PMID:23144874

Alpha-Lipoic Acid Downregulates IL-1β and IL-6 by DNA Hypermethylation in SK-N-BE Neuroblastoma Cells.

PubMed

Dinicola, Simona; Proietti, Sara; Cucina, Alessandra; Bizzarri, Mariano; Fuso, Andrea

2017-09-26

Alpha-lipoic acid (ALA) is a pleiotropic molecule with antioxidant and anti-inflammatory properties, of which the effects are exerted through the modulation of NF-kB. This nuclear factor, in fact, modulates different inflammatory cytokines, including IL-1b and IL-6, in different tissues and cell types. We recently showed that IL-1b and IL-6 DNA methylation is modulated in the brain of Alzheimer's disease patients, and that IL-1b expression is associated to DNA methylation in the brain of patients with tuberous sclerosis complex. These results prompted us to ask whether ALA-induced repression of IL-1b and IL-6 was dependent on DNA methylation. Therefore, we profiled DNA methylation in the 5'-flanking region of the two aforementioned genes in SK-N-BE human neuroblastoma cells cultured in presence of ALA 0.5 mM. Our experimental data pointed out that the two promoters are hypermethylated in cells supplemented with ALA, both at CpG and non-CpG sites. Moreover, the observed hypermethylation is associated with decreased mRNA expression and decreased cytokine release. These results reinforce previous findings indicating that IL-1b and IL-6 undergo DNA methylation-dependent modulation in neural models and pave the road to study the epigenetic mechanisms triggered by ALA.

Free energy profiles for two ubiquitous damaging agents: methylation and hydroxylation of guanine in B-DNA.

PubMed

Grüber, R; Aranda, J; Bellili, A; Tuñón, I; Dumont, E

2017-06-07

DNA methylation and hydroxylation are two ubiquitous reactions in DNA damage induction, yet insights are scarce concerning the free energy of activation within B-DNA. We resort to multiscale simulations to investigate the attack of a hydroxyl radical and of the primary diazonium onto a guanine embedded in a solvated dodecamer. Reaction free energy profiles characterize two strongly exergonic processes, yet allow unprecedented quantification of the barrier towards this damage reaction, not higher than 6 kcal mol -1 and sometimes inexistent, and of the exergonicities. In the case of the [G(C8)-OH]˙ intermediate, we challenge the functional dependence of such simulations: recently-proposed functionals, such as M06-2X and LC-BLYP, agree on a ∼4 kcal mol -1 barrier, whereas the hybrid GGA B3LYP functional predicts a barrier-less pathway. In the long term, multiscale approaches can help build up a unified panorama of DNA lesion induction. These results stress the importance of DFT/MM-MD simulations involving new functionals towards the sound modelling of biomolecule damage even in the ground state.

The DNA methylation profile of activated human natural killer cells.

PubMed

Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

2016-05-03

Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

DNA methylation and soy phytoestrogens: quantitative study in DU-145 and PC-3 human prostate cancer cell lines.

PubMed

Adjakly, Mawussi; Bosviel, Rémy; Rabiau, Nadège; Boiteux, Jean-Paul; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

2011-12-01

DNA hypermethylation is an epigenetic mechanism which induces silencing of tumor-suppressor genes in prostate cancer. Many studies have reported that specific components of food plants like soy phytoestrogens may have protective effects against prostate carcinogenesis or progression. Genistein and daidzein, the major phytoestrogens, have been reported to have the ability to reverse DNA hypermethylation in cancer cell lines. The aim of this study was to investigate the potential demethylating effects of these two soy compounds on BRCA1, GSTP1, EPHB2 and BRCA2 promoter genes. Prostate cell lines DU-145 and PC-3 were treated with genistein 40 µM, daidzein 110 µM, budesonide (methylating agent) 2 µM and 5-azacytidine (demethylating agent) 2 µM. In these two human prostate cancer cell lines we performed methylation quantification by using Methyl Profiler DNA methylation analysis. This technique is based on a methylation-specific digestion followed by quantitative PCR. We analyzed the corresponding protein expression by western blotting. Soy phytoestrogens induced a demethylation of all promoter regions studied except for BRCA2, which is not methylated in control cell lines. An increase in their protein expression was also demonstrated by western blot analysis and corroborated the potential demethylating effect of soy phytoestrogens. This study showed that the soy phytoestrogens, genistein and daidzein, induce a decrease of methylation of BRCA1, GSTP1 and EPHB2 promoters. Therefore, soy phytoestrogens may have a protective effect on prostate cancer. However, more studies are needed in order to understand the mechanism by which genistein and daidzein have an inhibiting action on DNA methylation.

LMethyR-SVM: Predict Human Enhancers Using Low Methylated Regions based on Weighted Support Vector Machines.

PubMed

Xu, Jingting; Hu, Hong; Dai, Yang

The identification of enhancers is a challenging task. Various types of epigenetic information including histone modification have been utilized in the construction of enhancer prediction models based on a diverse panel of machine learning schemes. However, DNA methylation profiles generated from the whole genome bisulfite sequencing (WGBS) have not been fully explored for their potential in enhancer prediction despite the fact that low methylated regions (LMRs) have been implied to be distal active regulatory regions. In this work, we propose a prediction framework, LMethyR-SVM, using LMRs identified from cell-type-specific WGBS DNA methylation profiles and a weighted support vector machine learning framework. In LMethyR-SVM, the set of cell-type-specific LMRs is further divided into three sets: reliable positive, like positive and likely negative, according to their resemblance to a small set of experimentally validated enhancers in the VISTA database based on an estimated non-parametric density distribution. Then, the prediction model is obtained by solving a weighted support vector machine. We demonstrate the performance of LMethyR-SVM by using the WGBS DNA methylation profiles derived from the human embryonic stem cell type (H1) and the fetal lung fibroblast cell type (IMR90). The predicted enhancers are highly conserved with a reasonable validation rate based on a set of commonly used positive markers including transcription factors, p300 binding and DNase-I hypersensitive sites. In addition, we show evidence that the large fraction of the LMethyR-SVM predicted enhancers are not predicted by ChromHMM in H1 cell type and they are more enriched for the FANTOM5 enhancers. Our work suggests that low methylated regions detected from the WGBS data are useful as complementary resources to histone modification marks in developing models for the prediction of cell-type-specific enhancers.

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information.

PubMed

Koelsche, Christian; Hartmann, Wolfgang; Schrimpf, Daniel; Stichel, Damian; Jabar, Susanne; Ranft, Andreas; Reuss, David E; Sahm, Felix; Jones, David T W; Bewerunge-Hudler, Melanie; Trautmann, Marcel; Klingebiel, Thomas; Vokuhl, Christian; Gessler, Manfred; Wardelmann, Eva; Petersen, Iver; Baumhoer, Daniel; Flucke, Uta; Antonescu, Cristina; Esteller, Manel; Fröhling, Stefan; Kool, Marcel; Pfister, Stefan M; Mechtersheimer, Gunhild; Dirksen, Uta; von Deimling, Andreas

2018-03-23

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

PubMed

Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

2018-01-01

Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

Genome-wide combination profiling of DNA copy number and methylation for deciphering biomarkers in non-small cell lung cancer patients.

PubMed

Son, Ji Woong; Jeong, Kang Jin; Jean, Woo-Sean; Park, Soon Young; Jheon, Sanghoon; Cho, Hyun Min; Park, Chang Gyo; Lee, Hoi Young; Kang, Jaeku

2011-12-01

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease's high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31-3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Differential DNA methylation patterns of polycystic ovarian syndrome in whole blood of Chinese women.

PubMed

Li, Shuxia; Zhu, Dongyi; Duan, Hongmei; Ren, Anran; Glintborg, Dorte; Andersen, Marianne; Skov, Vibe; Thomassen, Mads; Kruse, Torben; Tan, Qihua

2017-03-28

As a universally common endocrinopathy in women of reproductive age, the polycystic ovarian syndrome is characterized by composite clinical phenotypes reflecting the contributions of reproductive impact of ovarian dysfunction and metabolic abnormalities with widely varying symptoms resulting from interference of the genome with the environment through integrative biological mechanisms including epigenetics. We have performed a genome-wide DNA methylation analysis on polycystic ovarian syndrome and identified a substantial number of genomic sites differentially methylated in the whole blood of PCOS patients and healthy controls (52 sites, false discovery rate < 0.05 and corresponding p value < 5.68e-06), highly consistently replicating biological pathways extensively implicated in immunity and immunity-related inflammatory disorders (false discovery rate < 0.05) that were reportedly regulated in the DNA methylome from ovarian tissue under PCOS condition. Most importantly, our genome-wide profiling focusing on PCOS patients revealed a large number of DNA methylation sites and their enriched functional pathways significantly associated with diverse clinical features (levels of prolactin, estradiol, progesterone and menstrual cycle) that could serve as novel molecular basis of the clinical heterogeneity observed in PCOS women.

DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

PubMed Central

Slater, Amy A.; Alokail, Majed; Gentle, Dean; Yao, Masahiro; Kovacs, Gyula; Maher, Eamonn R.

2013-01-01

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors. PMID:23428843

Twenty-four-nucleotide siRNAs produce heritable trans-chromosomal methylation in F1 Arabidopsis hybrids.

PubMed

Greaves, Ian K; Eichten, Steven R; Groszmann, Michael; Wang, Aihua; Ying, Hua; Peacock, W James; Dennis, Elizabeth S

2016-11-01

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations.

Site-Dependent Differences in DNA Methylation and Their Impact on Plant Establishment and Phosphorus Nutrition in Populus trichocarpa.

PubMed

Schönberger, Brigitte; Chen, Xiaochao; Mager, Svenja; Ludewig, Uwe

2016-01-01

The propagation via clonal stem cuttings is a frequent practice in tree plantations. Despite their clonal origin, the trees establish differently according to weather, temperature and nutrient availability, as well as the presence of various stresses. Here, clonal Populus trichocarpa (cv. Muhle Larson) cuttings from different sites were transferred into a common, fully nutrient supplied environment. Despite identical underlying genetics, stem cuttings derived from sites with lower phosphorus availability established worse, independent of phosphorus (P) level after transplantation. Differential growth of material from the sites was reflected in differences in the whole genome DNA methylome. Methylation differences were sequence context-dependent, but differentially methylated regions (DMRs) were apparently unrelated to P nutrition genes. Despite the undisputed negative general correlation of DNA promoter methylation with gene repression, only few of the top-ranked DMRs resulted in differential gene expression in roots or shoots. However, differential methylation was associated with site-dependent, different total amounts of microRNAs (miRNAs), with few miRNAs sequences directly targeted by differential methylation. Interestingly, in roots and shoots, the miRNA amount was dependent on the previous habitat and changed in roots in a habitat-dependent way under phosphate starvation conditions. Differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression differences as a P-related epigenetic modification in poplar. Together with differences in DNA methylation, such epigenetic mechanisms may explain habitat or seasonal memory in perennials and site-dependent growth performances.

Epigenomics: dissecting hybridization and polyploidization.

PubMed

Jackson, Scott A

2017-06-19

Epigenetic profiling in diploid, allopolyploid, and domesticated cotton shows that despite most DNA methylation being conserved and stably inherited, alterations likely due to hybridization and domestication affect gene expression.

CMS: A Web-Based System for Visualization and Analysis of Genome-Wide Methylation Data of Human Cancers

PubMed Central

Huang, Yi-Wen; Roa, Juan C.; Goodfellow, Paul J.; Kizer, E. Lynette; Huang, Tim H. M.; Chen, Yidong

2013-01-01

Background DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters. Methodology/Principal Findings Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework. Conclusions/Significance CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible at: http://cbbiweb.uthscsa.edu/KMethylomes/. PMID:23630576

CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.

PubMed

Gu, Fei; Doderer, Mark S; Huang, Yi-Wen; Roa, Juan C; Goodfellow, Paul J; Kizer, E Lynette; Huang, Tim H M; Chen, Yidong

2013-01-01

DNA methylation of promoter CpG islands is associated with gene suppression, and its unique genome-wide profiles have been linked to tumor progression. Coupled with high-throughput sequencing technologies, it can now efficiently determine genome-wide methylation profiles in cancer cells. Also, experimental and computational technologies make it possible to find the functional relationship between cancer-specific methylation patterns and their clinicopathological parameters. Cancer methylome system (CMS) is a web-based database application designed for the visualization, comparison and statistical analysis of human cancer-specific DNA methylation. Methylation intensities were obtained from MBDCap-sequencing, pre-processed and stored in the database. 191 patient samples (169 tumor and 22 normal specimen) and 41 breast cancer cell-lines are deposited in the database, comprising about 6.6 billion uniquely mapped sequence reads. This provides comprehensive and genome-wide epigenetic portraits of human breast cancer and endometrial cancer to date. Two views are proposed for users to better understand methylation structure at the genomic level or systemic methylation alteration at the gene level. In addition, a variety of annotation tracks are provided to cover genomic information. CMS includes important analytic functions for interpretation of methylation data, such as the detection of differentially methylated regions, statistical calculation of global methylation intensities, multiple gene sets of biologically significant categories, interactivity with UCSC via custom-track data. We also present examples of discoveries utilizing the framework. CMS provides visualization and analytic functions for cancer methylome datasets. A comprehensive collection of datasets, a variety of embedded analytic functions and extensive applications with biological and translational significance make this system powerful and unique in cancer methylation research. CMS is freely accessible at: http://cbbiweb.uthscsa.edu/KMethylomes/.

Integrative Analysis of DNA Methylation and Gene Expression Data Identifies EPAS1 as a Key Regulator of COPD

PubMed Central

Yoo, Seungyeul; Takikawa, Sachiko; Geraghty, Patrick; Argmann, Carmen; Campbell, Joshua; Lin, Luan; Huang, Tao; Tu, Zhidong; Feronjy, Robert; Spira, Avrum; Schadt, Eric E.; Powell, Charles A.; Zhu, Jun

2015-01-01

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology. PMID:25569234

Choice of surrogate tissue influences neonatal EWAS findings.

PubMed

Lin, Xinyi; Teh, Ai Ling; Chen, Li; Lim, Ives Yubin; Tan, Pei Fang; MacIsaac, Julia L; Morin, Alexander M; Yap, Fabian; Tan, Kok Hian; Saw, Seang Mei; Lee, Yung Seng; Holbrook, Joanna D; Godfrey, Keith M; Meaney, Michael J; Kobor, Michael S; Chong, Yap Seng; Gluckman, Peter D; Karnani, Neerja

2017-12-05

Epigenomes are tissue specific and thus the choice of surrogate tissue can play a critical role in interpreting neonatal epigenome-wide association studies (EWAS) and in their extrapolation to target tissue. To develop a better understanding of the link between tissue specificity and neonatal EWAS, and the contributions of genotype and prenatal factors, we compared genome-wide DNA methylation of cord tissue and cord blood, two of the most accessible surrogate tissues at birth. In 295 neonates, DNA methylation was profiled using Infinium HumanMethylation450 beadchip arrays. Sites of inter-individual variability in DNA methylation were mapped and compared across the two surrogate tissues at birth, i.e., cord tissue and cord blood. To ascertain the similarity to target tissues, DNA methylation profiles of surrogate tissues were compared to 25 primary tissues/cell types mapped under the Epigenome Roadmap project. Tissue-specific influences of genotype on the variable CpGs were also analyzed. Finally, to interrogate the impact of the in utero environment, EWAS on 45 prenatal factors were performed and compared across the surrogate tissues. Neonatal EWAS results were tissue specific. In comparison to cord blood, cord tissue showed higher inter-individual variability in the epigenome, with a lower proportion of CpGs influenced by genotype. Both neonatal tissues were good surrogates for target tissues of mesodermal origin. They also showed distinct phenotypic associations, with effect sizes of the overlapping CpGs being in the same order of magnitude. The inter-relationship between genetics, prenatal factors and epigenetics is tissue specific, and requires careful consideration in designing and interpreting future neonatal EWAS. This birth cohort is a prospective observational study, designed to study the developmental origins of health and disease, and was retrospectively registered on 1 July 2010 under the identifier NCT01174875 .

Molecular Aging of Human Liver: An Epigenetic/Transcriptomic Signature.

PubMed

Bacalini, Maria Giulia; Franceschi, Claudio; Gentilini, Davide; Ravaioli, Francesco; Zhou, Xiaoyuan; Remondini, Daniel; Pirazzini, Chiara; Giuliani, Cristina; Marasco, Elena; Gensous, Noémie; Di Blasio, Anna Maria; Ellis, Ewa; Gramignoli, Roberto; Castellani, Gastone; Capri, Miriam; Strom, Stephen; Nardini, Christine; Cescon, Matteo; Grazi, Gian Luca; Garagnani, Paolo

2018-03-15

The feasibility of liver transplantation from old healthy donors suggests that this organ is able to preserve its functionality during aging. To explore the biological basis of this phenomenon, we characterized the epigenetic profile of liver biopsies collected from 45 healthy liver donors ranging from 13 to 90 years old using the Infinium HumanMethylation450 BeadChip. The analysis indicates that a large remodeling in DNA methylation patterns occurs, with 8823 age-associated differentially methylated CpG probes. Notably, these age-associated changes tended to level off after the age of 60, as confirmed by Horvath's clock. Using stringent selection criteria we further identified a DNA methylation signature of aging liver including 75 genomic regions. We demonstrated that this signature is specific for liver compared to other tissues and that it is able to detect biological age-acceleration effects associated with obesity. Finally we combined DNA methylation measurements with available expression data. Although the intersection between the two omic characterizations was low, both approaches suggested a previously unappreciated role of epithelial-mesenchymal transition and Wnt signaling pathways in the aging of human liver.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

PubMed Central

Baribault, Carl; Ehrlich, Kenneth C.; Ponnaluri, V. K. Chaithanya; Pradhan, Sriharsa; Lacey, Michelle; Ehrlich, Melanie

2018-01-01

ABSTRACT DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development. PMID:29498561

In vivo investigations of the effect of short- and long-term recombinant growth hormone treatment on DNA-methylation in humans.

PubMed

Kolarova, Julia; Ammerpohl, Ole; Gutwein, Jana; Welzel, Maik; Baus, Inka; Riepe, Felix G; Eggermann, Thomas; Caliebe, Almuth; Holterhus, Paul-Martin; Siebert, Reiner; Bens, Susanne

2015-01-01

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

Effects of the led therapy on the global DNA methylation and the expression of Dnmt1 and Dnmt3a genes in a rat model of skin wound healing.

