Minimal evidence for consistent changes in maize DNA methylation patterns following environmental stress.

PubMed

Eichten, Steven R; Springer, Nathan M

2015-01-01

DNA methylation is a chromatin modification that is sometimes associated with epigenetic regulation of gene expression. As DNA methylation can be reversible at some loci, it is possible that methylation patterns may change within an organism that is subjected to environmental stress. In order to assess the effects of abiotic stress on DNA methylation patterns in maize (Zea mays), seeding plants were subjected to heat, cold, and UV stress treatments. Tissue was later collected from individual adult plants that had been subjected to stress or control treatments and used to perform DNA methylation profiling to determine whether there were consistent changes in DNA methylation triggered by specific stress treatments. DNA methylation profiling was performed by immunoprecipitation of methylated DNA followed by microarray hybridization to allow for quantitative estimates of DNA methylation abundance throughout the low-copy portion of the maize genome. By comparing the DNA methylation profiles of each individual plant to the average of the control plants it was possible to identify regions of the genome with variable DNA methylation. However, we did not find evidence of consistent DNA methylation changes resulting from the stress treatments used in this study. Instead, the data suggest that there is a low-rate of stochastic variation that is present in both control and stressed plants.

Methylation variable position profiles of hMLH1 promoter CpG islands in human sporadic colorectal carcinoma.

PubMed

Huang, Qing; Huang, Jun-Fu; Zhang, Bo; Baum, Larry; Fu, Wei-Ling

2012-03-01

Aberrant hypermethylation of CpG islands (CGIs) in hMLH1 promoter regions has been well known to play an important role in the tumorigenesis of human sporadic colorectal carcinoma (SCRC). In this study, bisulfite sequencing was performed to analyze the methylation variable positions (MVPs) profiles of hMLH1 promoter CGIs in 30 clinical SCRC patients, and further analysis was carried out to evaluate the associations between the CGI methylation and the clinicopathological features in SCRC. Among the 2 CGIs in the hMLH1 promoter, that is, CGI-I and CGI-II, 20% (6/30) and 13% (4/30) of the patients had methylated CGI-I and CGI-II, respectively. Suppressed expression of hMLH1was significantly correlated with methylation of CGI-I but not CGI-II. Further analysis of the MVP profiles of CGI-I showed that most of the MVPs were hypermethylated and others were poorly methylated or unmethylated. The profiles could be classified into at least 4 groups based on the methylation status of 3 MVPs at positions 21 to 23 in CGI-I. All 6 patients with methylated CGI-I belonged to group I. This result suggests that the above 3 MVPs in CGI-I should be a targeted region to further analyze the epigenetic features of hMLH1 in human SCRC. Our results further suggest that MVP profiling is useful for identifying the aberrantly methylated CGIs associated with suppressed gene expression.

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

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

PubMed

Houshdaran, Sahar; Hawley, Sarah; Palmer, Chana; Campan, Mihaela; Olsen, Mari N; Ventura, Aviva P; Knudsen, Beatrice S; Drescher, Charles W; Urban, Nicole D; Brown, Patrick O; Laird, Peter W

2010-02-22

Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers.

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

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.

Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment

PubMed Central

Corley, Michael J.; Dye, Christian; D’Antoni, Michelle L.; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M.; Ndhlovu, Lishomwa C.; Maunakea, Alika K.

2016-01-01

Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation “signature” potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI. PMID:27629381

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.

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.

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.

Methylation profiling of choroid plexus tumors reveals 3 clinically distinct subgroups.

PubMed

Thomas, Christian; Sill, Martin; Ruland, Vincent; Witten, Anika; Hartung, Stefan; Kordes, Uwe; Jeibmann, Astrid; Beschorner, Rudi; Keyvani, Kathy; Bergmann, Markus; Mittelbronn, Michel; Pietsch, Torsten; Felsberg, Jörg; Monoranu, Camelia M; Varlet, Pascale; Hauser, Peter; Olar, Adriana; Grundy, Richard G; Wolff, Johannes E; Korshunov, Andrey; Jones, David T; Bewerunge-Hudler, Melanie; Hovestadt, Volker; von Deimling, Andreas; Pfister, Stefan M; Paulus, Werner; Capper, David; Hasselblatt, Martin

2016-06-01

Choroid plexus tumors are intraventricular neoplasms derived from the choroid plexus epithelium. A better knowledge of molecular factors involved in choroid plexus tumor biology may aid in identifying patients at risk for recurrence. Methylation profiles were examined in 29 choroid plexus papillomas (CPPs, WHO grade I), 32 atypical choroid plexus papillomas (aCPPs, WHO grade II), and 31 choroid plexus carcinomas (CPCs, WHO grade III) by Illumina Infinium HumanMethylation450 Bead Chip Array. Unsupervised hierarchical clustering identified 3 subgroups: methylation cluster 1 (pediatric CPP and aCPP of mainly supratentorial location), methylation cluster 2 (adult CPP and aCPP of mainly infratentorial location), and methylation cluster 3 (pediatric CPP, aCPP, and CPC of supratentorial location). In methylation cluster 3, progression-free survival (PFS) accounted for a mean of 72 months (CI, 55-89 mo), whereas only 1 of 42 tumors of methylation clusters 1 and 2 progressed (P< .001). On stratification of outcome data according to WHO grade, all CPCs clustered within cluster 3 and were associated with shorter overall survival (mean, 105 mo [CI, 81-128 mo]) and PFS (mean, 55 mo [CI, 36-73 mo]). The aCPP of methylation cluster 3 also progressed frequently (mean, 69 mo [CI, 44-93 mo]), whereas no tumor progression was observed in aCPP of methylation clusters 1 and 2 (P< .05). Only 1 of 29 CPPs recurred. Methylation profiling of choroid plexus tumors reveals 3 distinct subgroups (ie, pediatric low-risk choroid plexus tumors [cluster 1], adult low-risk choroid plexus tumors [cluster 2], and pediatric high-risk choroid plexus tumors [cluster 3]) and may provide useful prognostic information in addition to histopathology. Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

Identification of Gene Networks Associated with Acute Myeloid Leukemia by Comparative Molecular Methylation and Expression Profiling

PubMed Central

Dellett, Margaret; O’Hagan, Kathleen Ann; Colyer, Hilary Ann Alexandra; Mills, Ken I.

2010-01-01

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies. PMID:24179384

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.

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 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.

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/

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer.

PubMed

Wu, Yu; Davison, Jerry; Qu, Xiaoyu; Morrissey, Colm; Storer, Barry; Brown, Lisha; Vessella, Robert; Nelson, Peter; Fang, Min

2016-04-02

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

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.

EG-09EPIGENETIC PROFILING REVEALS A CpG HYPERMETHYLATION PHENOTYPE (CIMP) ASSOCIATED WITH WORSE PROGRESSION-FREE SURVIVAL IN MENINGIOMA

PubMed Central

Olar, Adriana; Wani, Khalida; Mansouri, Alireza; Zadeh, Gelareh; Wilson, Charmaine; DeMonte, Franco; Fuller, Gregory; Jones, David; Pfister, Stefan; von Deimling, Andreas; Sulman, Erik; Aldape, Kenneth

2014-01-01

BACKGROUND: Methylation profiling of solid tumors has revealed biologic subtypes, often with clinical implications. Methylation profiles of meningioma and their clinical implications are not well understood. METHODS: Ninety-two meningioma samples (n = 44 test set and n = 48 validation set) were profiled using the Illumina HumanMethylation450 BeadChip. Unsupervised clustering and analyses for recurrence-free survival (RFS) were performed. RESULTS: Unsupervised clustering of the test set using approximately 900 highly variable markers identified two clearly defined methylation subgroups. One of the groups (n = 19) showed global hypermethylation of a set of markers, analogous to CpG island methylator phenotype (CIMP). These findings were reproducible in the validation set, with 18/48 samples showing the CIMP-positive phenotype. Importantly, of 347 highly variable markers common to both the test and validation set analyses, 107 defined CIMP in the test set and 94 defined CIMP in the validation set, with an overlap of 83 markers between the two datasets. This number is much greater than expected by chance indicating reproducibly of the hypermethylated markers that define CIMP in meningioma. With respect to clinical correlation, the 37 CIMP-positive cases displayed significantly shorter RFS compared to the 55 non-CIMP cases (hazard ratio 2.9, p = 0.013). In an effort to develop a preliminary outcome predictor, a 155-marker subset correlated with RFS was identified in the test dataset. When interrogated in the validation dataset, this 155-marker subset showed a statistical trend (p < 0.1) towards distinguishing survival groups. CONCLUSIONS: This study defines the existence of a CIMP phenotype in meningioma, which involves a substantial proportion (37/92, 40%) of samples with clinical implications. Ongoing work will expand this cohort and examine identification of additional biologic differences (mutational and DNA copy number analysis) to further characterize the aberrant methylation subtype in meningioma. CIMP-positivity with aberrant methylation in recurrent/malignant meningioma suggests a potential therapeutic target for clinically aggressive cases.

The use of fatty acid methyl esters as biomarkers to determine aerobic, facultatively aerobic and anaerobic communities in wastewater treatment systems.

PubMed

Quezada, Maribel; Buitrón, Germán; Moreno-Andrade, Iván; Moreno, Gloria; López-Marín, Luz M

2007-01-01

The use of fatty acid methyl esters (FAME) as biomarkers to identify groups of microorganisms was studied. A database was constructed using previously published results that identify FAME biomarkers for aerobic, anaerobic and facultatively aerobic bacteria. FAME profiles obtained from pure cultures were utilized to confirm the predicted presence of biomarkers. Principal component analysis demonstrated that the FAME profiles can be used to determine the incidence of these bacterial groups. The presence of aerobic, anaerobic and facultatively aerobic bacteria in the communities, in four bioreactors being used to treat different wastewaters, was investigated by applying FAME biomarkers.

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.

[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.

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.

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

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.

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.

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.

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

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.

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.

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

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.

Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

PubMed Central

Hernández, Luis; Navarro, Alba; Beà, Sílvia; Pinyol, Magda; López-Guillermo, Armando; Rosenwald, Andreas; Ott, German; Campo, Elías; Jares, Pedro

2011-01-01

Background Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours. PMID:21603610

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.

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

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.

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.

Identification of the CIMP-like subtype and aberrant methylation of members of the chromosomal segregation and spindle assembly pathways in esophageal adenocarcinoma.

PubMed

Krause, Lutz; Nones, Katia; Loffler, Kelly A; Nancarrow, Derek; Oey, Harald; Tang, Yue Hang; Wayte, Nicola J; Patch, Ann Marie; Patel, Kalpana; Brosda, Sandra; Manning, Suzanne; Lampe, Guy; Clouston, Andrew; Thomas, Janine; Stoye, Jens; Hussey, Damian J; Watson, David I; Lord, Reginald V; Phillips, Wayne A; Gotley, David; Smithers, B Mark; Whiteman, David C; Hayward, Nicholas K; Grimmond, Sean M; Waddell, Nicola; Barbour, Andrew P

2016-04-01

The incidence of esophageal adenocarcinoma (EAC) has risen significantly over recent decades. Although survival has improved, cure rates remain poor, with <20% of patients surviving 5 years. This is the first study to explore methylome, transcriptome and ENCODE data to characterize the role of methylation in EAC. We investigate the genome-wide methylation profile of 250 samples including 125 EAC, 19 Barrett's esophagus (BE), 85 squamous esophagus and 21 normal stomach. Transcriptome data of 70 samples (48 EAC, 4 BE and 18 squamous esophagus) were used to identify changes in methylation associated with gene expression. BE and EAC showed similar methylation profiles, which differed from squamous tissue. Hypermethylated sites in EAC and BE were mainly located in CpG-rich promoters. A total of 18575 CpG sites associated with 5538 genes were differentially methylated, 63% of these genes showed significant correlation between methylation and mRNA expression levels. Pathways involved in tumorigenesis including cell adhesion, TGF and WNT signaling showed enrichment for genes aberrantly methylated. Genes involved in chromosomal segregation and spindle formation were aberrantly methylated. Given the recent evidence that chromothripsis may be a driver mechanism in EAC, the role of epigenetic perturbation of these pathways should be further investigated. The methylation profiles revealed two EAC subtypes, one associated with widespread CpG island hypermethylation overlapping H3K27me3 marks and binding sites of the Polycomb proteins. These subtypes were supported by an independent set of 89 esophageal cancer samples. The most hypermethylated tumors showed worse patient survival. © The Author 2016. Published by Oxford University Press.

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.

Variations in the Intragene Methylation Profiles Hallmark Induced Pluripotency

PubMed Central

Druzhkov, Pavel; Zolotykh, Nikolay; Meyerov, Iosif; Alsaedi, Ahmed; Shutova, Maria; Ivanchenko, Mikhail; Zaikin, Alexey

2015-01-01

We demonstrate the potential of differentiating embryonic and induced pluripotent stem cells by the regularized linear and decision tree machine learning classification algorithms, based on a number of intragene methylation measures. The resulting average accuracy of classification has been proven to be above 95%, which overcomes the earlier achievements. We propose a constructive and transparent method of feature selection based on classifier accuracy. Enrichment analysis reveals statistically meaningful presence of stemness group and cancer discriminating genes among the selected best classifying features. These findings stimulate the further research on the functional consequences of these differences in methylation patterns. The presented approach can be broadly used to discriminate the cells of different phenotype or in different state by their methylation profiles, identify groups of genes constituting multifeature classifiers, and assess enrichment of these groups by the sets of genes with a functionality of interest. PMID:26618180

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

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

Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

PubMed

Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

2016-01-19

Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

PubMed Central

Kamalakaran, Sitharthan; Kendall, Jude; Zhao, Xiaoyue; Tang, Chunlao; Khan, Sohail; Ravi, Kandasamy; Auletta, Theresa; Riggs, Michael; Wang, Yun; Helland, Åslaug; Naume, Bjørn; Dimitrova, Nevenka; Børresen-Dale, Anne-Lise; Hicks, Jim; Lucito, Robert

2009-01-01

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species. PMID:19474344

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

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

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.

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.

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...

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.

Epigenetics and human obesity.

PubMed

van Dijk, S J; Molloy, P L; Varinli, H; Morrison, J L; Muhlhausler, B S

2015-01-01

Recent technological advances in epigenome profiling have led to an increasing number of studies investigating the role of the epigenome in obesity. There is also evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may lead to an increased risk of obesity later in life. This paper provides a systematic review of studies investigating the association between obesity and either global, site-specific or genome-wide methylation of DNA. Studies on the impact of pre- and postnatal interventions on methylation and obesity are also reviewed. We discuss outstanding questions, and introduce EpiSCOPE, a multidisciplinary research program aimed at increasing the understanding of epigenetic changes in emergence of obesity. An electronic search for relevant articles, published between September 2008 and September 2013 was performed. From the 319 articles identified, 46 studies were included and reviewed. The studies provided no consistent evidence for a relationship between global methylation and obesity. The studies did identify multiple obesity-associated differentially methylated sites, mainly in blood cells. Extensive, but small, alterations in methylation at specific sites were observed in weight loss intervention studies, and several associations between methylation marks at birth and later life obesity were found. Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Eventually this may help in predicting an individual's obesity risk at a young age and opens possibilities for introducing targeted prevention strategies. It has also become clear that several epigenetic marks are modifiable, by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify unfavourable epigenomic profiles.

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.

Developmentally Programmed 3′ CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation

PubMed Central

Yu, Da-Hai; Ware, Carol; Waterland, Robert A.; Zhang, Jiexin; Chen, Miao-Hsueh; Gadkari, Manasi; Kunde-Ramamoorthy, Govindarajan; Nosavanh, Lagina M.

2013-01-01

During development, a small but significant number of CpG islands (CGIs) become methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here, we used genome-wide DNA methylation microarrays to identify epigenetic changes during human embryonic stem cell (hESC) differentiation. We discovered a group of CGIs associated with developmental genes that gain methylation after hESCs differentiate. Conversely, erasure of methylation was observed at the identified CGIs during subsequent reprogramming to induced pluripotent stem cells (iPSCs), further supporting a functional role for the CGI methylation. Both global gene expression profiling and quantitative reverse transcription-PCR (RT-PCR) validation indicated opposing effects of CGI methylation in transcriptional regulation during differentiation, with promoter CGI methylation repressing and 3′ CGI methylation activating transcription. By studying diverse human tissues and mouse models, we further confirmed that developmentally programmed 3′ CGI methylation confers tissue- and cell-type-specific gene activation in vivo. Importantly, luciferase reporter assays provided evidence that 3′ CGI methylation regulates transcriptional activation via a CTCF-dependent enhancer-blocking mechanism. These findings expand the classic view of mammalian CGI methylation as a mechanism for transcriptional silencing and indicate a functional role for 3′ CGI methylation in developmental gene regulation. PMID:23459939

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.

Clr4 specificity and catalytic activity beyond H3K9 methylation.

PubMed

Kusevic, Denis; Kudithipudi, Srikanth; Iglesias, Nahid; Moazed, Danesh; Jeltsch, Albert

2017-04-01

In fission yeast, the catalytic activity of the protein lysine methyltransferase (PKMT) Clr4, the sole homolog of the mammalian SUV39H1 and SUV39H2 enzymes, majorly contributes to the formation of heterochromatin. The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a central heterochromatic histone modification, and later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well. Herein, we have investigated the substrate specificity of Clr4 using custom made mutational scanning peptide arrays. Our data show, that Clr4 recognizes an RK core motif, showing high preference for R8. In addition, it exhibits specific contacts at the S10, T11, G12 and G13 positions of the H3 peptide recognizing an R-K-SKRT-TCS-G sequence. Based on the specificity profile and in vitro methyltransferase assay targeted searches, 11 putative methylation sites in S. pombe proteins were identified from reported Clr4 interacting proteins including Mlo3. Peptide methylation was observed on Mlo3 and 7 novel target sites with strongest methylation signals on Spbc28F2.11 (HMG box-containing protein) at lysine 292 and Hrp3 (Chromodomain ATP-dep DNA helicase) at lysine 89. These data suggest that Clr4 has additional methylation substrates and it will be important to study the biological function of these novel methylation events. Furthermore, the specificity profile of Clr4 has been used to develop a quantitative method to compare and cluster specificity profiles of PKMTs. It shows that the specificity profile of Clr4 is most similar to that of the SUV39H2 enzyme, one of its human homologs. This approach will be helpful in the comparison of the recognition profiles of other families of PKMTs as well. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Fatty acid methyl ester analysis to identify sources of soil in surface water.

PubMed

Banowetz, Gary M; Whittaker, Gerald W; Dierksen, Karen P; Azevedo, Mark D; Kennedy, Ann C; Griffith, Stephen M; Steiner, Jeffrey J

2006-01-01

Efforts to improve land-use practices to prevent contamination of surface waters with soil are limited by an inability to identify the primary sources of soil present in these waters. We evaluated the utility of fatty acid methyl ester (FAME) profiles of dry reference soils for multivariate statistical classification of soils collected from surface waters adjacent to agricultural production fields and a wooded riparian zone. Trials that compared approaches to concentrate soil from surface water showed that aluminum sulfate precipitation provided comparable yields to that obtained by vacuum filtration and was more suitable for handling large numbers of samples. Fatty acid methyl ester profiles were developed from reference soils collected from contrasting land uses in different seasons to determine whether specific fatty acids would consistently serve as variables in multivariate statistical analyses to permit reliable classification of soils. We used a Bayesian method and an independent iterative process to select appropriate fatty acids and found that variable selection was strongly impacted by the season during which soil was collected. The apparent seasonal variation in the occurrence of marker fatty acids in FAME profiles from reference soils prevented preparation of a standardized set of variables. Nevertheless, accurate classification of soil in surface water was achieved utilizing fatty acid variables identified in seasonally matched reference soils. Correlation analysis of entire chromatograms and subsequent discriminant analyses utilizing a restricted number of fatty acid variables showed that FAME profiles of soils exposed to the aquatic environment still had utility for classification at least 1 wk after submersion.

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.

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

Aroma profile and volatiles odor activity along gold cultivar pineapple flesh.

PubMed

Montero-Calderón, Marta; Rojas-Graü, María Alejandra; Martín-Belloso, Olga

2010-01-01

Physicochemical attributes, aroma profile, and odor contribution of pineapple flesh were studied for the top, middle, and bottom cross-sections cut along the central axis of Gold cultivar pineapple. Relationships between volatile and nonvolatile compounds were also studied. Aroma profile constituents were determined by headspace solid-phase microextraction at 30 °C, followed by gas chromatography/mass spectrometry analysis. A total of 20 volatile compounds were identified and quantified. Among them, esters were the major components which accounted for 90% of total extracted aroma. Methyl butanoate, methyl 2-methyl butanoate, and methyl hexanoate were the 3 most abundant components representing 74% of total volatiles in pineapple samples. Most odor active contributors were methyl and ethyl 2-methyl butanoate and 2,5-dimethyl 4-methoxy 3(2H)-furanone (mesifuran). Aroma profile components did not vary along the fruit, but volatile compounds content significantly varied (P < 0.05) along the fruit, from 7560 to 10910 μg/kg, from the top to the bottom cross-sections of the fruit, respectively. In addition, most odor-active volatiles concentration increased from the top to the bottom 3rd of the fruit, concurrently with soluble solids content (SSC) and titratable acidity (TA) differences attributed to fruitlets distinct degree of ripening. Large changes in SSC/TA ratio and volatiles content throughout the fruit found through this study are likely to provoke important differences among individual fresh-cut pineapple trays, compromising consumer perception and acceptance of the product. Such finding highlighted the need to include volatiles content and SSC/TA ratio and their variability along the fruit as selection criteria for pineapples to be processed and quality assessment of the fresh-cut fruit. © 2010 Institute of Food Technologists®

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

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

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.

Integrated analysis of dynamic FET PET/CT parameters, histology, and methylation profiling of 44 gliomas.

PubMed

Röhrich, Manuel; Huang, Kristin; Schrimpf, Daniel; Albert, Nathalie L; Hielscher, Thomas; von Deimling, Andreas; Schüller, Ulrich; Dimitrakopoulou-Strauss, Antonia; Haberkorn, Uwe

2018-05-07

Dynamic 18 F-FET PET/CT is a powerful tool for the diagnosis of gliomas. 18 F-FET PET time-activity curves (TAC) allow differentiation between histological low-grade gliomas (LGG) and high-grade gliomas (HGG). Molecular methods such as epigenetic profiling are of rising importance for glioma grading and subclassification. Here, we analysed dynamic 18 F-FET PET data, and the histological and epigenetic features of 44 gliomas. Dynamic 18 F-FET PET was performed in 44 patients with newly diagnosed, untreated glioma: 10 WHO grade II glioma, 13 WHO grade III glioma and 21 glioblastoma (GBM). All patients underwent stereotactic biopsy or tumour resection after 18 F-FET PET imaging. As well as histological analysis of tissue samples, DNA was subjected to epigenetic analysis using the Illumina 850 K methylation array. TACs, standardized uptake values corrected for background uptake in healthy tissue (SUVmax/BG), time to peak (TTP) and kinetic modelling parameters were correlated with histological diagnoses and with epigenetic signatures. Multivariate analyses were performed to evaluate the diagnostic accuracy of 18 F-FET PET in relation to the tumour groups identified by histological and methylation-based analysis. Epigenetic profiling led to substantial tumour reclassification, with six grade II/III gliomas reclassified as GBM. Overlap of HGG-typical TACs and LGG-typical TACs was dramatically reduced when tumours were clustered on the basis of their methylation profile. SUVmax/BG values of GBM were higher than those of LGGs following both histological diagnosis and methylation-based diagnosis. The differences in TTP between GBMs and grade II/III gliomas were greater following methylation-based diagnosis than following histological diagnosis. Kinetic modeling showed that relative K1 and fractal dimension (FD) values significantly differed in histology- and methylation-based GBM and grade II/III glioma between those diagnosed histologically and those diagnosed by methylation analysis. Multivariate analysis revealed slightly greater diagnostic accuracy with methylation-based diagnosis. IDH-mutant gliomas and GBM subgroups tended to differ in their 18 F-FET PET kinetics. The status of dynamic 18 F-FET PET as a biologically and clinically relevant imaging modality is confirmed in the context of molecular glioma diagnosis.

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

Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer

PubMed Central

Shen, Lanlan; Toyota, Minoru; Kondo, Yutaka; Lin, E; Zhang, Li; Guo, Yi; Hernandez, Natalie Supunpong; Chen, Xinli; Ahmed, Saira; Konishi, Kazuo; Hamilton, Stanley R.; Issa, Jean-Pierre J.

2007-01-01

Colon cancer has been viewed as the result of progressive accumulation of genetic and epigenetic abnormalities. However, this view does not fully reflect the molecular heterogeneity of the disease. We have analyzed both genetic (mutations of BRAF, KRAS, and p53 and microsatellite instability) and epigenetic alterations (DNA methylation of 27 CpG island promoter regions) in 97 primary colorectal cancer patients. Two clustering analyses on the basis of either epigenetic profiling or a combination of genetic and epigenetic profiling were performed to identify subclasses with distinct molecular signatures. Unsupervised hierarchical clustering of the DNA methylation data identified three distinct groups of colon cancers named CpG island methylator phenotype (CIMP) 1, CIMP2, and CIMP negative. Genetically, these three groups correspond to very distinct profiles. CIMP1 are characterized by MSI (80%) and BRAF mutations (53%) and rare KRAS and p53 mutations (16% and 11%, respectively). CIMP2 is associated with 92% KRAS mutations and rare MSI, BRAF, or p53 mutations (0, 4, and 31% respectively). CIMP-negative cases have a high rate of p53 mutations (71%) and lower rates of MSI (12%) or mutations of BRAF (2%) or KRAS (33%). Clustering based on both genetic and epigenetic parameters also identifies three distinct (and homogeneous) groups that largely overlap with the previous classification. The three groups are independent of age, gender, or stage, but CIMP1 and 2 are more common in proximal tumors. Together, our integrated genetic and epigenetic analysis reveals that colon cancers correspond to three molecularly distinct subclasses of disease. PMID:18003927

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

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

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

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.

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

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

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

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.

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.

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

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.

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.

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

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

PubMed

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

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

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.

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

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

Promoter Hypermethylation Profiling Identifies Subtypes of Head and Neck Cancer with Distinct Viral, Environmental, Genetic and Survival Characteristics

PubMed Central

Choudhury, Javed Hussain; Ghosh, Sankar Kumar

2015-01-01

Background Epigenetic and genetic alteration plays a major role to the development of head and neck squamous cell carcinoma (HNSCC). Consumption of tobacco (smoking/chewing) and human papilloma virus (HPV) are also associated with an increase the risk of HNSCC. Promoter hypermethylation of the tumor suppression genes is related with transcriptional inactivation and loss of gene expression. We investigated epigenetic alteration (promoter methylation of tumor-related genes/loci) in tumor tissues in the context of genetic alteration, viral infection, and tobacco exposure and survival status. Methodology The study included 116 tissue samples (71 tumor and 45 normal tissues) from the Northeast Indian population. Methylation specific polymerase chain reaction (MSP) was used to determine the methylation status of 10 tumor-related genes/loci (p16, DAPK, RASSF1, BRAC1, GSTP1, ECAD, MLH1, MINT1, MINT2 and MINT31). Polymorphisms of CYP1A1, GST (M1 & T1), XRCC1and XRCC2 genes were studied by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex-PCR respectively. Principal Findings Unsupervised hierarchical clustering analysis based on methylation pattern had identified two tumor clusters, which significantly differ by CpG island methylator phenotype (CIMP), tobacco, GSTM1, CYP1A1, HPV and survival status. Analyzing methylation of genes/loci individually, we have found significant higher methylation of DAPK, RASSF1, p16 and MINT31genes (P = 0.031, 0.013, 0.031 and 0.015 respectively) in HPV (+) cases compared to HPV (-). Furthermore, a CIMP-high and Cluster-1 characteristic was also associated with poor survival. Conclusions Promoter methylation profiles reflecting a correlation with tobacco, HPV, survival status and genetic alteration and may act as a marker to determine subtypes and patient outcome in HNSCC. PMID:26098903

A genome-wide methylation study on obesity: differential variability and differential methylation.

