Science.gov

Sample records for dna methylation contributes

  1. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  2. Transcriptional Repressive H3K9 and H3K27 Methylations Contribute to DNMT1-Mediated DNA Methylation Recovery

    PubMed Central

    Wong, Chun-Ming; Wong, Carmen Chak-Lui; Ng, Yeung-Lam; Au, Sandy Leung-Kuen; Ko, Frankie Chi-Fat; Ng, Irene Oi-Lin

    2011-01-01

    DNA methylation and histone modifications are two major epigenetic events regulating gene expression and chromatin structure, and their alterations are linked to human carcinogenesis. DNA methylation plays an important role in tumor suppressor gene inactivation, and can be revised by DNA methylation inhibitors. The reversible nature of DNA methylation forms the basis of epigenetic cancer therapy. However, it has been reported that DNA re-methylation and gene re-silencing could occur after removal of demethylation treatment and this may significantly hamper the therapeutic value of DNA methylation inhibitors. In this study we have provided detailed evidence demonstrating that mammalian cells possess a bona fide DNA methylation recovery system. We have also shown that DNA methylation recovery was mediated by the major human DNA methyltransferase, DNMT1. In addition, we found that H3K9-tri-methylation and H3K27-tri-methylation were closely associated with this DNA methylation recovery. These persistent transcriptional repressive histone modifications may have a crucial role in regulating DNMT1-mediated DNA methylation recovery. Our findings may have important implications towards a better understanding of epigenetic regulation and future development of epigenetic therapeutic intervention. PMID:21347439

  3. Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks

    PubMed Central

    Kobayashi, Hisato; Sakurai, Takayuki; Imai, Misaki; Takahashi, Nozomi; Fukuda, Atsushi; Yayoi, Obata; Sato, Shun; Nakabayashi, Kazuhiko; Hata, Kenichiro; Sotomaru, Yusuke; Suzuki, Yutaka; Kono, Tomohiro

    2012-01-01

    Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells. PMID:22242016

  4. [DNA methylation in thyroid carcinoma].

    PubMed

    Song, Xianyun; Shang, Xiaoling; Zhang, Yutuo

    2015-03-01

    Cancer has become clear that not merely gene variations but also epigenetic modifications may contribute to it. Epigenetic changes refer to stable alterations in gene expression with unrelated to changes in the underlying genetic sequence,resulting in heritable. DNA methylation is one of the common epigenetic changes. It control the gene expression through changing DNA conformation and stability, chromatin structer, DNA-protein interaction. The reversal of dysregulated DNA methylation has emerged as a potential strategy for the treatment of thyroid carcinoma. The artical will provide an overview of how DNA methylation contribute to thyroid carcinoma dissemination,invasion and metastasis and we will summarize the latest epigenetic therapies for thyroid carcinoma.

  5. DNA methylation contributes to the regulation of sclerostin expression in human osteocytes.

    PubMed

    Delgado-Calle, Jesús; Sañudo, Carolina; Bolado, Alfonso; Fernández, Agustín F; Arozamena, Jana; Pascual-Carra, María A; Rodriguez-Rey, José C; Fraga, Mario F; Bonewald, Lynda; Riancho, José A

    2012-04-01

    Sclerostin, encoded by the SOST gene, is a potent inhibitor of bone formation, produced by osteocytes, not by osteoblasts, but little is known about the molecular mechanisms controlling its expression. We aimed to test the hypothesis that epigenetic mechanisms, specifically DNA methylation, modulate SOST expression. We found two CpG-rich regions in SOST: region 1, located in the proximal promoter, and region 2, around exon 1. qMSP and pyrosequencing analysis of DNA methylation showed that region 2 was largely methylated in all samples analyzed. In contrast, marked differences were observed in region 1. Whereas the CpG-rich region 1 was hypermethylated in osteoblasts, this region was largely hypomethylated in microdissected human osteocytes. Bone lining cells showed a methylation profile between primary osteoblasts and osteocytes. Whereas SOST expression was detected at very low level or not at all by RT-qPCR in several human osteoblastic and nonosteoblastic cell lines, and human primary osteoblasts under basal conditions, it was dramatically upregulated (up to 1300-fold) by the demethylating agent AzadC. Experiments using reporter vectors demonstrated the functional importance of the region -581/+30 of the SOST gene, which contains the CpG-rich region 1. In vitro methylation of this CpG-island impaired nuclear protein binding and led to a 75 ± 12% inhibition of promoter activity. In addition, BMP-2-induced expression of SOST was markedly enhanced in cells demethylated by AzadC. Overall, these results strongly suggest that DNA methylation is involved in the regulation of SOST expression during osteoblast-osteocyte transition, presumably by preventing the binding of transcription factors to the proximal promoter. To our knowledge, our data provide first ever evidence of the involvement of DNA methylation in the regulation of SOST expression and may help to establish convenient experimental models for further studies of human sclerostin.