PubMed

Gomes, Marcus Vinícius de Matos; Manfredo, Marcelo Henrique; Toffoli, Leandro Vaz; Castro-Alves, Daniellen Christine; do Nascimento, Lucas Magnoni; da Silva, Wyllian Rafael; Kashimoto, Roberto Kiyoshi; Rodrigues, Gelson Marcos; Estrada, Viviane Batista; Andraus, Rodrigo Antonio; Pelosi, Gislaine Garcia

2016-09-01

The use of light emitting diodes (LED) as a therapeutic resource for wound healing has increased over the last years; however, little is still known about the molecular pathways associated to LED exposure. In the present study, we verified the effects of LED therapy on DNA methylation and expression of the DNA methyltransferase (Dnmt) genes, Dnmt1 and Dnmt3a, in an in vivo model of epithelial wound healing. Male Wistar rats were submitted to epithelial excision in the dorsal region and subsequently distributed within the experimental groups: group 1, animals that received irradiation of 0.8 J/cm(2) of LED (604 nm); group 2, animals that received 1.6 J/cm(2) of LED (604 nm); control (CTL), animals not submitted to therapeutic intervention. LED applications were performed during 7 days, and tissues from the periphery of the wound area were obtained for molecular analysis. The Image-J software was used for analysis of the wound area. DNA methylation was evaluated by ELISA-based method and gene expressions were quantified by real-time PCR. Decrease on global DNA methylation profile was observed in all experimental groups (CTL, 1, and 2) revealing the participation of DNA methylation in the healing process. Significant decrease in the wound area accompanied by increase in the Dnmt3a expression was associated to group 2. Based on our findings, we propose that DNA methylation is an important molecular mechanism associated to wound healing and that irradiation with 1.6 J/cm(2) of LED evokes an increase in the expression of the Dnmt3a that might associates to the efficiency of the epithelial wound healing.

Location matters: distinct DNA methylation patterns in GABAergic interneuronal populations from separate microcircuits within the human hippocampus.

PubMed

Ruzicka, W Brad; Subburaju, Sivan; Coyle, Joseph T; Benes, Francine M

2018-01-15

Recent studies describe distinct DNA methylomes among phenotypic subclasses of neurons in the human brain, but variation in DNA methylation between common neuronal phenotypes distinguished by their function within distinct neural circuits remains an unexplored concept. Studies able to resolve epigenetic profiles at the level of microcircuits are needed to illuminate chromatin dynamics in the regulation of specific neuronal populations and circuits mediating normal and abnormal behaviors. The Illumina HumanMethylation450 BeadChip was used to assess genome-wide DNA methylation in stratum oriens GABAergic interneurons sampled by laser-microdissection from two discrete microcircuits along the trisynaptic pathway in postmortem human hippocampus from eight control, eight schizophrenia, and eight bipolar disorder subjects. Data were analysed using the minfi Bioconductor package in R software version 3.3.2. We identified 11 highly significant differentially methylated regions associated with a group of genes with high construct-validity, including multiple zinc finger of the cerebellum gene family members and WNT signaling factors. Genomic locations of differentially methylated regions were highly similar between diagnostic categories, with a greater number of differentially methylated individual cytosine residues between circuit locations in bipolar disorder cases than in schizophrenia or control (42, 7, and 7 differentially methylated positions, respectively). These findings identify distinct DNA methylomes among phenotypically similar populations of GABAergic interneurons functioning within separate hippocampal subfields. These data compliment recent studies describing diverse epigenotypes among separate neuronal subclasses, extending this concept to distinct epigenotypes within similar neuronal phenotypes from separate microcircuits within the human brain. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genome-wide DNA methylation profile identified a unique set of differentially methylated immune genes in oral squamous cell carcinoma patients in India.

PubMed

Basu, Baidehi; Chakraborty, Joyeeta; Chandra, Aditi; Katarkar, Atul; Baldevbhai, Jadav Ritesh Kumar; Dhar Chowdhury, Debjit; Ray, Jay Gopal; Chaudhuri, Keya; Chatterjee, Raghunath

2017-01-01

Oral squamous cell carcinoma (OSCC) is one of the common malignancies in Southeast Asia. Epigenetic changes, mainly the altered DNA methylation, have been implicated in many cancers. Considering the varied environmental and genotoxic exposures among the Indian population, we conducted a genome-wide DNA methylation study on paired tumor and adjacent normal tissues of ten well-differentiated OSCC patients and validated in an additional 53 well-differentiated OSCC and adjacent normal samples. Genome-wide DNA methylation analysis identified several novel differentially methylated regions associated with OSCC. Hypermethylation is primarily enriched in the CpG-rich regions, while hypomethylation is mainly in the open sea. Distinct epigenetic drifts for hypo- and hypermethylation across CpG islands suggested independent mechanisms of hypo- and hypermethylation in OSCC development. Aberrant DNA methylation in the promoter regions are concomitant with gene expression. Hypomethylation of immune genes reflect the lymphocyte infiltration into the tumor microenvironment. Comparison of methylome data with 312 TCGA HNSCC samples identified a unique set of hypomethylated promoters among the OSCC patients in India. Pathway analysis of unique hypomethylated promoters indicated that the OSCC patients in India induce an anti-tumor T cell response, with mobilization of T lymphocytes in the neoplastic environment. Survival analysis of these epigenetically regulated immune genes suggested their prominent role in OSCC progression. Our study identified a unique set of hypomethylated regions, enriched in the promoters of immune response genes, and indicated the presence of a strong immune component in the tumor microenvironment. These methylation changes may serve as potential molecular markers to define risk and to monitor the prognosis of OSCC patients in India.

DNA methylation and potential multigenerational epigenetic effects linked to uranium chronic low-dose exposure in gonads of males and females rats.

PubMed

Elmhiri, G; Gloaguen, C; Grison, S; Kereselidze, D; Elie, C; Tack, K; Benderitter, M; Lestaevel, P; Legendre, A; Souidi, M

2018-01-05

An increased health problem in industrialised countries is the contemporary concern of public and scientific community as well. This has been attributed in part to accumulated environmental pollutants especially radioactive substances and the use of nuclear power plants worldwide. However, the outcome of chronic exposure to low doses of a radionuclide such as uranium remains unknown. Recently, a paradigm shift in the perception of risk of radiotoxicology has emerged through investigating the possibility of transmission of biological effects over generations, in particular by epigenetic pathways. These processes are known for their crucial roles associated with the development of several diseases. The current work investigates the epigenetic effect of chronic exposure to low doses of uranium and its inheritance across generations. Materials and Methods To test this proposition, a rodent multigenerational model, males and females, were exposed to a non-toxic concentration of uranium (40mgL -1 drinking water) for nine months. The uranium effects on were evaluated over three generations (F0, F1 and F2) by analysing the DNA methylation profile and DNMT genes expression in ovaries and testes tissues. Here we report a significant hypermethylation of testes DNA (p <0.005) whereas ovaries showed hypomethylated DNA (p <0.005). Interestingly, this DNA methylation profile was significantly maintained across generations F0, F1 and F2. Furthermore, qPCR results of both tissues imply a significant change in the expression of DNA methyltransferase genes (DNMT 1 and DNMT3a/b) as well. Altogether, our work demonstrates for the first time a sex-dependance and inheritance of epigenetic marks, DNA methylation, as a biological response to the exposure to low doses of uranium. However, it is not clear which type of reproductive cell type is more responsive in this context. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

PubMed

Li, Juan; Yao, Wu; Zhang, Lin; Bao, Lei; Chen, Huiting; Wang, Di; Yue, Zhongzheng; Li, Yiping; Zhang, Miao; Hao, Changfu

2017-05-12

Exposure to crystalline silica is considered to increase the risk of lung fibrosis. The primary effector cell, the myofibroblast, plays an important role in the deposition of extracellular matrix (ECM). DNA methylation change is considered to have a potential effect on myofibroblast differentiation. Therefore, the present study was designed to investigate the genome-wide DNA methylation profiles of lung fibroblasts co-cultured with alveolar macrophages exposed to crystalline silica in vitro. AM/fibroblast co-culture system was established. CCK8 was used to assess the toxicity of AMs. mRNA and protein expression of collagen I, α-SMA, MAPK9 and TGF-β1 of fibroblasts after AMs exposed to 100 μg /ml SiO 2 for 0-, 24-, or 48 h were determined by means of quantitative real-time PCR, immunoblotting and immunohistochemistry. Genomic DNA of fibroblasts was isolated using MeDIP-Seq to sequence. R software, GO, KEGG and Cytoscape were used to analyze the data. SiO 2 exposure increased the expression of collagen I and α-SMA in fibroblasts in co-culture system. Analysis of fibroblast methylome identified extensive methylation changes involved in several signaling pathways, such as the MAPK signaling pathway and metabolic pathways. Several candidates, including Tgfb1 and Mapk9, are hubs who can connect the gene clusters. MAPK9 mRNA expression was significantly higher in fibroblast exposed to SiO 2 in co-culture system for 48 h. MAPK9 protein expression was increased at both 24-h and 48-h treatment groups. TGF-β1 mRNA expression of fibroblast has a time-dependent manner, but we didn't observe the TGF-β1 protein expression. Tgfb1 and Mapk9 are helpful to explore the mechanism of myofibroblast differentiation. The genome-wide DNA methylation profiles of fibroblasts in this experimental silicosis model will be useful for future studies on epigenetic gene regulation during myofibroblast differentiation.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

PubMed

Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-31

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

PubMed Central

Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-01

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

Cancer Biomarkers from Genome-Scale DNA Methylation: Comparison of Evolutionary and Semantic Analysis Methods

PubMed Central

Valavanis, Ioannis; Pilalis, Eleftherios; Georgiadis, Panagiotis; Kyrtopoulos, Soterios; Chatziioannou, Aristotelis

2015-01-01

DNA methylation profiling exploits microarray technologies, thus yielding a wealth of high-volume data. Here, an intelligent framework is applied, encompassing epidemiological genome-scale DNA methylation data produced from the Illumina’s Infinium Human Methylation 450K Bead Chip platform, in an effort to correlate interesting methylation patterns with cancer predisposition and, in particular, breast cancer and B-cell lymphoma. Feature selection and classification are employed in order to select, from an initial set of ~480,000 methylation measurements at CpG sites, predictive cancer epigenetic biomarkers and assess their classification power for discriminating healthy versus cancer related classes. Feature selection exploits evolutionary algorithms or a graph-theoretic methodology which makes use of the semantics information included in the Gene Ontology (GO) tree. The selected features, corresponding to methylation of CpG sites, attained moderate-to-high classification accuracies when imported to a series of classifiers evaluated by resampling or blindfold validation. The semantics-driven selection revealed sets of CpG sites performing similarly with evolutionary selection in the classification tasks. However, gene enrichment and pathway analysis showed that it additionally provides more descriptive sets of GO terms and KEGG pathways regarding the cancer phenotypes studied here. Results support the expediency of this methodology regarding its application in epidemiological studies. PMID:27600245

Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

PubMed Central

Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

2012-01-01

Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

PubMed Central

Martens, Joost H A; O'Sullivan, Roderick J; Braunschweig, Ulrich; Opravil, Susanne; Radolf, Martin; Steinlein, Peter; Jenuwein, Thomas

2005-01-01

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints. PMID:15678104

Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer.

PubMed

White-Al Habeeb, Nicole M A; Ho, Linh T; Olkhov-Mitsel, Ekaterina; Kron, Ken; Pethe, Vaijayanti; Lehman, Melanie; Jovanovic, Lidija; Fleshner, Neil; van der Kwast, Theodorus; Nelson, Colleen C; Bapat, Bharati

2014-09-15

Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2'-deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.

DNA methylation patterns of genes related to immune response in the different clinical forms of oral lichen planus.

PubMed

Cruz, Aline Fernanda; de Resende, Renata Gonçalves; de Lacerda, Júlio César Tanos; Pereira, Núbia Braga; Melo, Leonardo Augusto; Diniz, Marina Gonçalves; Gomes, Carolina Cavalieri; Gomez, Ricardo Santiago

2018-01-01

The oral lichen planus is a chronic inflammatory disease. Although its aetiology is not well understood, the role of T lymphocytes in its inflammatory events is recognised. Identifying the epigenetic mechanisms involved in the pathogenesis of this immune-mediated condition is fundamental for understanding the inflammatory reaction that occurs in the disease. The purpose of this work was to evaluate the methylation pattern of 21 immune response-related genes in the different clinical forms of oral lichen planus. A cross-sectional study was performed to analyse the DNA methylation patterns in three distinct groups of oral lichen planus: (i) reticular/plaque lesions; (ii) erosive lesions; (iii) normal oral mucosa (control group). After DNA extraction from biopsies, the samples were submitted to digestions by methylation-sensitive and methylation-dependent enzymes and double digestion. The relative percentage of methylated DNA for each gene was provided using real-time polymerase chain reaction arrays. Hypermethylation of the STAT5A gene was observed only in the control group (59.0%). A higher hypermethylation of the ELANE gene was found in reticular/plaque lesions (72.1%) compared to the erosive lesions (50.0%). Our results show variations in the methylation profile of immune response-related genes, according to the clinical type of oral lichen planus after comparing with the normal oral mucosa. Further studies are necessary to validate these findings using gene expression analysis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of acute megakaryoblastic leukemia in Down syndrome is associated with sequential epigenetic changes

PubMed Central

Malinge, Sébastien; Chlon, Tim; Doré, Louis C.; Ketterling, Rhett P.; Tallman, Martin S.; Paietta, Elisabeth; Gamis, Alan S.; Taub, Jeffrey W.; Chou, Stella T.; Weiss, Mitchell J.

2013-01-01

Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development. PMID:23980066

Genome-wide DNA-(de)methylation is associated with Noninfectious Bud-failure exhibition in Almond (Prunus dulcis [Mill.] D.A.Webb)

PubMed Central

Fresnedo-Ramírez, Jonathan; Chan, Helen M.; Parfitt, Dan E.; Crisosto, Carlos H.; Gradziel, Thomas M.

2017-01-01

Noninfectious bud-failure (BF) remains a major threat to almond production in California, particularly with the recent rapid expansion of acreage and as more intensive cultural practices and modern cultivars are adopted. BF has been shown to be inherited in both vegetative and sexual progeny, with exhibition related to the age and propagation history of scion clonal sources. These characteristics suggest an epigenetic influence, such as the loss of juvenility mediated by DNA-(de)methylation. Various degrees of BF have been reported among cultivars as well as within sources of clonal propagation of the same cultivar. Genome-wide methylation profiles for different clones within almond genotypes were developed to examine their association with BF levels and association with the chronological time from initial propagation. The degree of BF exhibition was found to be associated with DNA-(de)methylation and clonal age, which suggests that epigenetic changes associated with ageing may be involved in the differential exhibition of BF within and among almond clones. Research is needed to investigate the potential of DNA-(de)methylation status as a predictor for BF as well as for effective strategies to improve clonal selection against age related deterioration. This is the first report of an epigenetic-related disorder threatening a major tree crop. PMID:28202904

Genome-wide DNA-(de)methylation is associated with Noninfectious Bud-failure exhibition in Almond (Prunus dulcis [Mill.] D.A.Webb).

PubMed

Fresnedo-Ramírez, Jonathan; Chan, Helen M; Parfitt, Dan E; Crisosto, Carlos H; Gradziel, Thomas M

2017-02-16

Noninfectious bud-failure (BF) remains a major threat to almond production in California, particularly with the recent rapid expansion of acreage and as more intensive cultural practices and modern cultivars are adopted. BF has been shown to be inherited in both vegetative and sexual progeny, with exhibition related to the age and propagation history of scion clonal sources. These characteristics suggest an epigenetic influence, such as the loss of juvenility mediated by DNA-(de)methylation. Various degrees of BF have been reported among cultivars as well as within sources of clonal propagation of the same cultivar. Genome-wide methylation profiles for different clones within almond genotypes were developed to examine their association with BF levels and association with the chronological time from initial propagation. The degree of BF exhibition was found to be associated with DNA-(de)methylation and clonal age, which suggests that epigenetic changes associated with ageing may be involved in the differential exhibition of BF within and among almond clones. Research is needed to investigate the potential of DNA-(de)methylation status as a predictor for BF as well as for effective strategies to improve clonal selection against age related deterioration. This is the first report of an epigenetic-related disorder threatening a major tree crop.

Epigenetic Changes in Response to Tai Chi Practice: A Pilot Investigation of DNA Methylation Marks

PubMed Central

Ren, Hua; Collins, Veronica; Clarke, Sandy J.; Han, Jin-Song; Lam, Paul; Clay, Fiona; Williamson, Lara M.; Andy Choo, K. H.

2012-01-01

Tai chi exercise has been shown to improve physiological and psychosocial functions, well-being, quality of life, and disease conditions. The biological mechanisms by which tai chi exerts its holistic effects remain unknown. We investigated whether tai chi practice results in positive epigenetic changes at the molecular level. Design. The DNA methylation profiles of sixty CpG-dinucleotide marks in female tai chi practitioners (N = 237; 45–88 years old) who have been practising tai chi for three or more years were compared with those of age-matched control females (N = 263) who have never practised tai chi. Results. Six CpG marks originating from three different chromosomes reveal a significant difference (P < 0.05) between the two cohorts. Four marks show losses while two marks show gains in DNA methylation with age in the controls. In the tai chi cohort all six marks demonstrate significant slowing (by 5–70%) of the age-related methylation losses or gains observed in the controls, suggesting that tai chi practice may be associated with measurable beneficial epigenetic changes. Conclusions. The results implicate the potential use of DNA methylation as an epigenetic biomarker to better understand the biological mechanisms and the health and therapeutic efficacies of tai chi. PMID:22719790

The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera

PubMed Central

Drewell, Robert A.; Bush, Eliot C.; Remnant, Emily J.; Wong, Garrett T.; Beeler, Suzannah M.; Stringham, Jessica L.; Lim, Julianne; Oldroyd, Benjamin P.