PubMed

Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

2013-05-01

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

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.

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.

Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis.

PubMed

Liang, Si-Cheng; Xia, Yang-Liu; Hou, Jie; Ge, Guang-Bo; Zhang, Jiang-Wei; He, Yu-Qi; Wang, Jia-Yue; Qi, Xiao-Yi; Yang, Ling

2016-02-01

Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

UV action spectroscopy of protonated PAH derivatives. Methyl substituted quinolines

NASA Astrophysics Data System (ADS)

Klærke, B.; Holm, A. I. S.; Andersen, L. H.

2011-08-01

Aims: We investigate the production of molecular photofragments upon UV excitation of PAH derivatives, relevant for the interstellar medium. Methods: The action absorption spectra of protonated gas-phase methyl-substituted quinolines (CH3 - C9H7NH+) have been recorded in the 215-338 nm spectral range using the electrostatic storage ring ELISA, an electrospray ion source and 3 ns UV laser pulses. Results: It is shown that the absorption profile is both redshifted and broadened when moving the methyl group from the heterocycle containing nitrogen to the homoatomic ring. The absorption profiles are explained by TD-DFT calculations. The dissociation time of the studied molecules is found to be of several milliseconds at 230 nm and it is shown that after redistribution of the absorbed energy the molecules dissociate in several channels. The dissociation time found is an order of magnitude faster than the estimated IR relaxation time. Photophysical properties of both nitrogen containing and methyl-substituted PAHs are interesting in an astrophysical context in connection with identifying the aromatic component of the interstellar medium.

MethylMeter®: bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples

PubMed Central

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-01-01

Aim: Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Materials & methods: Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter®. Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. Results: MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. Conclusion: MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas. PMID:27337298

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.

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.

MGMT-STP27 methylation status as predictive marker for response to PCV in anaplastic Oligodendrogliomas and Oligoastrocytomas. A report from EORTC study 26951.

PubMed

van den Bent, Martin J; Erdem-Eraslan, Lale; Idbaih, Ahmed; de Rooi, Johan; Eilers, Paul H C; Spliet, Wim G M; den Dunnen, Wilfred F A; Tijssen, Cees; Wesseling, Pieter; Sillevis Smitt, Peter A E; Kros, Johan M; Gorlia, Thierry; French, Pim J

2013-10-01

The long-term follow-up results from the EORTC-26951 trial showed that the addition of procarbazine, CCNU, and vincristine (PCV) after radiotherapy increases survival in anaplastic oligodendrogliomas/oligoastrocytomas (AOD/AOA). However, some patients appeared to benefit more from PCV treatment than others. We conducted genome-wide methylation profiling of 115 samples included in the EORTC-26951 trial and extracted the CpG island hypermethylated phenotype (CIMP) and MGMT promoter methylation (MGMT-STP27) status. We first show that methylation profiling can be conducted on archival tissues with a performance that is similar to snap-frozen tissue samples. We then conducted methylation profiling on EORTC-26951 clinical trial samples. Univariate analysis indicated that CIMP+ or MGMT-STP27 methylated tumors had an improved survival compared with CIMP- and/or MGMT-STP27 unmethylated tumors [median overall survival (OS), 1.05 vs. 6.46 years and 1.06 vs. 3.8 years, both P < 0.0001 for CIMP and MGMT-STP27 status, respectively]. Multivariable analysis indicates that CIMP and MGMT-STP27 are significant prognostic factors for survival in presence of age, sex, performance score, and review diagnosis in the model. CIMP+ and MGMT-STP27 methylated tumors showed a clear benefit from adjuvant PCV chemotherapy: the median OS of CIMP+ samples in the RT and RT-PCV arms was 3.27 and 9.51 years, respectively (P = 0.0033); for MGMT-STP27 methylated samples, it was 1.98 and 8.65 years. There was no such benefit for CIMP- or for MGMT-STP27 unmethylated tumors. MGMT-STP27 status remained significant in an interaction test (P = 0.003). Statistical analysis of microarray (SAM) identified 259 novel CpGs associated with treatment response. MGMT-STP27 may be used to guide treatment decisions in this tumor type. ©2013 AACR.

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 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.

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.

Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability.

PubMed

Min, Kyung-Won; Zealy, Richard W; Davila, Sylvia; Fomin, Mikhail; Cummings, James C; Makowsky, Daniel; Mcdowell, Catherine H; Thigpen, Haley; Hafner, Markus; Kwon, Sang-Ho; Georgescu, Constantin; Wren, Jonathan D; Yoon, Je-Hyun

2018-06-01

Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post-transcription, and post-translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6-methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A-modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady-state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady-state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2-depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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

Identification of diagnostic markers in colorectal cancer via integrative epigenomics and genomics data

PubMed Central

KOK-SIN, TEOW; MOKHTAR, NORFILZA MOHD; HASSAN, NUR ZARINA ALI; SAGAP, ISMAIL; ROSE, ISA MOHAMED; HARUN, ROSLAN; JAMAL, RAHMAN

2015-01-01

Apart from genetic mutations, epigenetic alteration is a common phenomenon that contributes to neoplastic transformation in colorectal cancer. Transcriptional silencing of tumor-suppressor genes without changes in the DNA sequence is explained by the existence of promoter hypermethylation. To test this hypothesis, we integrated the epigenome and transcriptome data from a similar set of colorectal tissue samples. Methylation profiling was performed using the Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent normal epithelial cells. Fifteen of the 55 paired tissues were used for gene expression profiling using the Affymetrix GeneChip Human Gene 1.0 ST array. Validation was carried out on 150 colorectal tissues using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique. PCA and supervised hierarchical clustering in the two microarray datasets showed good separation between cancer and normal samples. Significant genes from the two analyses were obtained based on a ≥2-fold change and a false discovery rate (FDR) P-value of <0.05. We identified 1,081 differentially hypermethylated CpG sites and 36 hypomethylated CpG sites. We also found 709 upregulated and 699 downregulated genes from the gene expression profiling. A comparison of the two datasets revealed 32 overlapping genes with 27 being hypermethylated with downregulated expression and 4 hypermethylated with upregulated expression. One gene was found to be hypomethylated and downregulated. The most enriched molecular pathway identified was cell adhesion molecules that involved 4 overlapped genes, JAM2, NCAM1, ITGA8 and CNTN1. In the present study, we successfully identified a group of genes that showed methylation and gene expression changes in well-defined colorectal cancer tissues with high purity. The integrated analysis gives additional insight regarding the regulation of colorectal cancer-associated genes and their underlying mechanisms that contribute to colorectal carcinogenesis. PMID:25997610

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

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.

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

Integrated Genomic and Epigenomic Analysis of Breast Cancer Brain Metastasis

PubMed Central

Salhia, Bodour; Kiefer, Jeff; Ross, Julianna T. D.; Metapally, Raghu; Martinez, Rae Anne; Johnson, Kyle N.; DiPerna, Danielle M.; Paquette, Kimberly M.; Jung, Sungwon; Nasser, Sara; Wallstrom, Garrick; Tembe, Waibhav; Baker, Angela; Carpten, John; Resau, Jim; Ryken, Timothy; Sibenaller, Zita; Petricoin, Emanuel F.; Liotta, Lance A.; Ramanathan, Ramesh K.; Berens, Michael E.; Tran, Nhan L.

2014-01-01

The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies. PMID:24489661

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 .

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.

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.

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.

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

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.

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.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling.

PubMed

Martín-Subero, José I; Kreuz, Markus; Bibikova, Marina; Bentink, Stefan; Ammerpohl, Ole; Wickham-Garcia, Eliza; Rosolowski, Maciej; Richter, Julia; Lopez-Serra, Lidia; Ballestar, Esteban; Berger, Hilmar; Agirre, Xabier; Bernd, Heinz-Wolfram; Calvanese, Vincenzo; Cogliatti, Sergio B; Drexler, Hans G; Fan, Jian-Bing; Fraga, Mario F; Hansmann, Martin L; Hummel, Michael; Klapper, Wolfram; Korn, Bernhard; Küppers, Ralf; Macleod, Roderick A F; Möller, Peter; Ott, German; Pott, Christiane; Prosper, Felipe; Rosenwald, Andreas; Schwaenen, Carsten; Schübeler, Dirk; Seifert, Marc; Stürzenhofecker, Benjamin; Weber, Michael; Wessendorf, Swen; Loeffler, Markus; Trümper, Lorenz; Stein, Harald; Spang, Rainer; Esteller, Manel; Barker, David; Hasenclever, Dirk; Siebert, Reiner

2009-03-12

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

Toward a comprehensive and systematic methylome signature in colorectal cancers.

PubMed

Ashktorab, Hassan; Rahi, Hamed; Wansley, Daniel; Varma, Sudhir; Shokrani, Babak; Lee, Edward; Daremipouran, Mohammad; Laiyemo, Adeyinka; Goel, Ajay; Carethers, John M; Brim, Hassan

2013-08-01

CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes' list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

PubMed Central

Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-01-01

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma. PMID:25971279

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

PubMed

Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-07-20

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

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.

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.

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

PubMed

Li, Zibo; Heng, Jianfu; Yan, Jinhua; Guo, Xinwu; Tang, Lili; Chen, Ming; Peng, Limin; Wu, Yepeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Wang, Jun

2016-11-01

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

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.

MLH1 Region Polymorphisms Show a Significant Association with CpG Island Shore Methylation in a Large Cohort of Healthy Individuals

PubMed Central

Savio, Andrea J.; Lemire, Mathieu; Mrkonjic, Miralem; Gallinger, Steven; Zanke, Brent W.; Hudson, Thomas J.; Bapat, Bharati

2012-01-01

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We previously demonstrated that SNPs (rs1800734, rs749072, and rs13098279) in the MLH1 gene region are associated with MLH1 promoter island methylation, loss of MLH1 protein expression, and microsatellite instability (MSI) in colorectal cancer (CRC) patients. Recent studies have identified less CpG-dense “shore” regions flanking many CpG islands. These shores often exhibit distinct methylation profiles between different tissues and matched normal versus tumor cells of patients. To date, most epigenetic studies have focused on somatic methylation events occurring within solid tumors; less is known of the contributions of peripheral blood cell (PBC) methylation to processes such as aging and tumorigenesis. To address whether MLH1 methylation in PBCs is correlated with tumorigenesis we utilized the Illumina 450 K microarrays to measure methylation in PBC DNA of 846 healthy controls and 252 CRC patients from Ontario, Canada. Analysis of a region of chromosome 3p21 spanning the MLH1 locus in healthy controls revealed that a CpG island shore 1 kb upstream of the MLH1 gene exhibits different methylation profiles when stratified by SNP genotypes (rs1800734, rs749072, and rs13098279). Individuals with wild-type genotypes incur significantly higher PBC shore methylation than heterozygous or homozygous variant carriers (p<1.1×10−6; ANOVA). This trend is also seen in CRC cases (p<0.096; ANOVA). Shore methylation also decreases significantly with increasing age in cases and controls. This is the first study of its kind to integrate PBC methylation at a CpG island shore with SNP genotype status in CRC cases and controls. These results indicate that CpG island shore methylation in PBCs may be influenced by genotype as well as the normal aging process. PMID:23240038

Genome-Wide RNA Polymerase II Profiles and RNA Accumulation Reveal Kinetics of Transcription and Associated Epigenetic Changes During Diurnal Cycles

PubMed Central

Gilardi, Federica; Liechti, Robin; Martin, Olivier; Harshman, Keith; Delorenzi, Mauro; Desvergne, Béatrice; Herr, Winship; Deplancke, Bart; Schibler, Ueli; Rougemont, Jacques; Guex, Nicolas; Hernandez, Nouria; Naef, Felix

2012-01-01

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver. PMID:23209382

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.

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

PubMed

Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y

2015-11-26

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

Biomarkers of Fatigue: Metabolomics Profiles Predictive of Cognitive Performance

DTIC Science & Technology

2013-05-01

metabolites. The latest version of the Human Metabolome Database (v. 2.5; released August , 2009) includes approximately 8,000 identified mammalian...monoamine oxidase; COMT , catechol-O-methyl transferase. (Modiefied from Rubí and Maechler, 2010). Ovals indicate metabolites found to be significantly

Methylomic profiling of cortex samples from completed suicide cases implicates a role for PSORS1C3 in major depression and suicide.

PubMed

Murphy, T M; Crawford, B; Dempster, E L; Hannon, E; Burrage, J; Turecki, G; Kaminsky, Z; Mill, J

2017-01-03

Major depressive disorder (MDD) represents a major social and economic health issue and constitutes a major risk factor for suicide. The molecular pathology of suicidal depression remains poorly understood, although it has been hypothesised that regulatory genomic processes are involved in the pathology of both MDD and suicidality. In this study, genome-wide patterns of DNA methylation were assessed in depressed suicide completers (n=20) and compared with non-psychiatric, sudden-death controls (n=20) using tissue from two cortical brain regions (Brodmann Area 11 (BA11) and Brodmann Area 25 (BA25)). Analyses focused on identifying differentially methylated regions (DMRs) associated with suicidal depression and epigenetic variation were explored in the context of polygenic risk scores for major depression and suicide. Weighted gene co-methylation network analysis was used to identify modules of co-methylated loci associated with depressed suicide completers and polygenic burden for MDD and suicide attempt. We identified a DMR upstream of the PSORS1C3 gene, subsequently validated using bisulfite pyrosequencing and replicated in a second set of suicide samples, which is characterised by significant hypomethylation in both cortical brain regions in MDD suicide cases. We also identified discrete modules of co-methylated loci associated with polygenic risk burden for suicide attempt, but not major depression. Suicide-associated co-methylation modules were enriched among gene networks implicating biological processes relevant to depression and suicidality, including nervous system development and mitochondria function. Our data suggest that there are coordinated changes in DNA methylation associated with suicide that may offer novel insights into the molecular pathology associated with depressed suicide completers.

Methylomic profiling of cortex samples from completed suicide cases implicates a role for PSORS1C3 in major depression and suicide

PubMed Central

Murphy, T M; Crawford, B; Dempster, E L; Hannon, E; Burrage, J; Turecki, G; Kaminsky, Z; Mill, J

2017-01-01

Major depressive disorder (MDD) represents a major social and economic health issue and constitutes a major risk factor for suicide. The molecular pathology of suicidal depression remains poorly understood, although it has been hypothesised that regulatory genomic processes are involved in the pathology of both MDD and suicidality. In this study, genome-wide patterns of DNA methylation were assessed in depressed suicide completers (n=20) and compared with non-psychiatric, sudden-death controls (n=20) using tissue from two cortical brain regions (Brodmann Area 11 (BA11) and Brodmann Area 25 (BA25)). Analyses focused on identifying differentially methylated regions (DMRs) associated with suicidal depression and epigenetic variation were explored in the context of polygenic risk scores for major depression and suicide. Weighted gene co-methylation network analysis was used to identify modules of co-methylated loci associated with depressed suicide completers and polygenic burden for MDD and suicide attempt. We identified a DMR upstream of the PSORS1C3 gene, subsequently validated using bisulfite pyrosequencing and replicated in a second set of suicide samples, which is characterised by significant hypomethylation in both cortical brain regions in MDD suicide cases. We also identified discrete modules of co-methylated loci associated with polygenic risk burden for suicide attempt, but not major depression. Suicide-associated co-methylation modules were enriched among gene networks implicating biological processes relevant to depression and suicidality, including nervous system development and mitochondria function. Our data suggest that there are coordinated changes in DNA methylation associated with suicide that may offer novel insights into the molecular pathology associated with depressed suicide completers. PMID:28045465

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.

Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage.

PubMed

Taguchi, Y-h

2015-01-01

Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study.

Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage

PubMed Central

2015-01-01

Background Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. Methods This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. Results The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Conclusions Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study. PMID:26677731

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.

Region-specific RNA m6A methylation represents a new layer of control in the gene regulatory network in the mouse brain.

PubMed

Chang, Mengqi; Lv, Hongyi; Zhang, Weilong; Ma, Chunhui; He, Xue; Zhao, Shunli; Zhang, Zhi-Wei; Zeng, Yi-Xin; Song, Shuhui; Niu, Yamei; Tong, Wei-Min

2017-09-01

N 6 -methyladenosine (m 6 A) is the most abundant epitranscriptomic mark found on mRNA and has important roles in various physiological processes. Despite the relatively high m 6 A levels in the brain, its potential functions in the brain remain largely unexplored. We performed a transcriptome-wide methylation analysis using the mouse brain to depict its region-specific methylation profile. RNA methylation levels in mouse cerebellum are generally higher than those in the cerebral cortex. Heterogeneity of RNA methylation exists across different brain regions and different types of neural cells including the mRNAs to be methylated, their methylation levels and methylation site selection. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. In addition, high methylation levels of fragile X mental retardation protein (FMRP) target mRNAs suggest that m 6 A methylation is likely to be used for selective recognition of target mRNAs by FMRP in the synapse. Overall, we provide a region-specific map of RNA m 6 A methylation and characterize the distinct features of specific and common methylation in mouse cerebellum and cerebral cortex. Our results imply that RNA m 6 A methylation is a newly identified element in the region-specific gene regulatory network in the mouse brain. © 2017 The Authors.

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

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.

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

Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development.

PubMed

Ikegami, Kohta; Ohgane, Jun; Tanaka, Satoshi; Yagi, Shintaro; Shiota, Kunio

2009-01-01

Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.

Hypoxia reduces the E-cadherin expression and increases OSCC cell migration regardless of the E-cadherin methylation profile.

PubMed

Domingos, Patrícia Luciana Batista; Souza, Marcela Gonçalves; Guimarães, Talita Antunes; Santos, Eliane Sobrinho; Farias, Lucyana Conceição; de Carvalho Fraga, Carlos Alberto; Jones, Kimberly Marie; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena

2017-05-01

The purpose of the current study is to investigate the association between E-cadherin methylation status, hypoxia and OSCC. HaCat and SCC9 cell lines were submitted to hypoxic treatment, followed by methylation profile analysis (MS-PCR) and analysis of the expression of mRNA gene E-cadherin (RT-PCR). Study group samples comprise individuals affected by potentially malignant lesions Potential Malignant Oral Lesion (PMOL, n=18) and oral squamous cell carcinoma (OSCC, n=28). The control group oral mucosa (OM, n=15) of patients with an oral mucocele. Cell migration ability was evaluated a scratch wound assay in SCC9 and HaCat cell lines RESULTS: E-cadherin mRNA expression in the cell lines SCC9 and HaCat was significantly reduced under hypoxia, regardless of the methylation profile, when compared to the control group. No differences in methylation profile of the E-cadherin were observed among the groups OM, PMOL and OSCC. HaCat and SCC9 presented increases in cell migration rates under hypoxia. The current study demonstrates that hypoxia reduces E-cadherin expression and increase cell migration, regardless of the methylation profile. Additionally, no differences in E-cadherin methylation patterns were observed among OM, PMOL and OSCC. Copyright © 2017 Elsevier GmbH. All rights reserved.

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.

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.

Discovery and validation of methylation markers for endometrial cancer

PubMed Central

Wentzensen, Nicolas; Bakkum-Gamez, Jamie N.; Killian, J. Keith; Sampson, Joshua; Guido, Richard; Glass, Andrew; Adams, Lisa; Luhn, Patricia; Brinton, Louise A.; Rush, Brenda; d’Ambrosio, Lori; Gunja, Munira; Yang, Hannah P.; Garcia-Closas, Montserrat; Lacey, James V.; Lissowska, Jolanta; Podratz, Karl; Meltzer, Paul; Shridhar, Viji; Sherman, Mark E.

2014-01-01

The prognosis of endometrial cancer is strongly associated with stage at diagnosis, suggesting that early detection may reduce mortality. Women who are diagnosed with endometrial carcinoma often have a lengthy history of vaginal bleeding, which offers an opportunity for early diagnosis and curative treatment. We performed DNA methylation profiling on population-based endometrial cancers to identify early detection biomarkers and replicated top candidates in two independent studies. We compared DNA methylation values of 1500 probes representing 807 genes in 148 population-based endometrial carcinoma samples and 23 benign endometrial tissues. Markers were replicated in another set of 69 carcinomas and 40 benign tissues profiled on the same platform. Further replication was conducted in The Cancer Genome Atlas and in prospectively collected endometrial brushings from women with and without endometrial carcinomas. We identified 114 CpG sites showing methylation differences with p-values of ≤10−7 between endometrial carcinoma and normal endometrium. Eight genes (ADCYAP1, ASCL2, HS3ST2, HTR1B, MME, NPY, and SOX1) were selected for further replication. Age-adjusted odds ratios for endometrial cancer ranged from 3.44 (95%-CI: 1.33–8.91) for ASCL2 to 18.61 (95%-CI: 5.50–62.97) for HTR1B. An area under the curve (AUC) of 0.93 was achieved for discriminating carcinoma from benign endometrium. Replication in The Cancer Genome Atlas and in endometrial brushings from an independent study confirmed the candidate markers. This study demonstrates that methylation markers may be used to evaluate women with abnormal vaginal bleeding to distinguish women with endometrial carcinoma from the majority of women without malignancy. PMID:24623538

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols

PubMed Central

Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λadjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12–1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λadjusted = 1.00–1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models. PMID:26799745

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

PubMed

Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

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.

Antioxidant capacity and chemical profiles of Satureja montana L. Honey: hotrienol and syringyl derivatives as biomarkers.

PubMed

Jerković, Igor; Tuberoso, Carlo I G; Marijanović, Zvonimir; Kranjac, Marina; Malenica-Staver, M

2015-07-01

The present study is focused on the antioxidant capacity and chemical profiling of eight Croatian Satureja montana L. honey samples. Among the 20 compounds obtained by headspace solid-phase microextraction (HS-SPME) and identified by GC-FID and GC/MS analyses, hotrienol was predominant (75.9-81.7%). The honey matrix volatile/semivolatile profile was investigated by ultrasonic solvent extraction (USE) followed by GC-FID and GC/MS analyses. The major compounds identified by this latter method were the sinapic-acid derivatives methyl syringate (36.2-72.8%) and syringaldehyde (2.2-43.1%). Direct, targeted HPLC-DAD analyses of the native honey samples revealed the presence of methyl syringate (7.10-39.60 mg/kg) and syringic acid (0.10-1.70 mg/kg). In addition, the total phenolic content of the samples was determined by the FolinCiocalteu assay (311.0-465.9 mg GAE/kg), and the antioxidant capacity was evaluated by the DPPH radical-scavenging activity (0.5-1.0 mmol TEAC/kg) and the ferric reducing antioxidant power (2.5-5.1 mmol Fe(2+) /kg). Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

PubMed Central

Down, Thomas A.; Rakyan, Vardhman K.; Turner, Daniel J.; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M.; Thorne, Natalie P.; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L.; Jackson, David K.; Miretti, Marcos M.; Marioni, John C.; Birney, Ewan; Hubbard, Tim J. P.; Durbin, Richard; Tavaré, Simon; Beck, Stephan

2009-01-01

DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation. PMID:18612301

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

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.

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

USDA-ARS?s Scientific Manuscript database

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylat...

CpG island methylation profile in non-invasive oral rinse samples is predictive of oral and pharyngeal carcinoma.

PubMed

Langevin, Scott M; Eliot, Melissa; Butler, Rondi A; Cheong, Agnes; Zhang, Xiang; McClean, Michael D; Koestler, Devin C; Kelsey, Karl T

2015-01-01

There are currently no screening tests in routine use for oral and pharyngeal cancer beyond visual inspection and palpation, which are provided on an opportunistic basis, indicating a need for development of novel methods for early detection, particularly in high-risk populations. We sought to address this need through comprehensive interrogation of CpG island methylation in oral rinse samples. We used the Infinium HumanMethylation450 BeadArray to interrogate DNA methylation in oral rinse samples collected from 154 patients with incident oral or pharyngeal carcinoma prior to treatment and 72 cancer-free control subjects. Subjects were randomly allocated to either a training or a testing set. For each subject, average methylation was calculated for each CpG island represented on the array. We applied a semi-supervised recursively partitioned mixture model to the CpG island methylation data to identify a classifier for prediction of case status in the training set. We then applied the resultant classifier to the testing set for validation and to assess the predictive accuracy. We identified a methylation classifier comprised of 22 CpG islands, which predicted oral and pharyngeal carcinoma with a high degree of accuracy (AUC = 0.92, 95 % CI 0.86, 0.98). This novel methylation panel is a strong predictor of oral and pharyngeal carcinoma case status in oral rinse samples and may have utility in early detection and post-treatment follow-up.

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 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.

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

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

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.

Arachidonic and oleic acid exert distinct effects on the DNA methylome

PubMed Central

Silva-Martínez, Guillermo A.; Rodríguez-Ríos, Dalia; Alvarado-Caudillo, Yolanda; Vaquero, Alejandro; Esteller, Manel; Carmona, F. Javier; Moran, Sebastian; Nielsen, Finn C.; Wickström-Lindholm, Marie; Wrobel, Katarzyna; Wrobel, Kazimierz; Barbosa-Sabanero, Gloria; Zaina, Silvio; Lund, Gertrud

2016-01-01

ABSTRACT Abnormal fatty acid metabolism and availability are landmarks of metabolic diseases, which in turn are associated with aberrant DNA methylation profiles. To understand the role of fatty acids in disease epigenetics, we sought DNA methylation profiles specifically induced by arachidonic (AA) or oleic acid (OA) in cultured cells and compared those with published profiles of normal and diseased tissues. THP-1 monocytes were stimulated with AA or OA and analyzed using Infinium HumanMethylation450 BeadChip (Illumina) and Human Exon 1.0 ST array (Affymetrix). Data were corroborated in mouse embryonic fibroblasts. Comparisons with publicly available data were conducted by standard bioinformatics. AA and OA elicited a complex response marked by a general DNA hypermethylation and hypomethylation in the 1–200 μM range, respectively, with a maximal differential response at the 100 μM dose. The divergent response to AA and OA was prominent within the gene body of target genes, where it correlated positively with transcription. AA-induced DNA methylation profiles were similar to the corresponding profiles described for palmitic acid, atherosclerosis, diabetes, obesity, and autism, but relatively dissimilar from OA-induced profiles. Furthermore, human atherosclerosis grade-associated DNA methylation profiles were significantly enriched in AA-induced profiles. Biochemical evidence pointed to β-oxidation, PPAR-α, and sirtuin 1 as important mediators of AA-induced DNA methylation changes. In conclusion, AA and OA exert distinct effects on the DNA methylome. The observation that AA may contribute to shape the epigenome of important metabolic diseases, supports and expands current diet-based therapeutic and preventive efforts. PMID:27088456

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.

Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder.

PubMed

Saeliw, Thanit; Tangsuwansri, Chayanin; Thongkorn, Surangrat; Chonchaiya, Weerasak; Suphapeetiporn, Kanya; Mutirangura, Apiwat; Tencomnao, Tewin; Hu, Valerie W; Sarachana, Tewarit

2018-01-01

Alu elements are a group of repetitive elements that can influence gene expression through CpG residues and transcription factor binding. Altered gene expression and methylation profiles have been reported in various tissues and cell lines from individuals with autism spectrum disorder (ASD). However, the role of Alu elements in ASD remains unclear. We thus investigated whether Alu elements are associated with altered gene expression profiles in ASD. We obtained five blood-based gene expression profiles from the Gene Expression Omnibus database and human Alu-inserted gene lists from the TranspoGene database. Differentially expressed genes (DEGs) in ASD were identified from each study and overlapped with the human Alu-inserted genes. The biological functions and networks of Alu-inserted DEGs were then predicted by Ingenuity Pathway Analysis (IPA). A combined bisulfite restriction analysis of lymphoblastoid cell lines (LCLs) derived from 36 ASD and 20 sex- and age-matched unaffected individuals was performed to assess the global DNA methylation levels within Alu elements, and the Alu expression levels were determined by quantitative RT-PCR. In ASD blood or blood-derived cells, 320 Alu-inserted genes were reproducibly differentially expressed. Biological function and pathway analysis showed that these genes were significantly associated with neurodevelopmental disorders and neurological functions involved in ASD etiology. Interestingly, estrogen receptor and androgen signaling pathways implicated in the sex bias of ASD, as well as IL-6 signaling and neuroinflammation signaling pathways, were also highlighted. Alu methylation was not significantly different between the ASD and sex- and age-matched control groups. However, significantly altered Alu methylation patterns were observed in ASD cases sub-grouped based on Autism Diagnostic Interview-Revised scores compared with matched controls. Quantitative RT-PCR analysis of Alu expression also showed significant differences between ASD subgroups. Interestingly, Alu expression was correlated with methylation status in one phenotypic ASD subgroup. Alu methylation and expression were altered in LCLs from ASD subgroups. Our findings highlight the association of Alu elements with gene dysregulation in ASD blood samples and warrant further investigation. Moreover, the classification of ASD individuals into subgroups based on phenotypes may be beneficial and could provide insights into the still unknown etiology and the underlying mechanisms of ASD.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

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.