  6. Limited Contribution of DNA Methylation Variation to Expression Regulation in Arabidopsis thaliana.

    PubMed

    Meng, Dazhe; Dubin, Manu; Zhang, Pei; Osborne, Edward J; Stegle, Oliver; Clark, Richard M; Nordborg, Magnus

    2016-07-01

    The extent to which epigenetic variation affects complex traits in natural populations is not known. We addressed this question using transcriptome and DNA methylation data from a sample of 135 sequenced A. thaliana accessions. Across individuals, expression was significantly associated with cis-methylation for hundreds of genes, and many of these associations remained significant after taking SNP effects into account. The pattern of correlations differed markedly between gene body methylation and transposable element methylation. The former was usually positively correlated with expression, and the latter usually negatively correlated, although exceptions were found in both cases. Finally, we developed graphical models of causality that adapt to a sample with heavy population structure, and used them to show that while methylation appears to affect gene expression more often than expression affects methylation, there is also strong support for both being independently controlled. In conclusion, although we find clear evidence for epigenetic regulation, both the number of loci affected and the magnitude of the effects appear to be small compared to the effect of SNPs. PMID:27398721

  7. Limited Contribution of DNA Methylation Variation to Expression Regulation in Arabidopsis thaliana

    PubMed Central

    Zhang, Pei; Osborne, Edward J.; Stegle, Oliver; Clark, Richard M.

    2016-01-01

    The extent to which epigenetic variation affects complex traits in natural populations is not known. We addressed this question using transcriptome and DNA methylation data from a sample of 135 sequenced A. thaliana accessions. Across individuals, expression was significantly associated with cis-methylation for hundreds of genes, and many of these associations remained significant after taking SNP effects into account. The pattern of correlations differed markedly between gene body methylation and transposable element methylation. The former was usually positively correlated with expression, and the latter usually negatively correlated, although exceptions were found in both cases. Finally, we developed graphical models of causality that adapt to a sample with heavy population structure, and used them to show that while methylation appears to affect gene expression more often than expression affects methylation, there is also strong support for both being independently controlled. In conclusion, although we find clear evidence for epigenetic regulation, both the number of loci affected and the magnitude of the effects appear to be small compared to the effect of SNPs. PMID:27398721

  8. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  9. DNA methylation in endometrial cancer

    PubMed Central

    Freudenheim, Jo L

    2010-01-01

    Endometrial cancer is the most commonly diagnosed gynecological cancer, and it has been shown to be a complex disease driven by abnormal genetic and epigenetic alterations, as well as environmental factors. Epigenetic changes resulting in aberrant gene expression are dynamic and modifiable features of many cancer types. A significant epigenetic change is aberrant DNA methylation. In this review, we review evidence on the role of aberrant DNA methylation, examining changes in relation to endometrial carcinogenesis, and report on recent advances in the understanding of the contribution of aberrant DNA methylation to endometrial cancer with the emphasis on the role of dietary/lifestyle and environmental factors, as well as opportunities and challenges of DNA methylation in endometrial cancer management and prevention. PMID:20543579

  10. DNA methylation contributes toward silencing of antioncogenic microRNA-203 in human and canine melanoma cells.

    PubMed

    Noguchi, Shunsuke; Mori, Takashi; Nakagawa, Takayuki; Itamoto, Kazuhito; Haraguchi, Tomoya; Mizuno, Takuya

    2015-10-01

    Melanoma is a poor-prognosis cancer in both humans and dogs. We have elucidated the antitumor mechanisms of antioncogenic microRNA (miR)-203 which is downregulated in human melanoma, as well as in canine melanoma. The aim of this study was to clarify the mechanism of this downregulation. We focused on epigenetic aberration of miR-203 transcription. Treatment with 5-aza-2'-deoxycitidine (5-aza) markedly upregulated the expression level of miR-203 in almost all of the cell lines tested. Furthermore, bisulfite sequencing or methylation-specific PCR showed DNA methylation of CpG islands upstream of the miR-203 coding region (MIR203) in both human and canine melanoma cells, as well as in canine clinical specimens, but not in human normal melanocytes. The results of a luciferase activity assay showed obvious suppression of the transcription of miR-203 by DNA methylation. The use of the luciferase activity assay for CREB1 and an inhibition assay of miR-203 function performed with an miR-203 inhibitor confirmed the contribution of miR-203 upregulation toward the negative regulation of the target gene of miR-203. These results indicate that canine melanoma might be a preclinical model of human melanoma for epigenetic studies. In addition, this study suggests that agents that can demethylate MIR203 could be a common promising therapeutic agent for the treatment of human and canine melanomas.