2014-01-01

In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species. PMID:24924193

Site-Dependent Differences in DNA Methylation and Their Impact on Plant Establishment and Phosphorus Nutrition in Populus trichocarpa

PubMed Central

Schönberger, Brigitte; Chen, Xiaochao; Mager, Svenja

2016-01-01

The propagation via clonal stem cuttings is a frequent practice in tree plantations. Despite their clonal origin, the trees establish differently according to weather, temperature and nutrient availability, as well as the presence of various stresses. Here, clonal Populus trichocarpa (cv. Muhle Larson) cuttings from different sites were transferred into a common, fully nutrient supplied environment. Despite identical underlying genetics, stem cuttings derived from sites with lower phosphorus availability established worse, independent of phosphorus (P) level after transplantation. Differential growth of material from the sites was reflected in differences in the whole genome DNA methylome. Methylation differences were sequence context-dependent, but differentially methylated regions (DMRs) were apparently unrelated to P nutrition genes. Despite the undisputed negative general correlation of DNA promoter methylation with gene repression, only few of the top-ranked DMRs resulted in differential gene expression in roots or shoots. However, differential methylation was associated with site-dependent, different total amounts of microRNAs (miRNAs), with few miRNAs sequences directly targeted by differential methylation. Interestingly, in roots and shoots, the miRNA amount was dependent on the previous habitat and changed in roots in a habitat-dependent way under phosphate starvation conditions. Differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression differences as a P-related epigenetic modification in poplar. Together with differences in DNA methylation, such epigenetic mechanisms may explain habitat or seasonal memory in perennials and site-dependent growth performances. PMID:27992519

Resource base influences genome-wide DNA methylation levels in wild baboons (Papio cynocephalus)

PubMed Central

Lea, Amanda J.; Altmann, Jeanne; Alberts, Susan C.; Tung, Jenny

2015-01-01

Variation in resource availability commonly exerts strong effects on fitness-related traits in wild animals. However, we know little about the molecular mechanisms that mediate these effects, or about their persistence over time. To address these questions, we profiled genome-wide whole blood DNA methylation levels in two sets of wild baboons: (i) ‘wild-feeding’ baboons that foraged naturally in a savanna environment and (ii) ‘Lodge’ baboons that had ready access to spatially concentrated human food scraps, resulting in high feeding efficiency and low daily travel distances. We identified 1,014 sites (0.20% of sites tested) that were differentially methylated between wild-feeding and Lodge baboons, providing the first evidence that resource availability shapes the epigenome in a wild mammal. Differentially methylated sites tended to occur in contiguous stretches (i.e., in differentially methylated regions or DMRs), in promoters and enhancers, and near metabolism-related genes, supporting their functional importance in gene regulation. In agreement, reporter assay experiments confirmed that methylation at the largest identified DMR, located in the promoter of a key glycolysis-related gene, was sufficient to causally drive changes in gene expression. Intriguingly, all dispersing males carried a consistent epigenetic signature of their membership in a wild-feeding group, regardless of whether males dispersed into or out of this group as adults. Together, our findings support a role for DNA methylation in mediating ecological effects on phenotypic traits in the wild, and emphasize the dynamic environmental sensitivity of DNA methylation levels across the life course. PMID:26508127

DNA Methylation Signature of Childhood Chronic Physical Aggression in T Cells of Both Men and Women

PubMed Central

Guillemin, Claire; Provençal, Nadine; Suderman, Matthew; Côté, Sylvana M.; Vitaro, Frank; Hallett, Michael; Tremblay, Richard E.; Szyf, Moshe

2014-01-01

Background High frequency of physical aggression is the central feature of severe conduct disorder and is associated with a wide range of social, mental and physical health problems. We have previously tested the hypothesis that differential DNA methylation signatures in peripheral T cells are associated with a chronic aggression trajectory in males. Despite the fact that sex differences appear to play a pivotal role in determining the development, magnitude and frequency of aggression, most of previous studies focused on males, so little is known about female chronic physical aggression. We therefore tested here whether or not there is a signature of physical aggression in female DNA methylation and, if there is, how it relates to the signature observed in males. Methodology/Principal Findings Methylation profiles were created using the method of methylated DNA immunoprecipitation (MeDIP) followed by microarray hybridization and statistical and bioinformatic analyses on T cell DNA obtained from adult women who were found to be on a chronic physical aggression trajectory (CPA) between 6 and 12 years of age compared to women who followed a normal physical aggression trajectory. We confirmed the existence of a well-defined, genome-wide signature of DNA methylation associated with chronic physical aggression in the peripheral T cells of adult females that includes many of the genes similarly associated with physical aggression in the same cell types of adult males. Conclusions This study in a small number of women presents preliminary evidence for a genome-wide variation in promoter DNA methylation that associates with CPA in women that warrant larger studies for further verification. A significant proportion of these associations were previously observed in men with CPA supporting the hypothesis that the epigenetic signature of early life aggression in females is composed of a component specific to females and another common to both males and females. PMID:24475181

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

PubMed Central

Kleb, Brittany; Estécio, Marcos R.H.; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M.; Tahir, Salahaldin; Marquez, Victor E.; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-01-01

ABSTRACT Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR−SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR+ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR− and AR+ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR−SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR−SCPC cell lines. We conclude that the epigenome of AR− is distinct from that of AR+ castration-resistant prostate carcinomas, and that the AR− phenotype can be reversed with epigenetic drugs. PMID:26890396

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

PubMed

Kleb, Brittany; Estécio, Marcos R H; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M; Tahir, Salahaldin; Marquez, Victor E; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-03-03

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

Twenty-four–nucleotide siRNAs produce heritable trans-chromosomal methylation in F1 Arabidopsis hybrids

PubMed Central

Greaves, Ian K.; Eichten, Steven R.; Groszmann, Michael; Wang, Aihua; Ying, Hua; Peacock, W. James; Dennis, Elizabeth S.

2016-01-01

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations. PMID:27791153

Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP-70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia

PubMed Central

Claus, Rainer; Lucas, David M.; Stilgenbauer, Stephan; Ruppert, Amy S.; Yu, Lianbo; Zucknick, Manuela; Mertens, Daniel; Bühler, Andreas; Oakes, Christopher C.; Larson, Richard A.; Kay, Neil E.; Jelinek, Diane F.; Kipps, Thomas J.; Rassenti, Laura Z.; Gribben, John G.; Döhner, Hartmut; Heerema, Nyla A.; Marcucci, Guido; Plass, Christoph; Byrd, John C.

2012-01-01

Purpose Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control. Patients and Methods High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies. Results Through this comprehensive analysis, we identified a small area in the 5′ regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points. Conclusion Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5′ regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL. PMID:22564988

DNA methylation profiling of asbestos-treated MeT5A cell line reveals novel pathways implicated in asbestos response.

PubMed

Casalone, E; Allione, A; Viberti, C; Pardini, B; Guarrera, S; Betti, M; Dianzani, I; Aldieri, E; Matullo, G

2018-05-01

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm 2 , 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm 2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose-response effect, Spearman's correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = - 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.

Differential DNA Methylation Regions in Cytokine and Transcription Factor Genomic Loci Associate with Childhood Physical Aggression

PubMed Central

Provençal, Nadine; Suderman, Matthew J.; Caramaschi, Doretta; Wang, Dongsha; Hallett, Michael; Vitaro, Frank

2013-01-01

Background Animal and human studies suggest that inflammation is associated with behavioral disorders including aggression. We have recently shown that physical aggression of boys during childhood is strongly associated with reduced plasma levels of cytokines IL-1α, IL-4, IL-6, IL-8 and IL-10, later in early adulthood. This study tests the hypothesis that there is an association between differential DNA methylation regions in cytokine genes in T cells and monocytes DNA in adult subjects and a trajectory of physical aggression from childhood to adolescence. Methodology/Principal Findings We compared the methylation profiles of the entire genomic loci encompassing the IL-1α, IL-6, IL-4, IL-10 and IL-8 and three of their regulatory transcription factors (TF) NFkB1, NFAT5 and STAT6 genes in adult males on a chronic physical aggression trajectory (CPA) and males with the same background who followed a normal physical aggression trajectory (control group) from childhood to adolescence. We used the method of methylated DNA immunoprecipitation with comprehensive cytokine gene loci and TF loci microarray hybridization, statistical analysis and false discovery rate correction. We found differentially methylated regions to associate with CPA in both the cytokine loci as well as in their transcription factors loci analyzed. Some of these differentially methylated regions were located in known regulatory regions whereas others, to our knowledge, were previously unknown as regulatory areas. However, using the ENCODE database, we were able to identify key regulatory elements in many of these regions that indicate that they might be involved in the regulation of cytokine expression. Conclusions We provide here the first evidence for an association between differential DNA methylation in cytokines and their regulators in T cells and monocytes and male physical aggression. PMID:23977113

Genome-scale analysis of aberrant DNA methylation in colorectal cancer

PubMed Central

Hinoue, Toshinori; Weisenberger, Daniel J.; Lange, Christopher P.E.; Shen, Hui; Byun, Hyang-Min; Van Den Berg, David; Malik, Simeen; Pan, Fei; Noushmehr, Houtan; van Dijk, Cornelis M.; Tollenaar, Rob A.E.M.; Laird, Peter W.

2012-01-01

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation–based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. PMID:21659424

Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

PubMed Central

Forn, Marta; Díez-Villanueva, Anna; Merlos-Suárez, Anna; Muñoz, Mar; Lois, Sergi; Carriò, Elvira; Jordà, Mireia; Bigas, Anna; Batlle, Eduard; Peinado, Miguel A.

2015-01-01

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart. PMID:25933092

Methylomic Analysis Identifies Frequent DNA Methylation of Zinc Finger Protein 582 (ZNF582) in Cervical Neoplasms

PubMed Central

Su, Po-Hsuan; Chen, Yu-Chih; Liao, Yu-Ping; Wang, Hui-Chen; Yo, Yi-Te; Chao, Tai-Kuang; Huang, Hsuan-Cheng; Lin, Ching-Yu; Chu, Tang-Yuan; Lai, Hung-Cheng

2012-01-01

Background Despite of the trend that the application of DNA methylation as a biomarker for cancer detection is promising, clinically applicable genes are few. Therefore, we looked for novel hypermethylated genes for cervical cancer screening. Methods and Findings At the discovery phase, we analyzed the methylation profiles of human cervical carcinomas and normal cervixes by methylated DNA immunoprecipitation coupled to promoter tiling arrays (MeDIP-on-chip). Methylation-specific PCR (MSP), quantitative MSP and bisulfite sequencing were used to verify the methylation status in cancer tissues and cervical scrapings from patients with different severities. Immunohistochemical staining of a cervical tissue microarray was used to confirm protein expression. We narrowed to three candidate genes: DBC1, PDE8B, and ZNF582; their methylation frequencies in tumors were 93%, 29%, and 100%, respectively. At the pre-validation phase, the methylation frequency of DBC1 and ZNF582 in cervical scraping correlated significantly with disease severity in an independent cohort (n = 330, both P<0.001). For the detection of cervical intraepithelial neoplasia 3 (CIN3) and worse, the area under the receiver operating characteristic curve (AUC) of ZNF582 was 0.82 (95% confidence interval  = 0.76–0.87). Conclusions Our study shows ZNF582 is frequently methylated in CIN3 and worse lesions, and it is demonstrated as a potential biomarker for the molecular screening of cervical cancer. PMID:22815913

Antipsychotic drugs attenuate aberrant DNA methylation of DTNBP1 (dysbindin) promoter in saliva and post-mortem brain of patients with schizophrenia and Psychotic bipolar disorder.

PubMed

Abdolmaleky, Hamid M; Pajouhanfar, Sara; Faghankhani, Masoomeh; Joghataei, Mohammad Taghi; Mostafavi, Ashraf; Thiagalingam, Sam

2015-12-01

Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance. © 2015 Wiley Periodicals, Inc.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

PubMed

Almeida, Luciana O; Neto, Marinaldo P C; Sousa, Lucas O; Tannous, Maryna A; Curti, Carlos; Leopoldino, Andreia M

2017-04-18

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

Integrated analysis of DNA methylation, immunohistochemistry and mRNA expression, data identifies a Methylation Expression Index (MEI) robustly associated with survival of ER-positive breast cancer patients

PubMed Central

Garcia-Closas, Montserrat; Davis, Sean; Meltzer, Paul; Lissowska, Jolanta; Horne, Hisani N.; Sherman, Mark E.; Lee, Maxwell

2015-01-01

Identification of prognostic gene expression signatures may enable improved decisions about management of breast cancer. To identify a prognostic signature for breast cancer, we performed DNA methylation profiling and identified methylation markers that were associated with expression of ER, PR, HER2, CK5/6 and EGFR proteins. Methylation markers that were correlated with corresponding mRNA expression levels were identified using 208 invasive tumors from a population-based case-control study conducted in Poland. Using this approach, we defined the Methylation Expression Index (MEI) signature that was based on a weighted sum of mRNA levels of 57 genes. Classification of cases as low or high MEI scores were related to survival using Cox regression models. In the Polish study, women with ER-positive low MEI cancers had reduced survival at a median of 5.20 years of follow-up, HR=2.85 95%CI=1.25-6.47. Low MEI was also related to decreased survival in four independent datasets totaling over 2500 ER-positive breast cancers. These results suggest that integrated analysis of tumor expression markers, DNA methylation, and mRNA data can be an important approach for identifying breast cancer prognostic signatures. Prospective assessment of MEI along with other prognostic signatures should be evaluated in future studies. PMID:25773928

Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus.

PubMed

Suzuki, Masako; Maekawa, Ryo; Patterson, Nicole E; Reynolds, David M; Calder, Brent R; Reznik, Sandra E; Heo, Hye J; Einstein, Francine Hughes; Greally, John M

2016-01-01

Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies. We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.

Genetic and epigenetic transgenerational implications related to omega-3 fatty acids. Part I: maternal FADS2 genotype and DNA methylation correlate with polyunsaturated fatty acid status in toddlers: an exploratory analysis.

PubMed

Lupu, Daniel S; Cheatham, Carol L; Corbin, Karen D; Niculescu, Mihai D

2015-11-01

Polyunsaturated fatty acid metabolism in toddlers is regulated by a complex network of interacting factors. The contribution of maternal genetic and epigenetic makeup to this milieu is not well understood. In a cohort of mothers and toddlers 16 months of age (n = 65 mother-child pairs), we investigated the association between maternal genetic and epigenetic fatty acid desaturase 2 (FADS2) profiles and toddlers' n-6 and n-3 fatty acid metabolism. FADS2 rs174575 variation and DNA methylation status were interrogated in mothers and toddlers, as well as food intake and plasma fatty acid concentrations in toddlers. A multivariate fit model indicated that maternal rs174575 genotype, combined with DNA methylation, can predict α-linolenic acid plasma concentration in all toddlers and arachidonic acid concentrations in boys. Arachidonic acid intake was predictive for its plasma concentration in girls, whereas intake of 3 major n-3 species (eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids) were predictive for their plasma concentrations in boys. FADS2 genotype and DNA methylation in toddlers were not related to plasma concentrations or food intakes, except for CpG8 methylation. Maternal FADS2 methylation was a predictor for the boys' α-linolenic acid intakes. This exploratory study suggests that maternal FADS2 genetic and epigenetic status could be related to toddlers' polyunsaturated fatty acid metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

PubMed

Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

2016-01-01

Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Epigenetics insights into chronic pain: DNA hypomethylation in fibromyalgia-a controlled pilot-study.

PubMed

Ciampi de Andrade, Daniel; Maschietto, Mariana; Galhardoni, Ricardo; Gouveia, Gisele; Chile, Thais; Victorino Krepischi, Ana C; Dale, Camila S; Brunoni, André R; Parravano, Daniella C; Cueva Moscoso, Ana S; Raicher, Irina; Kaziyama, Helena H S; Teixeira, Manoel J; Brentani, Helena P

2017-08-01

To evaluate changes in DNA methylation profiles in patients with fibromyalgia (FM) compared to matched healthy controls (HCs). All individuals underwent full clinical and neurophysiological assessment by cortical excitability (CE) parameters measured by transcranial magnetic stimulation. DNA from the peripheral blood of patients with FM (n = 24) and HC (n = 24) were assessed using the Illumina-HumanMethylation450 BeadChips. We identified 1610 differentially methylated positions (DMPs) in patients with FM displaying a nonrandom distribution in regions of the genome. Sixty-nine percent of DMP in FM were hypomethylated compared to HC. Differentially methylated positions were enriched in 5 genomic regions (1p34; 6p21; 10q26; 17q25; 19q13). The functional characterization of 960 genes related to DMPs revealed an enrichment for MAPK signaling pathway (n = 18 genes), regulation of actin cytoskeleton (n = 15 genes), and focal adhesion (n = 13 genes). A gene-gene interaction network enrichment analysis revealed the participation of DNA repair pathways, mitochondria-related processes, and synaptic signaling. Even though DNA was extracted from peripheral blood, this set of genes was enriched for disorders such as schizophrenia, mood disorders, bulimia, hyperphagia, and obesity. Remarkably, the hierarchical clusterization based on the methylation levels of the 1610 DMPs showed an association with neurophysiological measurements of CE in FM and HC. Fibromyalgia has a hypomethylation DNA pattern, which is enriched in genes implicated in stress response and DNA repair/free radical clearance. These changes occurred parallel to changes in CE parameters. New epigenetic insights into the pathophysiology of FM may provide the basis for the development of biomarkers of this disorder.

Using logic programming for modeling the one-carbon metabolism network to study the impact of folate deficiency on methylation processes.

PubMed

Gnimpieba, Etienne Z; Eveillard, Damien; Guéant, Jean-Louis; Chango, Abalo

2011-08-01

Dynamical modeling is an accurate tool for describing the dynamic regulation of one-carbon metabolism (1CM) with emphasis on the alteration of DNA methylation and/or dUMP methylation into dTMP. Using logic programming we present a comprehensive and adaptative mathematical model to study the impact of folate deficiency, including folate transport and enzymes activities. 5-Methyltetrahydrofolate (5mTHF) uptake and DNA and dUMP methylation were studied by simulating nutritional 5mTHF deficiency and methylenetetrahydrofolate reductase (MTHFR) gene defects. Both conditions had distinct effects on 1CM metabolite synthesis. Simulating severe 5mTHF deficiency (25% of normal levels) modulated 11 metabolites. However, simulating a severe decrease in MTHFR activity (25% of normal activity) modulated another set of metabolites. Two oscillations of varying amplitude were observed at the steady state for DNA methylation with severe 5mTHF deficiency, and the dUMP/dTMP ratio reached a steady state after 2 h, compared to 2.5 h for 100% 5mTHF. MTHFR activity with 25% of V(max) resulted in an increased methylated DNA pool after half an hour. We observed a deviation earlier in the profile compared to 50% and 100% V(max). For dUMP methylation, the highest level was observed with 25%, suggesting a low rate of dUMP methylation into dTMP with 25% of MTHFR activity. In conclusion, using logic programming we were able to construct the 1CM for analyzing the dynamic system behavior. This model may be used to refine biological interpretations of data or as a tool that can provide new hypotheses for pathogenesis.

How to interpret methylation sensitive amplified polymorphism (MSAP) profiles?