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.

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.

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.

DNA methylation profiles of long- and short-term glioblastoma survivors

PubMed Central

Shinawi, Thoraia; Hill, Victoria K.; Krex, Dietmar; Schackert, Gabriele; Gentle, Dean; Morris, Mark R.; Wei, Wenbin; Cruickshank, Garth; Maher, Eamonn R.; Latif, Farida

2013-01-01

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor in adults and prognosis of most GBM patients is poor. However, a small percentage of patients show a long term survival of 36 mo or longer after diagnosis. Epigenetic profiles can provide molecular markers for patient prognosis: recently, a G-CIMP positive phenotype associated with IDH1 mutations has been described for GBMs with good prognosis. In the present analysis we performed genome-wide DNA methylation profiling of short-term survivors (STS; overall survival < 1 y) and long-term survivors (LTS; overall survival > 3 y) by utilizing the HumanMethylation450K BeadChips to assess quantitative methylation at > 480,000 CpG sites. Cluster analysis has shown that a subset of LTS showed a G-CIMP positive phenotype that was tightly associated with IDH1 mutation status and was confirmed by analysis of the G-CIMP signature genes. Using high stringency criteria for differential hypermethylation between non-cancer brain and tumor samples, we identified 2,638 hypermethylated CpG loci (890 genes) in STS GBMs, 3,101 hypermethylated CpG loci (1,062 genes) in LTS (wild type IDH1) and 11,293 hypermethylated CpG loci in LTS (mutated for IDH1), reflecting the CIMP positive phenotype. The location of differentially hypermethylated CpG loci with respect to CpG content, neighborhood context and functional genomic distribution was similar in our sample set, with the majority of CpG loci residing in CpG islands and in gene promoters. Our preliminary study also identified a set of CpG loci differentially hypermethylated between STS and LTS cases, including members of the homeobox gene family (HOXD8, HOXD13 and HOXC4), the transcription factors NR2F2 and TFAP2A, and Dickkopf 2, a negative regulator of the wnt/β-catenin signaling pathway. PMID:23291739

Exhaustive methylation analysis revealed uneven profiles of methylation at IGF2/ICR1/H19 11p15 loci in Russell Silver syndrome.

PubMed

Azzi, Salah; Steunou, Virginie; Tost, Jörg; Rossignol, Sylvie; Thibaud, Nathalie; Das Neves, Cristina; Le Jule, Marilyne; Habib, Walid Abi; Blaise, Annick; Koudou, Yves; Busato, Florence; Le Bouc, Yves; Netchine, Irène

2015-01-01

The structural organisation of the human IGF2/ICR1/H19 11p15 domain is very complex, and the mechanisms underlying its regulation are poorly understood. The Imprinted Center Region 1 (ICR1) contains seven binding sites for the zinc-finger protein CTCF (CBS: CTCF Binding Sites); three additional differentially methylated regions (DMR) are located at the H19 promoter (H19DMR) and two in the IGF2 gene (DMR0 and DMR2), respectively. Loss of imprinting at the IGF2/ICR1/H19 domain results in two growth disorders with opposite phenotypes: Beckwith-Wiedemann syndrome and Russell Silver syndrome (RSS). Despite the IGF2/ICR1/H19 locus being widely studied, the extent of hypomethylation across the domain remains not yet addressed in patients with RSS. We assessed a detailed investigation of the methylation status of the 11p15 ICR1 CBS1-7, IGF2DMR0 and H19DMR (H19 promoter) in a population of controls (n=50) and RSS carrying (n=104) or not (n=65) carrying a hypomethylation at the 11p15 ICR1 region. The methylation indexes (MI) were balanced at all regions in the control population and patients with RSS without any as yet identified molecular anomaly. Interestingly, patients with RSS with ICR1 hypomethylation showed uneven profiles of methylation among the CBSs and DMRs. Furthermore, normal MIs at CBS1 and CBS7 were identified in 9% of patients. The hypomethylation does not spread equally throughout the IGF2/ICR1/H19 locus, and some loci could have normal MI, which may lead to underdiagnosis of patients with RSS with ICR1 hypomethylation. The uneven pattern of methylation suggests that some CBSs may play different roles in the tridimensional chromosomal looping regulation of this locus. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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.

Epigenetic Alteration by DNA Methylation of ESR1, MYOD1 and hTERT Gene Promoters is Useful for Prediction of Response in Patients of Locally Advanced Invasive Cervical Carcinoma Treated by Chemoradiation.

PubMed

Sood, S; Patel, F D; Ghosh, S; Arora, A; Dhaliwal, L K; Srinivasan, R

2015-12-01

Locally advanced invasive cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) IIB/III] is treated by chemoradiation. The response to treatment is variable within a given FIGO stage. Therefore, the aim of the present study was to evaluate the gene promoter methylation profile and corresponding transcript expression of a panel of six genes to identify genes which could predict the response of patients treated by chemoradiation. In total, 100 patients with invasive cervical cancer in FIGO stage IIB/III who underwent chemoradiation treatment were evaluated. Ten patients developed systemic metastases during therapy and were excluded. On the basis of patient follow-up, 69 patients were chemoradiation-sensitive, whereas 21 were chemoradiation-resistant. Gene promoter methylation and gene expression was determined by TaqMan assay and quantitative real-time PCR, respectively, in tissue samples. The methylation frequency of ESR1, BRCA1, RASSF1A, MLH1, MYOD1 and hTERT genes ranged from 40 to 70%. Univariate and hierarchical cluster analysis revealed that gene promoter methylation of MYOD1, ESR1 and hTERT could predict for chemoradiation response. A pattern of unmethylated MYOD1, unmethylated ESR1 and methylated hTERT promoter as well as lower ESR1 transcript levels predicted for chemoradiation resistance. Methylation profiling of a panel of three genes that includes MYOD1, ESR1 and hTERT may be useful to predict the response of invasive cervical carcinoma patients treated with standard chemoradiation therapy. Copyright © 2015 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

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

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.

Formation of complex natural flavours by biotransformation of apple pomace with basidiomycetes.

PubMed

Bosse, Andrea K; Fraatz, Marco A; Zorn, Holger

2013-12-01

Altogether 30 different basidiomycetes were grown submerged in liquid culture media using seven different by-products of the food industry as the only carbon source. Seven fungus/substrate combinations revealed interesting flavour profiles. Culture supernatants of Tyromyces chioneus grown on apple pomace were extracted, and the aroma compounds were analysed by gas chromatography-olfactometry (GC-O). Potent odorants were identified by aroma extract dilution analysis (AEDA), calculation of the odour activity values (OAV), and proven by confection of an aroma model. 3-Phenylpropanal, 3-phenyl-1-propanol, and benzyl alcohol were identified as potent aroma biotransformation products. Headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) experiments showed that 3-phenylpropanal, 3-phenyl-1-propanol, benzyl alcohol, methyl 3-phenylpropionate, methyl 2-phenylacetate, cinnamaldehyde and methyl cinnamate were produced during the cultivation period of eight days. By means of labelling experiments, (E)-cinnamic acid was identified as the precursor of 3-phenylpropanal and 3-phenyl-1-propanol. Basidiomycetes were able to biotransform food by-products to pleasant complex flavour mixtures. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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 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.

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

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.

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.

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.

Methylation profiling in individuals with Russell-Silver syndrome.

PubMed

Peñaherrera, Maria S; Weindler, Susanne; Van Allen, Margot I; Yong, Siu-Li; Metzger, Daniel L; McGillivray, Barbara; Boerkoel, Cornelius; Langlois, Sylvie; Robinson, Wendy P

2010-02-01

Russell-Silver syndrome (RSS) is a heterogeneous disorder associated with pre- and post-natal growth restriction and relative macrocephaly. Involvement of imprinted genes on both chromosome 7 and 11p15.5 has been reported. To further characterize the role of epimutations in RSS we evaluated the methylation status at both 11p15.5 imprinting control regions (ICRs): ICR1 associated with H19/IGF2 expression and ICR2 (KvDMR1) associated with CDKN1C expression in a series of 35 patients with RSS. We also evaluated methylation at the promoter regions of other imprinted genes involved in growth such as PLAGL1 (6q24), GCE (7q21), and PEG10 (7q21) in this series of 35 patients with RSS. Thirteen of the 35 patient samples, but none of 22 controls, showed methylation levels at ICR1 that were more than 2 SD below the mean for controls. Three RSS patients were highly methylated at the SCGE promoter, all of which were diagnosed with upd(7)mat. To identify further potential global methylation changes in RSS patients, a subset of 22 patients were evaluated at 1505 CpG sites by the Illumina GoldenGate methylation array. Among the few CpG sites displaying a significant difference between RSS patients and controls, was a CpG associated with the H19 promoter. No other sites associated with known imprinted genes were identified as abnormally methylated in RSS patients by this approach. While the association of hypomethylation of the H19/IGF2 ICR1 is clear, the continuous distribution of methylation values among the patients and controls complicates the establishment of clear cut-offs for clinical diagnosis. Copyright 2010 Wiley-Liss, Inc.

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.

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.

PHYLOGENETIC AFFILIATION OF WATER DISTRIBUTION SYSTEM BACTERIAL ISOLATES USING 16S RDNA SEQUENCE ANALYSIS

EPA Science Inventory

In a previously described study, only 15% of the bacterial strains isolated from a water distribution system (WDS) grown on R2A agar were identifiable using fatty acid methyl esthers (FAME) profiling. The lack of success was attributed to the use of fatty acid databases of bacter...

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

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.

Immortalization of T-cells is accompanied by gradual changes in CpG methylation resulting in a profile resembling a subset of T-cell leukemias.

PubMed

Degerman, Sofie; Landfors, Mattias; Siwicki, Jan Konrad; Revie, John; Borssén, Magnus; Evelönn, Emma; Forestier, Erik; Chrzanowska, Krystyna H; Rydén, Patrik; Keith, W Nicol; Roos, Göran

2014-07-01

We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

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

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.

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.

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

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

Placental epigenetic patterning of glucocorticoid response genes is associated with infant neurodevelopment

PubMed Central

Paquette, Alison G; Lester, Barry M; Lesseur, Corina; Armstrong, David A; Guerin, Dylan J; Appleton, Allison A; Marsit, Carmen J

2015-01-01

Aim: To determine associations between methylation of NR3C1, HSD11B2, FKBP5 and ADCYAP1R1 and newborn neurobehavioral outcomes. Methods: In 537 newborns, placental methylation was quantified using bisulfite pyrosequencing. Profiles of neurobehavior were derived via the Neonatal Intensive Care Unit Network Neurobehavioral Scales. Using exploratory factor analysis, the relationships between methylation factor scores and neurobehavioral profiles were examined. Results: Increased scores of the factor characterized by NR3C1 methylation were associated with membership in a reactive, poorly regulated profile (odds ratio: 1.47; 95% CI: 1.00–2.18), while increased scores of the factor characterized by HSD11B2 methylation reduced this risk. Conclusion: These results suggest that coordinated regulation of these genes influences neurobehavior and demonstrates the importance of examining these alterations in a harmonized fashion. PMID:26343289

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

Characterization of Aronia melanocarpa volatiles by headspace-solid-phase microextraction (HS-SPME), simultaneous distillation/extraction (SDE), and gas chromatography-olfactometry (GC-O) methods.

PubMed

Kraujalytė, Vilma; Leitner, Erich; Venskutonis, Petras Rimantas

2013-05-22

The profiles of volatile constituents of berry fruit of two Aronia melanocarpa genotypes were evaluated by headspace-solid-phase microextraction (HS-SPME), simultaneous distillation and extraction (SDE), and gas chromatography-olfactometry (GC-O). In total, 74 volatile compounds were identified in chokeberry juice, 3-penten-2-one, 3,9-epoxy-p-menth-1-ene, and benzaldehyde being the most abundant constituents; however, their percentage concentrations were remarkably different in the HS-SPME and SDE profiles. Twenty two aroma-active compounds were detected and characterized by the trained panelists in HS-SPME using GC-O detection frequency analysis. Olfactometry revealed that ethyl-2-methyl butanoate, ethyl-3-methyl butanoate, ethyl decanoate ("fruity" aroma notes), nonanal ("green" notes), unidentified compound possessing "moldy" odor, and some other volatiles may be very important constituents in formation of chokeberry aroma of both analyzed plant cultivars.

MIRA: An R package for DNA methylation-based inference of regulatory activity.

PubMed

Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

2018-03-01

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

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.

Comprehensive Analysis of DNA Methylation in Head and Neck Squamous Cell Carcinoma Indicates Differences by Survival and Clinicopathologic Characteristics

PubMed Central

Colacino, Justin A.; Dolinoy, Dana C.; Duffy, Sonia A.; Sartor, Maureen A.; Chepeha, Douglas B.; Bradford, Carol R.; McHugh, Jonathan B.; Patel, Divya A.; Virani, Shama; Walline, Heather M.; Bellile, Emily; Terrell, Jeffrey E.; Stoerker, Jay A.; Taylor, Jeremy M. G.; Carey, Thomas E.; Wolf, Gregory T.; Rozek, Laura S.

2013-01-01

Head and neck squamous cell carcinoma (HNSCC) is the eighth most commonly diagnosed cancer in the United States. The risk of developing HNSCC increases with exposure to tobacco, alcohol and infection with human papilloma virus (HPV). HPV-associated HNSCCs have a distinct risk profile and improved prognosis compared to cancers associated with tobacco and alcohol exposure. Epigenetic changes are an important mechanism in carcinogenic progression, but how these changes differ between viral- and chemical-induced cancers remains unknown. CpG methylation at 1505 CpG sites across 807 genes in 68 well-annotated HNSCC tumor samples from the University of Michigan Head and Neck SPORE patient population were quantified using the Illumina Goldengate Methylation Cancer Panel. Unsupervised hierarchical clustering based on methylation identified 6 distinct tumor clusters, which significantly differed by age, HPV status, and three year survival. Weighted linear modeling was used to identify differentially methylated genes based on epidemiological characteristics. Consistent with previous in vitro findings by our group, methylation of sites in the CCNA1 promoter was found to be higher in HPV(+) tumors, which was validated in an additional sample set of 128 tumors. After adjusting for cancer site, stage, age, gender, alcohol consumption, and smoking status, HPV status was found to be a significant predictor for DNA methylation at an additional 11 genes, including CASP8 and SYBL1. These findings provide insight into the epigenetic regulation of viral vs. chemical carcinogenesis and could provide novel targets for development of individualized therapeutic and prevention regimens based on environmental exposures. PMID:23358896

Comprehensive analysis of DNA methylation in head and neck squamous cell carcinoma indicates differences by survival and clinicopathologic characteristics.

PubMed

Colacino, Justin A; Dolinoy, Dana C; Duffy, Sonia A; Sartor, Maureen A; Chepeha, Douglas B; Bradford, Carol R; McHugh, Jonathan B; Patel, Divya A; Virani, Shama; Walline, Heather M; Bellile, Emily; Terrell, Jeffrey E; Stoerker, Jay A; Taylor, Jeremy M G; Carey, Thomas E; Wolf, Gregory T; Rozek, Laura S

2013-01-01

Head and neck squamous cell carcinoma (HNSCC) is the eighth most commonly diagnosed cancer in the United States. The risk of developing HNSCC increases with exposure to tobacco, alcohol and infection with human papilloma virus (HPV). HPV-associated HNSCCs have a distinct risk profile and improved prognosis compared to cancers associated with tobacco and alcohol exposure. Epigenetic changes are an important mechanism in carcinogenic progression, but how these changes differ between viral- and chemical-induced cancers remains unknown. CpG methylation at 1505 CpG sites across 807 genes in 68 well-annotated HNSCC tumor samples from the University of Michigan Head and Neck SPORE patient population were quantified using the Illumina Goldengate Methylation Cancer Panel. Unsupervised hierarchical clustering based on methylation identified 6 distinct tumor clusters, which significantly differed by age, HPV status, and three year survival. Weighted linear modeling was used to identify differentially methylated genes based on epidemiological characteristics. Consistent with previous in vitro findings by our group, methylation of sites in the CCNA1 promoter was found to be higher in HPV(+) tumors, which was validated in an additional sample set of 128 tumors. After adjusting for cancer site, stage, age, gender, alcohol consumption, and smoking status, HPV status was found to be a significant predictor for DNA methylation at an additional 11 genes, including CASP8 and SYBL1. These findings provide insight into the epigenetic regulation of viral vs. chemical carcinogenesis and could provide novel targets for development of individualized therapeutic and prevention regimens based on environmental exposures.

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.

Identification of methyl violet 2B as a novel blocker of focal adhesion kinase signaling pathway in cancer cells

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

Kim, Hwan; Translational Research Center for Protein Function Control; Kim, Nam Doo

2013-07-26

Highlights: •FAK signaling cascade in cancer cells is profoundly inhibited by methyl violet 2B. •Methyl violet 2B identified by virtual screening is a novel allosteric FAK inhibitor. •Methyl violet 2B possesses extremely high kinase selectivity. •Methyl violet 2B suppresses strongly the proliferation of cancer cells. •Methyl violet 2B inhibits focal adhesion, invasion and migration of cancer cells. -- Abstract: The focal adhesion kinase (FAK) signaling cascade in cancer cells was profoundly inhibited by methyl violet 2B identified with the structure-based virtual screening. Methyl violet 2B was shown to be a non-competitive inhibitor of full-length FAK enzyme vs. ATP. It turnedmore » out that methyl violet 2B possesses extremely high kinase selectivity in biochemical kinase profiling using a large panel of kinases. Anti-proliferative activity measurement against several different cancer cells and Western blot analysis showed that this substance is capable of suppressing significantly the proliferation of cancer cells and is able to strongly block FAK/AKT/MAPK signaling pathways in a dose dependent manner at low nanomolar concentration. Especially, phosphorylation of Tyr925-FAK that is required for full activation of FAK was nearly completely suppressed even with 1 nM of methyl violet 2B in A375P cancer cells. To the best of our knowledge, it has never been reported that methyl violet possesses anti-cancer effects. Moreover, methyl violet 2B significantly inhibited FER kinase phosphorylation that activates FAK in cell. In addition, methyl violet 2B was found to induce cell apoptosis and to exhibit strong inhibitory effects on the focal adhesion, invasion, and migration of A375P cancer cells at low nanomolar concentrations. Taken together, these results show that methyl violet 2B is a novel, potent and selective blocker of FAK signaling cascade, which displays strong anti-proliferative activities against a variety of human cancer cells and suppresses adhesion/migration/invasion of tumor cells.« less

A sedge plant as the source of Kangaroo Island propolis rich in prenylated p-coumarate ester and stilbenes.

PubMed

Duke, Colin C; Tran, Van H; Duke, Rujee K; Abu-Mellal, Abdallah; Plunkett, George T; King, Douglas I; Hamid, Kaiser; Wilson, Karen L; Barrett, Russell L; Bruhl, Jeremy J

2017-02-01

Propolis samples from Kangaroo Island, South Australia, were investigated for chemical constituents using high-field nuclear magnetic resonance spectral profiling. A type of propolis was found containing a high proportion of prenylated hydroxystilbenes. Subsequently, the botanical origin of this type of propolis was identified using a beehive propolis depletion method and analysis of flora. Ligurian honey bees, Apis mellifera ligustica Spinola, were found to produce propolis from resin exuded by the Australian native sedge plant Lepidosperma sp. Montebello (Cyperaceae). The plants, commonly known as sword sedge, were found to have resin that matched with the propolis samples identified as the most abundant propolis type on the island containing C- and O-prenylated tetrahydroxystilbenes (pTHOS) in addition to a small amount of prenylated p-coumarate. The isolation of five pTHOS not previously characterized are reported: (E)-4-(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene, (E)-2,4-bis(3-methyl-2-buten-1-yl)-3,3',4',5-tetrahydroxystilbene, (E)-2-(3-methyl-2-buten-1-yl)-3-(3-methyl-2-butenyloxy)-3',4',5-trihydroxystilbene, (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,3',5,5'-tetrahydroxystilbene and (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene. A National Cancer Institute 60 human cell line anticancer screen of three of these compounds showed growth inhibitory activity. The large Australasian genus Lepidosperma is identified as a valuable resource for the isolation of substances with medicinal potential. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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 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.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer.

PubMed

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-07-25

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer

PubMed Central

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-01-01

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach. PMID:28415743

Link between epigenomic alterations and genome-wide aberrant transcriptional response to allergen in dendritic cells conveying maternal asthma risk.

PubMed

Mikhaylova, Lyudmila; Zhang, Yiming; Kobzik, Lester; Fedulov, Alexey V

2013-01-01

We investigated the link between epigenome-wide methylation aberrations at birth and genomic transcriptional changes upon allergen sensitization that occur in the neonatal dendritic cells (DC) due to maternal asthma. We previously demonstrated that neonates of asthmatic mothers are born with a functional skew in splenic DCs that can be seen even in allergen-naïve pups and can convey allergy responses to normal recipients. However, minimal-to-no transcriptional or phenotypic changes were found to explain this alteration. Here we provide in-depth analysis of genome-wide DNA methylation profiles and RNA transcriptional (microarray) profiles before and after allergen sensitization. We identified differentially methylated and differentially expressed loci and performed manually-curated matching of methylation status of the key regulatory sequences (promoters and CpG islands) to expression of their respective transcripts before and after sensitization. We found that while allergen-naive DCs from asthma-at-risk neonates have minimal transcriptional change compared to controls, the methylation changes are extensive. The substantial transcriptional change only becomes evident upon allergen sensitization, when it occurs in multiple genes with the pre-existing epigenetic alterations. We demonstrate that maternal asthma leads to both hyper- and hypomethylation in neonatal DCs, and that both types of events at various loci significantly overlap with transcriptional responses to allergen. Pathway analysis indicates that approximately 1/2 of differentially expressed and differentially methylated genes directly interact in known networks involved in allergy and asthma processes. We conclude that congenital epigenetic changes in DCs are strongly linked to altered transcriptional responses to allergen and to early-life asthma origin. The findings are consistent with the emerging paradigm that asthma is a disease with underlying epigenetic changes.

Isolation and screening of heterocystous cyanobacterial strains for biodiesel production by evaluating the fuel properties from fatty acid methyl ester (FAME) profiles.

PubMed

Anahas, Antonyraj Matharasi Perianaika; Muralitharan, Gangatharan

2015-05-01

This study reports on the biodiesel quality parameters of eleven heterocystous cyanobacterial strains based on fatty acid methyl esters (FAME) profiles. The biomass productivity of the tested cyanobacterial strains ranged from 9.33 to 20.67 mg L(-1) d(-1) while the lipid productivity varied between 0.65 and 2.358 mg L(-1) d(-1). The highest biomass and lipid productivity was observed for Calothrix sp. MBDU 013 but its lipid content is only 11.221 in terms of percent dry weight, next to the Anabaena sphaerica MBDU 105, whose lipid content is high. To identify the most competent isolate, a multi-criteria decision analyses (MCDA) was performed by including the key chemical and physical parameters of biodiesel calculated from FAME profiles. The isolate A.sphaerica MBDU 105 is the most promising biodiesel feed stock based on decision vector through Preference Ranking Organisation Method for Enrichment Evaluation (PROMETHEE) and Graphical Analysis for Interactive Assistance (GAIA) analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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.

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

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.

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

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.

Use of MIDI-fatty acid methyl ester analysis to monitor the transmission of Campylobacter during commercial poultry processing.

PubMed

Hinton, Arthur; Cason, J A; Hume, Michael E; Ingram, Kimberly D

2004-08-01

The presence of Campylobacter spp. on broiler carcasses and in scald water taken from a commercial poultry processing facility was monitored on a monthly basis from January through June. Campylobacter agar, Blaser, was used to enumerate Campylobacter in water samples from a multiple-tank scalder; on prescalded, picked, eviscerated, and chilled carcasses; and on processed carcasses stored at 4 degrees C for 7 or 14 days. The MIDI Sherlock microbial identification system was used to identify Campylobacter-like isolates based on the fatty acid methyl ester profile of the bacteria. The dendrogram program of the Sherlock microbial identification system was used to compare the fatty acid methyl ester profiles of the bacteria and determine the degree of relatedness between the isolates. Findings indicated that no Campylobacter were recovered from carcasses or scald tank water samples collected in January or February, but the pathogen was recovered from samples collected in March, April, May, and June. Processing generally produced a significant (P < 0.05) decrease in the number of Campylobacter recovered from broiler carcasses, and the number of Campylobacter recovered from refrigerated carcasses generally decreased during storage. Significantly (P < 0.05) fewer Campylobacter were recovered from the final tank of the multiple-tank scald system than from the first tank. MIDI similarity index values ranged from 0.104 to 0.928 based on MIDI-fatty acid methyl ester analysis of Campylobacterjejuni and Campylobacter coli isolates. Dendrograms of the fatty acid methyl ester profile of the isolates indicated that poultry flocks may introduce several strains of C. jejuni and C. coli into processing plants. Different populations of the pathogen may be carried into the processing plant by successive broiler flocks, and the same Campylobacter strain may be recovered from different poultry processing operations. However, Campylobacter apparently is unable to colonize equipment in the processing facility and contaminate broilers from flocks processed at later dates in the facility.

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.

Low levels of serum serotonin and amino acids identified in migraine patients.

PubMed

Ren, Caixia; Liu, Jia; Zhou, Juntuo; Liang, Hui; Wang, Yayun; Sun, Yinping; Ma, Bin; Yin, Yuxin

2018-02-05

Migraine is a highly disabling primary headache associated with a high socioeconomic burden and a generally high prevalence. The clinical management of migraine remains a challenge. This study was undertaken to identify potential serum biomarkers of migraine. Using Liquid Chromatography coupled to Mass Spectrometry (LC-MS), the metabolomic profile of migraine was compared with healthy individuals. Principal component analysis (PCA) and Orthogonal partial least squares-discriminant analysis (orthoPLS-DA) showed the metabolomic profile of migraine is distinguishable from controls. Volcano plot analysis identified 10 serum metabolites significantly decreased during migraine. One of these was serotonin, and the other 9 were amino acids. Pathway analysis and enrichment analysis showed tryptophan metabolism (serotonin metabolism), arginine and proline metabolism, and aminoacyl-tRNA biosynthesis are the three most prominently altered pathways in migraine. ROC curve analysis indicated Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine are potential sensitive and specific biomarkers for migraine. Our results show Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine may be as specific or more specific for migraine than serotonin which is the traditional biomarker of migraine. We propose that therapeutic manipulation of these metabolites or metabolic pathways may be helpful in the prevention and treatment of migraine. Copyright © 2018 Elsevier Inc. All rights reserved.

Aberrant methylation of GCNT2 is tightly related to lymph node metastasis of primary CRC.