  11. DNA Methylation in Osteoarthritis.

    PubMed

    den Hollander, Wouter; Meulenbelt, Ingrid

    2015-12-01

    Osteoarthritis (OA) is a prevalent disease of articular joints and primarily characterized by degradation and calcification of articular cartilage. Presently, no effective treatment other than pain relief exists and patients ultimately need to undergo replacement surgery of the affected joint. During disease progression articular chondrocytes, the single cell type present in articular cartilage, show altered transcriptional profiles and undergo phenotypic changes that resemble the terminal differentiation route apparent in growth plate chondrocytes. Hence, given its prominent function in both regulating gene expression and maintaining cellular phenotypes, DNA methylation of CpG dinucleotides is intensively studied in the context of OA. An increasing number of studies have been published that employed a targeted approach on genes known to play a role in OA pathophysiology. As of such, it has become clear that OA responsive DNA methylation changes seem to mediate disease associated aberrant gene expression. Furthermore, established OA susceptibility alleles such as GDF5 and DIO2 appear to confer OA risk via DNA methylation and respective pathophysiological expression changes. In more recent years, genome wide profiling of DNA methylation in OA affected articular cartilage has emerged as a powerful tool to address the epigenetic changes in their entirety, which has resulted in the identification of putative patient subgroups as well as generic OA associated pathways. PMID:27019616

  12. DNA Methylation and Cancer Diagnosis

    PubMed Central

    Delpu, Yannick; Cordelier, Pierre; Cho, William C.; Torrisani, Jérôme

    2013-01-01

    DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results. PMID:23873296

  13. Stable loss of global DNA methylation in the radiation-target tissue-A possible mechanism contributing to radiation carcinogenesis?

    SciTech Connect

    Koturbash, Igor; Pogribny, Igor; Kovalchuk, Olga . E-mail: olga.kovalchuk@uleth.ca

    2005-11-18

    Radiation-induced lymphomagenesis and leukemogenesis are complex processes involving both genetic and epigenetic changes. Although genetic alterations during radiation-induced lymphoma- and leukemogenesis are fairly well studied, the role of epigenetic changes has been largely overlooked. Rodent models are valuable tools for identifying molecular mechanisms of lymphoma and leukemogenesis. A widely used mouse model of radiation-induced thymic lymphoma is characterized by a lengthy 'pre-lymphoma' period. Delineating molecular changes occurring during the pre-lymphoma period is crucial for understanding the mechanisms of radiation-induced leukemia/lymphoma development. In the present study, we investigated the role of radiation-induced DNA methylation changes in the radiation carcinogenesis target organ-thymus, and non-target organ-muscle. This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue. The presence of stable DNA hypomethylation in the radiation-target tissue, even though DNA damage resulting from initial genotoxic radiation insult was repaired, suggests of the importance of epigenetic mechanisms in the development of radiation-related pathologies. The possible role of radiation-induced DNA hypomethylation in radiation-induced genome

  14. Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis

    PubMed Central

    Wan, Wei; Yao, Feng; He, Ping-Ping; Xie, Wei; Mo, Zhong-Cheng; Shi, Jin-Feng; Wu, Jian-Feng; Peng, Juan; Liu, Dan; Cayabyab, Francisco S.; Zheng, Xi-Long; Tang, Xiang-Yang; Ouyang, Xin-Ping; Tang, Chao-Ke

    2016-01-01

    ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter. PMID:27295295

  15. DNA Methylation Screening and Analysis

    PubMed Central

    Sant, Karilyn E.; Nahar, Muna S.; Dolinoy, Dana C.

    2013-01-01

    DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations. PMID:22669678

  16. Regulation of DNA methylation of Rasgrf1

    PubMed Central

    Yoon, Bong June; Herman, Herry; Sikora, Aimee; Smith, Laura T.; Plass, Christoph; Soloway, Paul D.

    2009-01-01

    In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development1,2. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes3-10 or by activating growth-stimulating genes11-13. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive14,15, little is known about how DNA methylation is controlled16. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5′ of the promoter. Expression is exclusively from the paternal allele in neonatal brain17. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3′ of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted18. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus. PMID:11753386

  17. DNA damage, homology-directed repair, and DNA methylation.

    PubMed

    Cuozzo, Concetta; Porcellini, Antonio; Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Di Pardo, Alba; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-07-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  18. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  19. DNA methylation and hydroxymethylation in stem cells.

    PubMed

    Cheng, Ying; Xie, Nina; Jin, Peng; Wang, Tao

    2015-06-01

    In mammals, DNA methylation and hydroxymethylation are specific epigenetic mechanisms that can contribute to the regulation of gene expression and cellular functions. DNA methylation is important for the function of embryonic stem cells and adult stem cells (such as haematopoietic stem cells, neural stem cells and germline stem cells), and changes in DNA methylation patterns are essential for successful nuclear reprogramming. In the past several years, the rediscovery of hydroxymethylation and the TET enzymes expanded our insights tremendously and uncovered more dynamic aspects of cytosine methylation regulation. Here, we review the current knowledge and highlight the most recent advances in DNA methylation and hydroxymethylation in embryonic stem cells, induced pluripotent stem cells and several well-studied adult stems cells. Our current understanding of stem cell epigenetics and new advances in the field will undoubtedly stimulate further clinical applications of regenerative medicine in the future.