PubMed

Fulneček, Jaroslav; Kovařík, Aleš

2014-01-06

DNA methylation plays a key role in development, contributes to genome stability, and may also respond to external factors supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. Here, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII + MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile. We recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation.

DNA methylation in individuals with anorexia nervosa and in matched normal-eater controls: A genome-wide study.

PubMed

Booij, Linda; Casey, Kevin F; Antunes, Juliana M; Szyf, Moshe; Joober, Ridha; Israël, Mimi; Steiger, Howard

2015-11-01

Evidence associates anorexia nervosa (AN) with epigenetic alterations that could contribute to illness risk or entrenchment. We investigated the extent to which AN is associated with a distinct methylation profile compared to that seen in normal-eater women. Genome-wide methylation profiles, obtained using DNA from whole blood, were determined in 29 women currently ill with AN (10 with AN-restrictive type, 19 with AN-binge/purge type) and 15 normal-weight, normal-eater control women, using 450 K Illumina bead arrays. Regardless of type, AN patients showed higher and less-variable global methylation patterns than controls. False Discovery Rate corrected comparisons identified 14 probes that were hypermethylated in women with AN relative to levels obtained in normal-eater controls, representing genes thought to be associated with histone acetylation, RNA modification, cholesterol storage and lipid transport, and dopamine and glutamate signaling. Age of onset was significantly associated with differential methylation in gene pathways involved in development of the brain and spinal cord, while chronicity of illness was significantly linked to differential methylation in pathways involved with synaptogenesis, neurocognitive deficits, anxiety, altered social functioning, and bowel, kidney, liver and immune function. Although pre-existing differences cannot be ruled out, our findings are consistent with the idea of secondary alterations in methylation at genomic regions pertaining to social-emotional impairments and physical sequelae that are commonly seen in AN patients. Further investigation is needed to establish the clinical relevance of the affected genes in AN, and, importantly, reversibility of effects observed with nutritional rehabilitation and treatment. © 2015 Wiley Periodicals, Inc.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

PubMed

Zhang, Xiaoyang; Wang, Dongxu; Han, Yang; Duan, Feifei; Lv, Qinyan; Li, Zhanjun

2014-11-01

To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Differential DNA methylation and transcription profiles in date palm roots exposed to salinity

PubMed Central

Al-Harrasi, Ibtisam; Al-Yahyai, Rashid

2018-01-01

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress. PMID:29352281

Differential DNA methylation and transcription profiles in date palm roots exposed to salinity.

PubMed

Al-Harrasi, Ibtisam; Al-Yahyai, Rashid; Yaish, Mahmoud W

2018-01-01

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress.

Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

PubMed Central

Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2016-01-01

The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

DNA methylation markers for diagnosis and prognosis of common cancers

PubMed Central

Hao, Xiaoke; Luo, Huiyan; Krawczyk, Michal; Wei, Wei; Wang, Wenqiu; Wang, Juan; Flagg, Ken; Hou, Jiayi; Zhang, Heng; Yi, Shaohua; Jafari, Maryam; Lin, Danni; Chung, Christopher; Caughey, Bennett A.; Li, Gen; Dhar, Debanjan; Shi, William; Zheng, Lianghong; Hou, Rui; Zhu, Jie; Zhao, Liang; Fu, Xin; Zhang, Edward; Zhang, Charlotte; Zhu, Jian-Kang; Karin, Michael; Xu, Rui-Hua; Zhang, Kang

2017-01-01

The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis. PMID:28652331

Epigenome-wide association study of DNA methylation in narcolepsy: an integrated genetic and epigenetic approach.

PubMed

Shimada, Mihoko; Miyagawa, Taku; Toyoda, Hiromi; Tokunaga, Katsushi; Honda, Makoto

2018-04-01

Narcolepsy with cataplexy, which is a hypersomnia characterized by excessive daytime sleepiness and cataplexy, is a multifactorial disease caused by both genetic and environmental factors. Several genetic factors including HLA-DQB1*06:02 have been identified; however, the disease etiology is still unclear. Epigenetic modifications, such as DNA methylation, have been suggested to play an important role in the pathogenesis of complex diseases. Here, we examined DNA methylation profiles of blood samples from narcolepsy and healthy control individuals and performed an epigenome-wide association study (EWAS) to investigate methylation loci associated with narcolepsy. Moreover, data from the EWAS and a previously performed narcolepsy genome-wide association study were integrated to search for methylation loci with causal links to the disease. We found that (1) genes annotated to the top-ranked differentially methylated positions (DMPs) in narcolepsy were associated with pathways of hormone secretion and monocarboxylic acid metabolism. (2) Top-ranked narcolepsy-associated DMPs were significantly more abundant in non-CpG island regions and more than 95 per cent of such sites were hypomethylated in narcolepsy patients. (3) The integrative analysis identified the CCR3 region where both a single methylation site and multiple single-nucleotide polymorphisms were found to be associated with the disease as a candidate region responsible for narcolepsy. The findings of this study suggest the importance of future replication studies, using methylation technologies with wider genome coverage and/or larger number of samples, to confirm and expand on these results.

DNA methylation intratumor heterogeneity in localized lung adenocarcinomas.

PubMed

Quek, Kelly; Li, Jun; Estecio, Marcos; Zhang, Jiexin; Fujimoto, Junya; Roarty, Emily; Little, Latasha; Chow, Chi-Wan; Song, Xingzhi; Behrens, Carmen; Chen, Taiping; William, William N; Swisher, Stephen; Heymach, John; Wistuba, Ignacio; Zhang, Jianhua; Futreal, Andrew; Zhang, Jianjun

2017-03-28

Cancers are composed of cells with distinct molecular and phenotypic features within a given tumor, a phenomenon termed intratumor heterogeneity (ITH). Previously, we have demonstrated genomic ITH in localized lung adenocarcinomas; however, the nature of methylation ITH in lung cancers has not been well investigated. In this study, we generated methylation profiles of 48 spatially separated tumor regions from 11 localized lung adenocarcinomas and their matched normal lung tissues using Illumina Infinium Human Methylation 450K BeadChip array. We observed methylation ITH within the same tumors, but to a much less extent compared to inter-individual heterogeneity. On average, 25% of all differentially methylated probes compared to matched normal lung tissues were shared by all regions from the same tumors. This is in contrast to somatic mutations, of which approximately 77% were shared events amongst all regions of individual tumors, suggesting that while the majority of somatic mutations were early clonal events, the tumor-specific DNA methylation might be associated with later branched evolution of these 11 tumors. Furthermore, our data showed that a higher extent of DNA methylation ITH was associated with larger tumor size (average Euclidean distance of 35.64 (> 3cm, median size) versus 27.24 (<= 3cm), p = 0.014), advanced age (average Euclidean distance of 34.95 (above 65) verse 28.06 (below 65), p = 0.046) and increased risk of postsurgical recurrence (average Euclidean distance of 35.65 (relapsed patients) versus 29.03 (patients without relapsed), p = 0.039).

Characterization of non-CG genomic hypomethylation associated with gamma-ray-induced suppression of CMT3 transcription in Arabidopsis thaliana.

PubMed

Kim, Ji Eun; Lee, Min Hee; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin-Hong

2013-12-01

Ionizing radiation causes various epigenetic changes, as well as a variety of DNA lesions such as strand breaks, cross-links, oxidative damages, etc., in genomes. However, radiation-induced epigenetic changes have rarely been substantiated in plant genomes. The current study investigates whether DNA methylation of Arabidopsis thaliana genome is altered by gamma rays. We found that genomic DNA methylation decreased in wild-type plants with increasing doses of gamma rays (5, 50 and 200 Gy). Irradiation with 200 Gy significantly increased the expression of transcriptionally inactive centromeric 180-bp (CEN) and transcriptionally silent information (TSI) repeats. This increase suggested that there was a substantial release of transcriptional gene silencing by gamma rays, probably by induction of DNA hypomethylation. High expression of the DNA demethylase ROS1 and low expression of the DNA methyltransferase CMT3 supported this hypothesis. Moreover, Southern blot analysis following digestion of genomic DNA with methylation-sensitive enzymes revealed that the DNA hypomethylation occured preferentially at CHG or CHH sites rather than CG sites, depending on the radiation dose. Unlike CEN and TSI repeats, the number of Ta3, AtSN1 and FWA repeats decreased in transcription but increased in non-CG methylation. In addition, the cmt3-11 mutant showed neither DNA hypomethylation nor transcriptional activation of silenced repeats upon gamma irradiation. Furthermore, profiles of genome-wide transcriptomes in response to gamma rays differed between the wild-type and cmt3-11 mutant. These results suggest that gamma irradiation induced DNA hypomethylation preferentially at non-CG sites of transcriptionally inactive repeats in a locus-specific manner, which depends on CMT3 activity.

DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T-Cell Acute Lymphoblastic Leukemia.

PubMed

Borssén, Magnus; Haider, Zahra; Landfors, Mattias; Norén-Nyström, Ulrika; Schmiegelow, Kjeld; Åsberg, Ann E; Kanerva, Jukka; Madsen, Hans O; Marquart, Hanne; Heyman, Mats; Hultdin, Magnus; Roos, Göran; Forestier, Erik; Degerman, Sofie

2016-07-01

Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. Two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile had an inferior response to treatment compared to CIMP-positive patients (3-year cumulative incidence of relapse (CIR3y ) rate: 29% vs. 6%, P = 0.01). Most importantly, CIMP classification at diagnosis allowed subgrouping of high-risk T-ALL patients (MRD ≥0.1% at day 29) into two groups with significant differences in outcome (CIR3y rates: CIMP negative 50% vs. CIMP positive 12%; P = 0.02). These groups did not differ regarding ETP phenotype, but the CIMP-negative group was younger (P = 0.02) and had higher white blood cell count at diagnosis (P = 0.004) compared with the CIMP-positive group. CIMP classification at diagnosis in combination with MRD during induction therapy is a strong candidate for further risk classification and could confer important information in treatment decision making. © 2016 Wiley Periodicals, Inc.

Combined epigenetic and intraspecific variation of the DRD4 and SERT genes influence novelty seeking behavior in great tit Parus major

PubMed Central

Riyahi, Sepand; Sánchez-Delgado, Marta; Calafell, Francesc; Monk, David; Senar, Juan Carlos

2015-01-01

DNA methylation is one of the main epigenetic mechanisms that can regulate gene expression and is an important means for creating phenotypic variation. In the present study, we performed methylation profiling of 2 candidate genes for personality traits, namely DRD4 and SERT, in the great tit Parus major to ascertain whether personality traits and behavior within different habitats have evolved with the aid of epigenetic variation. We applied bisulphite PCR and strand-specific sequencing to determine the methylation profile of the CpG dinucleotides in the DRD4 and SERT promoters and also in the CpG island overlapping DRD4 exon 3. Furthermore, we performed pyrosequencing to quantify the total methylation levels at each CpG location. Our results indicated that methylation was ∼1–4% higher in urban than in forest birds, for all loci and tissues analyzed, suggesting that this epigenetic modification is influenced by environmental conditions. Screening of genomic DNA sequence revealed that the SERT promoter is CpG poor region. The methylation at a single CpG dinucleotide located 288 bp from the transcription start site was related to exploration score in urban birds. In addition, the genotypes of the SERT polymorphism SNP234 located within the minimal promoter were significantly correlated with novelty seeking behavior in captivity, with the allele increasing this behavior being more frequent in urban birds. As a conclusion, it seems that both genetic and methylation variability of the SERT gene have an important role in shaping personality traits in great tits, whereas genetic and methylation variation at the DRD4 gene is not strongly involved in behavior and personality traits. PMID:25933062

EG-13GENOME-WIDE METHYLATION ANALYSIS IDENTIFIES GENOMIC DNA DEMETHYLATION DURING MALIGNANT PROGRESSION OF GLIOMAS

PubMed Central

Saito, Kuniaki; Mukasa, Akitake; Nagae, Genta; Aihara, Koki; Otani, Ryohei; Takayanagi, Shunsaku; Omata, Mayu; Tanaka, Shota; Shibahara, Junji; Takahashi, Miwako; Momose, Toshimitsu; Shimamura, Teppei; Miyano, Satoru; Narita, Yoshitaka; Ueki, Keisuke; Nishikawa, Ryo; Nagane, Motoo; Aburatani, Hiroyuki; Saito, Nobuhito

2014-01-01

Low-grade gliomas often undergo malignant progression, and these transformations are a leading cause of death in patients with low-grade gliomas. However, the molecular mechanisms underlying malignant tumor progression are still not well understood. Recent evidence indicates that epigenetic deregulation is an important cause of gliomagenesis; therefore, we examined the impact of epigenetic changes during malignant progression of low-grade gliomas. Specifically, we used the Illumina Infinium Human Methylation 450K BeadChip to perform genome-wide DNA methylation analysis of 120 gliomas and four normal brains. This study sample included 25 matched-pairs of initial low-grade gliomas and recurrent tumors (temporal heterogeneity) and 20 of the 25 recurring tumors recurred as malignant progressions, and one matched-pair of newly emerging malignant lesions and pre-existing lesions (spatial heterogeneity). Analyses of methylation profiles demonstrated that most low-grade gliomas in our sample (43/51; 84%) had a CpG island methylator phenotype (G-CIMP). Remarkably, approximately 50% of secondary glioblastomas that had progressed from low-grade tumors with the G-CIMP status exhibited a characteristic partial demethylation of genomic DNA during malignant progression, but other recurrent gliomas showed no apparent change in DNA methylation pattern. Interestingly, we found that most loci that were demethylated during malignant progression were located outside of CpG islands. The information of histone modifications patterns in normal human astrocytes and embryonal stem cells also showed that the ratio of active marks at the site corresponding to DNA demethylated loci in G-CIMP-demethylated tumors was significantly lower; this finding indicated that most demethylated loci in G-CIMP-demethylated tumors were likely transcriptionally inactive. A small number of the genes that were upregulated and had demethylated CpG islands were associated with cell cycle-related pathway. In summary, we demonstrated that characteristic DNA demethylation occurred during malignant progression of a subset of low-grade gliomas. The mechanisms underlying and consequences of such DNA demethylation should be studied further.

The impact of methylation quantitative trait loci (mQTLs) on active smoking-related DNA methylation changes.

PubMed

Gao, Xu; Thomsen, Hauke; Zhang, Yan; Breitling, Lutz Philipp; Brenner, Hermann

2017-01-01

Methylation quantitative trait loci (mQTLs) are the genetic variants that may affect the DNA methylation patterns of CpG sites. However, their roles in influencing the disturbances of smoking-related epigenetic changes have not been well established. This study was conducted to address whether mQTLs exist in the vicinity of smoking-related CpG sites (± 50 kb) and to examine their associations with smoking exposure and all-cause mortality in older adults. We obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 BeadChip array of two independent subsamples of the ESTHER study (discovery set, n  = 581; validation set, n  = 368) and their corresponding genotyping data using the Illumina Infinium OncoArray BeadChip. After correction for multiple testing (FDR), we successfully identified that 70 out of 151 previously reported smoking-related CpG sites were significantly associated with 192 SNPs within the 50 kb search window of each locus. The 192 mQTLs significantly influenced the active smoking-related DNA methylation changes, with percentage changes ranging from 0.01 to 18.96%, especially for the weakly/moderately smoking-related CpG sites. However, these identified mQTLs were not directly associated with active smoking exposure or all-cause mortality. Our findings clearly demonstrated that if not dealt with properly, the mQTLs might impair the power of epigenetic-based models of smoking exposure to a certain extent. In addition, such genetic variants could be the key factor to distinguish between the heritable and smoking-induced impact on epigenome disparities. These mQTLs are of special importance when DNA methylation markers measured by Illumina Infinium assay are used for any comparative population studies related to smoking-related cancers and chronic diseases.

Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

PubMed Central

Skårn, Magne; Namløs, Heidi M.; Barragan-Polania, Ana H.; Cleton-Jansen, Anne-Marie; Serra, Massimo; Liestøl, Knut; Hogendoorn, Pancras C. W.; Hovig, Eivind; Myklebost, Ola; Meza-Zepeda, Leonardo A.

2012-01-01

Background Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies. Principal Findings The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2′-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes. Conclusions Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology. PMID:23144859

Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis

PubMed Central

Sun, Zhifu; Cunningham, Julie; Slager, Susan; Kocher, Jean-Pierre

2015-01-01

Bisulfite treatment-based methylation microarray (mainly Illumina 450K Infinium array) and next-generation sequencing (reduced representation bisulfite sequencing, Agilent SureSelect Human Methyl-Seq, NimbleGen SeqCap Epi CpGiant or whole-genome bisulfite sequencing) are commonly used for base resolution DNA methylome research. Although multiple tools and methods have been developed and used for the data preprocessing and analysis, confusions remains for these platforms including how and whether the 450k array should be normalized; which platform should be used to better fit researchers’ needs; and which statistical models would be more appropriate for differential methylation analysis. This review presents the commonly used platforms and compares the pros and cons of each in methylome profiling. We then discuss approaches to study design, data normalization, bias correction and model selection for differentially methylated individual CpGs and regions. PMID:26366945

Base-Resolution Analysis of DNA Methylation Patterns Downstream of Dnmt3a in Mouse Naïve B Cells.

PubMed

Duncan, Christopher G; Kondilis-Mangum, Hrisavgi D; Grimm, Sara A; Bushel, Pierre R; Chrysovergis, Kaliopi; Roberts, John D; Tyson, Frederick L; Merrick, B Alex; Wade, Paul A

2018-03-02

The DNA methyltransferase, Dnmt3a , is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a +/+ and Dnmt3a -/- naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a +/+ and Dnmt3a -/- naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a -/- naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development. Copyright © 2018 Duncan et al.

DNA methylation and inflammation marker profiles associated with a history of depression.