PubMed

Nakamura, Kazunori; Yamashita, Keishi; Sawaki, Hiromichi; Waraya, Mina; Katoh, Hiroshi; Nakayama, Nobukazu; Kawamata, Hiroshi; Nishimiya, Hiroshi; Ema, Akira; Narimatsu, Hisashi; Watanabe, Masahiko

2015-03-01

Glycoprotein expression profile is dramatically altered in human cancers; however, specific glycogenes have not been fully identified. A comprehensive real-time polymerase chain reaction (PCR) system for glycogenes (CRPS-G) identified several outstanding glycogenes. GCNT2 was of particular interest after GCNT2 expression and epigenetics were rigorously investigated in primary colorectal cancer (CRC). The highlights of this work can be summarized as follows: (i) Expression of GCNT2 was remarkably suppressed. (ii) Silenced expression of GCNT2 was reactivated by combined demethylating agents. (iii) Promoter DNA methylation of GCNT2 was silenced in CRC cell lines and tissues. Hypomethylation of GCNT2 variant 2 is tightly associated with lymph node metastasis in primary CRC. (iv) GCNT2 methylation level in the normal tissues also showed a close association with that in the tumor tissues and reflected lymph node metastasis. We identified aberrant expression of GCNT2, which can be explained by promoter DNA hypermethylation. Hypomethylation of the GCNT2 variant 2 reflected lymph node metastasis of CRC in the tumor and normal tissues. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[Effect of genetics, epigenetics and variations in the transcriptional expression of cadherin-E in breast cancer susceptibility].

PubMed

Aristizábal-Pachón, Andrés Felipe; Takahashi, Catarina Satie

2016-12-01

Cadherin-E (CDH1) is an important regulator of epithelial-mesenchymal transition, invasion and metastasis in many carcinomas. However, germinal epimutations and mutations effect in breast cancer susceptibility is not clear. To evaluate rs334558 polymorphism, promoter methylation status and CDH1 expression profile in breast cancer patients. We collected peripheral blood samples from 102 breast cancer patients and 102 healthy subjects. The identification of rs334558 polymorphism was performed using PCR-RFLP, while methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) were used to explore CDH1 methylation status; finally, CDH1 transcriptional expression profile was evaluated using RT-qPCR. We found no association between rs334558 polymorphism and breast cancer. Aberrant promoter methylation profile was found in breast cancer patients and it was related with early cancer stages. CDH1 down-regulation was significantly associated with metastasis and promoter methylation. CDH1 alterations were associated with invasion and metastasis in breast cancer. Our results offer further evidence of CDH1 relevance in breast cancer development and progression.

Quantitative, high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

PubMed Central

Ismail, Khaled MK; Haworth, Kim E; Mein, Charles; Carroll, William D

2011-01-01

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypomethylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period. PMID:20864804

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.

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.

Use of an injection port for thermochemolysis-gas chromatography/mass spectrometry: rapid profiling of biomaterials.

PubMed

Shadkami, Farzad; Helleur, Robert

2009-07-31

A simple and direct approach was developed for thermochemolytic analysis of a wide range of biomolecules present in plant materials using an injection port of a gas chromatograph/mass spectrometer (GC/MS) and a novel solids injector consisting of a coiled stainless steel wire placed inside a modified needle syringe. Optimum thermochemolysis (or Thermally Assisted Hydrolysis/Methylation) was achieved by using a suitable methanolic solution of trimethylsulfonium hydroxide (TMSH) or tetramethylammonium hydroxide (TMAH) with an injection port temperature of 350 degrees C. Intact, methylated flavonoids, saccharides, phenolic and fatty acids, lignin dimers and diterpene resin acids were identified. Samples include tea leaves, hemicelluloses, lignin isolates and herbal medicines. Unexpected chromatographic results using TMAH reagent revealed the presence of intact methylated trisaccharides (658 Da) and structurally informative dimer lignin markers.

miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes.

PubMed

Stojković, Vanja; Chu, Tongyue; Therizols, Gabriel; Weinberg, David E; Fujimori, Danica Galonić

2018-06-13

Although present across bacteria, the large family of radical SAM RNA methylating enzymes is largely uncharacterized. Escherichia coli RlmN, the founding member of the family, methylates an adenosine in 23S rRNA and several tRNAs to yield 2-methyladenosine (m 2 A). However, varied RNA substrate specificity among RlmN enzymes, combined with the ability of certain family members to generate 8-methyladenosine (m 8 A), makes functional predictions across this family challenging. Here, we present a method for unbiased substrate identification that exploits highly efficient, mechanism-based cross-linking between the enzyme and its RNA substrates. Additionally, by determining that the thermostable group II intron reverse transcriptase introduces mismatches at the site of the cross-link, we have identified the precise positions of RNA modification using mismatch profiling. These results illustrate the capability of our method to define enzyme-substrate pairs and determine modification sites of the largely uncharacterized radical SAM RNA methylating enzyme family.

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.

Mining pathway associations for disease-related pathway activity analysis based on gene expression and methylation data.

PubMed

Lee, Hyeonjeong; Shin, Miyoung

2017-01-01

The problem of discovering genetic markers as disease signatures is of great significance for the successful diagnosis, treatment, and prognosis of complex diseases. Even if many earlier studies worked on identifying disease markers from a variety of biological resources, they mostly focused on the markers of genes or gene-sets (i.e., pathways). However, these markers may not be enough to explain biological interactions between genetic variables that are related to diseases. Thus, in this study, our aim is to investigate distinctive associations among active pathways (i.e., pathway-sets) shown each in case and control samples which can be observed from gene expression and/or methylation data. The pathway-sets are obtained by identifying a set of associated pathways that are often active together over a significant number of class samples. For this purpose, gene expression or methylation profiles are first analyzed to identify significant (active) pathways via gene-set enrichment analysis. Then, regarding these active pathways, an association rule mining approach is applied to examine interesting pathway-sets in each class of samples (case or control). By doing so, the sets of associated pathways often working together in activity profiles are finally chosen as our distinctive signature of each class. The identified pathway-sets are aggregated into a pathway activity network (PAN), which facilitates the visualization of differential pathway associations between case and control samples. From our experiments with two publicly available datasets, we could find interesting PAN structures as the distinctive signatures of breast cancer and uterine leiomyoma cancer, respectively. Our pathway-set markers were shown to be superior or very comparable to other genetic markers (such as genes or gene-sets) in disease classification. Furthermore, the PAN structure, which can be constructed from the identified markers of pathway-sets, could provide deeper insights into distinctive associations between pathway activities in case and control samples.

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.

Distinct molecular profile of diffuse cerebellar gliomas.

PubMed

Nomura, Masashi; Mukasa, Akitake; Nagae, Genta; Yamamoto, Shogo; Tatsuno, Kenji; Ueda, Hiroki; Fukuda, Shiro; Umeda, Takayoshi; Suzuki, Tomonari; Otani, Ryohei; Kobayashi, Keiichi; Maruyama, Takashi; Tanaka, Shota; Takayanagi, Shunsaku; Nejo, Takahide; Takahashi, Satoshi; Ichimura, Koichi; Nakamura, Taishi; Muragaki, Yoshihiro; Narita, Yoshitaka; Nagane, Motoo; Ueki, Keisuke; Nishikawa, Ryo; Shibahara, Junji; Aburatani, Hiroyuki; Saito, Nobuhito

2017-12-01

Recent studies have demonstrated that tumor-driving alterations are often different among gliomas that originated from different brain regions and have underscored the importance of analyzing molecular characteristics of gliomas stratified by brain region. Therefore, to elucidate molecular characteristics of diffuse cerebellar gliomas (DCGs), 27 adult, mostly glioblastoma cases were analyzed. Comprehensive analysis using whole-exome sequencing, RNA sequencing, and Infinium methylation array (n = 17) demonstrated their distinct molecular profile compared to gliomas in other brain regions. Frequent mutations in chromatin-modifier genes were identified including, noticeably, a truncating mutation in SETD2 (n = 4), which resulted in loss of H3K36 trimethylation and was mutually exclusive with H3F3A K27M mutation (n = 3), suggesting that epigenetic dysregulation may lead to DCG tumorigenesis. Alterations that cause loss of p53 function including TP53 mutation (n = 9), PPM1D mutation (n = 2), and a novel type of PPM1D fusion (n = 1), were also frequent. On the other hand, mutations and copy number changes commonly observed in cerebral gliomas were infrequent. DNA methylation profile analysis demonstrated that all DCGs except for those with H3F3A mutations were categorized in the "RTK I (PDGFRA)" group, and those DCGs had a gene expression signature that was highly associated with PDGFRA. Furthermore, compared with the data of 315 gliomas derived from different brain regions, promoter methylation of transcription factors genes associated with glial development showed a characteristic pattern presumably reflecting their tumor origin. Notably, SOX10, a key transcription factor associated with oligodendroglial differentiation and PDGFRA regulation, was up-regulated in both DCG and H3 K27M-mutant diffuse midline glioma, suggesting their developmental and biological commonality. In contrast, SOX10 was silenced by promoter methylation in most cerebral gliomas. These findings may suggest potential tailored targeted therapy for gliomas according to their brain region, in addition to providing molecular clues to identify the region-related cellular origin of DCGs.

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.

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.

Polyphenolic profile as a useful tool to identify the wood used in wine aging.

PubMed

Sanz, Miriam; Fernández de Simón, Brígida; Cadahía, Estrella; Esteruelas, Enrique; Muñoz, Angel Ma; Hernández, Ma Teresa; Estrella, Isabel

2012-06-30

Although oak wood is the main material used in cooperage, other species are being considered as possible sources of wood for the production of wines and their derived products. In this work we have compared the phenolic composition of acacia (Robinia pseudoacacia), chestnut (Castanea sativa), cherry (Prunus avium) and ash (Fraxinus excelsior and F. americana) heartwoods, by using HPLC-DAD/ESI-MS/MS (some of these data have been showed in previous paper), as well as the changes that toasting intensity at cooperage produce in each polyphenolic profile. Before toasting, each wood shows a different and specific polyphenolic profile, with both qualitative and quantitative differences among them. Toasting notably changed these profiles, in general, proportionally to toasting intensity and led to a minor differentiation among species in toasted woods, although we also found phenolic markers in toasted woods. Thus, methyl syringate, benzoic acid, methyl vanillate, p-hydroxybenzoic acid, 3,4,5-trimethylphenol and p-coumaric acid, condensed tannins of the procyanidin type, and the flavonoids naringenin, aromadendrin, isosakuranetin and taxifolin will be a good tool to identify cherry wood. In acacia wood the chemical markers will be the aldehydes gallic and β-resorcylic and two not fully identified hydroxycinnamic compounds, condensed tannins of the prorobinetin type, and when using untoasted wood, dihydrorobinetin, and in toasted acacia wood, robinetin. In untoasted ash wood, the presence of secoiridoids, phenylethanoid glycosides, or di and oligolignols will be a good tool, especially oleuropein, ligstroside and olivil, together verbascoside and isoverbascoside in F. excelsior, and oleoside in F. americana. In toasted ash wood, tyrosol, syringaresinol, cyclolovil, verbascoside and olivil, could be used to identify the botanical origin. In addition, in ash wood, seasoned and toasted, neither hydrolysable nor condensed tannins were detected. Lastly, in chestnut wood, gallic and ellagic acids and hydrolysable tannins of both the gallotannin and ellagitannin type, can be used as chemical markers. Copyright © 2011 Elsevier B.V. All rights reserved.

Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

PubMed

Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

2016-02-25

Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

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.

Genome Wide Methylome Alterations in Lung Cancer.

PubMed

Mullapudi, Nandita; Ye, Bin; Suzuki, Masako; Fazzari, Melissa; Han, Weiguo; Shi, Miao K; Marquardt, Gaby; Lin, Juan; Wang, Tao; Keller, Steven; Zhu, Changcheng; Locker, Joseph D; Spivack, Simon D

2015-01-01

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.

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.

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.

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.

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

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

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.

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.

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

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.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions.

PubMed

Schmidt, Martin; Van Bel, Michiel; Woloszynska, Magdalena; Slabbinck, Bram; Martens, Cindy; De Block, Marc; Coppens, Frederik; Van Lijsebettens, Mieke

2017-07-06

Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Effects of soluble and particulate Cr(VI) on genome-wide DNA methylation in human B lymphoblastoid cells.

PubMed

Lou, Jianlin; Wang, Yu; Chen, Junqiang; Ju, Li; Yu, Min; Jiang, Zhaoqiang; Feng, Lingfang; Jin, Lingzhi; Zhang, Xing

2015-10-01

Several previous studies highlighted the potential epigenetic effects of Cr(VI), especially DNA methylation. However, few studies have compared the effects of Cr(VI) on DNA methylation profiles between soluble and particulate chromate in vitro. Accordingly, Illumina Infinium Human Methylation 450K BeadChip array was used to analyze DNA methylation profiles of human B lymphoblastoid cells exposed to potassium dichromate or lead chromate, and the cell viability was also studied. Array based DNA methylation analysis showed that the impacts of Cr(VI) on DNA methylation were limited, only about 40 differentially methylated CpG sites, with an overlap of 15CpG sites, were induced by both potassium dichromate and lead chromate. The results of mRNA expression showed that after Cr(VI) treatment, mRNA expression changes of four genes (TBL1Y, FZD5, IKZF2, and KIAA1949) were consistent with their DNA methylation alteration, but DNA methylation changes of other six genes did not correlate with mRNA expression. In conclusion, both of soluble and particulate Cr(VI) could induce a small amount of differentially methylated sites in human B lymphoblastoid cells, and the correlations between DNA methylation changes and mRNA expression varied between different genes. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

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

Islam, Kabirul; Chen, Yuling; Wu, Hong

2013-11-18

Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. Withmore » two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.« less

Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma.

PubMed

Zhang, Xiaoying; Li, Hiu Ming; Liu, Zhiyan; Zhou, Gengyin; Zhang, Qinghui; Zhang, Tingguo; Zhang, Jianping; Zhang, Cuijuan

2013-01-01

Genetic and epigenetic alterations are the two key mechanisms in the development of hepatocellular carcinoma (HCC). However, how they contribute to hepatocarcinogenesis and the correlation between them has not been fully elucidated. A total of 48 paired HCCs and noncancerous tissues were used to detect loss of heterozygosity (LOH) and the methylation profiles of five tumor suppressor genes (RASSF1A, BLU, FHIT, CRBP1, and HLTF) on chromosome 3 by using polymerase chain reaction (PCR) and methylation-specific PCR. Gene expression was analyzed by immunohistochemistry and reverse transcription (RT)-PCR. Sixteen of 48 (33.3 %) HCCs had LOH on at least one locus on chromosome 3, and two smallest common deleted regions (3p22.3-24.3 and 3p12.3-14.2) were identified. RASSF1A, BLU, and FHIT showed very high frequencies of methylation in HCCs (100, 81.3, and 64.6 %, respectively) and noncancerous tissues, but not in liver tissues from control patients. Well-differentiated HCCs showed high methylation frequencies of these genes but very low frequencies of LOH. Furthermore, BLU methylation was associated with an increased level of alpha-fetoprotein, and FHIT methylation was inversely correlated with HCC recurrence. In comparison, CRBP1 showed moderate frequencies of methylation, while HLTF showed low frequencies of methylation, and CRBP1 methylation occurred mainly in elderly patients. Treatment with 5-aza-2'-deoxycytidine demethylated at least one of these genes and restored their expression in a DNA methylation-dependent or -independent manner. Hypermethylation of RASSF1A, BLU, and FHIT is a common and very early event in hepatocarcinogenesis; CRBP1 methylation may also be involved in the later stage. Although LOH was not too frequent on chromosome 3, it may play a role as another mechanism in hepatocarcinogenesis.

DM-BLD: differential methylation detection using a hierarchical Bayesian model exploiting local dependency.

PubMed

Wang, Xiao; Gu, Jinghua; Hilakivi-Clarke, Leena; Clarke, Robert; Xuan, Jianhua

2017-01-15

The advent of high-throughput DNA methylation profiling techniques has enabled the possibility of accurate identification of differentially methylated genes for cancer research. The large number of measured loci facilitates whole genome methylation study, yet posing great challenges for differential methylation detection due to the high variability in tumor samples. We have developed a novel probabilistic approach, D: ifferential M: ethylation detection using a hierarchical B: ayesian model exploiting L: ocal D: ependency (DM-BLD), to detect differentially methylated genes based on a Bayesian framework. The DM-BLD approach features a joint model to capture both the local dependency of measured loci and the dependency of methylation change in samples. Specifically, the local dependency is modeled by Leroux conditional autoregressive structure; the dependency of methylation changes is modeled by a discrete Markov random field. A hierarchical Bayesian model is developed to fully take into account the local dependency for differential analysis, in which differential states are embedded as hidden variables. Simulation studies demonstrate that DM-BLD outperforms existing methods for differential methylation detection, particularly when the methylation change is moderate and the variability of methylation in samples is high. DM-BLD has been applied to breast cancer data to identify important methylated genes (such as polycomb target genes and genes involved in transcription factor activity) associated with breast cancer recurrence. A Matlab package of DM-BLD is available at http://www.cbil.ece.vt.edu/software.htm CONTACT: Xuan@vt.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Metabolic engineering for the high-yield production of isoprenoid-based C 5 alcohols in E. coli

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

George, Kevin W.; Thompson, Mitchell G.; Kang, Aram

2015-06-08

Branched five carbon (C 5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C 5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphatemore » (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Lastly, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.« less

Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli

PubMed Central

George, Kevin W.; Thompson, Mitchell G.; Kang, Aram; Baidoo, Edward; Wang, George; Chan, Leanne Jade G.; Adams, Paul D.; Petzold, Christopher J.; Keasling, Jay D.; Soon Lee, Taek

2015-01-01

Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol. PMID:26052683

Forensic DNA methylation profiling from evidence material for investigative leads

PubMed Central

Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

2016-01-01

DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369] PMID:27099236

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

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.

Anthocyanin Profiles in Flowers of Grape Hyacinth.

PubMed

Lou, Qian; Wang, Lin; Liu, Hongli; Liu, Yali

2017-04-26

Grape hyacinth ( Muscari spp.) is a popular ornamental bulbous perennial famous for its blue flowers. To understand the chemical basis of the rich blue colors in this plant, anthocyanin profiles of six blue flowering grape hyacinths as well as one pink and one white cultivar were determined using high-performance liquid chromatography and mass spectrometry. Along with two known compounds, eight putative anthocyanins were identified in the tepals of grape hyacinth for the first time. The accumulation and distribution of anthocyanins in the plant showed significant cultivar and flower development specificity. Violet-blue flowers mainly contained simple delphinidin-type anthocyanins bearing one or two methyl-groups but no acyl groups, whereas white and pink flowers synthesised more complex pelargonidin/cyanidin-derivatives with acyl-moieties but no methyl-groups. The results partially reveal why solid blue, orange or red flowers are rare in this plant in nature. In addition, pelargonidin-type anthocyanins were found for the first time in the genus, bringing more opportunities in terms of breeding of flower color in grape hyacinth.

Quantification and characterisation of fatty acid methyl esters in microalgae: Comparison of pretreatment and purification methods.

PubMed

Lage, Sandra; Gentili, Francesco G

2018-06-01

A systematic qualitative and quantitative analysis of fatty acid methyl esters (FAMEs) is crucial for microalgae species selection for biodiesel production. The aim of this study is to identify the best method to assess microalgae FAMEs composition and content. A single-step method, was tested with and without purification steps-that is, separation of lipid classes by thin-layer chromatography (TLC) or solid-phase extraction (SPE). The efficiency of a direct transesterification method was also evaluated. Additionally, the yield of the FAMEs and the profiles of the microalgae samples with different pretreatments (boiled in isopropanol, freezing, oven-dried and freeze-dried) were compared. The application of a purification step after lipid extraction proved to be essential for an accurate FAMEs characterisation. The purification methods, which included TLC and SPE, provided superior results compared to not purifying the samples. Freeze-dried microalgae produced the lowest FAMEs yield. However, FAMEs profiles were generally equivalent among the pretreatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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.

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

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.

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 .

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

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...

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.

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.

Cigarette smoke condensate induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: short-term and chronic exposure.

PubMed

Word, Beverly; Lyn-Cook, Lascelles E; Mwamba, Bibi; Wang, Honggang; Lyn-Cook, Beverly; Hammons, George

2013-01-01

Establishing early diagnostic markers of harm is critical for effective prevention programs and regulation of tobacco products. This study examined effects of cigarette smoke condensate (CSC) on expression and promoter methylation profile of critical genes (DAPK, ECAD, MGMT, and RASSF1A) involved in lung cancer development in different human lung cell lines. NL-20 cells were treated with 0.1-100 μg/ml of CSC for 24 to 72 hrs for short-term exposures. DAPK expression or methylation status was not significantly affected. However, CSC treatment resulted in changes in expression and promoter methylation profile of ECAD, MGMT, and RASSF1A. For chronic studies, cells were exposed to 1 or 10 μg/ml CSC up to 28 days. Cells showed morphological changes associated with transformation and changes in invasion capacities and global methylation status. This study provides critical data suggesting that epigenetic changes could serve as an early biomarker of harm due to exposure to cigarette smoke.

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...

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.

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

EG-01EPIGENETIC INACTIVATION OF ARGININE BIOSYNTHESIS PATHWAY IN PAEDIATRIC HIGH GRADE GLIOMA

PubMed Central

Channathodiyil, Prasanna; Kardooni, Hoda; Khozoie, Combiz; Nelofer, Syed; Darling, John; Morris, Mark; Warr, Tracy

2014-01-01

Aberrant cellular metabolism contributes significantly to the growth and proliferation of several tumour types. Identification of genes that control critical metabolic pathways is a major factor in the development of novel therapies that target metabolic defects in tumour cells. Our aim is to identify such genes in paediatric high grade glioma that are altered due to promoter hyper-methylation of cytosine residues in CpG dinucleotides. Genome wide DNA methylation profiling using Illumina infinium methylation 450K bead chip array was performed on 18 well-characterised short term cultures derived from paediatric high grade astrocytoma including 3 from diffuse intrinsic pontine glioma. Data analyses were based on beta scores of probes for each gene as measures of intensities of methylation. Genes were selected with beta scores of tumour > =0.70 and that of normal human astrocytes < =0.30. We identified that two vital genes involved in the regulation of arginine biosynthetic pathway, argininosuccinate synthetase 1(ASS1) and argininosuccinate lyase (ASL) were methylated in 9/18 (50%) cases. Hyper methylation was confirmed by methylation-specific PCR and up-regulation of gene expression following treatment with 2 µM 5-aza-2'-deoxyctidine. Down-regulation of ASS1 in hyper methylated samples was confirmed by Western blot analysis. Our findings report epigenetic deregulation of ASS1 and ASL in a subset of paediatric high grade glioma. The enzymes encoded by these genes are essential elements of urea cycle that function together in the de novo synthesis of arginine from citrulline. Tumour cells with deficient ASS1/ASL depend on external sources of arginine for survival and have been reported to be sensitive to autophagic cell death induced by arginine starvation. Therefore, further investigation may render the possibility of arginine-deprivation therapy in such sub type of paediatric high grade glioma. This therapeutic approach is of interest as tumour cells with abnormal expression of ASS1/ASL can be specifically targeted, while the normal cells can be spared.

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

Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex

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 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (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)more » 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. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular transformation.« less

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

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

He, Yupeng; Gorkin, David U.; Dickel, Diane E.

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulator y element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared withmore » existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types.« less

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

DOE PAGES

He, Yupeng; Gorkin, David U.; Dickel, Diane E.; ...

2017-02-13

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulator y element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared withmore » existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types.« less

[Analyze on volatile compounds of Antrodia camphorata using HS-SPME-GC-MS].

PubMed

He, Zhe; Lu, Zhen-Ming; Xu, Hong-Yu; Shi, Jing-Song; Xu, Zheng-Hong

2011-11-01

To analyze the volatile compounds of Antrodia camphorata in solid-state and submerged cultures. A headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry(GC-MS) were used to evaluate the profile of the volatile compounds. 49 volatile compounds were identified in A. camphorata mycelia in submerged culture, while 43 volatile compounds were identified in mycelia in solid-state culture. 1-octen-3-ol, 3-octanone, 1-octen-3-ylacetate, acetic acid octyl ester and ethanol were the main volatile compounds in A. camphorata mycelia in submerged culture, while 1-octen-3-ol, 3-octanone, 3-methyl-butyraldenhyde, gamma-podecalactone and methyl 2-furozte were the most potent key volatile compounds in mycelia in solid-state culture. The volatile compounds in the mycelia of A. camphorata in solid-state and submerged cultures are similar but their relative contents are different.

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

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.

Silencing of GATA3 defines a novel stem cell-like subgroup of ETP-ALL.

PubMed

Fransecky, L; Neumann, M; Heesch, S; Schlee, C; Ortiz-Tanchez, J; Heller, S; Mossner, M; Schwartz, S; Mochmann, L H; Isaakidis, K; Bastian, L; Kees, U R; Herold, T; Spiekermann, K; Gökbuget, N; Baldus, C D

2016-09-22

GATA3 is pivotal for the development of T lymphocytes. While its effects in later stages of T cell differentiation are well recognized, the role of GATA3 in the generation of early T cell precursors (ETP) has only recently been explored. As aberrant GATA3 mRNA expression has been linked to cancerogenesis, we investigated the role of GATA3 in early T cell precursor acute lymphoblastic leukemia (ETP-ALL). We analyzed GATA3 mRNA expression by RT-PCR (n = 182) in adult patients with T-ALL. Of these, we identified 70 of 182 patients with ETP-ALL by immunophenotyping. DNA methylation was assessed genome wide (Illumina Infinium® HumanMethylation450 BeadChip platform) in 12 patients and GATA3-specifically by pyrosequencing in 70 patients with ETP-ALL. The mutational landscape of ETP-ALL with respect to GATA3 expression was investigated in 18 patients and validated by Sanger sequencing in 65 patients with ETP-ALL. Gene expression profiles (Affymetrix Human genome U133 Plus 2.0) of an independent cohort of adult T-ALL (n = 83) were used to identify ETP-ALL and investigate GATA3 low and GATA3 high expressing T-ALL patients. In addition, the ETP-ALL cell line PER-117 was investigated for cytotoxicity, apoptosis, GATA3 mRNA expression, DNA methylation, and global gene expression before and after treatment with decitabine. In our cohort of 70 ETP-ALL patients, 33 % (23/70) lacked GATA3 expression and were thus defined as GATA3 low . DNA methylation analysis revealed a high degree of GATA3 CpG island methylation in GATA3 low compared with GATA3 high ETP-ALL patients (mean 46 vs. 21 %, p < 0.0001). Genome-wide expression profiling of GATA3 low ETP-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T cell-specific signatures were downregulated compared to GATA3 high ETP-ALL. Among others, FLT3 expression was upregulated and mutational analyses demonstrated a high rate (79 %) of FLT3 mutations. Hypomethylating agents induced reversal of GATA3 silencing, and gene expression profiling revealed downregulation of hematopoietic stem cell genes and upregulation of T cell differentiation. We propose GATA3 low ETP-ALL as a novel stem cell-like leukemia with implications for the use of myeloid-derived therapies.