  20. Methods of DNA methylation detection

    NASA Technical Reports Server (NTRS)

    Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

    2010-01-01

    The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

  1. DNA methylation biomarkers: cancer and beyond.

    PubMed

    Mikeska, Thomas; Craig, Jeffrey M

    2014-01-01

    Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient's response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

  2. DNA Methylation Biomarkers: Cancer and Beyond

    PubMed Central

    Mikeska, Thomas; Craig, Jeffrey M.

    2014-01-01

    Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease. PMID:25229548

  3. DNA methylation and carcinogenesis.

    PubMed

    Lichtenstein, A V; Kisseljova, N P

    2001-03-01

    In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

  4. A context dependent role for DNA methylation in bivalves.

    PubMed

    Gavery, Mackenzie R; Roberts, Steven B

    2014-05-01

    The function of DNA methylation in species such as bivalves where the limited amount of DNA methylation is predominantly found in gene bodies remains unclear. An emerging possible explanation is that the role of gene body DNA methylation is dependent on gene function, a potential phenomenon that has arisen from selective pressure on lineage-specific life history traits. In genes contributing to phenotypes that benefit from increased plasticity, the absence of DNA methylation could contribute to stochastic transcriptional opportunities and increased transposable element activity. In genes where regulated control of activity is essential, DNA methylation may also play a role in targeted, predictable genome regulation. Here, we review the current knowledge concerning DNA methylation in bivalves and explore the putative role of DNA methylation in both an evolutionary and ecological context.

  5. DNA methylation and application in forensic sciences.

    PubMed

    Kader, Farzeen; Ghai, Meenu

    2015-04-01

    DNA methylation of cytosine residues is a stable epigenetic alteration, beginning as early as foetal development in the uterus and continuously evolving throughout life. DNA methylation as well as other epigenetic modifications such as chromatin remodelling and histone modifications are indispensable in mammalian development. Methylation is to a large extent influenced by the ageing process, diets and lifestyle choices. Our understanding of this crucial modification may even contribute to the treatment and prevention of age-related illnesses in the very near future. Genome-wide methylation analysis using high throughput DNA technologies has discovered numerous differentially methylated regions (tDMRs) which differ in levels of methylation in various cell types and tissues. TDMRs have been useful in various applications, particularly medicine and forensic sciences. Forensic scientists are constantly seeking exciting and novel methods to aid in the reconstruction of crime scenes, and the analysis of tDMRs represents a new and reliable technique to identify biological fluids and tissues found at the scene of a violent act. Not only has research been able to unequivocally identify various fluids and tissues, but methods to determine the sex, age and phenotype of donors has been developed. New tDMRs in genes are being searched for consistently to serve as novel markers in forensic DNA analysis. PMID:25732744

  6. DNA methylation and application in forensic sciences.

    PubMed

    Kader, Farzeen; Ghai, Meenu

    2015-04-01

    DNA methylation of cytosine residues is a stable epigenetic alteration, beginning as early as foetal development in the uterus and continuously evolving throughout life. DNA methylation as well as other epigenetic modifications such as chromatin remodelling and histone modifications are indispensable in mammalian development. Methylation is to a large extent influenced by the ageing process, diets and lifestyle choices. Our understanding of this crucial modification may even contribute to the treatment and prevention of age-related illnesses in the very near future. Genome-wide methylation analysis using high throughput DNA technologies has discovered numerous differentially methylated regions (tDMRs) which differ in levels of methylation in various cell types and tissues. TDMRs have been useful in various applications, particularly medicine and forensic sciences. Forensic scientists are constantly seeking exciting and novel methods to aid in the reconstruction of crime scenes, and the analysis of tDMRs represents a new and reliable technique to identify biological fluids and tissues found at the scene of a violent act. Not only has research been able to unequivocally identify various fluids and tissues, but methods to determine the sex, age and phenotype of donors has been developed. New tDMRs in genes are being searched for consistently to serve as novel markers in forensic DNA analysis.