PubMed

Crawford, Bethany; Craig, Zoe; Mansell, Georgina; White, Isobel; Smith, Adam; Spaull, Steve; Imm, Jennifer; Hannon, Eilis; Wood, Andrew; Yaghootkar, Hanieh; Ji, Yingjie; Mullins, Niamh; Lewis, Cathryn M; Mill, Jonathan; Murphy, Therese M

2018-05-22

Depression is a common and disabling disorder, representing a major social and economic health issue. Moreover, depression is associated with the progression of diseases with an inflammatory etiology including many inflammatory-related disorders. At the molecular level, the mechanisms by which depression might promote the onset of these diseases and associated immune-dysfunction are not well understood. In this study we assessed genome-wide patterns of DNA methylation in whole blood-derived DNA obtained from individuals with a self-reported history of depression (n = 100) and individuals without a history of depression (n = 100) using the Illumina 450K microarray. Our analysis identified 6 significant (Sidak corrected P < 0.05) depression-associated differentially methylated regions (DMRs); the top-ranked DMR was located in exon 1 of the LTB4R2 gene (Sidak corrected P = 1.27 x 10-14). Polygenic risk scores (PRS) for depression were generated and known biological markers of inflammation, telomere length (TL) and IL-6, were measured in DNA and serum samples respectively. Next, we employed a systems-level approach to identify networks of co-methylated loci associated with a history of depression, in addition to depression PRS, TL and IL-6 levels. Our analysis identified one depression-associated co-methylation module (P = 0.04). Interestingly, the depression-associated module was highly enriched for pathways related to immune function and was also associated with TL and IL-6 cytokine levels. In summary, our genome-wide DNA methylation analysis of individuals with and without a self-reported history of depression identified several candidate DMRs of potential relevance to the pathogenesis of depression and its associated immune-dysfunction phenotype.

Sage Gene Expression Profiles Characterizing Cure

DTIC Science & Technology

2005-10-01

achieved in haematological malignancies. In addition, tumour cell lines showed a log-linear dose re- sponse when exposed to alkylating agents [26,27...direct carcinogen, N-methyl nitrosourea (NMU) and an indirect carcinogen, 7,12- dimethylbenzanthracene each depend upon the estrous cycle phase at the...a single dose of N-methyl-N- nitrosourea . Cancer Res 45: 3042–3047, 1995 8. Nagasawa H, Yanai R, Taniguchi H: Importance of mammary gland DNA synthesis

Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia

PubMed Central

Meldi, Kristen; Qin, Tingting; Buchi, Francesca; Droin, Nathalie; Sotzen, Jason; Micol, Jean-Baptiste; Selimoglu-Buet, Dorothée; Masala, Erico; Allione, Bernardino; Gioia, Daniela; Poloni, Antonella; Lunghi, Monia; Solary, Eric; Abdel-Wahab, Omar; Santini, Valeria; Figueroa, Maria E.

2015-01-01

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34+ and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance. PMID:25822018

Reprogramming the Maternal Zebrafish Genome after Fertilization to Match the Paternal Methylation Pattern

PubMed Central

Potok, Magdalena E.; Nix, David A.; Parnell, Timothy J.; Cairns, Bradley R.

2014-01-01

SUMMARY Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germ-line functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming. PMID:23663776

Detecting methylation patterns of p16, MGMT, DAPK and E-cadherin genes in multiple myeloma patients.

PubMed

Yuregir, O Ozalp; Yurtcu, E; Kizilkilic, E; Kocer, N E; Ozdogu, H; Sahin, F I

2010-04-01

Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing.

PubMed

Ye, Pohao; Luan, Yizhao; Chen, Kaining; Liu, Yizhi; Xiao, Chuanle; Xie, Zhi

2017-01-04

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

PubMed Central

Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

2015-01-01

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

Epigenetic Diversity of Clonal White Poplar (Populus alba L.) Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

PubMed

Guarino, Francesco; Cicatelli, Angela; Brundu, Giuseppe; Heinze, Berthold; Castiglione, Stefano

2015-01-01

The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i) to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii) to assess if and how methylation status influences population clustering; iii) to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

PubMed Central

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

2017-01-01

Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

Genome-wide screen of ovary-specific DNA methylation in polycystic ovary syndrome.

PubMed

Yu, Ying-Ying; Sun, Cui-Xiang; Liu, Yin-Kun; Li, Yan; Wang, Li; Zhang, Wei

2015-07-01

To compare genome-wide DNA methylation profiles in ovary tissue from women with polycystic ovary syndrome (PCOS) and healthy controls. Case-control study matched for age and body mass index. University-affiliated hospital. Ten women with PCOS who underwent ovarian drilling to induce ovulation and 10 healthy women who were undergoing laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain, or oophorectomy for nonovarian indications. None. Genome-wide DNA methylation patterns determined by immunoprecipitation and microarray (MeDIP-chip) analysis. The methylation levels were statistically significantly higher in CpG island shores (CGI shores), which lie outside of core promoter regions, and lower within gene bodies in women with PCOS relative to the controls. In addition, high CpG content promoters were the most frequently hypermethylated promoters in PCOS ovaries but were more often hypomethylated in controls. Second, 872 CGIs, specifically methylated in PCOS, represented 342 genes that could be associated with various molecular functions, including protein binding, hormone activity, and transcription regulator activity. Finally, methylation differences were validated in seven genes by methylation-specific polymerase chain reaction. These genes correlated to several functional families related to the pathogenesis of PCOS and may be potential biomarkers for this disease. Our results demonstrated that epigenetic modification differs between PCOS and normal ovaries, which may help to further understand the pathophysiology of this disease. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of temperature and salinity on survival, growth and DNA methylation of juvenile Pacific abalone, Haliotis discus hannai Ino

NASA Astrophysics Data System (ADS)

Kong, Ning; Liu, Xiao; Li, Junyuan; Mu, Wendan; Lian, Jianwu; Xue, Yanjie; Li, Qi

2017-09-01

Temperature and salinity are two of the most potent abiotic factors influencing marine mollusks. In this study, we investigated the individual and combined effects of temperature and salinity on the survival and growth of juvenile Pacific abalone, Haliotis discus hannai Ino, and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to different rearing conditions. The single-factor data showed that the suitable ranges of temperature and salinity were 16-28°C at a constant salinity of 32, and 24-40 at a constant temperature of 20°C, respectively. The two-factor data indicated that both survival and growth were significantly affected by temperature, salinity and their interaction. The optimal temperature-salinity combination for juveniles was 23-25°C and 30-36. To explore environment-induced DNA methylation alteration, the methylation-sensitive amplified polymorphism (MSAP) technique was used to analyze the genomic methylation profiles of abalone reared in optimal and adverse conditions. Neither temperature nor salinity induced evident changes in the global methylation level, but 67 and 63 differentially methylated loci were identified in temperature and salinity treatments, respectively. The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic differentiation in H. discus hannai Ino. The results of our study provide optimal rearing conditions for juvenile H. discus hannai Ino, and represent the first step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.

Whole-genome scale identification of methylation markers specific for cerebral palsy in monozygotic discordant twins

PubMed Central

Jiao, Zhe; Jiang, Zhimei; Wang, Jingtao; Xu, Hui; Zhang, Qiang; Liu, Shuang; Du, Ning; Zhang, Yuanyuan; Qiu, Hongbin

2017-01-01

Cerebral palsy (CP) is a severe type of brain disease affecting movement and posture. Although CP has strong genetic and environmental components, considerable differences in the methylome between monozygotic (MZ) twins discordant for CP implicates epigenetic contributors as well. In order to determine the differences in methylation in patients with CP without interference of the interindividual genomic variation, four pairs of MZ twins discordant for CP were profiled for DNA methylation changes using reduced representation bisulfite sequencing on the genomic-scale. Similar DNA methylation patterns were observed in all samples. However, MZ twins demonstrated higher correlations and closer evolutionary associations compared with the other samples, indicating a stable methylome of MZ twins. A total of 190 differentially methylated genes (DMGs) were identified using Student's t-test, of which 37 genes were hypermethylated in the CP group while the remainders were hypomethylated compared with control group. The identified DMGs were enriched in several cerebral abnormalities, including cerebral cortical atrophy and cerebral atrophy, suggesting that the occurrence of CP may be associated with the methylation alterations. The neighboring genes of DMGs in the protein-protein interaction network were enriched in numerous important functions in essential processes. The results of the present study identified important genes that may epigenetically contribute to the occurrence and development of CP in MZ twins, suggesting that the different prevalence of CP in identical twins may be associated with DNA methylation alterations. PMID:29039597

Concordance in hippocampal and fecal Nr3c1 methylation is moderated by maternal behavior in the mouse

PubMed Central

Liberman, Shayna A; Mashoodh, Rahia; Thompson, Robert C; Dolinoy, Dana C; Champagne, Frances A

2012-01-01

Recent advances in genomic technologies now enable a reunion of molecular and evolutionary biology. Researchers investigating naturally living animal populations are thus increasingly able to capitalize upon genomic technologies to connect molecular findings with multiple levels of biological organization. Using this vertical approach in the laboratory, epigenetic gene regulation has emerged as an important mechanism integrating genotype and phenotype. To connect phenotype to population fitness, however, this same vertical approach must now be applied to naturally living populations. A major obstacle to studying epigenetics in noninvasive samples is tissue specificity of epigenetic marks. Here, using the mouse as a proof-of-principle model, we present the first known attempt to validate an epigenetic assay for use in noninvasive samples. Specifically, we compare DNA methylation of the NGFI-A (nerve growth factor-inducible protein A) binding site in the promoter of the glucocorticoid receptor (Nr3c1) gene between central (hippocampal) and peripheral noninvasive (fecal) tissues in juvenile Balb/c mice that had received varying levels of postnatal maternal care. Our results indicate that while hippocampal DNA methylation profiles correspond to maternal behavior, fecal DNA methylation levels do not. Moreover, concordance in methylation levels between these tissues within individuals only emerges after accounting for the effects of postnatal maternal care. Thus, although these findings may be specific to the Nr3c1 gene, we urge caution when interpreting DNA methylation patterns from noninvasive tissues, and offer suggestions for further research in this field. PMID:23301177

Aging effects on DNA methylation modules in human brain and blood tissue

PubMed Central

2012-01-01

Background Several recent studies reported aging effects on DNA methylation levels of individual CpG dinucleotides. But it is not yet known whether aging-related consensus modules, in the form of clusters of correlated CpG markers, can be found that are present in multiple human tissues. Such a module could facilitate the understanding of aging effects on multiple tissues. Results We therefore employed weighted correlation network analysis of 2,442 Illumina DNA methylation arrays from brain and blood tissues, which enabled the identification of an age-related co-methylation module. Module preservation analysis confirmed that this module can also be found in diverse independent data sets. Biological evaluation showed that module membership is associated with Polycomb group target occupancy counts, CpG island status and autosomal chromosome location. Functional enrichment analysis revealed that the aging-related consensus module comprises genes that are involved in nervous system development, neuron differentiation and neurogenesis, and that it contains promoter CpGs of genes known to be down-regulated in early Alzheimer's disease. A comparison with a standard, non-module based meta-analysis revealed that selecting CpGs based on module membership leads to significantly increased gene ontology enrichment, thus demonstrating that studying aging effects via consensus network analysis enhances the biological insights gained. Conclusions Overall, our analysis revealed a robustly defined age-related co-methylation module that is present in multiple human tissues, including blood and brain. We conclude that blood is a promising surrogate for brain tissue when studying the effects of age on DNA methylation profiles. PMID:23034122

Maturation of the developing human fetal prostate in a rodent xenograft model

PubMed Central

Saffarini, Camelia M.; McDonnell, Elizabeth V.; Amin, Ali; Spade, Daniel J.; Huse, Susan M.; Kostadinov, Stefan; Hall, Susan J.; Boekelheide, Kim

2015-01-01

Background Prostate cancer is the most commonly diagnosed non-skin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. Methods We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. Results Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture micro-dissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30 and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. Conclusion This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease. PMID:24038131

DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects

PubMed Central

Rochtus, Anne; Izzi, Benedetta; Vangeel, Elise; Louwette, Sophie; Wittevrongel, Christine; Lambrechts, Diether; Moreau, Yves; Winand, Raf; Verpoorten, Carla; Jansen, Katrien; Van Geet, Chris; Freson, Kathleen

2015-01-01

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; β-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9–16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways. PMID:25565354

How to interpret Methylation Sensitive Amplified Polymorphism (MSAP) profiles?

PubMed Central

2014-01-01

Background DNA methylation plays a key role in development, contributes to genome stability, and may also respond to external factors supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. Results Here, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII + MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile. Conclusions We recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation. PMID:24393618

Regulation and function of DNA methylation in plants and animals

PubMed Central

He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

2011-01-01

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

Epigenetic modifications in multiple myeloma: recent advances on the role of DNA and histone methylation.

PubMed

Amodio, Nicola; D'Aquila, Patrizia; Passarino, Giuseppe; Tassone, Pierfrancesco; Bellizzi, Dina

2017-01-01

Multiple Myeloma (MM) is a clonal late B-cell disorder accounting for about 13% of hematological cancers and 1% of all neoplastic diseases. Recent studies on the molecular pathogenesis and biology of MM have highlighted a complex epigenomic landscape contributing to MM onset, prognosis and high individual variability. Areas covered: We describe here the current knowledge on epigenetic events characterizing MM initiation and progression, focusing on the role of DNA and histone methylation and on the most promising epi-therapeutic approaches targeting the methylation pathway. Expert opinion: Data published so far indicate that alterations of the epigenetic framework, which include aberrant global or gene/non-coding RNA specific methylation profiles, feature prominently in the pathobiology of MM. Indeed, the aberrant expression of components of the epigenetic machinery as well as the reversibility of the epigenetic marks make this pathway druggable, providing the basis for the design of epigenetic therapies against this still fatal malignancy.

Sleep quality and methylation status of selected tumor suppressor genes among nurses and midwives.

PubMed

Bukowska-Damska, Agnieszka; Reszka, Edyta; Kaluzny, Pawel; Wieczorek, Edyta; Przybek, Monika; Zienolddiny, Shanbeh; Peplonska, Beata

2018-01-01

Chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited reports suggest also epigenetic effects, such as changes in DNA methylation profiles. The study aims to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of selected tumor suppressor genes. A cross-sectional study was conducted on 710 nurses and midwives aged 40-60 years. Data from interviews regarding sleep habits and potential confounders were used. The methylation status of tumor suppressor genes was determined via qMSP reactions using DNA samples derived from leucocytes. No significant findings were observed in the total study population or in the two subgroups of women stratified by the current system of work. A borderline significance association was observed between a shorter duration of sleep and an increased methylation level in CDKN2A among day working nurses and midwives. Further studies are warranted to explore this under-investigated topic.

Acetylation of hMOF Modulates H4K16ac to Regulate DNA Repair Genes in Response to Oxidative Stress.

PubMed

Zhong, Jianing; Ji, Liying; Chen, Huiqian; Li, Xianfeng; Zhang, Jian'an; Wang, Xingxing; Wu, Weilin; Xu, Ying; Huang, Fei; Cai, Wanshi; Sun, Zhong Sheng

2017-01-01

Oxidative stress is considered to be a key risk state for a variety of human diseases. In response to oxidative stress, the regulation of transcriptional expression of DNA repair genes would be important to DNA repair and genomic stability. However, the overall pattern of transcriptional expression of DNA repair genes and the underlying molecular response mechanism to oxidative stress remain unclear. Here, by employing colorectal cancer cell lines following exposure to hydrogen peroxide, we generated expression profiles of DNA repair genes via RNA-seq and identified gene subsets that are induced or repressed following oxidative stress exposure. RRBS-seq analyses further indicated that transcriptional regulation of most of the DNA repair genes that were induced or repressed is independent of their DNA methylation status. Our analyses also indicate that hydrogen peroxide induces deacetylase SIRT1 which decreases chromatin affinity and the activity of histone acetyltransferase hMOF toward H4K16ac and results in decreased transcriptional expression of DNA repair genes. Taken together, our findings provide a potential mechanism by which oxidative stress suppresses DNA repair genes which is independent of the DNA methylation status of their promoters.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

PubMed Central

Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

2016-01-01

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy

PubMed Central

Declerck, Ken; Traen, Sophie; Koppen, Gudrun; Van Camp, Guy; Schoeters, Greet; Vanden Berghe, Wim; De Boever, Patrick

2016-01-01

The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (PAdj<0.001 and |Δβ|>0.2), though the methylation status of about 96% of the cg-sites was comparable between peripheral blood mononuclear cells and saliva. When comparing RA cases with healthy controls, the number of differentially methylated sites (DMS) in saliva and blood were 485 and 437 (P<0.05 and |Δβ|>0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy. PMID:26999364

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells.

PubMed

Swathy, Babu; Banerjee, Moinak

2017-01-01

Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission were observed to be upregulated while CHRM2 gene expression was down regulated. Haloperidol can influence methylation traits thereby inducing a pharmacoepigenomic response, which seems to be regulated by DNMTs and their putative miRNA expression. Increased methylation seems to influence CHRM2 gene expression while microRNA could influence neurotransmission, pharmacogene expression and methylation events. Altered expression of various therapeutically relevant genes and miRNA expression, could account for their role in therapeutic response or side effects.

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells

PubMed Central

Swathy, Babu

2017-01-01

Introduction Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. Methods SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Results Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission were observed to be upregulated while CHRM2 gene expression was down regulated. Conclusions Haloperidol can influence methylation traits thereby inducing a pharmacoepigenomic response, which seems to be regulated by DNMTs and their putative miRNA expression. Increased methylation seems to influence CHRM2 gene expression while microRNA could influence neurotransmission, pharmacogene expression and methylation events. Altered expression of various therapeutically relevant genes and miRNA expression, could account for their role in therapeutic response or side effects. PMID:28886082

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

PubMed

Kuo, Tony Chien-Yen; Chen, Chuan-Hung; Chen, Shu-Hwa; Lu, I-Hsuan; Chu, Mei-Ju; Huang, Li-Chun; Lin, Chung-Yen; Chen, Chien-Yu; Lo, Hsiao-Feng; Jeng, Shih-Tong; Chen, Long-Fang O

2015-06-12

Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies.

PubMed

O'Doherty, Alan M; McGettigan, Paul; Irwin, Rachelle E; Magee, David A; Gagne, Dominic; Fournier, Eric; Al-Naib, Abdullah; Sirard, Marc-André; Walsh, Colum P; Robert, Claude; Fair, Trudee

2018-06-05

Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

Identification of pathogenic genes related to rheumatoid arthritis through integrated analysis of DNA methylation and gene expression profiling.

PubMed

Zhang, Lei; Ma, Shiyun; Wang, Huailiang; Su, Hang; Su, Ke; Li, Longjie

2017-11-15

The purpose of our study was to identify new pathogenic genes used for exploring the pathogenesis of rheumatoid arthritis (RA). To screen pathogenic genes of RA, an integrated analysis was performed by using the microarray datasets in RA derived from the Gene Expression Omnibus (GEO) database. The functional annotation and potential pathways of differentially expressed genes (DEGs) were further discovered by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Afterwards, the integrated analysis of DNA methylation and gene expression profiling was used to screen crucial genes. In addition, we used RT-PCR and MSP to verify the expression levels and methylation status of these crucial genes in 20 synovial biopsy samples obtained from 10 RA model mice and 10 normal mice. BCL11B, CCDC88C, FCRLA and APOL6 were both up-regulated and hypomethylated in RA according to integrated analysis, RT-PCR and MSP verification. Four crucial genes (BCL11B, CCDC88C, FCRLA and APOL6) identified and analyzed in this study might be closely connected with the pathogenesis of RA. Copyright © 2017. Published by Elsevier B.V.

Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats

PubMed Central

López-Álvarez, Guadalupe S.; Wojdacz, Tomasz K.; García-Cuellar, Claudia M.; Monroy-Ramírez, Hugo C.; Rodríguez-Segura, Miguel A.; Pacheco-Rivera, Ruth A.; Valencia-Antúnez, Carlos A.; Cervantes-Anaya, Nancy; Soto-Reyes, Ernesto; Vásquez-Garzón, Verónica R.; Sánchez-Pérez, Yesennia; Villa-Treviño, Saúl

2017-01-01

ABSTRACT The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats. PMID:27895046

Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells

PubMed Central

Kim, Kitai; Zhao, Rui; Doi, Akiko; Ng, Kitwa; Unternaehrer, Juli; Cahan, Patrick; Hongguang, Huo; Loh, Yuin-Han; Aryee, Martin J.; Lensch, M. William; Li, Hu; Collins, James J.; Feinberg, Andrew P.; Daley, George Q.

2012-01-01

We compared bona-fide human induced pluripotent stem cells (iPSC) derived from umbilical cord blood (CB) and neonatal keratinocytes (K). As a consequence of both incomplete erasure of tissue-specific methylation and aberrant de novo methylation, CB-iPSC and K-iPSC are distinct in genome-wide DNA methylation profiles and differentiation potential. Extended passage of some iPSC clones in culture didn't improve their epigenetic resemblance to ESC, implying that some human iPSC retain a residual “epigenetic memory” of their tissue of origin. PMID:22119740

Evolution of DNA Methylation across Insects

PubMed Central

Vogel, Kevin J.; Moore, Allen J.; Schmitz, Robert J.

2017-01-01

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. PMID:28025279

Delineation of Methyl-DNA Binding Protein Interactions in the Prostate Cancer Genome

DTIC Science & Technology

2013-07-01

smaller portion was used for isolation of mRNAs - see below.) Chromatin immunoprecipitations were carried out using antibodies that recognize MeCP2...tissues were performed with antibodies directed against MeCP2, MBD1, MBD2 and MBD4 (methyl-CpG binding domain containing proteins) - “ChIP DNA” from...the matched tissues and these antibodies . - The analysis of NGS data was initiated. Differences in the MBD association profiles between matched

Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

PubMed Central

Pangeson, Tanapat; Sanguansermsri, Phanchana; Sanguansermsri, Torpong; Seeratanachot, Teerapat; Suwanakhon, Narutchala; Srikummool, Metawee; Kaewkong, Worasak; Mahingsa, Khwanruedee

2017-01-01

In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA. PMID:29162979

Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

PubMed

Steenbergen, Renske D M; Ongenaert, Maté; Snellenberg, Suzanne; Trooskens, Geert; van der Meide, Wendy F; Pandey, Deeksha; Bloushtain-Qimron, Noga; Polyak, Kornelia; Meijer, Chris J L M; Snijders, Peter J F; Van Criekinge, Wim

2013-09-01

Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV-positive women. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring

PubMed Central

Zhang, Qian; Sun, Xiaofang; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

2017-01-01

Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet) or a low chromium diet (LC, 0.14 mg chromium/kg diet) during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON), while others remained on the same diet (CON-CON, or LC-LC) for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA methylation which is associated with the insulin signaling pathway in the mice livers. PMID:28072825

Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring.

PubMed

Zhang, Qian; Sun, Xiaofang; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

2017-01-01

Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet) or a low chromium diet (LC, 0.14 mg chromium/kg diet) during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON), while others remained on the same diet (CON-CON, or LC-LC) for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA methylation which is associated with the insulin signaling pathway in the mice livers.

Perinatal lead (Pb) exposure results in sex and tissue-dependent adult DNA methylation alterations in murine IAP transposons.

PubMed

Montrose, L; Faulk, C; Francis, J; Dolinoy, D C

2017-10-01

Epidemiological and animal data suggest that adult chronic disease is influenced by early-life exposure-induced changes to the epigenome. Previously, we observed that perinatal lead (Pb) exposure results in persistent murine metabolic- and activity-related effects. Using phylogenetic and DNA methylation analysis, we have also identified novel intracisternal A particle (IAP) retrotransposons exhibiting regions of variable methylation as candidate loci for environmental effects on the epigenome. Here, we now evaluate brain and kidney DNA methylation profiles of four representative IAPs in adult mice exposed to human physiologically relevant levels of Pb two weeks prior to mating through lactation. When IAPs across the genome were evaluated globally, average (sd) methylation levels were 92.84% (3.74) differing by tissue (P < 0.001), but not sex or dose. By contrast, the four individual IAPs displayed tissue-specific Pb and sex effects. Medium Pb-exposed mice had 3.86% less brain methylation at IAP 110 (P < 0.01), while high Pb-exposed mice had 2.83% less brain methylation at IAP 236 (P = 0.01) and 1.77% less at IAP 506 (P = 0.05). Individual IAP DNA methylation differed by sex for IAP 110 in the brain and kidney, IAP 236 in the kidney, and IAP 1259 in the kidney. Using Tomtom, we identified three binding motifs that matched to each of our novel IAPs impacted by Pb, one of which (HMGA2) has been linked to metabolic-related conditions in both mice and humans. Thus, these recently identified IAPs display tissue-specific environmental lability as well as sex-specific differences supporting an epigenetic link between early exposure to Pb and later-in-life health outcomes. Environ. Mol. Mutagen. 58:540-550, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Altered DNA methylation profile in Norwegian patients with Autoimmune Addison's Disease.

PubMed

Bjanesoy, Trine E; Andreassen, Bettina Kulle; Bratland, Eirik; Reiner, Andrew; Islam, Shahinul; Husebye, Eystein S; Bakke, Marit

2014-06-01

Autoimmune Addison's Disease (AAD) is an endocrine and immunological disease of uncertain pathogenesis resulting from the immune system's destruction of the hormone producing cells of the adrenal cortex. The underlying molecular mechanisms are largely unknown, but it is commonly accepted that a combination of genetic susceptibility and environmental impact is critical. In the present study, we identified multiple hypomethylated gene promoter regions in patients with isolated AAD using DNA isolated from CD4+ T cells. The identified differentially methylated regions were distributed evenly across the 10.5-kb-promoter regions covered by the array, and a substantial number localized to promoters of genes involved in immune regulation and autoimmunity. This study reveals a hypomethylated status in CD4+ T cells from AAD patients and indicates differential methylation of promoters of key genes involved in immune responses. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

The epigenomic landscape of African rainforest hunter-gatherers and farmers.

PubMed

Fagny, Maud; Patin, Etienne; MacIsaac, Julia L; Rotival, Maxime; Flutre, Timothée; Jones, Meaghan J; Siddle, Katherine J; Quach, Hélène; Harmant, Christine; McEwen, Lisa M; Froment, Alain; Heyer, Evelyne; Gessain, Antoine; Betsem, Edouard; Mouguiama-Daouda, Patrick; Hombert, Jean-Marie; Perry, George H; Barreiro, Luis B; Kobor, Michael S; Quintana-Murci, Lluis

2015-11-30

The genetic history of African populations is increasingly well documented, yet their patterns of epigenomic variation remain uncharacterized. Moreover, the relative impacts of DNA sequence variation and temporal changes in lifestyle and habitat on the human epigenome remain unknown. Here we generate genome-wide genotype and DNA methylation profiles for 362 rainforest hunter-gatherers and sedentary farmers. We find that the current habitat and historical lifestyle of a population have similarly critical impacts on the methylome, but the biological functions affected strongly differ. Specifically, methylation variation associated with recent changes in habitat mostly concerns immune and cellular functions, whereas that associated with historical lifestyle affects developmental processes. Furthermore, methylation variation--particularly that correlated with historical lifestyle--shows strong associations with nearby genetic variants that, moreover, are enriched in signals of natural selection. Our work provides new insight into the genetic and environmental factors affecting the epigenomic landscape of human populations over time.

G-cimp status prediction of glioblastoma samples using mRNA expression data.

PubMed

Baysan, Mehmet; Bozdag, Serdar; Cam, Margaret C; Kotliarova, Svetlana; Ahn, Susie; Walling, Jennifer; Killian, Jonathan K; Stevenson, Holly; Meltzer, Paul; Fine, Howard A

2012-01-01

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

G-Cimp Status Prediction Of Glioblastoma Samples Using mRNA Expression Data

PubMed Central

Baysan, Mehmet; Bozdag, Serdar; Cam, Margaret C.; Kotliarova, Svetlana; Ahn, Susie; Walling, Jennifer; Killian, Jonathan K.; Stevenson, Holly; Meltzer, Paul; Fine, Howard A.

2012-01-01

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data. PMID:23139755

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

PubMed

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

2017-06-02

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

PubMed Central

Luo, Judong; Wang, Wenjie; Tang, Yiting; Zhou, Dandan; Gao, Yi; Zhang, Qi; Zhou, Xifa; Zhu, Hui; Xing, Ligang; Yu, Jinming

2017-01-01

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignancies worldwide. Radiotherapy plays a critical role in the curative management of inoperable ESCC patients. However, radioresistance restricts the efficacy of radiotherapy for ESCC patients. The molecules involved in radioresistance remain largely unknown, and new approaches to sensitize cells to irradiation are in demand. Technical advances in analysis of mRNA and methylation have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathways of ESCC radioresistance. In this study, we constructed radioresistant TE-1 and Eca-109 cell lines (TE-1/R and Eca-109/R, respectively). The radioresistant cells showed an increased migration ability but reduced apoptosis and cisplatin sensitivity compared with their parent cells. mRNA and methylation profiling by microarray revealed 1192 preferentially expressed mRNAs and 8841 aberrantly methylated regions between TE-1/R and TE-1 cells. By integrating the mRNA and methylation profiles, we related the decreased expression of transcription factor Sall2 with a corresponding increase in its methylation in TE-1/R cells, indicating its involvement in radioresistance. Upregulation of Sall2 decreased the growth and migration advantage of radioresistant ESCC cells. Taken together, our present findings illustrate the mRNA and DNA methylation changes during the radioresistance of ESCC and the important role of Sall2 in esophageal cancer malignancy. PMID:28367244

Salinity induced differential methylation patterns in contrasting cultivars of foxtail millet (Setaria italica L.).

PubMed

Pandey, Garima; Yadav, Chandra Bhan; Sahu, Pranav Pankaj; Muthamilarasan, Mehanathan; Prasad, Manoj

2017-05-01

Genome-wide methylation analysis of foxtail millet cultivars contrastingly differing in salinity tolerance revealed DNA demethylation events occurring in tolerant cultivar under salinity stress, eventually modulating the expression of stress-responsive genes. Reduced productivity and significant yield loss are the adverse effects of environmental conditions on physiological and biochemical pathways in crop plants. In this context, understanding the epigenetic machinery underlying the tolerance traits in a naturally stress tolerant crop is imperative. Foxtail millet (Setaria italica) is known for its better tolerance to abiotic stresses compared to other cereal crops. In the present study, methylation-sensitive amplified polymorphism (MSAP) technique was used to quantify the salt-induced methylation changes in two foxtail millet cultivars contrastingly differing in their tolerance levels to salt stress. The study highlighted that the DNA methylation level was significantly reduced in tolerant cultivar compared to sensitive cultivar. A total of 86 polymorphic MSAP fragments were identified, sequenced and functionally annotated. These fragments showed sequence similarity to several genes including ABC transporter, WRKY transcription factor, serine threonine-protein phosphatase, disease resistance, oxidoreductases, cell wall-related enzymes and retrotransposon and transposase like proteins, suggesting salt stress-induced methylation in these genes. Among these, four genes were chosen for expression profiling which showed differential expression pattern between both cultivars of foxtail millet. Altogether, the study infers that salinity stress induces genome-wide DNA demethylation, which in turn, modulates expression of corresponding genes.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

PubMed

Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

2013-03-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage

PubMed Central

Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E.; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M.; Song, Lingyun; Crawford, Gregory E.; Myers, Richard M.; Lacey, Michelle; Ehrlich, Melanie

2013-01-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues. PMID:23417056

Epigenetic discrimination of identical twins from blood under the forensic scenario.

PubMed

Vidaki, Athina; Díez López, Celia; Carnero-Montoro, Elena; Ralf, Arwin; Ward, Kirsten; Spector, Timothy; Bell, Jordana T; Kayser, Manfred

2017-11-01

Monozygotic (MZ) twins share the same STR profile, demonstrating a practical problem in forensic casework. DNA methylation has provided a suitable resource for MZ twin differentiation; however, studies addressing the forensic feasibility are lacking. Here, we investigated epigenetic MZ twin differentiation from blood under the forensic scenario comprising i) the discovery of candidate markers in reference-type blood DNA via genome-wide analysis, ii) the technical validation of candidate markers in reference-type blood DNA using a suitable targeted method, and iii) the analysis of the validated markers in trace-type DNA. Genome-wide methylation analysis in blood DNA from 10 MZ twin pairs resulted in 19-111 twin-differentially methylated sites (tDMSs) per pair with >0.3 twin-to-twin differences. Considering all top three candidate tDMSs across all pairs in the technical validation based on methylation-specific qPCR, 67.85% generated >0.1 twin-to-twin differences. Of the validated tDMSs, 68.4% showed >0.1 twin-to-twin differences with qPCR in trace-type DNA across 8 pairs. Using an updated marker selection strategy, 8 additional candidate tDMSs were obtained for an example MZ pair, of which 7 showed >0.1 twin-to-twin differences in both reference- and trace-type DNA. Lastly, we introduce a high-resolution melting curve analysis of the entire fragment that can complement the proposed approach. Overall, our study demonstrates the general feasibility of epigenetic twin differentiation in the forensic context and highlights that the number of informative tDMSs in the final trace DNA analysis is crucial, as some candidate markers identified in reference DNA were shown not informative in the trace DNA due to various, including technical, reasons. Future studies will need to address the optimal number of epigenetic markers required for reliable identification of MZ twin individuals including statistical considerations. Copyright © 2017 Elsevier B.V. All rights reserved.

Prostate tumor DNA methylation is associated with cigarette smoking and adverse prostate cancer outcomes.

PubMed

Shui, Irene M; Wong, Chao-Jen; Zhao, Shanshan; Kolb, Suzanne; Ebot, Ericka M; Geybels, Milan S; Rubicz, Rohina; Wright, Jonathan L; Lin, Daniel W; Klotzle, Brandy; Bibikova, Marina; Fan, Jian-Bing; Ostrander, Elaine A; Feng, Ziding; Stanford, Janet L

2016-07-15

DNA methylation has been hypothesized as a mechanism for explaining the association between smoking and adverse prostate cancer (PCa) outcomes. This study was aimed at assessing whether smoking is associated with prostate tumor DNA methylation and whether these alterations may explain in part the association of smoking with PCa recurrence and mortality. A total of 523 men had radical prostatectomy as their primary treatment, detailed smoking history data, long-term follow-up for PCa outcomes, and tumor tissue profiled for DNA methylation. Ninety percent of the men also had matched tumor gene expression data. A methylome-wide analysis was conducted to identify differentially methylated regions (DMRs) by smoking status. To select potential functionally relevant DMRs, their correlation with the messenger RNA (mRNA) expression of corresponding genes was evaluated. Finally, a smoking-related methylation score based on the top-ranked DMRs was created to assess its association with PCa outcomes. Forty DMRs were associated with smoking status, and 10 of these were strongly correlated with mRNA expression (aldehyde oxidase 1 [AOX1], claudin 5 [CLDN5], early B-cell factor 1 [EBF1], homeobox A7 [HOXA7], lectin galactoside-binding soluble 3 [LGALS3], microtubule-associated protein τ [MAPT], protocadherin γ A [PCDHGA]/protocadherin γ B [PCDHGB], paraoxonase 3 [PON3], synaptonemal complex protein 2 like [SYCP2L], and zinc finger and SCAN domain containing 12 [ZSCAN12]). Men who were in the highest tertile for the smoking-methylation score derived from these DMRs had a higher risk of recurrence (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.42-3.72) and lethal disease (OR, 4.21; 95% CI, 1.65-11.78) in comparison with men in the lower 2 tertiles. This integrative molecular epidemiology study supports the hypothesis that smoking-associated tumor DNA methylation changes may explain at least part of the association between smoking and adverse PCa outcomes. Future studies are warranted to confirm these findings and understand the implications for improving patient outcomes. Cancer 2016;122:2168-77. © 2016 American Cancer Society. © 2016 American Cancer Society.

Obesity and menopause modify the epigenomic profile of breast cancer.

PubMed

Crujeiras, Ana B; Diaz-Lagares, Angel; Stefansson, Olafur A; Macias-Gonzalez, Manuel; Sandoval, Juan; Cueva, Juan; Lopez-Lopez, Rafael; Moran, Sebastian; Jonasson, Jon G; Tryggvadottir, Laufey; Olafsdottir, Elinborg; Tinahones, Francisco J; Carreira, Marcos C; Casanueva, Felipe F; Esteller, Manel

2017-07-01

Obesity is a high risk factor for breast cancer. This relationship could be marked by a specific methylome. The current work was aimed to explore the impact of obesity and menopausal status on variation in breast cancer methylomes. Data from Infinium 450K array-based methylomes of 64 breast tumors were coupled with information on BMI and menopausal status. Additionally, DNA methylation results were validated in 18 non-tumor and 81 tumor breast samples. Breast tumors arising in either pre- or postmenopausal women stratified by BMI or menopausal status alone were not associated with a specific DNA methylation pattern. Intriguingly, the DNA methylation pattern identified in association with the high-risk group (postmenopausal women with high BMI (>25) and premenopausal women with normal or low BMI < 25) exclusively characterized by hypermethylation of 1287 CpG sites as compared with the low-risk group. These CpG sites included the promoter region of fourteen protein-coding genes of which CpG methylation over the ZNF577 promoter region represents the top scoring associated event. In an independent cohort, the ZNF577 promoter methylation remained statistically significant in association with the high-risk group. Additionally, the impact of ZNF577 promoter methylation on mRNA expression levels was demonstrated in breast cancer cell lines after treatment with a demethylating agent (5-azacytidine). In conclusion, the epigenome of breast tumors is affected by a complex interaction between BMI and menopausal status. The ZNF577 methylation quantification is clearly relevant for the development of novel biomarkers of precision therapy in breast cancer. © 2017 Society for Endocrinology.