Nuclear localisation and epigenetic inactivation of the ras effector/tumour suppressor RASSF2A in multiple human cancers

PubMed Central

Cooper, Wendy N.; Dickinson, Rachel E.; Dallol, Ashraf; Grigorieva, Elvira V.; Pavlova, Tatiana V.; Hesson, Luke B.; Bieche, Ivan; Broggini, Massimo; Maher, Eamonn R; Zabarovsky, Eugene R.; Clark, Geoffrey J; Latif, Farida

2010-01-01

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras association domain. We previously demonstrated that the A isoform of RASSF2, is frequently inactivated by promoter region hypermethylation in colorectal tumours and adenomas, methylation was tumour specific and that expression in methylated tumour lines could be reactivated by treatment with 5-aza-2dc. RASSF2 resides at 20p13, this region has been demonstrated to be frequently lost in human cancers. In this report we investigated methylation status of the RASSF2A promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2A was frequently methylated in breast tumour cell lines 65% (13/20) and in primary breast tumours 38% (15/40). RASSF2A gene expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC, whilst unmethylated lines showed no difference in level of expression before and after 5-aza-2dC treatment. RASSF2A was also frequently methylated in NSCLC tumours 44% (22/50). Methylation in breast tumours and NSCLC was tumour specific. We did not detect RASSF2A methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2A in these ovarian tumours. RASSF2A suppressed breast tumour cell growth in vitro (through colony formation and soft agar assays) and in vivo. We identified a highly conserved putative bipartite nuclear localisation signal (NLS) between amino acids 151 and 167 in the RASSF2A sequence and demonstrated that endogenous RASSF2A localised to the nucleus. Mutation of the putative nuclear localisation signal abolished the nuclear localisation so RASSF2A became predominantly cytoplasmic. Our data indicates that RASSF2A is frequently methylated in colorectal, breast and NSCLC tumours, furthermore, the methylation is tumour specific. Hence we have identified RASSF2A as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles. We also demonstrate that RASSF2 has a functional NLS signal. Furthermore this is the first report demonstrating that RASSF2 suppresses growth of cancer cells in vivo. Hence providing further evidence for its role as a tumour suppressor gene located at 20p13. PMID:17891178

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

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

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

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.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture.

PubMed

Ross, Heather A; Wright, Kathryn M; McDougall, Gordon J; Roberts, Alison G; Chapman, Sean N; Morris, Wayne L; Hancock, Robert D; Stewart, Derek; Tucker, Gregory A; James, Euan K; Taylor, Mark A

2011-01-01

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture

PubMed Central

Ross, Heather A.; Wright, Kathryn M.; McDougall, Gordon J.; Roberts, Alison G.; Chapman, Sean N.; Morris, Wayne L.; Hancock, Robert D.; Stewart, Derek; Tucker, Gregory A.; James, Euan K.; Taylor, Mark A.

2011-01-01

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties. PMID:20855456

Epigenome-Wide Tumor DNA Methylation Profiling Identifies Novel Prognostic Biomarkers of Metastatic-Lethal Progression in Men Diagnosed with Clinically Localized Prostate Cancer.

PubMed

Zhao, Shanshan; Geybels, Milan S; Leonardson, Amy; Rubicz, Rohina; Kolb, Suzanne; Yan, Qingxiang; Klotzle, Brandy; Bibikova, Marina; Hurtado-Coll, Antonio; Troyer, Dean; Lance, Raymond; Lin, Daniel W; Wright, Jonathan L; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L

2017-01-01

Aside from Gleason sum, few factors accurately identify the subset of prostate cancer patients at high risk for metastatic progression. We hypothesized that epigenetic alterations could distinguish prostate tumors with life-threatening potential. Epigenome-wide DNA methylation profiling was performed in surgically resected primary tumor tissues from a population-based (n = 430) and a replication (n = 80) cohort of prostate cancer patients followed prospectively for at least 5 years. Metastasis was confirmed by positive bone scan, MRI, CT, or biopsy, and death certificates confirmed cause of death. AUC, partial AUC (pAUC, 95% specificity), and P value criteria were used to select differentially methylated CpG sites that robustly stratify patients with metastatic-lethal from nonrecurrent tumors, and which were complementary to Gleason sum. Forty-two CpG biomarkers stratified patients with metastatic-lethal versus nonrecurrent prostate cancer in the discovery cohort, and eight of these CpGs replicated in the validation cohort based on a significant (P < 0.05) AUC (range, 0.66-0.75) or pAUC (range, 0.007-0.009). The biomarkers that improved discrimination of patients with metastatic-lethal prostate cancer include CpGs in five genes (ALKBH5, ATP11A, FHAD1, KLHL8, and PI15) and three intergenic regions. In the validation dataset, the AUC for Gleason sum alone (0.82) significantly increased with the addition of four individual CpGs (range, 0.86-0.89; all P <0.05). Eight differentially methylated CpGs that distinguish patients with metastatic-lethal from nonrecurrent tumors were validated. These novel epigenetic biomarkers warrant further investigation as they may improve prognostic classification of patients with clinically localized prostate cancer and provide new insights on tumor aggressiveness. Clin Cancer Res; 23(1); 311-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Effect of methyl salicylate and methyl jasmonate pre-treatment on the volatile profile in tomato fruit subjected to chilling temperature

USDA-ARS?s Scientific Manuscript database

Tomato fruits exposed to chilling temperatures suffer aroma loss prior to visual chilling injury (CI) symptoms. Methyl salicylate (MeSA) and methyl jasmonate (MeJA) treatments were reported to alleviate the development of visual CI, however, it is unknown if the treatments alleviate internal CI in t...

Identification of an episignature of human colorectal cancer associated with obesity by genome-wide DNA methylation analysis.

PubMed

Crujeiras, Ana B; Morcillo, Sonsoles; Diaz-Lagares, Angel; Sandoval, Juan; Castellano-Castillo, Daniel; Torres, Esperanza; Hervas, David; Moran, Sebastian; Esteller, Manel; Macias-Gonzalez, Manuel; Casanueva, Felipe F; Tinahones, Francisco J

2018-05-01

Obesity was established as a relevant modifiable risk factor in the onset and progression of colorectal cancer (CRC). This relationship could be mediated by an epigenetic regulation. The current work aimed to explore the effects of excess body weight on the DNA methylation profile of CRC using a genome-wide DNA methylation approach and to identify an epigenetic signature of obesity-related CRC. Fifty-six CRC-diagnosed patients (50 years) were included in the study and categorized according to their body mass index (BMI) as non-obese (BMI ≤ 25 kg/m 2 ) or overweight/obese (BMI > 25 kg/m 2 ). Data from Infinium 450k array-based methylomes of 28 CRC tumor samples were coupled with information on BMI categories. Additionally, DNA methylation results were validated in 28 CRC tumor samples. The analysis revealed statistically significant differences at 299 CpG sites, and they were mostly characterized as changes towards CpG hypermethylation occurring in the obese group. The 152 identified genes were involved in inflammatory and metabolic functional processes. Among these genes, novel genes were identified as epigenetically regulated in CRC depending on adiposity. ZNF397OS and ZNF543 represented the top scoring associated events that were further validated in an independent cohort and exhibited strong correlation with BMI and excellent and statistically significant efficiency in the discrimination of obese from non-obese CRC patients (area under the curve >0.80; p < 0.05). The present study identifies a potential epigenome mark of obesity-related CRC that could be useful for precision medicine in the management of this disease taking into account adiposity as a relevant risk factor.

The volatile profiles of a rare apple (Malus domestica Borkh.) honey: shikimic acid-pathway derivatives, terpenes, and others.

PubMed

Kuś, Piotr Marek; Jerković, Igor; Tuberoso, Carlo Ignazio Giovanni; Šarolić, Mladenka

2013-09-01

The volatile profiles of rare Malus domestica Borkh. honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44-45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid-pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H-indole-3-acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car-2-en-4-one (10.2%). CH2 Cl2 and pentane/Et2 O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9-3.9%), benzoic acid (2.0-11.2%), terpendiol I (0.3-7.4%), coumaran (0.0-2.8%), 2-phenylacetic acid (2.0-26.4%), methyl syringate (3.9-13.1%), vomifoliol (5.0-31.8%), and methyl 1H-indole-3-acetate (1.9-10.2%). Apple honey contained also benzyl alcohol, 2-phenylethanol, (E)-cinnamaldehyde, (E)-cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.

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

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 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.

Methylation signature of lymph node metastases in breast cancer patients

PubMed Central

2012-01-01

Background Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers. Methods The quantitative methylation analysis was performed using the SEQUENOM’s EpiTYPER™ assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis. Conclusions The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis. PMID:22695536

Use of fatty acid methyl ester profiles for discrimination of Bacillus cereus T-strain spores grown on different media.

PubMed

Ehrhardt, Christopher J; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L; Swan, Brandon K; Bannan, Jason; Robertson, James M

2010-03-01

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 omega9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media▿

PubMed Central

Ehrhardt, Christopher J.; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L.; Swan, Brandon K.; Bannan, Jason; Robertson, James M.

2010-01-01

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation. PMID:20097814

Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

PubMed

Wong, Michael H L; Bryan, Holly K; Copple, Ian M; Jenkins, Rosalind E; Chiu, Pak Him; Bibby, Jaclyn; Berry, Neil G; Kitteringham, Neil R; Goldring, Christopher E; O'Neill, Paul M; Park, B Kevin

2016-03-24

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

Polyphenolic profile and bioactivity study of Oenothera speciosa Nutt. aerial parts.

PubMed

Marzouk, Mohamed S; Moharram, Fatma A; El Dib, Rabab A; El-Shenawy, Siham M; Tawfike, Ahmed F

2009-04-07

Two new flavonol glycosides, myricetin 4'-O-alpha-L-rhamnopyranoside (1) and quercetin 3'-O-alpha-L-rhamnopyranoside (2), together with a novel biflavonol compound, speciin (3), as well as eleven phenolic metabolites, namely myricitrin (4), europetin 3-O-alpha-L-(1)C(4)-rhamnopyranoside (5), quercitrin (6), hyperin (7), rhamnetin 3-O-beta-galacto-pyranoside (8), caffeic acid (9), caffeic acid methyl ester (10), chlorogenic acid (11), chlorogenic acid methyl ester (12), gallic acid (13) and gallic acid methyl ester (14), were identified from the 80 % methanol extract of the aerial parts (leaves and stems) of Oenothera speciosa Nutt. (Onagraceae). In addition myricetin (15), quercetin (16) and ellagic acid (17) were identified from the chloroform extract. The structures were established depending on their chemical and physical analyses (UV, HR-ESIMS, 1D and 2D NMR). It was found that 80 % aqueous methanol extract of O. speciosa is non-toxic to mice up to 5 g kg(-1)b wt. The investigated extract exhibited significant antihyperglycaemic and anti-inflammatory activities in a dose dependant manner. Also, the 80 % methanol extract, myricitrin(4) and hyperin(7) showed potent antioxidant activity in vitro using 1,1-diphenyl 2-picryl hydrazyl (DPPH) radical assay.

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

Effect of cysteine and cystine addition on sensory profile and potent odorants of extruded potato snacks.

PubMed

Majcher, Małgorzata A; Jeleń, Henryk H

2007-07-11

Aromas generated in extruded potato snacks without and with addition of 0.25, 0.5, and 1% (w/w) of flavor precursors, cysteine and cystine, were compared and evaluated by descriptive sensory profiling. The results showed that high addition of cysteine (0.5 and 1%) resulted in the formation of undesirable odor and taste described as mercaptanic/sulfur, onion-like, and bitter; on the contrary, addition of cystine even at high concentration gave product with pleasant odor and taste, slightly changed into breadlike notes. GC/O analysis showed cysteine to be a much more reactive flavor precursor than cystine, stimulating formation of 12 compounds with garlic, sulfury, burnt, pungent/beer, cabbage/mold, meatlike, roasted, and popcorn odor notes. Further analysis performed by the AEDA technique identified 2-methyl-3-furanthiol (FD 2048) as a most potent odorant of extruded potato snacks with 1% addition of cysteine. Other identified compounds with high FD were butanal, 3-methyl-2-butenethiol, 2-methylthiazole, methional, 2-acetyl-1-pyrroline, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone. In the case of cystine addition (1%) the highest FD factors were calculated for butanal, 2-acetyl-1-pyrroline, benzenemethanethiol, methional, phenylacetaldehyde, dimethyltrisulfide, 1-octen-3-ol, 1,5-octadien-3-one, and 2-acetylpyrazine.

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.

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.

Causal network analysis of head and neck keloid tissue identifies potential master regulators.

PubMed

Garcia-Rodriguez, Laura; Jones, Lamont; Chen, Kang Mei; Datta, Indrani; Divine, George; Worsham, Maria J

2016-10-01

To generate novel insights and hypotheses in keloid development from potential master regulators. Prospective cohort. Six fresh keloid and six normal skin samples from 12 anonymous donors were used in a prospective cohort study. Genome-wide profiling was done previously on the cohort using the Infinium HumanMethylation450 BeadChip (Illumina, San Diego, CA). The 190 statistically significant CpG islands between keloid and normal tissue mapped to 152 genes (P < .05). The top 10 statistically significant genes (VAMP5, ACTR3C, GALNT3, KCNAB2, LRRC61, SCML4, SYNGR1, TNS1, PLEKHG5, PPP1R13-α, false discovery rate <.015) were uploaded into the Ingenuity Pathway Analysis software's Causal Network Analysis (QIAGEN, Redwood City, CA). To reflect expected gene expression direction in the context of methylation changes, the inverse of the methylation ratio from keloid versus normal tissue was used for the analysis. Causal Network Analysis identified disease-specific master regulator molecules based on downstream differentially expressed keloid-specific genes and expected directionality of expression (hypermethylated vs. hypomethylated). Causal Network Analysis software identified four hierarchical networks that included four master regulators (pyroxamide, tributyrin, PRKG2, and PENK) and 19 intermediate regulators. Causal Network Analysis of differentiated methylated gene data of keloid versus normal skin demonstrated four causal networks with four master regulators. These hierarchical networks suggest potential driver roles for their downstream keloid gene targets in the pathogenesis of the keloid phenotype, likely triggered due to perturbation/injury to normal tissue. NA Laryngoscope, 126:E319-E324, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

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.

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.

Histone deacetylation, as opposed to promoter methylation, results in epigenetic BIM silencing and resistance to EGFR TKI in NSCLC.

PubMed

Zhao, Mingchuan; Zhang, Yishi; Li, Jiayu; Li, Xuefei; Cheng, Ningning; Wang, Qi; Cai, Weijing; Zhao, Chao; He, Yayi; Chang, Jianhua; Zhou, Caicun

2018-01-01

Drug resistance remains a major challenge in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. Bcl-2-like protein 11 (BIM), a B-cell lymphoma 2 family pro-apoptotic protein, is a prime target for specific anti-cancer therapeutics. However, the epigenetic regulation of BIM in non-small cell lung cancer (NSCLC) cell lines and patients with NSCLC in association with EGFR-TKI resistance requires investigation. Methylation-specific PCR (MSP), pyrosequencing, and nested quantitative (q)-MSP were conducted to explore the methylation status of BIM in NSCLC cell lines. In addition, the methylation profile of BIM in patients with NSCLC was assessed by nested q-MSP using circulating free DNA. Cell lines, treated with methylation inhibitor 5-Aza-2'-deoxycytidine (AZA) or histone deacetylation inhibitor trichostatin A (TSA) prior to gefitinib treatment, were examined for BIM gene expression and resistance to gefitinib. All cell lines used in the present study presented with hypo-methylated BIM . Treatment with AZA had no effect on BIM RNA expression in PC9 cells or the gefitinib-resistant cell lines PC9/R and PC9/G2, nor did it reverse their resistance to gefitinib. In contrast, TSA treatment produced the opposite result. In the present study, 25 (78.1%) patients with hypo-methylated BIM and 7 patients (21.9%) with partial or hyper-methylated BIM were identified. The clinicopathological data revealed a random hypo-methylated BIM distribution amongst patients with NSCLC. In the overall study group and EGFR mutant group, hypo-methylated BIM carriers presented with no significant differences in progression free survival compared with patients with partial or hyper-methylated BIM . All cell lines in the present study and the majority of patients with NSCLC carried hypo-methylated BIM . Histone deacetylation, as opposed to promoter methylation, may contribute to the epigenetic silencing of BIM and lead to EGFR TKI resistance in NSCLC.

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

Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors.

PubMed

Martin-Trujillo, Alex; Vidal, Enrique; Monteagudo-Sa Nchez, Ana; Sanchez-Delgado, Marta; Moran, Sebastian; Hernandez Mora, Jose Ramon; Heyn, Holger; Guitart, Miriam; Esteller, Manel; Monk, David

2017-09-07

It has been postulated that imprinting aberrations are common in tumors. To understand the role of imprinting in cancer, we have characterized copy-number and methylation in over 280 cancer cell lines and confirm our observations in primary tumors. Imprinted differentially methylated regions (DMRs) regulate parent-of-origin monoallelic expression of neighboring transcripts in cis. Unlike single-copy CpG islands that may be prone to hypermethylation, imprinted DMRs can either loose or gain methylation during tumorigenesis. Here, we show that methylation profiles at imprinted DMRs often not represent genuine epigenetic changes but simply the accumulation of underlying copy-number aberrations (CNAs), which is independent of the genome methylation state inferred from cancer susceptible loci. Our results reveal that CNAs also influence allelic expression as loci with copy-number neutral loss-of-heterozygosity or amplifications may be expressed from the appropriate parental chromosomes, which is indicative of maintained imprinting, although not observed as a single expression foci by RNA FISH.Altered genomic imprinting is frequently reported in cancer. Here, the authors analyze copy number and methylation in cancer cell lines and primary tumors to show that imprinted methylation profiles represent the accumulation of copy number alteration, rather than epigenetic alterations.

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

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.

Epigenetic estimation of age in humpback whales

PubMed Central

Polanowski, Andrea M; Robbins, Jooke; Chandler, David; Jarman, Simon N

2014-01-01

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R2 of 0.787 (P = 3.04e−16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent–offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms. PMID:24606053

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

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence

PubMed Central

Wilting, Saskia M.; Boon, Debby; Sørgård, Hanne; Lando, Malin; Snoek, Barbara C.; van Wieringen, Wessel N.; Meijer, Chris J.L.M.; Lyng, Heidi; Snijders, Peter J.F.; Steenbergen, Renske D.M.

2016-01-01

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence. Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2′-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings. In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets. PMID:27270309

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence.

PubMed

Wilting, Saskia M; Miok, Viktorian; Jaspers, Annelieke; Boon, Debby; Sørgård, Hanne; Lando, Malin; Snoek, Barbara C; van Wieringen, Wessel N; Meijer, Chris J L M; Lyng, Heidi; Snijders, Peter J F; Steenbergen, Renske D M

2016-07-12

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2'-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.

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.

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.

Identification of potent odorants in different cultivars of snake fruit [Salacca zalacca (Gaert.) Voss] using gas chromatography-olfactometry.

PubMed

Wijaya, C H; Ulrich, D; Lestari, R; Schippel, K; Ebert, G

2005-03-09

Three cultivars of snake fruits, Pondoh Hitam, Pondoh Super, and Gading, were freshly extracted using liquid-liquid extraction. The aroma compounds of the three samples were analyzed by GC-MS and GC-olfactometry using the nasal impact frequency (NIF) method. A total of 24 odor-active compounds were associated with the aroma of snake fruit. Methyl 3-methylpentanoate was regarded as the character impact odorant of typical snake fruit aroma. 2-Methylbutanoic acid, 3-methylpentanoic acid, and an unknown odorant with very high intensity were found to be responsible for the snake fruit's sweaty odor. Other odorants including methyl 3-methyl-2-butenoate (overripe fruity, ethereal), methyl 3-methyl-2-pentenoate (ethereal, strong green, woody), and 2,5-dimethyl-4-hydroxy-3[2]-furanone (caramel, sweet, cotton candy-like) contribute to the overall aroma of snake fruit. Methyl dihydrojasmonate and isoeugenol, which also have odor impact, were identified for the first time as snake fruit volatiles. The main differences between the aroma of Pondoh and Gading cultivars could be attributed to the olfactory attributes (metallic, chemical, rubbery, strong green, and woody), which were perceived by most of the panelists in the Pondoh samples but were not detected in the Gading samples. This work is a prerequisite for effective selection of salak genotypes with optimal aroma profiles for high consumer acceptance.

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...

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

PubMed

Wang, Li-jia; Bai, Yu; Bao, Zhao-shi; Chen, Yan; Yan, Zhuo-hong; Zhang, Wei; Zhang, Quan-geng

2013-01-01

Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

Epigenetic Variability in the Genetically Uniform Forest Tree Species Pinus pinea L

PubMed Central

Sáez-Laguna, Enrique; Guevara, María-Ángeles; Díaz, Luis-Manuel; Sánchez-Gómez, David; Collada, Carmen; Aranda, Ismael; Cervera, María-Teresa

2014-01-01

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees. PMID:25084460

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

PubMed

Sáez-Laguna, Enrique; Guevara, María-Ángeles; Díaz, Luis-Manuel; Sánchez-Gómez, David; Collada, Carmen; Aranda, Ismael; Cervera, María-Teresa

2014-01-01

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

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...

Molecular profiling of aged neural progenitors identifies Dbx2 as a candidate regulator of age-associated neurogenic decline.

PubMed

Lupo, Giuseppe; Nisi, Paola S; Esteve, Pilar; Paul, Yu-Lee; Novo, Clara Lopes; Sidders, Ben; Khan, Muhammad A; Biagioni, Stefano; Liu, Hai-Kun; Bovolenta, Paola; Cacci, Emanuele; Rugg-Gunn, Peter J

2018-06-01

Adult neurogenesis declines with aging due to the depletion and functional impairment of neural stem/progenitor cells (NSPCs). An improved understanding of the underlying mechanisms that drive age-associated neurogenic deficiency could lead to the development of strategies to alleviate cognitive impairment and facilitate neuroregeneration. An essential step towards this aim is to investigate the molecular changes that occur in NSPC aging on a genomewide scale. In this study, we compare the transcriptional, histone methylation and DNA methylation signatures of NSPCs derived from the subventricular zone (SVZ) of young adult (3 months old) and aged (18 months old) mice. Surprisingly, the transcriptional and epigenomic profiles of SVZ-derived NSPCs are largely unchanged in aged cells. Despite the global similarities, we detect robust age-dependent changes at several hundred genes and regulatory elements, thereby identifying putative regulators of neurogenic decline. Within this list, the homeobox gene Dbx2 is upregulated in vitro and in vivo, and its promoter region has altered histone and DNA methylation levels, in aged NSPCs. Using functional in vitro assays, we show that elevated Dbx2 expression in young adult NSPCs promotes age-related phenotypes, including the reduced proliferation of NSPC cultures and the altered transcript levels of age-associated regulators of NSPC proliferation and differentiation. Depleting Dbx2 in aged NSPCs caused the reverse gene expression changes. Taken together, these results provide new insights into the molecular programmes that are affected during mouse NSPC aging, and uncover a new functional role for Dbx2 in promoting age-related neurogenic decline. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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.

Genomic phenotyping by barcode sequencing broadly distinguishes between alkylating agents, oxidizing agents, and non-genotoxic agents, and reveals a role for aromatic amino acids in cellular recovery after quinone exposure.

PubMed

Svensson, J Peter; Quirós Pesudo, Laia; McRee, Siobhan K; Adeleye, Yeyejide; Carmichael, Paul; Samson, Leona D

2013-01-01

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic 'barcode', were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput 'barcode' sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.

Genomic Phenotyping by Barcode Sequencing Broadly Distinguishes between Alkylating Agents, Oxidizing Agents, and Non-Genotoxic Agents, and Reveals a Role for Aromatic Amino Acids in Cellular Recovery after Quinone Exposure

PubMed Central

Svensson, J. Peter; Quirós Pesudo, Laia; McRee, Siobhan K.; Adeleye, Yeyejide; Carmichael, Paul; Samson, Leona D.

2013-01-01

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised. PMID:24040048

Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.

PubMed

Ceccarelli, Michele; Barthel, Floris P; Malta, Tathiane M; Sabedot, Thais S; Salama, Sofie R; Murray, Bradley A; Morozova, Olena; Newton, Yulia; Radenbaugh, Amie; Pagnotta, Stefano M; Anjum, Samreen; Wang, Jiguang; Manyam, Ganiraju; Zoppoli, Pietro; Ling, Shiyun; Rao, Arjun A; Grifford, Mia; Cherniack, Andrew D; Zhang, Hailei; Poisson, Laila; Carlotti, Carlos Gilberto; Tirapelli, Daniela Pretti da Cunha; Rao, Arvind; Mikkelsen, Tom; Lau, Ching C; Yung, W K Alfred; Rabadan, Raul; Huse, Jason; Brat, Daniel J; Lehman, Norman L; Barnholtz-Sloan, Jill S; Zheng, Siyuan; Hess, Kenneth; Rao, Ganesh; Meyerson, Matthew; Beroukhim, Rameen; Cooper, Lee; Akbani, Rehan; Wrensch, Margaret; Haussler, David; Aldape, Kenneth D; Laird, Peter W; Gutmann, David H; Noushmehr, Houtan; Iavarone, Antonio; Verhaak, Roel G W

2016-01-28

Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Aberrant DNA methylation at genes associated with a stem cell-like phenotype in cholangiocarcinoma tumours

PubMed Central

Dai, Wei; Siddiq, Afshan; Walley, Andrew J; Limpaiboon, Temduang; Brown, Robert

2013-01-01

Genetic abnormalities of cholangiocarcinoma have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterised. We have profiled the DNA methylomes of 28 primary cholangiocarcinoma and six matched adjacent normal tissues using Infinium’s HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly epigenetically regulated in this tumour type. Using a linear model for microarray data we identified 1610 differentially methylated autosomal CpG sites with 809 CpG sites (representing 603 genes) being hypermethylated and 801 CpG sites (representing 712 genes) being hypomethylated in cholangiocarcinoma versus adjacent normal tissues (false discovery rate ≤ 0.05). Gene ontology and gene set enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in cholangiocarcinoma including homeobox genes and target genes of PRC2, EED, SUZ12 and histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of cholangiocarcinoma (n = 102). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in cholangiocarcinoma. These data have implications for cholangiocarcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. PMID:24089088

Identification and validation of FaP1D7, a putative marker associated with the biosynthesis of methyl butanoate in cultivated strawberry (Fragaria x ananassa).

PubMed

Gor, Mian Chee; Candappa, Chrishani; de Silva, Thishakya; Mantri, Nitin; Pang, Edwin

2017-12-12

Breeding strawberry (Fragaria x ananassa) with enhanced fruit flavour is one of the top breeding goals of many strawberry-producing countries. Although several genes involved in the biosynthetic pathways of key aroma compounds have been identified, the development and application of molecular markers associated with fruit flavour remain limited. This study aims to identify molecular markers closely linked to genes controlling strawberry aroma. A purpose-built Subtracted Diversity Array (SDA) known as Fragaria Discovery Panel (FDP) was used for marker screening. Polymorphic sequences associated with key aroma compounds were identified from two DNA bulks with extreme phenotypes, established using 50 F 1 progeny plants derived from Juliette X 07-102-41 cross, two strawberry genotypes differing in aroma profile. A total of 49 polymorphic markers for eight key aroma compounds were detected using genotypic data of the extreme DNA bulks and phenotypic data obtained from gas chromatography-mass spectrometry (GC-MS). A similarity search against the physical maps of Fragaria vesca revealed that FaP1D7 is linked to genes potentially involved in the synthesis of methyl butanoate. A C/T SNP was detected within the feature, which could possibly be converted to a molecular tool for rapid screening of the strawberry accessions for their methyl butanoate production capacity.

Discovery of a novel class of potent coumarin monoamine oxidase B inhibitors: development and biopharmacological profiling of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate (NW-1772) as a highly potent, selective, reversible, and orally active monoamine oxidase B inhibitor.

PubMed

Pisani, Leonardo; Muncipinto, Giovanni; Miscioscia, Teresa Fabiola; Nicolotti, Orazio; Leonetti, Francesco; Catto, Marco; Caccia, Carla; Salvati, Patricia; Soto-Otero, Ramon; Mendez-Alvarez, Estefania; Passeleu, Celine; Carotti, Angelo

2009-11-12

In an effort to discover novel selective monoamine oxidase (MAO) B inhibitors with favorable physicochemical and pharmacokinetic profiles, 7-[(m-halogeno)benzyloxy]coumarins bearing properly selected polar substituents at position 4 were designed, synthesized, and evaluated as MAO inhibitors. Several compounds with MAO-B inhibitory activity in the nanomolar range and excellent MAO-B selectivity (selectivity index SI > 400) were identified. Structure-affinity relationships and docking simulations provided valuable insights into the enzyme-inhibitor binding interactions at position 4, which has been poorly explored. Furthermore, computational and experimental studies led to the identification and biopharmacological characterization of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate 22b (NW-1772) as an in vitro and in vivo potent and selective MAO-B inhibitor, with rapid blood-brain barrier penetration, short-acting and reversible inhibitory activity, slight inhibition of selected cytochrome P450s, and low in vitro toxicity. On the basis of this preliminary preclinical profile, inhibitor 22b might be viewed as a promising clinical candidate for the treatment of neurodegenerative diseases.