  7. Aberrant DNA Methylation in Keratoacanthoma

    PubMed Central

    Nakagawa, Hidemi

    2016-01-01

    Background Keratoacanthoma (KA) is a self-limiting epidermal tumor for which histopathological examination sometimes suggests malignancy. Based on inconsistent clinical views, KA can be regarded as both a benign tumor and a variant of squamous cell carcinoma (SCC). Aberrant DNA methylation frequently occurs in malignant tumors but it scarcely occurs in benign tumors. Whether aberrant methylation occurs in KA has not been previously examined. Objective The aim is to elucidate whether aberrant methylation of CpG islands (CGI) containing a high density of cytosine-guanine dinucleotide (CpG) sites occurs in KA. Methods Five SCC cell lines, two cultured samples of normal human epidermal keratinocytes (NHEKs), 18 clinical SCC samples, and 21 clinical KA samples were analyzed with Infinium HumanMethylation450 BeadChips, quantitative real-time methylation-specific PCR (RT-MSP) and/or bisulfite sequencing. Results Genome-wide analyses of NHEK, KA, and SCC indicated that there was a greater number of aberrantly hypermethylated CGIs in SCC than in KA and there were aberrantly hypermethylated CGIs which are common in both. Among the common hypermethylated CGIs, RT-MSP and bisulfite sequencing targeting CGIs located on CCDC17, PVR, and MAP3K11 gene bodies also showed that methylation levels were significantly higher in KA than in normal epidermis. Statistical analyses suggested that the methylation level of CGI located on PVR in SCC might be correlated to lymph node metastasis (P = 0.013, Mann-Whitney U test) and that the methylation level of CGI in MAP3K11 in KA might be correlated to age (P = 0.031, linear regression analysis). Conclusion Aberrant DNA methylation occurs in KA. PMID:27788211

  8. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.

  9. DNA methylation and healthy human aging.

    PubMed

    Jones, Meaghan J; Goodman, Sarah J; Kobor, Michael S

    2015-12-01

    The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies. PMID:25913071

  10. 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.

  11. 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

  12. Forensic DNA methylation profiling from evidence material for investigative leads.

    PubMed

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

    2016-07-01

    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].

  13. DNA and histone methylation in gastric carcinogenesis

    PubMed Central

    Calcagno, Danielle Queiroz; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Burbano, Rommel Rodriguez; Smith, Marília de Arruda Cardoso

    2013-01-01

    Epigenetic alterations contribute significantly to the development and progression of gastric cancer, one of the leading causes of cancer death worldwide. Epigenetics refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches that target DNA methylation and histone modifications have emerged. A greater understanding of epigenetics and the therapeutic potential of manipulating these processes is necessary for gastric cancer treatment. Here, we review recent research on the effects of aberrant DNA and histone methylation on the onset and progression of gastric tumors and the development of compounds that target enzymes that regulate the epigenome. PMID:23482412

  14. Electronic transport in methylated fragments of DNA

    SciTech Connect

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L. Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  15. Electronic transport in methylated fragments of DNA

    NASA Astrophysics Data System (ADS)

    de Almeida, M. L.; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; de Moura, F. A. B. F.; Lyra, M. L.

    2015-11-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  16. DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

    PubMed Central

    Chamberlain, Alyssa A.; Lin, Mingyan; Lister, Rolanda L.; Maslov, Alex A.; Wang, Yidong; Suzuki, Masako; Wu, Bingruo; Greally, John M.; Zheng, Deyou; Zhou, Bin

    2014-01-01

    Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease. PMID:24947998

  17. Increased DNA methylation in the suicide brain.

    PubMed

    Haghighi, Fatemeh; Xin, Yurong; Chanrion, Benjamin; O'Donnell, Anne H; Ge, Yongchao; Dwork, Andrew J; Arango, Victoria; Mann, J John

    2014-09-01

    Clinical studies find that childhood adversity and stressful life events in adulthood increase the risk for major depression and for suicide. The predispositions to either major depression or suicide are thought to depend on genetic risk factors or epigenetic effects. We investigated DNA methylation signatures postmortem in brains of suicides with diagnosis of major depressive disorder. DNA methylation levels were determined at single C-phosphate-G (CpG) resolution sites within ventral prefrontal cortex of 53 suicides and nonpsychiatric controls, aged 16 to 89 years. We found that DNA methylation increases throughout the lifespan. Suicides showed an 8-fold greater number of methylated CpG sites relative to controls (P < 2.2 x 10(-16)), with greater DNA methylation changes over and above the increased methylation observed in normal aging. This increased DNA methylation may be a significant contributor to the neuropathology and psychopathology underlying the risk of suicide in depression.

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

    PubMed

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

    2013-05-01

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

  19. DNA Methylation as a Biomarker for Preeclampsia

    SciTech Connect

    Anderson, Cindy M.; Ralph, Jody L.; Wright, Michelle L.; Linggi, Bryan E.; Ohm, Joyce E.

    2014-10-01

    Background: Preeclampsia contributes significantly to pregnancy-associated morbidity and mortality as well as future risk of cardiovascular disease in mother and offspring, and preeclampsia in offspring. The lack of reliable methods for early detection limits the opportunities for prevention, diagnosis, and timely treatment. Purpose: The purpose of this study was to explore distinct DNA methylation patterns associated with preeclampsia in both maternal cells and fetal-derived tissue that represent potential biomarkers to predict future preeclampsia and inheritance in children. Method: A convenience sample of nulliparous women (N = 55) in the first trimester of pregnancy was recruited for this prospective study. Genome-wide DNA methylation was quantified in first-trimester maternal peripheral white blood cells and placental chorionic tissue from normotensive women and those with preeclampsia (n = 6/group). Results: Late-onset preeclampsia developed in 12.7% of women. Significant differences in DNA methylation were identified in 207 individual linked cytosine and guanine (CpG) sites in maternal white blood cells collected in the first trimester (132 sites with gain and 75 sites with loss of methylation), which were common to approximately 75% of the differentially methylated CpG sites identified in chorionic tissue of fetal origin. Conclusion: This study is the first to identify maternal epigenetic targets and common targets in fetal-derived tissue that represent putative biomarkers for early detection and heritable risk of preeclampsia. Findings may pave the way for diagnosis of preeclampsia prior to its clinical presentation and acute damaging effects, and the potential for prevention of the detrimental long-term sequelae.