Evolution of DNA Methylation across Insects.

PubMed

Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

2017-03-01

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Controlling DNA methylation: many roads to one modification.

PubMed

Freitag, Michael; Selker, Eric U

2005-04-01

Genetic, biochemical and cytological studies on DNA methylation in several eukaryotic organisms have resulted in leaps of understanding in the past three years. Discoveries of mechanistic links between DNA methylation and histone methylation, and between these processes and RNA interference (RNAi) machineries have reinvigorated the field. The details of the connections between DNA methylation, histone modifications and RNA silencing remain to be elucidated, but it is already clear that no single pathway accounts for all DNA methylation found in eukaryotes. Rather, different taxa use one or more of several general mechanisms to control methylation. Despite recent progress, classic questions remain, including: What are the signals for DNA methylation? Are "de novo" and "maintenance" methylation truly separate processes? How is DNA methylation regulated?

Salicylic acid and nitric oxide alleviate high temperature induced oxidative damage in Lablab purpureus L plants by regulating bio-physical processes and DNA methylation.

PubMed

Rai, Krishna Kumar; Rai, Nagendra; Rai, Shashi Pandey

2018-07-01

Salicylic acid (SA) and sodium nitroprusside (SNP, NO donor) modulates plant growth and development processes and recent findings have also revealed their involvement in the regulation of epigenetic factors under stress condition. In the present study, some of these factors were comparatively studied in hyacinth bean plants subjected to high temperature (HT) environment (40-42 °C) with and without exogenous application of SA and SNP under field condition. Exogenous application of SA and SNP substantially modulated the growth and biophysical process of hyacinth bean plants under HT environment. Exogenous application of SA and SNP also remarkably regulated the activities of antioxidant enzymes, modulated mRNA level of certain enzymes, improves plant water relation, enhance photosynthesis and thereby increasing plant defence under HT. Coupled restriction enzyme digestion-random amplification (CRED-RA) technique revealed that many methylation changes were "dose dependent" and HT significantly increased DNA damages as evidenced by both increase and decrease in bands profiles, methylation and de-methylation pattern. Thus, the result of the present study clearly shows that exogenous SA and SNP regulates DNA methylation pattern, modulates stress-responsive genes and can impart transient HT tolerance by synchronizing growth and physiological acclimatization of plants, thus narrowing the gaps between physio-biochemical and molecular events in addressing HT tolerance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Genome-wide DNA Methylation Profiles and Their Relationships with mRNA and the microRNA Transcriptome in Bovine Muscle Tissue (Bos taurine)

PubMed Central

Huang, Yong-Zhen; Sun, Jia-Jie; Zhang, Liang-Zhi; Li, Cong-Jun; Womack, James E.; Li, Zhuan-Jian; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

2014-01-01

DNA methylation is a key epigenetic modification in mammals and plays important roles in muscle development. We sampled longissimus dorsi muscle (LDM) from a well-known elite native breed of Chinese Qinchuan cattle living within the same environment but displaying distinct skeletal muscle at the fetal and adult stages. We generated and provided a genome-wide landscape of DNA methylomes and their relationship with mRNA and miRNA for fetal and adult muscle studies. Integration analysis revealed a total of 77 and 1,054 negatively correlated genes with methylation in the promoter and gene body regions, respectively, in both the fetal and adult bovine libraries. Furthermore, we identified expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from nine different tissues at multiple developmental stages of bovine muscle-related tissue or organs. In addition, we validated the MeDIP-Seq results by bisulfite sequencing PCR (BSP) in some of the differentially methylated promoters. Together, these results provide valuable data for future biomedical research and genomic and epigenomic studies of bovine skeletal muscle that may help uncover the molecular basis underlying economically valuable traits in cattle. This comprehensive map also provides a solid basis for exploring the epigenetic mechanisms of muscle growth and development. PMID:25306978

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

PubMed

Rivière, Guillaume; Lienhard, Daniel; Andrieu, Thomas; Vieau, Didier; Frey, Brigitte M; Frey, Felix J

2011-04-01

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Prognostic relevance of aberrant DNA methylation in g1 and g2 pancreatic neuroendocrine tumors.

PubMed

Stefanoli, Michele; La Rosa, Stefano; Sahnane, Nora; Romualdi, Chiara; Pastorino, Roberta; Marando, Alessandro; Capella, Carlo; Sessa, Fausto; Furlan, Daniela

2014-01-01

The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. In a well-characterized series of 56 sporadic G1 and G2 PanNETs, methylation-sensitive multiple ligation-dependent probe amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNAs) of 53 chromosomal regions. Long interspersed nucleotide element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene-specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. 12 tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only 5 PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene-specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis, low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression. © 2014 S. Karger AG, Basel.

DNA methylation dynamics in plants and mammals: overview of regulation and dysregulation.

PubMed

Elhamamsy, Amr Rafat

2016-07-01

DNA methylation is a major epigenetic marking mechanism regulating various biological functions in mammals and plant. The crucial role of DNA methylation has been observed in cellular differentiation, embryogenesis, genomic imprinting and X-chromosome inactivation. Furthermore, DNA methylation takes part in disease susceptibility, responses to environmental stimuli and the biodiversity of natural populations. In plant, different types of environmental stress have demonstrated the ability to alter the archetype of DNA methylation through the genome, change gene expression and confer a mechanism of adaptation. DNA methylation dynamics are regulated by three processes de novo DNA methylation, methylation maintenance and DNA demethylation. These processes have their similarities and differences between mammals and plants. Furthermore, the dysregulation of DNA methylation dynamics represents one of the primary molecular mechanisms of developing diseases in mammals. This review discusses the regulation and dysregulation of DNA methylation in plants and mammals. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Targeting Siah2 as Novel Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2015-10-01

research proposed here will provide a novel insight into previously unexplored cross-talking that occurs between hedgehog -signalling and DNA... hedgehog signaling pathway, DNMTs and CpG methylation loci profiles in different stages of prostate. (2) Characterize mechanism(s) of Gli regulation of

The epigenetic effects of a high prenatal folate intake in male mouse fetuses exposed in utero to arsenic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsang, Verne; Fry, Rebecca C.; Niculescu, Mihai D.

Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never beenmore » thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring. Highlights: ► We used transplacental CD1 mice model for inorganic arsenic (iAs) carcinogenesis. ► We examined the effects of gestational iAs and high folate exposure on DNA methylation. ► iAs–folate interaction resulted in low fetal weights and changes in DNA methylation. ► Epigenetically altered genes were associated with cancer and neurodevelopment. ► We showed that in utero iAs–folate interaction negatively affects fetal development.« less

CB5C affects the glucosinolate profile in Arabidopsis thaliana

PubMed Central

Vik, Daniel; Crocoll, Christoph; Andersen, Tonni Grube; Burow, Meike; Halkier, Barbara Ann

2016-01-01

ABSTRACT Cytochrome b5 (CB5) proteins are small heme-binding proteins, that influence cytochrome P450 activity. While only one CB5 isoform is found in mammals, higher plants have several isoforms of these proteins. The roles of the many CB5 isoforms in plants remain unknown. We hypothesized that CB5 proteins support the cytochrome P450 enzymes of plant specialized metabolism and found CB5C from Arabidopsis thaliana to co-express with glucosinolate biosynthetic genes. We characterized the glucosinolate profiles of 2 T-DNA insertion mutants of CB5C, and found that long-chained aliphatic glucosinolates were reduced in one of the mutant lines – a phenotype that was exaggerated upon methyl-jasmonate treatment. These results support the hypothesis, that CB5C influences glucosinolate biosynthesis, however, the mode of action remains unknown. Furthermore, the mutants differed in their biomass response to methyl jasmonate treatment. Thereby, our results highlight the varying effects of T-DNA insertion sites, as the 2 analyzed alleles show different phenotypes. PMID:27454255

Effect of DNA methylation on identification of aggressive prostate cancer.

PubMed

Alumkal, Joshi J; Zhang, Zhe; Humphreys, Elizabeth B; Bennett, Christina; Mangold, Leslie A; Carducci, Michael A; Partin, Alan W; Garrett-Mayer, Elizabeth; DeMarzo, Angelo M; Herman, James G

2008-12-01

Biochemical (prostate-specific antigen) recurrence of prostate cancer after radical prostatectomy remains a major problem. Better biomarkers are needed to identify high-risk patients. DNA methylation of promoter regions leads to gene silencing in many cancers. In this study, we assessed the effect of DNA methylation on the identification of recurrent prostate cancer. We studied the methylation status of 15 pre-specified genes using methylation-specific polymerase chain reaction on tissue samples from 151 patients with localized prostate cancer and at least 5 years of follow-up after prostatectomy. On multivariate logistic regression analysis, a high Gleason score and involvement of the capsule, lymph nodes, seminal vesicles, or surgical margin were associated with an increased risk of biochemical recurrence. Methylation of CDH13 by itself (odds ratio 5.50, 95% confidence interval [CI] 1.34 to 22.67; P = 0.02) or combined with methylation of ASC (odds ratio 5.64, 95% CI 1.47 to 21.7; P = 0.01) was also associated with an increased risk of biochemical recurrence. The presence of methylation of ASC and/or CDH13 yielded a sensitivity of 72.3% (95% CI 57% to 84.4%) and negative predictive value of 79% (95% CI 66.8% to 88.3%), similar to the weighted risk of recurrence (determined from the lymph node status, seminal vesicle status, surgical margin status, and postoperative Gleason score), a powerful clinicopathologic prognostic score. However, 34% (95% CI 21% to 49%) of the patients with recurrence were identified by the methylation profile of ASC and CDH13 rather than the weighted risk of recurrence. The results of our study have shown that methylation of CDH13 alone or combined with methylation of ASC is independently associated with an increased risk of biochemical recurrence after radical prostatectomy even considering the weighted risk of recurrence score. These findings should be validated in an independent, larger cohort of patients with prostate cancer who have undergone radical prostatectomy.

Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes and Tissues

PubMed Central

Osabe, Kenji; Clement, Jenny D.; Bedon, Frank; Pettolino, Filomena A.; Ziolkowski, Lisa; Llewellyn, Danny J.; Finnegan, E. Jean; Wilson, Iain W.

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP. PMID:24465864

Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues.

PubMed

Osabe, Kenji; Clement, Jenny D; Bedon, Frank; Pettolino, Filomena A; Ziolkowski, Lisa; Llewellyn, Danny J; Finnegan, E Jean; Wilson, Iain W

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

A simple modification to the luminometric methylation assay to control for the effects of DNA fragmentation.

PubMed

Duman, Elif Aysimi; Kriaucionis, Skirmantas; Dunn, John J; Hatchwell, Eli

2015-05-01

Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently described methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequencing technology for quantification. Here we provide evidence suggesting that the global methylation level reported by LUMA is affected by the integrity of the DNA being analyzed. The less intact the DNA, the lower the global methylation levels reported by LUMA. In order to overcome this problem, we propose the use of undigested DNA alongside digested samples. Finally, we demonstrate that this results in a more accurate assessment of global DNA methylation levels.

Two-stage Genome-wide Methylation Profiling in Childhood-onset Crohn's Disease Implicates Epigenetic Alterations at the VMP1/MIR21 and HLA Loci

PubMed Central

Adams, Alex T.; Kennedy, Nicholas A.; Hansen, Richard; Ventham, Nicholas T.; O'Leary, Kate R.; Drummond, Hazel E.; Noble, Colin L.; El-Omar, Emad; Russell, Richard K.; Wilson, David C.; Nimmo, Elaine R.; Hold, Georgina L.

2014-01-01

Background: As a result of technological and analytical advances, genome-wide characterization of key epigenetic alterations is now feasible in complex diseases. We hypothesized that this may provide important insights into gene-environmental interactions in Crohn's disease (CD) and is especially pertinent to early onset disease. Methods: The Illumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leukocyte DNA in discovery and replication pediatric CD cohorts and controls. Data were corrected for differential leukocyte proportions. Targeted replication was performed in adults using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa. Results: We identified 65 individual CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P < 1.1 × 10−7), and 19 differently methylated regions displaying unidirectional methylation change. There was a highly significant enrichment of methylation changes around GWAS single nucleotide polymorphisms (P = 3.7 × 10−7), notably the HLA region and MIR21. Two-locus discriminant analysis in the discovery cohort predicted disease in the pediatric replication cohort with high accuracy (area under the curve, 0.98). The findings strongly implicate the transcriptional start site of MIR21 as a region of extended epigenetic alteration, containing the most significant individual probes (P = 1.97 × 10−15) within a GWAS risk locus. In extension studies, we confirmed hypomethylation of MIR21 in adults (P = 6.6 × 10−5, n = 172) and show increased mRNA expression in leukocytes (P < 0.005, n = 66) and in the inflamed intestine (P = 1.4 × 10−6, n = 99). Conclusions: We demonstrate highly significant and replicable differences in DNA methylation in CD, defining the disease-associated epigenome. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21 and the HLA region. PMID:25144570

From forensic epigenetics to forensic epigenomics: broadening DNA investigative intelligence.

PubMed

Vidaki, Athina; Kayser, Manfred

2017-12-21

Human genetic variation is a major resource in forensics, but does not allow all forensically relevant questions to be answered. Some questions may instead be addressable via epigenomics, as the epigenome acts as an interphase between the fixed genome and the dynamic environment. We envision future forensic applications of DNA methylation analysis that will broaden DNA-based forensic intelligence. Together with genetic prediction of appearance and biogeographic ancestry, epigenomic lifestyle prediction is expected to increase the ability of police to find unknown perpetrators of crime who are not identifiable using current forensic DNA profiling.

Alteration of gene expression and DNA methylation in drug-resistant gastric cancer.

PubMed

Maeda, Osamu; Ando, Takafumi; Ohmiya, Naoki; Ishiguro, Kazuhiro; Watanabe, Osamu; Miyahara, Ryoji; Hibi, Yoko; Nagai, Taku; Yamada, Kiyofumi; Goto, Hidemi

2014-04-01

The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term 'extracellular region' and 'p53 signaling pathway' in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

Effect of smoking on gene expression profile - overall mechanism, impact on respiratory system function, and reference to electronic cigarettes.

PubMed

Kopa, Paulina Natalia; Pawliczak, Rafał

2018-07-01

Cigarette smoke has a crucial impact on transcriptome alteration by its effect on chromatin remodeling and DNA methylation status. The first mechanism is associated with the histone acetylation/deacetylation balance damage as a result of increased activity of NFĸB and lipid peroxidation products, which lead to an increased activity of HATs and DNMTs and reduced HDACs. The second mechanism is connected with direct damaging of DNA by smoke components, activation of downstream repair mechanism and recruitment of DNMTs into the breakage site, 'nicotine effect' and carbon monoxide (CO) activity on gene transcription and DNA methylation reduction. Cigarette smoking activates oxidative and inflammatory response and leads to uncontrolled structural changes in airways and alters gene expression. Such changes have a characteristic similar to that for COPD patients. Therefore, smoking is determined as a key risk factor for chronic respiratory disease development. Furthermore, electronic cigarettes, an alternative of tobacco cigarettes, also affect gene expression profile, which suggests some similarities in action mechanisms for both conventional and electronic cigarettes. However, there is only a limited number of trials discussing this issue and future investigations are needed.

Blood as a surrogate marker for tissue-specific DNA methylation and changes due to folate depletion in post-partum female mice.

PubMed

McKay, Jill A; Xie, Long; Harris, Sarah; Wong, Yi K; Ford, Dianne; Mathers, John C

2011-07-01

DNA methylation patterns are tissue specific and may influence tissue-specific gene regulation. Human studies investigating DNA methylation in relation to environmental factors primarily use blood-derived DNA as a surrogate for DNA from target tissues. It is therefore important to know if DNA methylation changes in blood in response to environmental changes reflect those in target tissues. Folate intake can influence DNA methylation, via altered methyl donor supply. Previously, manipulations of maternal folate intake during pregnancy altered the patterns of DNA methylation in offspring but, to our knowledge, the consequences for maternal DNA methylation are unknown. Given the increased requirement for folate during pregnancy, mothers may be susceptible to aberrant DNA methylation due to folate depletion. Female mice were fed folate-adequate (2 mg folic acid/kg diet) or folate-deplete (0.4 mg folic acid/kg diet) diets prior to mating and during pregnancy and lactation. Following weaning, dams were killed and DNA methylation was assessed by pyrosequencing® in blood, liver, and kidney at the Esr1, Igf2 differentially methylated region (DMR)1, Igf2 DMR2, Slc39a4CGI1, and Slc39a4CGI2 loci. We observed tissue-specific differences in methylation at all loci. Folate depletion reduced Igf2 DMR1 and Slc39a4CGI1 methylation across all tissues and altered Igf2 DMR2 methylation in a tissue-specific manner (p<0.05). Blood-derived DNA methylation measurements may not always reflect methylation within other tissues. Further measurements of blood-derived and tissue-specific methylation patterns are warranted to understand the complexity of tissue-specific responses to altered nutritional exposure. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA methylation in amphioxus: from ancestral functions to new roles in vertebrates.

PubMed

Albalat, Ricard; Martí-Solans, Josep; Cañestro, Cristian

2012-03-01

In vertebrates, DNA methylation is an epigenetic mechanism that modulates gene transcription, and plays crucial roles during development, cell fate maintenance, germ cell pluripotency and inheritable genome imprinting. DNA methylation might also play a role as a genome defense mechanism against the mutational activity derived from transposon mobility. In contrast to the heavily methylated genomes in vertebrates, most genomes in invertebrates are poorly or just moderately methylated, and the function of DNA methylation remains unclear. Here, we review the DNA methylation system in the cephalochordate amphioxus, which belongs to the most basally divergent group of our own phylum, the chordates. First, surveys of the amphioxus genome database reveal the presence of the DNA methylation machinery, DNA methyltransferases and methyl-CpG-binding domain proteins. Second, comparative genomics and analyses of conserved synteny between amphioxus and vertebrates provide robust evidence that the DNA methylation machinery of amphioxus represents the ancestral toolkit of chordates, and that its expansion in vertebrates was originated by the two rounds of whole-genome duplication that occurred in stem vertebrates. Third, in silico analysis of CpGo/e ratios throughout the amphioxus genome suggests a bimodal distribution of DNA methylation, consistent with a mosaic pattern comprising domains of methylated DNA interspersed with domains of unmethylated DNA, similar to the situation described in ascidians, but radically different to the globally methylated vertebrate genomes. Finally, we discuss potential roles of the DNA methylation system in amphioxus in the context of chordate genome evolution and the origin of vertebrates.