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

PubMed

Smith, E N; Ghia, E M; DeBoever, C M; Rassenti, L Z; Jepsen, K; Yoon, K-A; Matsui, H; Rozenzhak, S; Alakus, H; Shepard, P J; Dai, Y; Khosroheidari, M; Bina, M; Gunderson, K L; Messer, K; Muthuswamy, L; Hudson, T J; Harismendy, O; Barrett, C L; Jamieson, C H M; Carson, D A; Kipps, T J; Frazer, K A

2015-04-10

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

ViralEpi v1.0: a high-throughput spectrum of viral epigenomic methylation profiles from diverse diseases.

PubMed

Khan, Mohd Shoaib; Gupta, Amit Kumar; Kumar, Manoj

2016-01-01

To develop a computational resource for viral epigenomic methylation profiles from diverse diseases. Methylation patterns of Epstein-Barr virus and hepatitis B virus genomic regions are provided as web platform developed using open source Linux-Apache-MySQL-PHP (LAMP) bundle: programming and scripting languages, that is, HTML, JavaScript and PERL. A comprehensive and integrated web resource ViralEpi v1.0 is developed providing well-organized compendium of methylation events and statistical analysis associated with several diseases. Additionally, it also facilitates 'Viral EpiGenome Browser' for user-affable browsing experience using JavaScript-based JBrowse. This web resource would be helpful for research community engaged in studying epigenetic biomarkers for appropriate prognosis and diagnosis of diseases and its various stages.

Changes in urinary amino acids excretion in relationship with muscle activity markers over a professional cycling stage race: in search of fatigue markers.

PubMed

Corsetti, Roberto; Barassi, Alessandra; Perego, Silvia; Sansoni, Veronica; Rossi, Alessandra; Damele, Clara Anna Linda; Melzi D'Eril, Gianlodovico; Banfi, Giuseppe; Lombardi, Giovanni

2016-01-01

The aim of this study was to identify the relationship between metabolic effort, muscular damage/activity indices, and urinary amino acids profile over the course of a strenuous prolonged endurance activity, as a cycling stage race is, in order to identify possible fatigue markers. Nine professional cyclists belonging to a single team, competing in the Giro d'Italia cycling stage race, were anthropometrically characterized and sampled for blood and urine the day before the race started, and on days 12 and 23 of the race. Diet was kept the same over the race, and power output and energy expenditure were recorded. Sera were assayed for muscle markers (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase activities, and blood urea nitrogen), and creatinine, all corrected for plasma volume changes. Urines were profiled for amino acid concentrations, normalized on creatinine excretion. Renal function, in terms of glomerular filtration rate, was monitored by MDRD equation corrected on body surface area. Creatine kinase activity and blood urea were increased during the race as did serum creatinine while kidney function remained stable. Among the amino acids, taurine, glycine, cysteine, leucine, carnosine, 1-methyl histidine, and 3-methyl histidine showed a net decreased, while homocysteine was increased. Taurine and the dipeptide carnosine (β-alanyl-L-histidine) were significantly correlated with the muscle activity markers and the indices of effort. In conclusion, the metabolic profile is modified strikingly due to the effort. Urinary taurine and carnosine seem useful tools to evaluate the muscle damage and possibly the fatigue status on a long-term basis.

Omic personality: implications of stable transcript and methylation profiles for personalized medicine.

PubMed

Tabassum, Rubina; Sivadas, Ambily; Agrawal, Vartika; Tian, Haozheng; Arafat, Dalia; Gibson, Greg

2015-08-13

Personalized medicine is predicated on the notion that individual biochemical and genomic profiles are relatively constant in times of good health and to some extent predictive of disease or therapeutic response. We report a pilot study quantifying gene expression and methylation profile consistency over time, addressing the reasons for individual uniqueness, and its relation to N = 1 phenotypes. Whole blood samples from four African American women, four Caucasian women, and four Caucasian men drawn from the Atlanta Center for Health Discovery and Well Being study at three successive 6-month intervals were profiled by RNA-Seq, miRNA-Seq, and Illumina Methylation 450 K arrays. Standard regression approaches were used to evaluate the proportion of variance for each type of omic measure among individuals, and to quantify correlations among measures and with clinical attributes related to wellness. Longitudinal omic profiles were in general highly consistent over time, with an average of 67 % variance in transcript abundance, 42 % in CpG methylation level (but 88 % for the most differentiated CpG per gene), and 50 % in miRNA abundance among individuals, which are all comparable to 74 % variance among individuals for 74 clinical traits. One third of the variance could be attributed to differential blood cell type abundance, which was also fairly stable over time, and a lesser amount to expression quantitative trait loci (eQTL) effects. Seven conserved axes of covariance that capture diverse aspects of immune function explained over half of the variance. These axes also explained a considerable proportion of individually extreme transcript abundance, namely approximately 100 genes that were significantly up-regulated or down-regulated in each person and were in some cases enriched for relevant gene activities that plausibly associate with clinical attributes. A similar fraction of genes had individually divergent methylation levels, but these did not overlap with the transcripts, and fewer than 20 % of genes had significantly correlated methylation and gene expression. People express an "omic personality" consisting of peripheral blood transcriptional and epigenetic profiles that are constant over the course of a year and reflect various types of immune activity. Baseline genomic profiles can provide a window into the molecular basis of traits that might be useful for explaining medical conditions or guiding personalized health decisions.

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

Identification of key regulators of pancreatic cancer progression through multidimensional systems-level analysis.

PubMed

Rajamani, Deepa; Bhasin, Manoj K

2016-05-03

Pancreatic cancer is an aggressive cancer with dismal prognosis, urgently necessitating better biomarkers to improve therapeutic options and early diagnosis. Traditional approaches of biomarker detection that consider only one aspect of the biological continuum like gene expression alone are limited in their scope and lack robustness in identifying the key regulators of the disease. We have adopted a multidimensional approach involving the cross-talk between the omics spaces to identify key regulators of disease progression. Multidimensional domain-specific disease signatures were obtained using rank-based meta-analysis of individual omics profiles (mRNA, miRNA, DNA methylation) related to pancreatic ductal adenocarcinoma (PDAC). These domain-specific PDAC signatures were integrated to identify genes that were affected across multiple dimensions of omics space in PDAC (genes under multiple regulatory controls, GMCs). To further pin down the regulators of PDAC pathophysiology, a systems-level network was generated from knowledge-based interaction information applied to the above identified GMCs. Key regulators were identified from the GMC network based on network statistics and their functional importance was validated using gene set enrichment analysis and survival analysis. Rank-based meta-analysis identified 5391 genes, 109 miRNAs and 2081 methylation-sites significantly differentially expressed in PDAC (false discovery rate ≤ 0.05). Bimodal integration of meta-analysis signatures revealed 1150 and 715 genes regulated by miRNAs and methylation, respectively. Further analysis identified 189 altered genes that are commonly regulated by miRNA and methylation, hence considered GMCs. Systems-level analysis of the scale-free GMCs network identified eight potential key regulator hubs, namely E2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1, associated with important pathways driving cancer progression. Survival analysis on individual key regulators revealed that higher expression of IRS1 and DLL1 and lower expression of HMGA2, ACTN1 and SKI were associated with better survival probabilities. It is evident from the results that our hierarchical systems-level multidimensional analysis approach has been successful in isolating the converging regulatory modules and associated key regulatory molecules that are potential biomarkers for pancreatic cancer progression.

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.

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

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

PubMed Central

Kleihues, P.; Magee, P. N.

1973-01-01

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2–3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[14C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA. PMID:4774397

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.

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.

Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma

PubMed Central

2013-01-01

Background Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Methods Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Results and discussion Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene. Conclusions Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis. PMID:23419152

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…

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

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

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

PubMed Central

He, Yupeng; Gorkin, David U.; Dickel, Diane E.; Nery, Joseph R.; Castanon, Rosa G.; Lee, Ah Young; Shen, Yin; Visel, Axel; Pennacchio, Len A.; Ren, Bing; Ecker, Joseph R.

2017-01-01

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/. PMID:28193886

Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent.

PubMed

Theoclitou, Maria-Elena; Aquila, Brian; Block, Michael H; Brassil, Patrick J; Castriotta, Lillian; Code, Erin; Collins, Michael P; Davies, Audrey M; Deegan, Tracy; Ezhuthachan, Jayachandran; Filla, Sandra; Freed, Ellen; Hu, Haiqing; Huszar, Dennis; Jayaraman, Muthusamy; Lawson, Deborah; Lewis, Paula M; Nadella, Murali V P; Oza, Vibha; Padmanilayam, Maniyan; Pontz, Timothy; Ronco, Lucienne; Russell, Daniel; Whitston, David; Zheng, Xiaolan

2011-10-13

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

Exclusive mutation in epidermal growth factor receptor gene, HER-2, and KRAS, and synchronous methylation of nonsmall cell lung cancer.

PubMed

Suzuki, Makoto; Shigematsu, Hisayuki; Iizasa, Toshihiko; Hiroshima, Kenzo; Nakatani, Yukio; Minna, John D; Gazdar, Adi F; Fujisawa, Takehiko

2006-05-15

Both genetic and epigenetic changes in nonsmall cell lung cancer (NSCLC) are known to be a common event. Mutations in the epidermal growth factor receptor gene (EGFR), HER-2, and KRAS and the methylation profile of 9 genes for NSCLC were analyzed and correlated with clinical and histologic data. Thirty-nine EGFR, 4 HER-2, and 6 KRAS mutations were found in 150 NSCLC cases, with the methylation percentages of the genes ranging from 13% to 54%. Most mutations were present in adenocarcinomas, but mutations of the 3 genes were never found to be present in individual tumors. The frequency of methylation for all the genes was correlated with the Methylation Index, a reflection of the overall methylation pattern (all genes, P< or = .01), supporting the presence of the CpG island methylator phenotype (CIMP) in NSCLC. On the basis of the methylation profile, CRBP1 and CDH13 methylation were good indicators of CIMP in NSCLC, and were correlated with a poorer prognosis in adenocarcinomas. Mutations in EGFR, HER-2, and KRAS were found to be present exclusively, whereas methylation tended to be present synchronously. A comparison of mutation and methylation demonstrated that the EGFR mutation had an inverse correlation with methylation of SPARC (secreted protein acidic and rich in cysteine), an extracellular Ca2+-binding matricellular glycoprotein associated with the regulation of cell adhesion and growth, and the p16INK4A gene. The findings of the current study suggest that adenocarcinoma cases with CIMP have a poorer prognosis than adenocarcinoma cases without CIMP, and the EGFR mutation was shown to have an inverse correlation with methylation of SPARC and the p16INK4A gene in NSCLC. Copyright 2006 American Cancer Society

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

A review on environmental factors regulating arsenic methylation in humans.

PubMed

Tseng, Chin-Hsiao

2009-03-15

Subjects exposed to arsenic show significant inter-individual variation in urinary patterns of arsenic metabolites but insignificant day-to-day intra-individual variation. The inter-individual variation in arsenic methylation can be partly responsible for the variation in susceptibility to arsenic toxicity. Wide inter-ethnic variation and family correlation in urinary arsenic profile suggest a genetic effect on arsenic metabolism. In this paper the environmental factors affecting arsenic metabolism are reviewed. Methylation capacity might reduce with increasing dosage of arsenic exposure. Furthermore, women, especially at pregnancy, have better methylation capacity than their men counterparts, probably due to the effect of estrogen. Children might have better methylation capacity than adults and age shows inconsistent relevance in adults. Smoking and alcohol consumption might be associated with a poorer methylation capacity. Nutritional status is important in the methylation capacity and folate may facilitate the methylation and excretion of arsenic. Besides, general health conditions and medications might influence the arsenic methylation capacity; and technical problems can cause biased estimates. The consumption of seafood, seaweed, rice and other food with high arsenic contents and the extent of cooking and arsenic-containing water used in food preparation may also interfere with the presentation of the urinary arsenic profile. Future studies are necessary to clarify the effects of the various arsenic metabolites including the trivalent methylated forms on the development of arsenic-induced human diseases with the consideration of the effects of confounding factors and the interactions with other effect modifiers.

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

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.

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.

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 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.

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.

Epigenetic estimation of age in humpback whales.

PubMed

Polanowski, Andrea M; Robbins, Jooke; Chandler, David; Jarman, Simon N

2014-09-01

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R(2) of 0.787 (P = 3.04e-16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent-offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms. © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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.

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.

System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination.

PubMed

Zimmermann, Michael T; Oberg, Ann L; Grill, Diane E; Ovsyannikova, Inna G; Haralambieva, Iana H; Kennedy, Richard B; Poland, Gregory A

2016-01-01

Failure to achieve a protected state after influenza vaccination is poorly understood but occurs commonly among aged populations experiencing greater immunosenescence. In order to better understand immune response in the elderly, we studied epigenetic and transcriptomic profiles and humoral immune response outcomes in 50-74 year old healthy participants. Associations between DNA methylation and gene expression reveal a system-wide regulation of immune-relevant functions, likely playing a role in regulating a participant's propensity to respond to vaccination. Our findings show that sites of methylation regulation associated with humoral response to vaccination impact known cellular differentiation signaling and antigen presentation pathways. We performed our analysis using per-site and regionally average methylation levels, in addition to continuous or dichotomized outcome measures. The genes and molecular functions implicated by each analysis were compared, highlighting different aspects of the biologic mechanisms of immune response affected by differential methylation. Both cis-acting (within the gene or promoter) and trans-acting (enhancers and transcription factor binding sites) sites show significant associations with measures of humoral immunity. Specifically, we identified a group of CpGs that, when coordinately hypo-methylated, are associated with lower humoral immune response, and methylated with higher response. Additionally, CpGs that individually predict humoral immune responses are enriched for polycomb-group and FOXP2 transcription factor binding sites. The most robust associations implicate differential methylation affecting gene expression levels of genes with known roles in immunity (e.g. HLA-B and HLA-DQB2) and immunosenescence. We believe our data and analysis strategy highlight new and interesting epigenetic trends affecting humoral response to vaccination against influenza; one of the most common and impactful viral pathogens.

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

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.

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

PubMed Central

Calvello, Mariarosaria; Tabano, Silvia; Colapietro, Patrizia; Maitz, Silvia; Pansa, Alessandra; Augello, Claudia; Lalatta, Faustina; Gentilin, Barbara; Spreafico, Filippo; Calzari, Luciano; Perotti, Daniela; Larizza, Lidia; Russo, Silvia; Selicorni, Angelo; Sirchia, Silvia M; Miozzo, Monica

2013-01-01

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD. PMID:23917791

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.

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.

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

PubMed Central

Brait, Mariana; Ling, Shizhang; Nagpal, Jatin K.; Chang, Xiaofei; Park, Hannah Lui; Lee, Juna; Okamura, Jun; Yamashita, Keishi; Sidransky, David; Kim, Myoung Sook

2012-01-01

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. PMID:23028699

Epigenetic determinants of ovarian clear cell carcinoma biology

PubMed Central

Yamaguchi, Ken; Huang, Zhiqing; Matsumura, Noriomi; Mandai, Masaki; Okamoto, Takako; Baba, Tsukasa; Konishi, Ikuo; Berchuck, Andrew; Murphy, Susan K.

2015-01-01

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC, and 4 corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1 binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p<0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process. PMID:24382740

Increased Expression and Altered Methylation of HERVWE1 in the Human Placentas of Smaller Fetuses from Monozygotic, Dichorionic, Discordant Twins

PubMed Central

Wang, Zilian; Luo, Yanmin; Sun, Hongyu; Zhou, Yi; Huang, Linhuan; Li, Manchao; Fang, Qun; Jiang, Shiwen

2012-01-01

Background The human endogenous retroviral family W, Env(C7), member 1 gene (HERVWE1) is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. Methodology/Principal Findings Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as “smaller” or “larger” according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC) staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT) transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC) was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P<0.01). Whereas the mean methylation level of the HERVWE1 promoter region was diminished in the smaller group in discordant twins(P<0.01), increased mRNA and protein levels of HERVWE1 were found in smaller fetuses compared with larger fetuses in discordant twins(P<0.01). There was no significant difference in 5-MC staining intensity between discordant twins (P>0.05). The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05), whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05). Conclusions/Significance In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies. PMID:22457770

Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

PubMed Central

Guo, Ailan; Gu, Hongbo; Zhou, Jing; Mulhern, Daniel; Wang, Yi; Lee, Kimberly A.; Yang, Vicky; Aguiar, Mike; Kornhauser, Jon; Jia, Xiaoying; Ren, Jianmin; Beausoleil, Sean A.; Silva, Jeffrey C.; Vemulapalli, Vidyasiri; Bedford, Mark T.; Comb, Michael J.

2014-01-01

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins. PMID:24129315

Different profiles of quercetin metabolites in rat plasma: comparison of two administration methods.

PubMed

Kawai, Yoshichika; Saito, Satomi; Nishikawa, Tomomi; Ishisaka, Akari; Murota, Kaeko; Terao, Junji

2009-03-23

The bioavailability of polyphenols in human and rodents has been discussed regarding their biological activity. We found different metabolite profiles of quercetin in rat plasma between two administration procedures. A single intragastric administration (50 mg/kg) resulted in the appearance of a variety of metabolites in the plasma, whereas only a major fraction was detected by free access (1% quercetin). The methylated/non-methylated metabolites ratio was much higher in the free access group. Mass spectrometric analyses showed that the fraction from free access contained highly conjugated quercetin metabolites such as sulfo-glucuronides of quercetin and methylquercetin. The metabolite profile of human plasma after an intake of onion was similar to that with intragastric administration in rats. In vitro oxidation of human low-density lipoprotein showed that methylation of the catechol moiety of quercetin significantly attenuated the antioxidative activity. These results might provide information about the bioavailability of quercetin when conducting animal experiments.

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

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.

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/.

Clinical significance of miRNA host gene promoter methylation in prostate cancer.

PubMed

Daniunaite, Kristina; Dubikaityte, Monika; Gibas, Povilas; Bakavicius, Arnas; Rimantas Lazutka, Juozas; Ulys, Albertas; Jankevicius, Feliksas; Jarmalaite, Sonata

2017-07-01

Only a part of prostate cancer (PCa) patients has aggressive malignancy requiring adjuvant treatment after radical prostatectomy (RP). Biomarkers capable to predict biochemical PCa recurrence (BCR) after RP would significantly improve preoperative risk stratification and treatment decisions. MicroRNA (miRNA) deregulation has recently emerged as an important phenomenon in tumor development and progression, however, the mechanisms remain largely unstudied. In the present study, based on microarray profiling of DNA methylation in 9 pairs of PCa and noncancerous prostate tissues (NPT), host genes of miR-155-5p, miR-152-3p, miR-137, miR-31-5p, and miR-642a, -b were analyzed for promoter methylation in 129 PCa, 35 NPT, and 17 benign prostatic hyperplasia samples (BPH) and compared to the expression of mature miRNAs and their selected targets (DNMT1, KDM1A, and KDM5B). The Cancer Genome Atlas dataset was utilized for validation. Methylation of mir-155, mir-152, and mir-137 host genes was PCa-specific, and downregulation of miR-155-5p significantly correlated with promoter methylation. Higher KDM5B expression was observed in samples with methylated mir-155 or mir-137 promoters, whereas upregulation of KDM1A and DNMT1 was associated with mir-155 and mir-152 methylation status, respectively. Promoter methylation of mir-155, mir-152, and mir-31 was predictive of BCR-free survival in various Cox models and increased the prognostic value of clinicopathologic factors. In conclusion, methylated mir-155, mir-152, mir-137, and mir-31 host genes are promising diagnostic and/or prognostic biomarkers of PCa. Methylation status of particular miRNA host genes as independent variables or in combinations might assist physicians in identifying poor prognosis PCa patients preoperatively. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

PubMed Central

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-01-01

Background Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine–phosphate–guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine–phosphate–guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation–specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. PMID:24997139

A novel in vitro metabolomics approach for neurotoxicity testing, proof of principle for methyl mercury chloride and caffeine.

PubMed

van Vliet, Erwin; Morath, Siegfried; Eskes, Chantra; Linge, Jens; Rappsilber, Juri; Honegger, Paul; Hartung, Thomas; Coecke, Sandra

2008-01-01

There is a need for more efficient methods giving insight into the complex mechanisms of neurotoxicity. Testing strategies including in vitro methods have been proposed to comply with this requirement. With the present study we aimed to develop a novel in vitro approach which mimics in vivo complexity, detects neurotoxicity comprehensively, and provides mechanistic insight. For this purpose we combined rat primary re-aggregating brain cell cultures with a mass spectrometry (MS)-based metabolomics approach. For the proof of principle we treated developing re-aggregating brain cell cultures for 48 h with the neurotoxicant methyl mercury chloride (0.1-100 microM) and the brain stimulant caffeine (1-100 microM) and acquired cellular metabolic profiles. To detect toxicant-induced metabolic alterations the profiles were analysed using commercial software which revealed patterns in the multi-parametric dataset by principal component analyses (PCA), and recognised the most significantly altered metabolites. PCA revealed concentration-dependent cluster formations for methyl mercury chloride (0.1-1 microM), and treatment-dependent cluster formations for caffeine (1-100 microM) at sub-cytotoxic concentrations. Four relevant metabolites responsible for the concentration-dependent alterations following methyl mercury chloride treatment could be identified using MS-MS fragmentation analysis. These were gamma-aminobutyric acid, choline, glutamine, creatine and spermine. Their respective mass ion intensities demonstrated metabolic alterations in line with the literature and suggest that the metabolites could be biomarkers for mechanisms of neurotoxicity or neuroprotection. In addition, we evaluated whether the approach could identify neurotoxic potential by testing eight compounds which have target organ toxicity in the liver, kidney or brain at sub-cytotoxic concentrations. PCA revealed cluster formations largely dependent on target organ toxicity indicating possible potential for the development of a neurotoxicity prediction model. With such results it could be useful to perform a validation study to determine the reliability, relevance and applicability of this approach to neurotoxicity screening. Thus, for the first time we show the benefits and utility of in vitro metabolomics to comprehensively detect neurotoxicity and to discover new biomarkers.

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

Fatty acid methyl ester from Neurospora intermedia N-1 isolated from Indonesian red peanut cake (oncom merah).

PubMed

Priatni, S; Hartati, S; Dewi, P; Kardono, L B S; Singgih, M; Gusdinar, T

2010-08-01

The objective of this study was to identify the Fatty Acid Methyl Ester (FAME) from Neurospora intermedia N-1 that isolated from Indonesian red peanut cake (oncom). FAME profiles have been used as biochemical characters to study many different groups of organisms, such as bacteria and yeasts. FAME from N. intermedia N-1 was obtained by some stages of extraction the orange spores and fractination using a chromatotron. The pure compound (1) was characterized by 500 mHz NMR (1H and 13C), FTIR and LC-MS. Summarized data's of 1H and 13C NMR spectra of compound 1 contained 19 Carbon, 34 Hydrogen and 2 Oxygen (C19H34O2). The position of the double bonds at carbon number 8 and 12 were indicated in the HMBC spectrum (2D-NMR). LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is visible by the presence of protonated molecular ion [M+H] at m/z 295. Methyl esters of long chain fatty acids was presented by a 3 band pattern of IR spectrum with bands near 1249, 1199 and 1172 cm(-1). We suggested that the structure of the pure compound 1 is methyl octadeca-8,12-dienoate. The presence methyl octadeca-8,12-dienoate in N. intermedia is the first report.

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

Systems Genetics Reveals the Functional Context of PCOS Loci and Identifies Genetic and Molecular Mechanisms of Disease Heterogeneity.

PubMed

Jones, Michelle R; Brower, Meredith A; Xu, Ning; Cui, Jinrui; Mengesha, Emebet; Chen, Yii-Der I; Taylor, Kent D; Azziz, Ricardo; Goodarzi, Mark O

2015-08-01

Genome wide association studies (GWAS) have revealed 11 independent risk loci for polycystic ovary syndrome (PCOS), a common disorder in young women characterized by androgen excess and oligomenorrhea. To put these risk loci and the single nucleotide polymorphisms (SNPs) therein into functional context, we measured DNA methylation and gene expression in subcutaneous adipose tissue biopsies to identify PCOS-specific alterations. Two genes from the LHCGR region, STON1-GTF2A1L and LHCGR, were overexpressed in PCOS. In analysis stratified by obesity, LHCGR was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, INSR was underexpressed in obese PCOS subjects only. Alterations in gene expression in the LHCGR, RAB5B and INSR regions suggest that SNPs in these loci may be functional and could affect gene expression directly or indirectly via epigenetic alterations. We identified reduced methylation in the LHCGR locus and increased methylation in the INSR locus, changes that are concordant with the altered gene expression profiles. Complex patterns of meQTL and eQTL were identified in these loci, suggesting that local genetic variation plays an important role in gene regulation. We propose that non-obese PCOS women possess significant alterations in LH receptor expression, which drives excess androgen secretion from the ovary. Alternatively, obese women with PCOS possess alterations in insulin receptor expression, with underexpression in metabolic tissues and overexpression in the ovary, resulting in peripheral insulin resistance and excess ovarian androgen production. These studies provide a genetic and molecular basis for the reported clinical heterogeneity of PCOS.

Genome-wide histone state profiling of fibroblasts from the opossum, Monodelphis domestica, identifies the first marsupial-specific imprinted gene

PubMed Central

2014-01-01

Background Imprinted genes have been extensively documented in eutherian mammals and found to exhibit significant interspecific variation in the suites of genes that are imprinted and in their regulation between tissues and developmental stages. Much less is known about imprinted loci in metatherian (marsupial) mammals, wherein studies have been limited to a small number of genes previously known to be imprinted in eutherians. We describe the first ab initio search for imprinted marsupial genes, in fibroblasts from the opossum, Monodelphis domestica, based on a genome-wide ChIP-seq strategy to identify promoters that are simultaneously marked by mutually exclusive, transcriptionally opposing histone modifications. Results We identified a novel imprinted gene (Meis1) and two additional monoallelically expressed genes, one of which (Cstb) showed allele-specific, but non-imprinted expression. Imprinted vs. allele-specific expression could not be resolved for the third monoallelically expressed gene (Rpl17). Transcriptionally opposing histone modifications H3K4me3, H3K9Ac, and H3K9me3 were found at the promoters of all three genes, but differential DNA methylation was not detected at CpG islands at any of these promoters. Conclusions In generating the first genome-wide histone modification profiles for a marsupial, we identified the first gene that is imprinted in a marsupial but not in eutherian mammals. This outcome demonstrates the practicality of an ab initio discovery strategy and implicates histone modification, but not differential DNA methylation, as a conserved mechanism for marking imprinted genes in all therian mammals. Our findings suggest that marsupials use multiple epigenetic mechanisms for imprinting and support the concept that lineage-specific selective forces can produce sets of imprinted genes that differ between metatherian and eutherian lines. PMID:24484454

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.

Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins.

PubMed

Banfi, Cristina; Baetta, Roberta; Gianazza, Erica; Tremoli, Elena

2017-06-01

Proteomic-based techniques provide a powerful tool for identifying the full spectrum of protein targets of a drug, elucidating its mechanism(s) of action, and identifying biomarkers of its efficacy and safety. Herein, we outline the technological advancements in the field, and illustrate the contribution of proteomics to the definition of the pharmacological profile of statins, which represent the cornerstone of the prevention and treatment of cardiovascular diseases (CVDs). Statins act by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, thus reducing cholesterol biosynthesis and consequently enhancing the clearance of low-density lipoproteins from the blood; however, HMG-CoA reductase inhibition can result in a multitude of additional effects beyond lipid lowering, known as 'pleiotropic effects'. The case of statins highlights the unique contribution of proteomics to the target profiling of a drug molecule. Copyright © 2017 Elsevier Ltd. All rights reserved.

Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

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

Putnik, Milica, E-mail: milica.putnik@ki.se; Zhao, Chunyan, E-mail: chunyan.zhao@ki.se; Gustafsson, Jan-Ake, E-mail: jan-ake.gustafsson@ki.se

Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between thesemore » two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment showed no effect on the methylation status of these promoters. Additionally, we show that the ER{alpha} recruitment occurs at the FHL2 promoter in an E2- and DAC-independent fashion. In conclusion, we identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes.« less

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.

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

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...

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.

Examining post-translational modification-mediated protein–protein interactions using a chemical proteomics approach

PubMed Central

Li, Xiang; Foley, Emily A; Kawashima, Shigehiro A; Molloy, Kelly R; Li, Yinyin; Chait, Brian T; Kapoor, Tarun M

2013-01-01

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me3)-dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me3). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs. PMID:23281010

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.

Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach.

PubMed

Kupcinskaite-Noreikiene, Rita; Ugenskiene, Rasa; Noreika, Alius; Rudzianskas, Viktoras; Gedminaite, Jurgita; Skieceviciene, Jurgita; Juozaityte, Elona

2016-01-26

There is considerable information on the methylation of the promoter regions of different genes involved in gastric carcinogenesis. However, there is a lack of information on how this epigenetic process differs in tumors originating at different sites in the stomach. The aim of this study is to assess the methylation profiles of the MLH1, MGMT, and DAPK-1 genes in cancerous tissues from different stomach sites. Samples were acquired from 81 patients suffering stomach adenocarcinoma who underwent surgery for gastric cancer in the Lithuanian University of Health Sciences Hospital Kaunas Clinics in 2009-2012. Gene methylation was investigated with methylation-specific PCR. The study was approved by the Lithuanian Biomedical Research Ethics Committee. The frequencies of methylation in cancerous tissues from the upper, middle, and lower thirds of the stomach were 11.1, 23.1, and 45.4%, respectively, for MLH1; 22.2, 30.8, and 57.6%, respectively, for MGMT; and 44.4, 48.7, and 51.5%, respectively, for DAPK-1. MLH1 and MGMT methylation was observed more often in the lower third of the stomach than in the upper third (p < 0.05). In the middle third, DAPK-1 promoter methylation was related to more-advanced disease in the lymph nodes (N2-3 compared with N0-1 [p = 0.02]) and advanced tumor stage (stage III rather than stages I-II [p = 0.05]). MLH1 and MGMT methylation correlated inversely when the tumor was located in the lower third of the stomach (coefficient, -0.48; p = 0.01). DAPK-1 and MLH1 methylation correlated inversely in tumors in the middle-third of the stomach (coefficient, -0.41; p = 0.01). Gene promoter methylation depends on the gastric tumor location.

Methylation-based classification of benign and malignant peripheral nerve sheath tumors.

PubMed

Röhrich, Manuel; Koelsche, Christian; Schrimpf, Daniel; Capper, David; Sahm, Felix; Kratz, Annekathrin; Reuss, Jana; Hovestadt, Volker; Jones, David T W; Bewerunge-Hudler, Melanie; Becker, Albert; Weis, Joachim; Mawrin, Christian; Mittelbronn, Michel; Perry, Arie; Mautner, Victor-Felix; Mechtersheimer, Gunhild; Hartmann, Christian; Okuducu, Ali Fuat; Arp, Mirko; Seiz-Rosenhagen, Marcel; Hänggi, Daniel; Heim, Stefanie; Paulus, Werner; Schittenhelm, Jens; Ahmadi, Rezvan; Herold-Mende, Christel; Unterberg, Andreas; Pfister, Stefan M; von Deimling, Andreas; Reuss, David E

2016-06-01

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.

Profiling post-translational modifications of histones in human monocyte-derived macrophages.

PubMed

Olszowy, Pawel; Donnelly, Maire Rose; Lee, Chanho; Ciborowski, Pawel

2015-01-01

Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.

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

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.

Classification of juices and fermented beverages made from unripe, ripe and senescent apples based on the aromatic profile using chemometrics.

PubMed

Braga, Cíntia Maia; Zielinski, Acácio Antonio Ferreira; Silva, Karolline Marques da; de Souza, Frederico Koch Fernandes; Pietrowski, Giovana de Arruda Moura; Couto, Marcelo; Granato, Daniel; Wosiacki, Gilvan; Nogueira, Alessandro

2013-11-15

The aim of this study was to assess differences between apple juices and fermented apple beverages elaborated with fruits from different varieties and at different ripening stages in the aroma profile by using chemometrics. Ripening influenced the aroma composition of the apple juice and fermented apple. For all varieties, senescent fruits provided more aromatic fermented apple beverages. However, no significant difference was noticed in samples made of senescent or ripe fruits of the Lisgala variety. Regarding the juices, ripe Gala apple had the highest total aroma concentration. Ethanal was the major compound identified in all the samples, with values between 11.83mg/L (unripe Lisgala juice) and 81.05mg/L (ripe Gala juice). 3-Methyl-1-butanol was the major compound identified in the fermented juices. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied and classified the juices and fermented juices based on physicochemical and aroma profile, demonstrating their applicability as tools to monitor the quality of apple-based products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bisphenol A-associated epigenomic changes in prepubescent girls: a cross-sectional study in Gharbiah, Egypt

PubMed Central

2013-01-01

Background There is now compelling evidence that epigenetic modifications link adult disease susceptibility to environmental exposures during specific life stages, including pre-pubertal development. Animal studies indicate that bisphenol A (BPA), the monomer used in epoxy resins and polycarbonate plastics, may impact health through epigenetic mechanisms, and epidemiological data associate BPA levels with metabolic disorders, behavior changes, and reproductive effects. Thus, we conducted an environmental epidemiology study of BPA exposure and CpG methylation in pre-adolescent girls from Gharbiah, Egypt hypothesizing that methylation profiles exhibit exposure-dependent trends. Methods Urinary concentrations of total (free plus conjugated) species of BPA in spot samples were quantified for 60 girls aged 10 to 13. Genome-wide CpG methylation was concurrently measured in bisulfite-converted saliva DNA using the Infinium HumanMethylation27 BeadChip (N = 46). CpG sites from four candidate genes were validated via quantitative bisulfite pyrosequencing. Results CpG methylation varied widely among girls, and higher urinary BPA concentrations were generally associated with less genomic methylation. Based on pathway analyses, genes exhibiting reduced methylation with increasing urinary BPA were involved in immune function, transport activity, metabolism, and caspase activity. In particular, hypomethylation of CpG targets on chromosome X was associated with higher urinary BPA. Using the Comparative Toxicogenomics Database, we identified a number of candidate genes in our sample that previously have been associated with BPA-related expression change. Conclusions These data indicate that BPA may affect human health through specific epigenomic modification of genes in relevant pathways. Thus, epigenetic epidemiology holds promise for the identification of biomarkers from previous exposures and the development of epigenetic-based diagnostic strategies. PMID:23590724

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.

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

Methyl Mercury Production In Tropical Hydromorphic Soils: Impact Of Gold Mining.

NASA Astrophysics Data System (ADS)

Guedron, S.; Charlet, L.; Harris, J.; Grimaldi, M.; Cossa, D.

2007-12-01

Artisanal alluvial gold mining is important in many tropical developing countries and several million people are involved worldwide. The dominant use of mercury for gold amalgamation in this activity leads to mercury accumulation in soils, to sediment contamination and to methyl mercury (MMHg) bioaccumulation along the food chain. In this presentation we will present recent data on methyl mercury production in hydromorphic soils and tailing ponds from a former gold mining area located in French Guiana (South America). Comparison of specific fluxes between a pristine sub watershed and the contaminated watershed shows that former mining activities lead to a large enhancement of dissolved and particulate MMHg emissions at least by a factor of 4 and 6, respectively. MMHg production was identified in sediments from tailing ponds and in surrounding hydromorphic soils. Moreover, interstitial soil water and tailing pond water profiles sampled in an experimental tailing pond demonstrate the presence of a large MMHg production in the suboxic areas. Both tailing ponds and hydromorphic soils present geochemical conditions that are favorable to bacterial mercury methylation (high soil Hg content, high aqueous ferric iron and dissolved organic carbon concentrations). Although sulfate-reducing bacteria have been described as being the principal mercury methylating bacteria, the positive correlation between dissolved MMHg and ferrous iron concentrations argue for a significant role of iron-reducing bacteria. Identifications by sequencing fragments of 16S rRNA from total soil DNA support these interpretations. This study demonstrates that current and past artisanal gold mining in the tropics lead to methyl mercury production in contaminated areas. As artisanal activities are increasing with increasing gold prices, the bio- magnification of methyl mercury in fish presents an increasing threat to local populations whose diet relies on fish consumption.

[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.

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.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

PubMed

Kitimu, Shedrack R; Taylor, Julian; March, Timothy J; Tairo, Fred; Wilkinson, Mike J; Rodríguez López, Carlos M

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation

PubMed Central

Kitimu, Shedrack R.; Taylor, Julian; March, Timothy J.; Tairo, Fred; Wilkinson, Mike J.; Rodríguez López, Carlos M.

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops. PMID:26322052

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

MS-HRM assay identifies high levels of epigenetic heterogeneity in human immortalized cell lines.

PubMed

Putnik, Milica; Wojdacz, Tomasz K; Pournara, Angeliki; Vahter, Marie; Wallberg, Annika E

2015-04-15

Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Aroma Profile of Rubus ulmifolius Flowers and Fruits During Different Ontogenetic Phases.

PubMed

Bandeira Reidel, Rose Vanessa; Melai, Bernardo; Cioni, Pierluigi; Flamini, Guido; Pistelli, Luisa

2016-12-01

The chemical composition of spontaneous volatile emission from Rubus ulmifolius flowers and fruits during different stages of development was evaluated by HS-SPME-GC/MS. In total, 155 chemical compounds were identified accounting 84.6 - 99.4% of whole aroma profile of flowers samples and 92.4 - 96.6% for fruit samples. The main constituents were α-copaene, β-caryophyllene, germacrene D, (E,E)-α-farnesene, 1,7-octadien-3-one,2-methyl-6-methylene, tridecane, (E)-2-hexenol acetate, (E)-3-hexenol acetate and cyperene. The results give a chemotaxonomic contribution to the characterization of the VOCs emitted from flowers and fruits during their ontogenic development. © 2016 Wiley-VHCA AG, Zurich, Switzerland.

Comprehensive molecular tumor profiling in radiation oncology: How it could be used for precision medicine.

PubMed

Eke, Iris; Makinde, Adeola Y; Aryankalayil, Molykutty J; Ahmed, Mansoor M; Coleman, C Norman

2016-11-01

New technologies enabling the analysis of various molecules, including DNA, RNA, proteins and small metabolites, can aid in understanding the complex molecular processes in cancer cells. In particular, for the use of novel targeted therapeutics, elucidation of the mechanisms leading to cell death or survival is crucial to eliminate tumor resistance and optimize therapeutic efficacy. While some techniques, such as genomic analysis for identifying specific gene mutations or epigenetic testing of promoter methylation, are already in clinical use, other "omics-based" assays are still evolving. Here, we provide an overview of the current status of molecular profiling methods, including promising research strategies, as well as possible challenges, and their emerging role in radiation oncology. Published by Elsevier Ireland Ltd.

Genetic mechanisms underlying the methylation level of anthocyanins in grape (Vitis vinifera L.)

PubMed Central

2011-01-01

Background Plant color variation is due not only to the global pigment concentration but also to the proportion of different types of pigment. Variation in the color spectrum may arise from secondary modifications, such as hydroxylation and methylation, affecting the chromatic properties of pigments. In grapes (Vitis vinifera L.), the level of methylation modifies the stability and reactivity of anthocyanin, which directly influence the color of the berry. Anthocyanin methylation, as a complex trait, is controlled by multiple molecular factors likely to involve multiple regulatory steps. Results In a Syrah × Grenache progeny, two QTLs were detected for variation in level of anthocyanin methylation. The first one, explaining up to 27% of variance, colocalized with a cluster of Myb-type transcription factor genes. The second one, explaining up to 20% of variance, colocalized with a cluster of O-methyltransferase coding genes (AOMT). In a collection of 32 unrelated cultivars, MybA and AOMT expression profiles correlated with the level of methylated anthocyanin. In addition, the newly characterized AOMT2 gene presented two SNPs associated with methylation level. These mutations, probably leading to a structural change of the AOMT2 protein significantly affected the enzyme specific catalytic efficiency for the 3'-O-methylation of delphinidin 3-glucoside. Conclusion We demonstrated that variation in methylated anthocyanin accumulation is susceptible to involve both transcriptional regulation and structural variation. We report here the identification of novel AOMT variants likely to cause methylated anthocyanin variation. The integration of QTL mapping and molecular approaches enabled a better understanding of how variation in gene expression and catalytic efficiency of the resulting enzyme may influence the grape anthocyanin profile. PMID:22171701

Toward a Unified Understanding of Mercury and Methylated Mercury from the World's Oceans

NASA Astrophysics Data System (ADS)

McNutt, M. K.; Krabbenhoft, D. P.; Landing, W. M.; Sunderland, E. M.

2012-12-01

Marine fish and shellfish are the main source of toxic methylmercury exposure for humans. As recently as decade ago, very limited aqueous methylated mercury data were available from marine settings, resulting in a generally poor understanding of the processes controlling mercury in pelagic marine food webs. Recent oceanographic cruises have significantly improved availability of reliable measurements of methylated mercury and total mercury in seawater. This presentation will focus on vertical seawater profiles collected to depths 1000 m from three recent sampling efforts in collaboration with the CLIVAR Repeat Hydrography Program sponsored by NOAA including: 1) the northeastern Pacific (P16N cruise from Honolulu, Hawaii to Kodiak, Alaska); (2) the southern Indian Ocean (I5 cruise from Cape Town, South Africa, to Fremantle, Australia); and, (3) the Southern Ocean cruise (S4P from McMurdo, Antarctica, to Punta Arenas, Chile). Analytical results presented were all derived from the USGS Mercury Research Lab (http://wi.water.usgs.gov/mercury-lab). Supporting data derived from these cruises on water mass ages, nutrients, carbon and dissolved oxygen provide an opportunity to develop a stronger understanding of the biogeochemical factors controlling oceanic distributions of mercury and methylated mercury. Whole-water, median total mercury, and methylated mercury concentrations for the northern Pacific, southern Indian, and Southern Ocean were 1.10, 0.80, and 1.65 pM, , and 0.11, 0.08, and 0.32 pM, respectively. For all three oceans, vertical profiles of total mercury generally show the lowest concentrations in the surface mixed layer, and concentration maxima at the 700-1000 m depths. Surface depletion of total mercury is attributed to photo-chemical reduction and evasion of gaseous elemental mercury as well as scavenging by settling particulate matter, the main vector of transport to the subsurface ocean. Methylated mercury in all the ocean profiles reveal distinct mid-profile concentration maxima, however, the depth of the maxima are more varied than the total mercury profiles (150 - 700m). Also, our observed distribution of methylated mercury highly correlated with organic carbon remineralization rates (OCRR) in the North Pacific and Indian Oceans. Interestingly, we find the highest methylated mercury concentrations in the Southern Ocean, suggesting the possibility of unique mechanisms for methylmercury production, preservation, and degradation in polar ecosystems such as cold water temperatures, extended periods of sea ice cover, and annual atmospheric mercury depletion events. We are using these data to better link oceanic production of bioaccumulative mercury to models for atmospheric and oceanic transport and bioaccumulation. This will ultimately lead to a better understanding of mercury levels in consumable fish and shell fish.

Methylation of HPA axis related genes in men with hypersexual disorder.

PubMed

Jokinen, Jussi; Boström, Adrian E; Chatzittofis, Andreas; Ciuculete, Diana M; Öberg, Katarina Görts; Flanagan, John N; Arver, Stefan; Schiöth, Helgi B

2017-06-01

Hypersexual Disorder (HD) defined as non-paraphilic sexual desire disorder with components of compulsivity, impulsivity and behavioral addiction, and proposed as a diagnosis in the DSM 5, shares some overlapping features with substance use disorder including common neurotransmitter systems and dysregulated hypothalamic-pituitary-adrenal (HPA) axis function. In this study, comprising 67 HD male patients and 39 male healthy volunteers, we aimed to identify HPA-axis coupled CpG-sites, in which modifications of the epigenetic profile are associated with hypersexuality. The genome-wide methylation pattern was measured in whole blood using the Illumina Infinium Methylation EPIC BeadChip, measuring the methylation state of over 850K CpG sites. Prior to analysis, the global DNA methylation pattern was pre-processed according to standard protocols and adjusted for white blood cell type heterogeneity. We included CpG sites located within 2000bp of the transcriptional start site of the following HPA-axis coupled genes: Corticotropin releasing hormone (CRH), corticotropin releasing hormone binding protein (CRHBP), corticotropin releasing hormone receptor 1 (CRHR1), corticotropin releasing hormone receptor 2 (CRHR2), FKBP5 and the glucocorticoid receptor (NR3C1). We performed multiple linear regression models of methylation M-values to a categorical variable of hypersexuality, adjusting for depression, dexamethasone non-suppression status, Childhood Trauma Questionnaire total score and plasma levels of TNF-alpha and IL-6. Of 76 tested individual CpG sites, four were nominally significant (p<0.05), associated with the genes CRH, CRHR2 and NR3C1. Cg23409074-located 48bp upstream of the transcription start site of the CRH gene - was significantly hypomethylated in hypersexual patients after corrections for multiple testing using the FDR-method. Methylation levels of cg23409074 were positively correlated with gene expression of the CRH gene in an independent cohort of 11 healthy male subjects. The methylation levels at the identified CRH site, cg23409074, were significantly correlated between blood and four different brain regions. CRH is an important integrator of neuroendocrine stress responses in the brain, with a key role in the addiction processes. Our results show epigenetic changes in the CRH gene related to hypersexual disorder in men. Copyright © 2017 Elsevier Ltd. All rights reserved.

Apple juice inhibits human low density lipoprotein oxidation.

PubMed

Pearson, D A; Tan, C H; German, J B; Davis, P A; Gershwin, M E

1999-01-01

Dietary phenolic compounds, ubiquitous in vegetables and fruits and their juices possess antioxidant activity that may have beneficial effects on human health. The phenolic composition of six commercial apple juices, and of the peel (RP), flesh (RF) and whole fresh Red Delicious apples (RW), was determined by high performance liquid chromatography (HPLC), and total phenols were determined by the Folin-Ciocalteau method. HPLC analysis identified and quantified several classes of phenolic compounds: cinnamates, anthocyanins, flavan-3-ols and flavonols. Phloridzin and hydroxy methyl furfural were also identified. The profile of phenolic compounds varied among the juices. The range of concentrations as a percentage of total phenolic concentration was: hydroxy methyl furfural, 4-30%; phloridzin, 22-36%; cinnamates, 25-36%; anthocyanins, n.d.; flavan-3-ols, 8-27%; flavonols, 2-10%. The phenolic profile of the Red Delicious apple extracts differed from those of the juices. The range of concentrations of phenolic classes in fresh apple extracts was: hydroxy methyl furfural, n.d.; phloridzin, 11-17%; cinnamates, 3-27%; anthocyanins, n.d.-42%; flavan-3-ols, 31-54%; flavonols, 1-10%. The ability of compounds in apple juices and extracts from fresh apple to protect LDL was assessed using an in vitro copper catalyzed human LDL oxidation system. The extent of LDL oxidation was determined as hexanal production using static headspace gas chromatography. The apple juices and extracts, tested at 5 microM gallic acid equivalents (GAE), all inhibited LDL oxidation. The inhibition by the juices ranged from 9 to 34%, and inhibition by RF, RW and RP was 21, 34 and 38%, respectively. Regression analyses revealed no significant correlation between antioxidant activity and either total phenolic concentration or any specific class of phenolics. Although the specific components in the apple juices and extracts that contributed to antioxidant activity have yet to be identified, this study found that both fresh apple and commercial apple juices inhibited copper-catalyzed LDL oxidation. The in vitro antioxidant activity of apples support the inclusion of this fruit and its juice in a healthy human diet.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer

PubMed Central

Ha, Ye-Na; Sung, Hye Youn; Yang, San-Duk; Chae, Yun Ju

2018-01-01

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer. PMID:29302211

Epigenetic differences between monozygotic twins discordant for amyotrophic lateral sclerosis (ALS) provide clues to disease pathogenesis.

PubMed

Young, Paul E; Kum Jew, Stephen; Buckland, Michael E; Pamphlett, Roger; Suter, Catherine M

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating late-onset neurodegenerative disorder in which only a small proportion of patients carry an identifiable causative genetic lesion. Despite high heritability estimates, a genetic etiology for most sporadic ALS remains elusive. Here we report the epigenetic profiling of five monozygotic twin pairs discordant for ALS, four with classic ALS and one with the progressive muscular atrophy ALS variant, in whom previous whole genome sequencing failed to uncover a genetic basis for their disease discordance. By studying cytosine methylation patterns in peripheral blood DNA we identified thousands of large between-twin differences at individual CpGs. While the specific sites of differences were mostly idiosyncratic to a twin pair, a proportion involving GABA signalling were common to all ALS individuals. For both idiosyncratic and common sites the differences occurred within genes and pathways related to neurobiological functions or dysfunctions, some of particular relevance to ALS such as glutamate metabolism and the Golgi apparatus. All four classic ALS patients were epigenetically older than their unaffected co-twins, suggesting accelerated aging in multiple tissues in this disease. In conclusion, widespread changes in methylation patterns were found in ALS-affected co-twins, consistent with an epigenetic contribution to disease. These DNA methylation findings could be used to develop blood-based ALS biomarkers, gain insights into disease pathogenesis, and provide a reference for future large-scale ALS epigenetic studies.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

PubMed

Ha, Ye-Na; Sung, Hye Youn; Yang, San-Duk; Chae, Yun Ju; Ju, Woong; Ahn, Jung-Hyuck

2018-01-01

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α- N acetylgalactosaminidase ( NAGA ) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

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...

Epigenetic regulation of depot-specific gene expression in adipose tissue.

PubMed

Gehrke, Sandra; Brueckner, Bodo; Schepky, Andreas; Klein, Johannes; Iwen, Alexander; Bosch, Thomas C G; Wenck, Horst; Winnefeld, Marc; Hagemann, Sabine

2013-01-01

In humans, adipose tissue is distributed in subcutaneous abdominal and subcutaneous gluteal depots that comprise a variety of functional differences. Whereas energy storage in gluteal adipose tissue has been shown to mediate a protective effect, an increase of abdominal adipose tissue is associated with metabolic disorders. However, the molecular basis of depot-specific characteristics is not completely understood yet. Using array-based analyses of transcription profiles, we identified a specific set of genes that was differentially expressed between subcutaneous abdominal and gluteal adipose tissue. To investigate the role of epigenetic regulation in depot-specific gene expression, we additionally analyzed genome-wide DNA methylation patterns in abdominal and gluteal depots. By combining both data sets, we identified a highly significant set of depot-specifically expressed genes that appear to be epigenetically regulated. Interestingly, the majority of these genes form part of the homeobox gene family. Moreover, genes involved in fatty acid metabolism were also differentially expressed. Therefore we suppose that changes in gene expression profiles might account for depot-specific differences in lipid composition. Indeed, triglycerides and fatty acids of abdominal adipose tissue were more saturated compared to triglycerides and fatty acids in gluteal adipose tissue. Taken together, our results uncover clear differences between abdominal and gluteal adipose tissue on the gene expression and DNA methylation level as well as in fatty acid composition. Therefore, a detailed molecular characterization of adipose tissue depots will be essential to develop new treatment strategies for metabolic syndrome associated complications.

Integrated Molecular Profiling of Human Gastric Cancer Identifies DDR2 as a Potential Regulator of Peritoneal Dissemination.

PubMed

Kurashige, Junji; Hasegawa, Takanori; Niida, Atsushi; Sugimachi, Keishi; Deng, Niantao; Mima, Kosuke; Uchi, Ryutaro; Sawada, Genta; Takahashi, Yusuke; Eguchi, Hidetoshi; Inomata, Masashi; Kitano, Seigo; Fukagawa, Takeo; Sasako, Mitsuru; Sasaki, Hiroki; Sasaki, Shin; Mori, Masaki; Yanagihara, Kazuyoshi; Baba, Hideo; Miyano, Satoru; Tan, Patrick; Mimori, Koshi

2016-03-03

Peritoneal dissemination is the most frequent, incurable metastasis occurring in patients with advanced gastric cancer (GC). However, molecular mechanisms driving peritoneal dissemination still remain poorly understood. Here, we aimed to provide novel insights into the molecular mechanisms that drive the peritoneal dissemination of GC. We performed combined expression analysis with in vivo-selected metastatic cell lines and samples from 200 GC patients to identify driver genes of peritoneal dissemination. The driver-gene functions associated with GC dissemination were examined using a mouse xenograft model. We identified a peritoneal dissemination-associated expression signature, whose profile correlated with those of genes related to development, focal adhesion, and the extracellular matrix. Among the genes comprising the expression signature, we identified that discoidin-domain receptor 2 (DDR2) as a potential regulator of peritoneal dissemination. The DDR2 was upregulated by the loss of DNA methylation and that DDR2 knockdown reduced peritoneal metastasis in a xenograft model. Dasatinib, an inhibitor of the DDR2 signaling pathway, effectively suppressed peritoneal dissemination. DDR2 was identified as a driver gene for GC dissemination from the combined expression signature and can potentially serve as a novel therapeutic target for inhibiting GC peritoneal dissemination.

Identification of volatiles from the secretions and excretions of African wild dogs (Lycaon pictus).

PubMed

Apps, Peter; Mmualefe, Lesego; McNutt, J Weldon

2012-11-01

Gas chromatography/mass spectrometry was used to identify 103 organic compounds from urine, feces, anal glands, and preputial glands of free-ranging African wild dogs, Lycaon pictus. Aliphatic acids were the dominant class of compound in all materials. In addition to aliphatic acids, urine contained dimethyl sulfone, 1,3-propanediol, benzoic acid, 1-methyl-2,4-imidazolidinedione, and squalene as major components: feces contained indole and cholesterol; and both contained 2-piperidone, phenol, 4-methyl phenol, benzeneacetic acid, and benzenepropanoic acid and other compounds. Anal gland secretion was particularly rich in cholesterol and fatty acids, and preputial gland secretion rich in squalene. A large majority of the identified compounds have been reported from other mammals, including species sympatric with African wild dogs. Eleven of the African wild dog components have not been reported previously from mammals and have not been found in sympatric species; one component, 1-methylimidazole-5-carboxaldehyde has not been reported previously as a natural product. In the chemical profiles of their urine, feces, and anal gland secretion African wild dogs differ markedly from other canids.

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.

Differential transmission of the molecular signature of RBSP3, LIMD1 and CDC25A in basal/ parabasal versus spinous of normal epithelium during head and neck tumorigenesis: A mechanistic study.

PubMed

Sarkar, Shreya; Alam, Neyaz; Mandal, Syam Sundar; Chatterjee, Kabita; Ghosh, Supratim; Roychoudhury, Susanta; Panda, Chinmay Kumar

2018-01-01

Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.

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.

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.

Promoter Methylation and BDNF and DAT1 Gene Expression Profiles in Patients with Drug Addiction.