  20. P1 plasmid replication requires methylated DNA.

    PubMed Central

    Abeles, A L; Austin, S J

    1987-01-01

    Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo. PMID:2826133

  1. DNA Methylation Landscapes of Human Fetal Development.

    PubMed

    Slieker, Roderick C; Roost, Matthias S; van Iperen, Liesbeth; Suchiman, H Eka D; Tobi, Elmar W; Carlotti, Françoise; de Koning, Eelco J P; Slagboom, P Eline; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2015-10-01

    Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.

  2. Conformation-selective methylation of geminivirus DNA.

    PubMed

    Paprotka, T; Deuschle, K; Metzler, V; Jeske, H

    2011-11-01

    Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially. PMID:21835804

  3. DNA methylation: old dog, new tricks?

    PubMed

    Spruijt, Cornelia G; Vermeulen, Michiel

    2014-11-01

    DNA methylation is an epigenetic modification that is generally associated with repression of transcription initiation at CpG-island promoters. Here we argue that, on the basis of recent high-throughput genomic and proteomic screenings, DNA methylation can also have different outcomes, including activation of transcription. This is evidenced by the fact that transcription factors can interact with methylated DNA sequences. Furthermore, in certain cellular contexts, genes containing methylated promoters are highly transcribed. Interestingly, this uncoupling between methylated DNA and repression of transcription seems to be particularly evident in germ cells and pluripotent cells. Thus, contrary to previous assumptions, DNA methylation is not exclusively associated with repression of transcription initiation. PMID:25372310

  4. Increased DNA methylation of scavenger receptor class B type I contributes to inhibitory effects of prenatal caffeine ingestion on cholesterol uptake and steroidogenesis in fetal adrenals

    SciTech Connect

    Wu, Dong-Mei; He, Zheng; Ma, Liang-Peng; Wang, Lin-Long; Ping, Jie; Wang, Hui

    2015-06-01

    Steroid hormones synthesized from cholesterol in the fetal adrenal are crucial for fetal development. We have observed the inhibited fetal adrenal corticosterone synthesis and increased intrauterine growth retardation (IUGR) rate in rats under prenatal caffeine ingestion. The aim of this study is to evaluate the effects of prenatal caffeine ingestion on cholesterol supply in fetal adrenal steroidogenesis in rats and explore the underlying epigenetic mechanisms. Pregnant Wistar rats were treated with 60 mg/kg·d caffeine from gestational day (GD) 7 to GD17. Histological changes of fetal adrenals and increased IUGR rates were observed in the caffeine group. There were significantly decreased steroid hormone contents and cholesterol supply in caffeine-treated fetal adrenals. Data from the gene expression array suggested that prenatal caffeine ingestion caused increased expression of genes related to DNA methylation and decreased expression of genes related to cholesterol uptake. The following conjoint analysis of DNA methylation array with these differentially expressed genes suggested that scavenger receptor class B type I (SR-BI) may play an important role in caffeine-induced cholesterol supply deficiency. Moreover, real-time RT-PCR and immunohistochemical detection certified the inhibitory effects of caffeine on both mRNA expression and protein expression of SR-BI in the fetal adrenal. And the increased DNA methylation frequency in the proximal promoter of SR-BI was confirmed by bisulfite-sequencing PCR. In conclusion, prenatal caffeine ingestion can induce DNA hypermethylation of the SR-BI promoter in the rat fetal adrenal. These effects may lead to decreased SR-BI expression and cholesterol uptake, which inhibits steroidogenesis in the fetal adrenal. - Highlights: • Prenatal caffeine ingestion inhibits steroid hormone production in the fetal adrenal. • Prenatal caffeine ingestion inhibits cholesterol uptake in the fetal adrenal. • Prenatal caffeine

  5. Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

    PubMed Central

    Yu, Yinni; Blair, Steve; Gillespie, David; Jensen, Randy; Myszka, David G.; Badran, Ahmed H.; Ghosh, Indraneel; Chagovetz, Alexander

    2010-01-01

    Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemi-methylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 nM to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for non-symmetrical methylation motifs were consistently greater (> 2 µM), but the binding selectivity between symmetric and hemi-methylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density. PMID:20507169

  6. An integrated workflow for DNA methylation analysis.

    PubMed

    Li, Pingchuan; Demirci, Feray; Mahalingam, Gayathri; Demirci, Caghan; Nakano, Mayumi; Meyers, Blake C