Predicting aberrant CpG island methylation

PubMed Central

Feltus, F. A.; Lee, E. K.; Costello, J. F.; Plass, C.; Vertino, P. M.

2003-01-01

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context. PMID:14519846

Predicting aberrant CpG island methylation.

PubMed

Feltus, F A; Lee, E K; Costello, J F; Plass, C; Vertino, P M

2003-10-14

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.

Current and Emerging Technologies for the Analysis of the Genome-Wide and Locus-Specific DNA Methylation Patterns.

PubMed

Tost, Jörg

2016-01-01

DNA methylation is the most studied epigenetic modification, and altered DNA methylation patterns have been identified in cancer and more recently also in many other complex diseases. Furthermore, DNA methylation is influenced by a variety of environmental factors, and the analysis of DNA methylation patterns might allow deciphering previous exposure. Although a large number of techniques to study DNA methylation either genome-wide or at specific loci have been devised, they all are based on a limited number of principles for differentiating the methylation state, viz., methylation-specific/methylation-dependent restriction enzymes, antibodies or methyl-binding proteins, chemical-based enrichment, or bisulfite conversion. Second-generation sequencing has largely replaced microarrays as readout platform and is also becoming more popular for locus-specific DNA methylation analysis. In this chapter, the currently used methods for both genome-wide and locus-specific analysis of 5-methylcytosine and as its oxidative derivatives, such as 5-hydroxymethylcytosine, are reviewed in detail, and the advantages and limitations of each approach are discussed. Furthermore, emerging technologies avoiding PCR amplification and allowing a direct readout of DNA methylation are summarized, together with novel applications, such as the detection of DNA methylation in single cells or in circulating cell-free DNA.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

PubMed Central

Lei, Mingguang; Zhang, Huiming; Julian, Russell; Tang, Kai; Xie, Shaojun; Zhu, Jian-Kang

2015-01-01

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression. PMID:25733903

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

PubMed Central

Nwaobi, Sinifunanya E.; Olsen, Michelle L.

2015-01-01

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity. PMID:26436772

Direct detection of methylation in genomic DNA

PubMed Central

Bart, A.; van Passel, M. W. J.; van Amsterdam, K.; van der Ende, A.

2005-01-01

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote. PMID:16091626

DNA-Methylation: Master or Slave of Neural Fate Decisions?

PubMed Central

Stricker, Stefan H.; Götz, Magdalena

2018-01-01

The pristine formation of complex organs depends on sharp temporal and spatial control of gene expression. Therefore, epigenetic mechanisms have been frequently attributed a central role in controlling cell fate determination. A prime example for this is the first discovered and still most studied epigenetic mark, DNA methylation, and the development of the most complex mammalian organ, the brain. Recently, the field of epigenetics has advanced significantly: new DNA modifications were discovered, epigenomic profiling became widely accessible, and methods for targeted epigenomic manipulation have been developed. Thus, it is time to challenge established models of epigenetic gene regulation. Here, we review the current state of knowledge about DNA modifications, their epigenomic distribution, and their regulatory role. We will summarize the evidence suggesting they possess crucial roles in neurogenesis and discuss whether this likely includes lineage choice regulation or rather effects on differentiation. Finally, we will attempt an outlook on how questions, which remain unresolved, could be answered soon. PMID:29449798

DNA methylation: the future of crime scene investigation?

PubMed

Gršković, Branka; Zrnec, Dario; Vicković, Sanja; Popović, Maja; Mršić, Gordan

2013-07-01

Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.

DNA methylation profiles correlated to striped bass sperm fertility

USDA-ARS?s Scientific Manuscript database

Striped bass (Morone saxatilis) spermatozoa are used to fertilize in vitro the eggs of white bass (Morone chrysops) to produce the preferred hybrid for the striped bass aquaculture industry. Currently, only one source of domestic striped bass juveniles are available to growers that are not obtained ...

Inheritance and Variation of Genomic DNA Methylation in Diploid and Triploid Pacific Oyster (Crassostrea gigas).

PubMed

Jiang, Qun; Li, Qi; Yu, Hong; Kong, Lingfeng

2016-02-01

DNA methylation is an important epigenetic mechanism that could be responsive to environmental changes indicating a potential role in natural selection and adaption. In order to evaluate an evolutionary role of DNA methylation, it is essential to first gain a better insight into inheritability. To address this question, this study investigated DNA methylation variation from parents to offspring in the Pacific oyster Crassostrea gigas using fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis. Most of parental methylated loci were stably transmitted to offspring segregating following Medelian expectation. However, methylated loci deviated more often than non-methylated loci and offspring showed a few de novo methylated loci indicating DNA methylation changes from parents to offspring. Interestingly, some male-specific methylated loci were found in this study which might help to explore sex determination in oyster. Despite environmental stimuli, genomic stresses such as polyploidization also can induce methylation changes. This study also compared global DNA methylation level and individual methylated loci between diploid and triploid oysters. Results showed no difference in global methylation state but a few ploidy-specific loci were detected. DNA methylation variation during polyploidization was less than autonomous methylation variation from parents to offspring.

Detection of regional DNA methylation using DNA-graphene affinity interactions.

PubMed

Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

2017-01-15

We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation.

PubMed

Gao, Lexuan; Geng, Yupeng; Li, Bo; Chen, Jiakuan; Yang, Ji

2010-11-01

Alternanthera philoxeroides (alligator weed) is an invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation in its introduced range. The mechanisms underpinning the wide range of phenotypic variation and rapid adaptation to novel and changing environments remain uncharacterized. In this study, we examined the epigenetic variation and its correlation with phenotypic variation in plants exposed to natural and manipulated environmental variability. Genome-wide methylation profiling using methylation-sensitive amplified fragment length polymorphism (MSAP) revealed considerable DNA methylation polymorphisms within and between natural populations. Plants of different source populations not only underwent significant morphological changes in common garden environments, but also underwent a genome-wide epigenetic reprogramming in response to different treatments. Methylation alterations associated with response to different water availability were detected in 78.2% (169/216) of common garden induced polymorphic sites, demonstrating the environmental sensitivity and flexibility of the epigenetic regulatory system. These data provide evidence of the correlation between epigenetic reprogramming and the reversible phenotypic response of alligator weed to particular environmental factors. © 2010 Blackwell Publishing Ltd.

TEA: the epigenome platform for Arabidopsis methylome study.

PubMed

Su, Sheng-Yao; Chen, Shu-Hwa; Lu, I-Hsuan; Chiang, Yih-Shien; Wang, Yu-Bin; Chen, Pao-Yang; Lin, Chung-Yen

2016-12-22

Bisulfite sequencing (BS-seq) has become a standard technology to profile genome-wide DNA methylation at single-base resolution. It allows researchers to conduct genome-wise cytosine methylation analyses on issues about genomic imprinting, transcriptional regulation, cellular development and differentiation. One single data from a BS-Seq experiment is resolved into many features according to the sequence contexts, making methylome data analysis and data visualization a complex task. We developed a streamlined platform, TEA, for analyzing and visualizing data from whole-genome BS-Seq (WGBS) experiments conducted in the model plant Arabidopsis thaliana. To capture the essence of the genome methylation level and to meet the efficiency for running online, we introduce a straightforward method for measuring genome methylation in each sequence context by gene. The method is scripted in Java to process BS-Seq mapping results. Through a simple data uploading process, the TEA server deploys a web-based platform for deep analysis by linking data to an updated Arabidopsis annotation database and toolkits. TEA is an intuitive and efficient online platform for analyzing the Arabidopsis genomic DNA methylation landscape. It provides several ways to help users exploit WGBS data. TEA is freely accessible for academic users at: http://tea.iis.sinica.edu.tw .

Epigenetics and obesity cardiomyopathy: From pathophysiology to prevention and management.

PubMed

Zhang, Yingmei; Ren, Jun

2016-05-01

Uncorrected obesity has been associated with cardiac hypertrophy and contractile dysfunction. Several mechanisms for this cardiomyopathy have been identified, including oxidative stress, autophagy, adrenergic and renin-angiotensin aldosterone overflow. Another process that may regulate effects of obesity is epigenetics, which refers to the heritable alterations in gene expression or cellular phenotype that are not encoded on the DNA sequence. Advances in epigenome profiling have greatly improved the understanding of the epigenome in obesity, where environmental exposures during early life result in an increased health risk later on in life. Several mechanisms, including histone modification, DNA methylation and non-coding RNAs, have been reported in obesity and can cause transcriptional suppression or activation, depending on the location within the gene, contributing to obesity-induced complications. Through epigenetic modifications, the fetus may be prone to detrimental insults, leading to cardiac sequelae later in life. Important links between epigenetics and obesity include nutrition, exercise, adiposity, inflammation, insulin sensitivity and hepatic steatosis. Genome-wide studies have identified altered DNA methylation patterns in pancreatic islets, skeletal muscle and adipose tissues from obese subjects compared with non-obese controls. In addition, aging and intrauterine environment are associated with differential DNA methylation. Given the intense research on the molecular mechanisms of the etiology of obesity and its complications, this review will provide insights into the current understanding of epigenetics and pharmacological and non-pharmacological (such as exercise) interventions targeting epigenetics as they relate to treatment of obesity and its complications. Particular focus will be on DNA methylation, histone modification and non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata.

PubMed

Fraga, Mario F; Rodríguez, Roberto; Cañal, Maria Jesús

2002-08-01

In animals, DNA methylation is related to gene silencing during ontogenic development. Little is known about DNA methylation in plants, although occasional changes in the DNA methylation state of specific gene promoters have been reported in angiosperms during some developmental processes. We found large differences in the extent of DNA methylation between meristematic areas of juvenile and mature Pinus radiata D. Don. trees, whereas differences in the extent of DNA methylation between differentiated tissues of juvenile and mature trees were small. In meristematic areas, there was a gradual decrease in extent of DNA methylation as the degree of reinvigoration increased. The observed changes in extent of DNA methylation during aging and reinvigoration indicate that reinvigoration could be a consequence of epigenetic modifications opposite in direction to those that occur during aging.

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

PubMed

Joo, Jihoon E; Hiden, Ursula; Lassance, Luciana; Gordon, Lavinia; Martino, David J; Desoye, Gernot; Saffery, Richard

2013-07-15

The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

DNA Methylation and Cancer Diagnosis

PubMed Central

Delpu, Yannick; Cordelier, Pierre; Cho, William C.; Torrisani, Jérôme

2013-01-01

DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results. PMID:23873296

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

PubMed

Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Signatures of DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola

PubMed Central

Provataris, Panagiotis; Meusemann, Karen; Niehuis, Oliver; Grath, Sonja; Misof, Bernhard

2018-01-01

Abstract It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects. PMID:29697817

Comparative Methylation of ERVWE1/Syncytin-1 and Other Human Endogenous Retrovirus LTRs in Placenta Tissues

PubMed Central

Gimenez, Juliette; Montgiraud, Cécile; Oriol, Guy; Pichon, Jean-Philippe; Ruel, Karine; Tsatsaris, Vassilis; Gerbaud, Pascale; Frendo, Jean-Louis; Evain-Brion, Danièle; Mallet, François

2009-01-01

Human endogenous retroviruses (HERVs) are globally silent in somatic cells. However, some HERVs display high transcription in physiological conditions. In particular, ERVWE1, ERVFRDE1 and ERV3, three proviruses of distinct families, are highly transcribed in placenta and produce envelope proteins associated with placenta development. As silencing of repeated elements is thought to occur mainly by DNA methylation, we compared the methylation of ERVWE1 and related HERVs to appreciate whether HERV methylation relies upon the family, the integration site, the tissue, the long terminal repeat (LTR) function or the associated gene function. CpG methylation of HERV-W LTRs in placenta-associated tissues was heterogeneous but a joint epigenetic control was found for ERVWE1 5′LTR and its juxtaposed enhancer, a mammalian apparent LTR retrotransposon. Additionally, ERVWE1, ERVFRDE1 and ERV3 5′LTRs were all essentially hypomethylated in cytotrophoblasts during pregnancy, but showed distinct and stage-dependent methylation profiles. In non-cytotrophoblastic cells, they also exhibited different methylation profiles, compatible with their respective transcriptional activities. Comparative analyses of transcriptional activity and LTR methylation in cell lines further sustained a role for methylation in the control of functional LTRs. These results suggest that HERV methylation might not be family related but copy-specific, and related to the LTR function and the tissue. In particular, ERVWE1 and ERV3 could be developmentally epigenetically regulated HERVs. PMID:19561344

Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation

PubMed Central

Zhang, Qingzhu; Wang, Dong; Lang, Zhaobo; He, Li; Yang, Lan; Zeng, Liang; Li, Yanqiang; Zhao, Cheng; Huang, Huan; Zhang, Heng; Zhang, Huiming; Zhu, Jian-Kang

2016-01-01

DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. PMID:27382183

Final Report - Epigenetics of low dose radiation effects in an animal model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovalchuk, Olga

This project sought mechanistic understanding of the epigenetic response of tissues as well as the consequences of those responses, when induced by low dose irradiation in a well-established model system (mouse). Based on solid and extensive preliminary data we investigated the molecular epigenetic mechanisms of in vivo radiation responses, particularly – effects of low, occupationally relevant radiation exposures on the genome stability and adaptive response in mammalian tissues and organisms. We accumulated evidence that low dose irradiation altered epigenetic profiles and impacted radiation target organs of the exposed animals. The main long-term goal was to dissect the epigenetic basis ofmore » induction of the low dose radiation-induced genome instability and adaptive response and the specific fundamental roles of epigenetic changes (i.e. DNA methylation, histone modifications and miRNAs) in their generation. We hypothesized that changes in global and regional DNA methylation, global histone modifications and regulatory microRNAs played pivotal roles in the generation and maintenance low-dose radiation-induced genome instability and adaptive response. We predicted that epigenetic changes influenced the levels of genetic rearrangements (transposone reactivation). We hypothesized that epigenetic responses from low dose irradiation were dependent on exposure regimes, and would be greatest when organisms are exposed in a protracted/fractionated manner: fractionated exposures > acute exposures. We anticipated that the epigenetic responses were correlated with the gene expression levels. Our immediate objectives were: • To investigate the exact nature of the global and locus-specific DNA methylation changes in the LDR exposed cells and tissues and dissect their roles in adaptive response • To investigate the roles of histone modifications in the low dose radiation effects and adaptive response • To dissect the roles of regulatory microRNAs and their targets in low dose radiation effects and adaptive response • To correlate the levels of epigenetic changes with genetic rearrangement levels and gene expression patterns. In sum, we determined the precise global and locus-specific DNA methylation patterns in the LDR-exposed cells and tissues of mice, and to correlated DNA methylation changes with the gene expression patterns and manifestations of genome instability. We also determined the alterations of global histone modification pattern in the LDR exposed tissues. Additionally, we established the nature of microRNAome changes in the LDR exposed tissue. In this study we for the first time found that LDR exposure caused profound tissue-specific epigenetic changes in the exposed tissues. We established that LDR exposure affect methylation of repetitive elements in the murine genome, causes changes in histone methylation, acetylation and phosphorylation. Importantly, we found that LDR causes profound and persistent effects on small RNA profiles and gene expression, and that miRNAs are excellent biomarkers of LDR exposure. Furthermore, we extended our analysis and studied LDR effects in rat tissues and human tissues and cell lines. There we also analyzed LDR-induced gene expression, DNA methylation and miRNA changes. Our datasets laid foundation for several new research projects aimed to understand molecular underpinnings of low dose radiation responses, and biological repercussions of low dose radiation effects and radiation carcinogenesis.« less

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

PubMed Central

Wertheim, Gerald B.W.; Smith, Catherine; Figueroa, Maria E.; Kalos, Michael; Bagg, Adam; Carroll, Martin; Master, Stephen R.

2015-01-01

Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid leukemia can be predicted by the DNA methylation pattern of 18 loci. We have developed a novel strategy, termed microsphere HpaII tiny fragment enrichment by ligation-mediated PCR (MELP), to simultaneously analyze the DNA methylation pattern at these loci using methylation-specific DNA digestion, fluorescently labeled microspheres, and branched DNA hybridization. The method uses techniques that are inexpensive and easily performed in a molecular laboratory. MELP accurately reflects the methylation levels at each locus analyzed and segregates patients with acute myeloid leukemia into prognostic subgroups. Our results demonstrate the usefulness of MELP as a platform for simultaneous evaluation of DNA methylation of multiple loci. PMID:24373919

TET2 Mutations Are Associated with Specific 5-Methylcytosine and 5-Hydroxymethylcytosine Profiles in Patients with Chronic Myelomonocytic Leukemia

PubMed Central

Pérez, Cristina; Martínez-Calle, Nicolas; Martín-Subero, José Ignacio; Segura, Victor; Delabesse, Eric; Fernandez-Mercado, Marta; Garate, Leire; Alvarez, Sara; Rifon, José; Varea, Sara; Boultwood, Jacqueline; Wainscoat, James S.; Cigudosa, Juan Cruz; Calasanz, María José; Cross, Nicholas C. P.

2012-01-01

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has beenrecently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile. PMID:22328940

Collaborations between CpG sites in DNA methylation

NASA Astrophysics Data System (ADS)

Song, You; Ren, Honglei; Lei, Jinzhi

2017-08-01

DNA methylation patterns have profound impacts on genome stability, gene expression and development. The molecular base of DNA methylation patterns has long been focused at single CpG sites level. Here, we construct a kinetic model of DNA methylation with collaborations between CpG sites, from which a correlation function was established based on experimental data. The function consists of three parts that suggest three possible sources of the correlation: movement of enzymes along DNA, collaboration between DNA methylation and nucleosome modification, and global enzyme concentrations within a cell. Moreover, the collaboration strength between DNA methylation and nucleosome modification is universal for mouse early embryo cells. The obtained correlation function provides insightful understanding for the mechanisms of inheritance of DNA methylation patterns.

Genome-Wide Negative Feedback Drives Transgenerational DNA Methylation Dynamics in Arabidopsis

PubMed Central

Kassam, Mohamed; Duvernois-Berthet, Evelyne; Cortijo, Sandra; Takashima, Kazuya; Saze, Hidetoshi; Toyoda, Atsushi; Fujiyama, Asao; Colot, Vincent; Kakutani, Tetsuji

2015-01-01

Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3’ regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome. PMID:25902052