PubMed

Kordi-Tamandani, Dor Mohammad; Tajoddini, Shahrad; Salimi, Farzaneh

2015-01-01

Drug addiction is a brain disorder that has negative consequences for individuals and society. Addictions are chronic relapsing diseases of the brain that are caused by direct drug-induced effects and persevering neuroadaptations at the epigenetic, neuropeptide and neurotransmitter levels. Because the dopaminergic system has a significant role in drug abuse, the purpose of this study was to analyze the methylation and expression profile of brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in individuals with drug addiction. BDNF and DAT1 promoter methylation were investigated with a methylation-specific polymerase chain reaction (PCR) technique in blood samples from 75 individuals with drug addiction and 65 healthy controls. The expression levels of BDNF and DAT1 were assessed in 12 mRNA samples from the blood of patients and compared to the samples of healthy controls (n = 12) with real-time quantitative reverse transcription PCR. No significant differences were found in the methylation of BDNF and DAT1 between patients and controls, but the relative levels of expression of BDNF and DAT1 mRNA differed significantly in the patients compared to controls (p < 0.0001). These results showed that the methylation status of the BDNF and DAT1 genes had no significant function in the processes of drug addiction.

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.

Systems Genetics Reveals the Functional Context of PCOS Loci and Identifies Genetic and Molecular Mechanisms of Disease Heterogeneity

PubMed Central

Xu, Ning; Cui, Jinrui; Mengesha, Emebet; Chen, Yii-Der I.; Taylor, Kent D.; Azziz, Ricardo; Goodarzi, Mark O.

2015-01-01

Genome wide association studies (GWAS) have revealed 11 independent risk loci for polycystic ovary syndrome (PCOS), a common disorder in young women characterized by androgen excess and oligomenorrhea. To put these risk loci and the single nucleotide polymorphisms (SNPs) therein into functional context, we measured DNA methylation and gene expression in subcutaneous adipose tissue biopsies to identify PCOS-specific alterations. Two genes from the LHCGR region, STON1-GTF2A1L and LHCGR, were overexpressed in PCOS. In analysis stratified by obesity, LHCGR was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, INSR was underexpressed in obese PCOS subjects only. Alterations in gene expression in the LHCGR, RAB5B and INSR regions suggest that SNPs in these loci may be functional and could affect gene expression directly or indirectly via epigenetic alterations. We identified reduced methylation in the LHCGR locus and increased methylation in the INSR locus, changes that are concordant with the altered gene expression profiles. Complex patterns of meQTL and eQTL were identified in these loci, suggesting that local genetic variation plays an important role in gene regulation. We propose that non-obese PCOS women possess significant alterations in LH receptor expression, which drives excess androgen secretion from the ovary. Alternatively, obese women with PCOS possess alterations in insulin receptor expression, with underexpression in metabolic tissues and overexpression in the ovary, resulting in peripheral insulin resistance and excess ovarian androgen production. These studies provide a genetic and molecular basis for the reported clinical heterogeneity of PCOS. PMID:26305227

Species-Specific 5 mC and 5 hmC Genomic Landscapes Indicate Epigenetic Contribution to Human Brain Evolution

PubMed Central

Madrid, Andy; Chopra, Pankaj; Alisch, Reid S.

2018-01-01

Human evolution from non-human primates has seen substantial change in the central nervous system, with the molecular mechanisms underlying human brain evolution remaining largely unknown. Methylation of cytosine at the fifth carbon (5-methylcytosine; 5 mC) is an essential epigenetic mark linked to neurodevelopment, as well as neurological disease. The emergence of another modified form of cytosine (5-hydroxymethylcytosine; 5 hmC) that is enriched in the brain further substantiates a role for these epigenetic marks in neurodevelopment, yet little is known about the evolutionary importance of these marks in brain development. Here, human and monkey brain tissue were profiled, identifying 5,516 and 4,070 loci that were differentially methylated and hydroxymethylated, respectively, between the species. Annotation of these loci to the human genome revealed genes critical for the development of the nervous system and that are associated with intelligence and higher cognitive functioning, such as RELN and GNAS. Moreover, ontological analyses of these differentially methylated and hydroxymethylated genes revealed a significant enrichment of neuronal/immunological–related processes, including neurogenesis and axon development. Finally, the sequences flanking the differentially methylated/hydroxymethylated loci contained a significant enrichment of binding sites for neurodevelopmentally important transcription factors (e.g., OTX1 and PITX1), suggesting that DNA methylation may regulate gene expression by mediating transcription factor binding on these transcripts. Together, these data support dynamic species-specific epigenetic contributions in the evolution and development of the human brain from non-human primates. PMID:29491831

UHPLC-(ESI)QTOF MS/MS profiling of quercetin metabolites in human plasma postconsumption of applesauce enriched with apple peel and onion.

PubMed

Lee, Jihyun; Ebeler, Susan E; Zweigenbaum, Jerry A; Mitchell, Alyson E

2012-08-29

An ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-(ESI)QTOF MS/MS) method was developed for measuring individual quercetin metabolites in human plasma with high sensitivity and high selectivity. Quercetin (3,3',4',5,7-pentahydroxyflavone) occurs as glycosides in foods. The composition of glycosides is species and cultivar specific. In humans, quercetin undergoes extensive biotransformation, resulting in a range of metabolites. The bioactivity of quercetin metabolites will depend on the type and position of the conjugates. Herein, individual quercetin metabolites (i.e., sulfate, glucuronide or methyl conjugates) were identified by accurate mass MS in human plasma (females = 8 and males = 8) over 24 h after consumption of applesauce enriched with either micronized apple peel (AP) or onion powder (OP). The AP and OP contained ~180 μmol of quercetin glycosides. The relative amounts of quercetin metabolites were quantified in plasma. The complement of identified quercetin metabolites was similar after consumption of AP and OP. Primary metabolites included the following: quercetin sulfate, quercetin glucuronide, and quercetin diglucuronide. A quercetin glutathione adduct was identified in negative ion mode but not apparent in positive ion mode. The pharmacokinetic parameters for AUC0-24 h and Cmax were significantly different for AP and OP. For example, consumption of the AP resulted in Cmax of quercetin sulfate, 4.6 ng/mL; quercetin glucuronide, 15.5 ng/mL; quercetin diglucuronide, 9.3 ng/mL; quercetin glucuronide sulfate, 1.3 ng/mL; methyl quercetin glucuronide, 7.5 ng/mL; and methyl quercetin diglucuronide, 3.6 ng/mL, whereas the OP resulted in Cmax of quercetin sulfate, 37.3 ng/mL; quercetin glucuronide, 212.8 ng/mL; quercetin diglucuronide, 168.8 ng/mL; quercetin glucuronide sulfate, 43.0 ng/mL; methyl quercetin glucuronide, 90.1 ng/mL; methyl quercetin diglucuronide, 65.4 ng/mL. Gender-related differences in the AUC0-24 h for quercetin sulfate and quercetin sulfate glucuronide metabolites were also observed.

Multimodal molecular analysis of astroblastoma enables reclassification of most cases into more specific molecular entities.

PubMed

Wood, Matthew D; Tihan, Tarik; Perry, Arie; Chacko, Geeta; Turner, Clinton; Pu, Cunfeng; Payne, Christopher; Yu, Alexander; Bannykh, Serguei I; Solomon, David A

2018-03-01

Astroblastoma is a rare and controversial glioma with variable clinical behavior. The diagnosis currently rests on histologic findings of a circumscribed glioma with astroblastomatous pseudorosettes and vascular hyalinization. Immunohistochemical studies have suggested different oncogenic drivers, such as BRAF p.V600E, but very few cases have been studied using genome-wide methodologies. Recent genomic profiling identified a subset of CNS embryonal tumors with astroblastoma-like morphology that harbored MN1 gene fusions, termed "CNS high-grade neuroepithelial tumors with MN1 alteration" (CNS-HGNET-MN1). To further characterize the genetic alterations that drive astroblastomas, we performed targeted next-generation sequencing (NGS) of 500 cancer-associated genes in a series of eight cases. We correlated these findings with break-apart fluorescence in situ hybridization (FISH) analysis of the MN1 locus and genome-wide DNA methylation profiling. Four cases showed MN1 alteration by FISH, including two pediatric cases that lacked other pathogenic alterations, and two adult cases that harbored other cancer-associated gene mutations or copy number alterations (eg, CDKN2A/B homozygous deletion, TP53, ATM and TERT promoter mutations). Three of these cases grouped with the CNS-HGNET-MN1 entity by methylation profiling. Two of four MN1 intact cases by FISH showed genetic features of either anaplastic pleomorphic xanthoastrocytoma (BRAF p.V600E mutation, CDKN2A/B homozygous deletion and TERT promoter mutation) or IDH-wildtype glioblastoma (trisomy 7, monosomy 10, CDK4 amplification and TP53, NRAS and TERT promoter mutations) and these cases had an aggressive clinical course. Two clinically indolent cases remained unclassifiable despite multimodal molecular analysis. We conclude that astroblastoma histology is not specific for any entity including CNS-HGNET-MN1, and that additional genetic characterization should be considered for astroblastomas, as a number of these tumors likely contain a methylation profile or genetic alterations that suggest classification as other tumor entities. Our heterogeneous molecular findings help to explain the clinical unpredictability of astroblastoma. © 2017 International Society of Neuropathology.

Low-resolution structure of Drosophila translin

PubMed Central

Kumar, Vinay; Gupta, Gagan D.

2012-01-01

Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P21 with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to Rwork (Rfree) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers. PMID:23650579

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

Heritable DNA methylation marks associated with susceptibility to breast cancer.

PubMed

Joo, Jihoon E; Dowty, James G; Milne, Roger L; Wong, Ee Ming; Dugué, Pierre-Antoine; English, Dallas; Hopper, John L; Goldgar, David E; Giles, Graham G; Southey, Melissa C

2018-02-28

Mendelian-like inheritance of germline DNA methylation in cancer susceptibility genes has been previously reported. We aimed to scan the genome for heritable methylation marks associated with breast cancer susceptibility by studying 25 Australian multiple-case breast cancer families. Here we report genome-wide DNA methylation measured in 210 peripheral blood DNA samples provided by family members using the Infinium HumanMethylation450. We develop and apply a new statistical method to identify heritable methylation marks based on complex segregation analysis. We estimate carrier probabilities for the 1000 most heritable methylation marks based on family structure, and we use Cox proportional hazards survival analysis to identify 24 methylation marks with corresponding carrier probabilities significantly associated with breast cancer. We replicate an association with breast cancer risk for four of the 24 marks using an independent nested case-control study. Here, we report a novel approach for identifying heritable DNA methylation marks associated with breast cancer risk.

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.

Methyl-CpG island-associated genome signature tags

DOEpatents

Dunn, John J

2014-05-20

Disclosed is a method for analyzing the organismic complexity of a sample through analysis of the nucleic acid in the sample. In the disclosed method, through a series of steps, including digestion with a type II restriction enzyme, ligation of capture adapters and linkers and digestion with a type IIS restriction enzyme, genome signature tags are produced. The sequences of a statistically significant number of the signature tags are determined and the sequences are used to identify and quantify the organisms in the sample. Various embodiments of the invention described herein include methods for using single point genome signature tags to analyze the related families present in a sample, methods for analyzing sequences associated with hyper- and hypo-methylated CpG islands, methods for visualizing organismic complexity change in a sampling location over time and methods for generating the genome signature tag profile of a sample of fragmented DNA.

Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.

PubMed

Campana, Wendy M; Mantuano, Elisabetta; Azmoon, Pardis; Henry, Kenneth; Banki, Michael A; Kim, John H; Pizzo, Donald P; Gonias, Steven L

2017-04-01

In the peripheral nervous system, Schwann cells (SCs) demonstrate surveillance activity, detecting injury and undergoing trans -differentiation to support repair. SC receptors that detect peripheral nervous system injury remain incompletely understood. We used RT-PCR to profile ionotropic glutamate receptor expression in cultured SCs. We identified subunits required for assembly of N -methyl-d-aspartic acid (NMDA) receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and kainate receptors. Treatment of SCs with 40-100 µM glutamate or with 0.5-1.0 µM NMDA robustly activated Akt and ERK1/2. The response was transient and bimodal; glutamate concentrations that exceeded 250 µM failed to activate cell signaling. Phosphoprotein profiling identified diverse phosphorylated proteins in glutamate-treated SCs in addition to ERK1/2 and Akt, including p70 S6-kinase, glycogen synthase kinase-3, ribosomal S6 kinase, c-Jun, and cAMP response element binding protein. Activation of SC signaling by glutamate was blocked by EGTA and dizocilpine and by silencing expression of the NMDA-R NR1 subunit. Phosphoinositide 3-kinase/PI3K functioned as an essential upstream activator of Akt and ERK1/2 in glutamate-treated SCs. When glutamate or NMDA was injected directly into crush-injured rat sciatic nerves, ERK1/2 phosphorylation was observed in myelinated and nonmyelinating SCs. Glutamate promoted SC migration by a pathway that required PI3K and ERK1/2. These results identified ionotropic glutamate receptors and NMDA-Rs, specifically, as potentially important cell signaling receptors in SCs.-Campana, W. M., Mantuano, E., Azmoon, P., Henry, K., Banki, M. A., Kim, J. H., Pizzo, D. P., Gonias, S. L. Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells. © FASEB.

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.

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.

Simulation of human plasma concentration-time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling.

PubMed

Kamimura, Hidetaka; Ito, Satoshi; Chijiwa, Hiroyuki; Okuzono, Takeshi; Ishiguro, Tomohiro; Yamamoto, Yosuke; Nishinoaki, Sho; Ninomiya, Shin-Ichi; Mitsui, Marina; Kalgutkar, Amit S; Yamazaki, Hiroshi; Suemizu, Hiroshi

2017-05-01

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes.

PubMed

Nectoux, J; Fichou, Y; Rosas-Vargas, H; Cagnard, N; Bahi-Buisson, N; Nusbaum, P; Letourneur, F; Chelly, J; Bienvenu, T

2010-07-01

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene that encodes the methyl-CpG-binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild-type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome-wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild-type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non-sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT-PCR indicated four up-regulated genes and five down-regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up-regulated candidate genes and sequencing of bisulphite-converted DNA indicated that MeCP2 preferentially binds to methylated-DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.

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

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.

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.

Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia

PubMed Central

Geng, Huimin; Brennan, Sarah; Milne, Thomas A.; Chen, Wei-Yi; Li, Yushan; Hurtz, Christian; Kweon, Soo-Mi; Zickl, Lynette; Shojaee, Seyedmehdi; Neuberg, Donna; Huang, Chuanxin; Biswas, Debabrata; Xin, Yuan; Racevskis, Janis; Ketterling, Rhett P.; Luger, Selina M.; Lazarus, Hillard; Tallman, Martin S.; Rowe, Jacob M.; Litzow, Mark R.; Guzman, Monica L.; Allis, C. David; Roeder, Robert G.; Müschen, Markus; Paietta, Elisabeth; Elemento, Olivier; Melnick, Ari M.

2012-01-01

Genetic lesions such as BCR-ABL1, E2A-PBX1 and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point towards disease mechanisms and useful biomarkers and therapeutic targets. We therefore performed DNA methylation and gene expression profiling on a cohort of 215 adult B-ALL patients enrolled in a single phase III clinical trial (ECOG E2993) and normal control B-cells. In BCR-ABL1-positive B-ALL, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in ALL patients regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALL. In E2A-PBX1-positive B-ALL, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 ChIP sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6 targeted therapy as a new therapeutic strategy for MLLr B-ALL. PMID:23107779

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

A preliminary evaluation of bardoxolone methyl for the treatment of diabetic nephropathy.

PubMed

Thomas, Merlin

2012-08-01

The coordinated activation of Nrf-2-dependent signaling pathway is currently being investigated in a range of chronic diseases. Bardoxolone methyl is a potent, orally bioavailable Nrf-2 agonist. In a recent 52-week study, treatment with bardoxolone methyl improved renal function in patients with chronic kidney disease (CKD) and type 2 diabetes. This improvement was sustained for the duration of the treatment. Such agonists potentially offer new options for the complex management of renal impairment. A literature search was performed to analyze the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of bardoxolone methyl in both healthy volunteers and patients. Updated information about bardoxolone methyl, either after single administration or after chronic administration is also included. A special focus has been put on the putative mechanisms of action and potential toxicity profiles as well as an ongoing trials in patients with CKD and type 2 diabetes. The development of an agent that leads to sustained improvement in renal function comes as a welcome relief to the millions of individuals with diabetes and CKD. However, much remains to be established regarding its actions in a complex and pleiotropic signalling cascade. Other triterpenoids with different PK/PD profiles are currently under development.

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

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.

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.

Proteomic Identification and Analysis of Arginine-Methylated Proteins of Plasmodium falciparum at Asexual Blood Stages.

PubMed

Zeeshan, Mohammad; Kaur, Inderjeet; Joy, Joseph; Saini, Ekta; Paul, Gourab; Kaushik, Abhinav; Dabral, Surbhi; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

2017-02-03

Plasmodium falciparum undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with Plasmodium phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of Plasmodium falciparum to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC-MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as GRx/RGx, RxG, GxxR, or WxxxR. We identified Plasmodium homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in Plasmodium, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host-pathogen interactions.

Metabolomic Profiling of Plasma from Patients with Tuberculosis by Use of Untargeted Mass Spectrometry Reveals Novel Biomarkers for Diagnosis.

PubMed

Lau, Susanna K P; Lee, Kim-Chung; Curreem, Shirly O T; Chow, Wang-Ngai; To, Kelvin K W; Hung, Ivan F N; Ho, Deborah T Y; Sridhar, Siddharth; Li, Iris W S; Ding, Vanessa S Y; Koo, Eleanor W F; Wong, Chi-Fong; Tam, Sidney; Lam, Ching-Wan; Yuen, Kwok-Yung; Woo, Patrick C Y

2015-12-01

Although tuberculosis (TB) is a reemerging disease that affects people in developing countries and immunocompromised populations in developed countries, the current diagnostic methods are far from optimal. Metabolomics is increasingly being used for studies on infectious diseases. We performed metabolome profiling of plasma samples to identify potential biomarkers for diagnosing TB. We compared the plasma metabolome profiles of TB patients (n = 46) with those of community-acquired pneumonia (CAP) patients (n = 30) and controls without active infection (n = 30) using ultrahigh-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (UHPLC-ESI-QTOFMS). Using multivariate and univariate analyses, four metabolites, 12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(R)-HETE], ceramide (d18:1/16:0), cholesterol sulfate, and 4α-formyl-4β-methyl-5α-cholesta-8-en-3β-ol, were identified and found to have significantly higher levels in TB patients than those in CAP patients and controls. In a comparison of TB patients and controls, the four metabolites demonstrated area under the receiver operating characteristic curve (AUC) values of 0.914, 0.912, 0.905, and 0.856, sensitivities of 84.8%, 84.8%, 87.0%, and 89.1%, specificities of 90.0%, 86.7%, 86.7%, and 80.0%, and fold changes of 4.19, 26.15, 6.09, and 1.83, respectively. In a comparison of TB and CAP patients, the four metabolites demonstrated AUC values of 0.793, 0.717, 0.802, and 0.894, sensitivities of 89.1%, 71.7%, 80.4%, and 84.8%, specificities of 63.3%, 66.7%, 70.0%, and 83.3%, and fold changes of 4.69, 3.82, 3.75, and 2.16, respectively. 4α-Formyl-4β-methyl-5α-cholesta-8-en-3β-ol combined with 12(R)-HETE or cholesterol sulfate offered ≥70% sensitivity and ≥90% specificity for differentiating TB patients from controls or CAP patients. These novel plasma biomarkers, especially 12(R)-HETE and 4α-formyl-4β-methyl-5α-cholesta-8-en-3β-ol, alone or in combination, are potentially useful for rapid and noninvasive diagnosis of TB. The present findings may offer insights into the pathogenesis and host response in TB. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Metabolomic Profiling of Plasma from Patients with Tuberculosis by Use of Untargeted Mass Spectrometry Reveals Novel Biomarkers for Diagnosis

PubMed Central

Lee, Kim-Chung; Curreem, Shirly O. T.; To, Kelvin K. W.; Hung, Ivan F. N.; Ho, Deborah T. Y.; Sridhar, Siddharth; Li, Iris W. S.; Ding, Vanessa S. Y.; Koo, Eleanor W. F.; Wong, Chi-Fong; Tam, Sidney; Lam, Ching-Wan; Yuen, Kwok-Yung

2015-01-01

Although tuberculosis (TB) is a reemerging disease that affects people in developing countries and immunocompromised populations in developed countries, the current diagnostic methods are far from optimal. Metabolomics is increasingly being used for studies on infectious diseases. We performed metabolome profiling of plasma samples to identify potential biomarkers for diagnosing TB. We compared the plasma metabolome profiles of TB patients (n = 46) with those of community-acquired pneumonia (CAP) patients (n = 30) and controls without active infection (n = 30) using ultrahigh-performance liquid chromatography–electrospray ionization-quadrupole time of flight mass spectrometry (UHPLC-ESI-QTOFMS). Using multivariate and univariate analyses, four metabolites, 12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(R)-HETE], ceramide (d18:1/16:0), cholesterol sulfate, and 4α-formyl-4β-methyl-5α-cholesta-8-en-3β-ol, were identified and found to have significantly higher levels in TB patients than those in CAP patients and controls. In a comparison of TB patients and controls, the four metabolites demonstrated area under the receiver operating characteristic curve (AUC) values of 0.914, 0.912, 0.905, and 0.856, sensitivities of 84.8%, 84.8%, 87.0%, and 89.1%, specificities of 90.0%, 86.7%, 86.7%, and 80.0%, and fold changes of 4.19, 26.15, 6.09, and 1.83, respectively. In a comparison of TB and CAP patients, the four metabolites demonstrated AUC values of 0.793, 0.717, 0.802, and 0.894, sensitivities of 89.1%, 71.7%, 80.4%, and 84.8%, specificities of 63.3%, 66.7%, 70.0%, and 83.3%, and fold changes of 4.69, 3.82, 3.75, and 2.16, respectively. 4α-Formyl-4β-methyl-5α-cholesta-8-en-3β-ol combined with 12(R)-HETE or cholesterol sulfate offered ≥70% sensitivity and ≥90% specificity for differentiating TB patients from controls or CAP patients. These novel plasma biomarkers, especially 12(R)-HETE and 4α-formyl-4β-methyl-5α-cholesta-8-en-3β-ol, alone or in combination, are potentially useful for rapid and noninvasive diagnosis of TB. The present findings may offer insights into the pathogenesis and host response in TB. PMID:26378277

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.

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

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.

Genome-wide DNA methylation profiling in infants born to gestational diabetes mellitus.

PubMed

Weng, Xiaoling; Liu, Fatao; Zhang, Hong; Kan, Mengyuan; Wang, Ting; Dong, Mingyue; Liu, Yun

2018-03-26

Offspring exposed to gestational diabetes mellitus (GDM) are at a high risk for metabolic diseases. The mechanisms behind the association between offspring exposed to GDM in utero and an increased risk of health consequences later in life remain unclear. The aim of this study was to clarify the changes in methylation levels in the foetuses of women with GDM and to explore the possible mechanisms linking maternal GDM with a high risk of metabolic diseases in offspring later in life. A genome-wide comparative methylome analysis on the umbilical cord blood of infants born to 30 women with GDM and 33 women with normal pregnancy was performed using Infinium HumanMethylation 450 BeadChip assays. A quantitative methylation analysis of 18 CpG dinucleotides was verified in the validation umbilical cord blood samples from 102 newborns exposed to GDM and 103 newborns who experienced normal pregnancy by MassARRAY EpiTYPER. A total of 4485 differentially methylated sites (DMSs), including 2150 hypermethylated sites and 2335 hypomethylated sites, with a mean β-value difference of >0.05, were identified by the 450k array. Good agreement was observed between the massarray validation data and the 450k array data (R 2 > 0.99; P < 0.0001). Thirty-seven CpGs (representing 20 genes) with a β-value difference of >0.15 between the GDM and healthy groups were identified and showed potential as clinical biomarkers for GDM. "hsa04940: Type I diabetes mellitus" was the most significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, with a P-value = 3.20E-07 and 1.36E-02 in the hypermethylated and hypomethylated genepathway enrichment analyses, respectively. In the Gene Ontology (GO) pathway analyses, immune MHC-related pathways and neuron development-related pathways were significantly enriched. Our results suggest that GDM has epigenetic effects on genes that are preferentially involved in the Type I diabetes mellitus pathway, immune MHC (major histocompatibility complex)-related pathways and neuron development-related pathways, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming. Copyright © 2018. Published by Elsevier B.V.

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.

Unraveling Additional O-Methylation Steps in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

PubMed

Purwanto, Ratmoyo; Hori, Kentaro; Yamada, Yasuyuki; Sato, Fumihiko

2017-09-01

California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Global Proteome Response to Deletion of Genes Related to Mercury Methylation and Dissimilatory Metal Reduction Reveals Changes in Respiratory Metabolism in Geobacter sulfurreducens PCA

DOE PAGES

Qian, Chen; Johs, Alexander; Chen, Hongmei; ...

2016-07-27

Geobacter sulfurreducens PCA can reduce, sorb, and methylate mercury (Hg); however, the underlying biochemical mechanisms of these processes and interdependent metabolic pathways remain unknown. In this study, shotgun proteomics was used to compare global proteome profiles between wild-type G. sulfurreducens PCA and two mutant strains: a ΔhgcAB mutant, which is deficient in two genes known to be essential for Hg methylation and a ΔomcBESTZ mutant, which is deficient in five outer membrane c-type cytochromes and thus impaired in its ability for dissimilatory metal ion reduction. We were able to delineate the global response of G. sulfurreducens PCA in both mutantsmore » and identify cellular networks and metabolic pathways that were affected by the loss of these genes. Deletion of hgcAB increased the relative abundances of proteins implicated in extracellular electron transfer, including most of the c-type cytochromes, PilA-C, and OmpB, and is consistent with a previously observed increase in Hg reduction in the hgcAB mutant. Deletion of omcBESTZ was found to significantly increase relative abundances of various methyltransferases, suggesting that a loss of dissimilatory reduction capacity results in elevated activity among one-carbon metabolic pathways and thus increased methylation. We show that G. sulfurreducens PCA encodes only the folate branch of the Wood Ljungdahl pathway, and proteins associated with the folate branch were found at lower abundance in the ΔhgcAB mutant strain than the wild type. In conclusion, this observation supports the hypothesis that the function of HgcA and HgcB may be linked to one carbon metabolism through the folate branch of the Wood-Ljungdahl pathway by providing methyl groups required for Hg methylation.« less

MethylMix 2.0: an R package for identifying DNA methylation genes. | Office of Cancer Genomics

Cancer.gov

DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes.

COMPARISON OF ACUTE NEUROBEHAVIORAL AND CHOLINESTERASE INHIBITORY EFFECTS OF N-METHYL CARBAMATES IN RAT

EPA Science Inventory

There are few studies evaluating direct functional and biochemical consequences of exposure. In the present study of the acute toxicity of seven N-methyl carbamate pesticides, we evaluated the dose-response profiles of cholinesterase (ChE) inhibition in brain and erythrocytes (R...

EXPRESSION OF AS3MT ALTERS TRANSCRIPTIONAL PROFILES IN HUMAN UROTHELIAL CELLS EXPOSED TO ARSENITE

EPA Science Inventory

Inorganic arsenic (iAs) is an environmental toxin and human carcinogen. The enzymatic methylation of iAs that is catalyzed by arsenic (+3 oxidation state)-methyltransferase (AS3MT) generates reactive methylated intermediates that contribute to the toxic and carcinogenic effects ...

Methyl esters (biodiesel) from and fatty acid profile of Gliricidia sepium seed oil

USDA-ARS?s Scientific Manuscript database

Increasing the supply of biodiesel by defining and developing additional feedstocks is important to overcome the still limited amounts available of this alternative fuel. In this connection, the methyl esters of the seed oil of Gliricidia sepium were synthesized and the significant fuel-related prop...

Low-temperature phase behavior of fatty acid methyl esters by differential scanning calorimetry (DSC)

USDA-ARS?s Scientific Manuscript database

Fatty acid methyl ester (FAME) mixtures have many uses including biodiesel, lubricants, metal-working fluids, surfactants, polymers, coatings, green solvents and phase-change materials. The physical properties of a FAME mixture depends on the fatty acid concentration (FAC) profile. Some products hav...

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation.

PubMed

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-12-01

Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.