    2013-05-20

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes. DNA methylation has a demonstrated role in the genome stability and protection, regulation of gene expression and many other aspects of genome function and maintenance. BS-seq is a relatively unbiased method for profiling the DNA methylation, with a resolution capable of measuring methylation at individual cytosines. Here we describe, as an example, a workflow to handle DNA methylation analysis, from BS-seq library preparation to the data visualization. We describe some applications for the analysis and interpretation of these data. Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu), along with small RNA, RNA-seq and other data types. PMID:23706300

  7. Epigenetic regulation of hematopoiesis by DNA methylation

    PubMed Central

    Gore, Aniket V; Athans, Brett; Iben, James R; Johnson, Kristin; Russanova, Valya; Castranova, Daniel; Pham, Van N; Butler, Matthew G; Williams-Simons, Lisa; Nichols, James T; Bresciani, Erica; Feldman, Bejamin; Kimmel, Charles B; Liu, Paul P; Weinstein, Brant M

    2016-01-01

    During embryonic development, cell type-specific transcription factors promote cell identities, while epigenetic modifications are thought to contribute to maintain these cell fates. Our understanding of how genetic and epigenetic modes of regulation work together to establish and maintain cellular identity is still limited, however. Here, we show that DNA methyltransferase 3bb.1 (dnmt3bb.1) is essential for maintenance of hematopoietic stem and progenitor cell (HSPC) fate as part of an early Notch-runx1-cmyb HSPC specification pathway in the zebrafish. Dnmt3bb.1 is expressed in HSPC downstream from Notch1 and runx1, and loss of Dnmt3bb.1 activity leads to reduced cmyb locus methylation, reduced cmyb expression, and gradual reduction in HSPCs. Ectopic overexpression of dnmt3bb.1 in non-hematopoietic cells is sufficient to methylate the cmyb locus, promote cmyb expression, and promote hematopoietic development. Our results reveal an epigenetic mechanism supporting the maintenance of hematopoietic cell fate via DNA methylation-mediated perdurance of a key transcription factor in HSPCs. DOI: http://dx.doi.org/10.7554/eLife.11813.001 PMID:26814702

  8. Clinical potential of DNA methylation in organ transplantation.

    PubMed

    Peters, Fleur S; Manintveld, Olivier C; Betjes, Michiel G H; Baan, Carla C; Boer, Karin

    2016-07-01

    Identification of patients at risk for post-transplant complications is a major challenge, but it will improve clinical care and patient health after organ transplantation. The poor predictive value of the current biomarkers highlights the need to explore novel and innovative methods, such as epigenetics, for the discovery of new biomarkers. Cell differentiation and function of immune cells is dependent on epigenetic mechanisms, which regulate gene expression without altering the original DNA sequence. These epigenetic mechanisms are dynamic, potentially heritable, change with age, and can be regulated and influenced by environmental conditions. One of the most well-known epigenetic mechanisms is DNA methylation, which comprises the methylation of a cytosine (C) next to a guanine (G; CpG dinucleotides). Aberrant DNA methylation is increasingly associated with disease, including immune-mediated diseases, and these alterations precede the clinical phenotype. The impact of DNA methylation profiles on transplant acceptance and rejection as well as on other post-transplant complications is unknown. In this study we examine the current evidence of the functional role of recipient and donor DNA methylation on outcome after organ transplantation. Changes in DNA methylation may predict the risk of developing post-transplant complications, such as infections, malignancies and allograft rejection. We speculate that identification of these changes in DNA methylation contributes to earlier diagnosis and prevention of post-transplant complications, leading to improved patient care. PMID:27085975

  9. Clinical potential of DNA methylation in organ transplantation.

    PubMed

    Peters, Fleur S; Manintveld, Olivier C; Betjes, Michiel G H; Baan, Carla C; Boer, Karin

    2016-07-01

    Identification of patients at risk for post-transplant complications is a major challenge, but it will improve clinical care and patient health after organ transplantation. The poor predictive value of the current biomarkers highlights the need to explore novel and innovative methods, such as epigenetics, for the discovery of new biomarkers. Cell differentiation and function of immune cells is dependent on epigenetic mechanisms, which regulate gene expression without altering the original DNA sequence. These epigenetic mechanisms are dynamic, potentially heritable, change with age, and can be regulated and influenced by environmental conditions. One of the most well-known epigenetic mechanisms is DNA methylation, which comprises the methylation of a cytosine (C) next to a guanine (G; CpG dinucleotides). Aberrant DNA methylation is increasingly associated with disease, including immune-mediated diseases, and these alterations precede the clinical phenotype. The impact of DNA methylation profiles on transplant acceptance and rejection as well as on other post-transplant complications is unknown. In this study we examine the current evidence of the functional role of recipient and donor DNA methylation on outcome after organ transplantation. Changes in DNA methylation may predict the risk of developing post-transplant complications, such as infections, malignancies and allograft rejection. We speculate that identification of these changes in DNA methylation contributes to earlier diagnosis and prevention of post-transplant complications, leading to improved patient care.

  10. Determining DNA methylation profiles using sequencing.

    PubMed

    Feng, Suhua; Rubbi, Liudmilla; Jacobsen, Steven E; Pellegrini, Matteo

    2011-01-01

    Cytosine methylation is an epigenetic mark that has a significant impact on the regulation of transcription and replication of DNA. DNA methylation patterns are highly conserved across cell divisions and are therefore highly heritable. Furthermore, in multicellular organisms, DNA methylation patterning is a key determinant of cellular differentiation and tissue-specific expression patterns. Lastly, DNA demethylases can affect global levels of DNA methylation during specific stages of development. Bisulfite sequencing is considered the gold standard for measuring the methylation state of cytosines. Sodium bisulfite -converts unmethylated cytosines to uracils (which after PCR are converted to thymines), while leaving methylated cytosines unconverted. By mapping bisulfite treated DNA back to the original reference genome, it is then possible to determine the methylation state of individual cytosines. With the advent of next-generation sequencers during the past few years, it is now possible to determine the methylation state of an entire genome. Here, we describe in detail two protocols for preparing bisulfite treated libraries, which may be sequenced using Illumina GAII sequencers. The first of these uses premethylated adapters, which are not affected by bisulfite treatments, while the second uses a two-stage adapter strategy and does not require premethylation of the adapters. We also describe the specialized protocol for mapping bisulfite converted reads. These approaches allow one to determine the methylation state of each cytosine in the genome. PMID:21431774

  11. DNA methylation pathways and their crosstalk with histone methylation

    PubMed Central

    Du, Jiamu; Johnson, Lianna M.; Jacobsen, Steven E.; Patel, Dinshaw J.

    2015-01-01

    Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk. PMID:26296162

  12. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines.

    PubMed

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I (2) statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating "hub genes" - heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  13. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility.

  14. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  15. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

    PubMed

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. PMID:27595565

  16. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    PubMed Central

    Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

    2016-01-01

    The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001 PMID:27595565

  17. DNA methylation changes in epithelial ovarian cancer histotypes

    PubMed Central

    Earp, Madalene A.; Cunningham, Julie M.

    2016-01-01

    Survival after a diagnosis of ovarian cancer has not improved, and despite histological differences, treatment is similar for all cases. Understanding the molecular basis for ovarian cancer risk and prognosis is fundamental, and to this end much has been gleaned about genetic changes contributing to risk, and to a lesser extent, survival. There’s considerable evidence for genetic differences between the four pathologically defined histological subtypes; however, the contribution of epigenetics is less well documented. In this report, we review alterations in DNA methylation in ovarian cancer, focusing on histological subtypes, and studies examining the roles of methylation in determining therapy response. As epigenetics is making its way into clinical care, we review the application of cell free DNA methylation to ovarian cancer diagnosis and care. Finally, we comment on recurrent limitations in the DNA methylation literature for ovarian cancer, which can and should be addressed to mature this field. PMID:26363302

  18. Increased DNA methylation of scavenger receptor class B type I contributes to inhibitory effects of prenatal caffeine ingestion on cholesterol uptake and steroidogenesis in fetal adrenals.

    PubMed

    Wu, Dong-Mei; He, Zheng; Ma, Liang-Peng; Wang, Lin-Long; Ping, Jie; Wang, Hui

    2015-06-01

    Steroid hormones synthesized from cholesterol in the fetal adrenal are crucial for fetal development. We have observed the inhibited fetal adrenal corticosterone synthesis and increased intrauterine growth retardation (IUGR) rate in rats under prenatal caffeine ingestion. The aim of this study is to evaluate the effects of prenatal caffeine ingestion on cholesterol supply in fetal adrenal steroidogenesis in rats and explore the underlying epigenetic mechanisms. Pregnant Wistar rats were treated with 60 mg/kg · d caffeine from gestational day (GD) 7 to GD17. Histological changes of fetal adrenals and increased IUGR rates were observed in the caffeine group. There were significantly decreased steroid hormone contents and cholesterol supply in caffeine-treated fetal adrenals. Data from the gene expression array suggested that prenatal caffeine ingestion caused increased expression of genes related to DNA methylation and decreased expression of genes related to cholesterol uptake. The following conjoint analysis of DNA methylation array with these differentially expressed genes suggested that scavenger receptor class B type I (SR-BI) may play an important role in caffeine-induced cholesterol supply deficiency. Moreover, real-time RT-PCR and immunohistochemical detection certified the inhibitory effects of caffeine on both mRNA expression and protein expression of SR-BI in the fetal adrenal. And the increased DNA methylation frequency in the proximal promoter of SR-BI was confirmed by bisulfite-sequencing PCR. In conclusion, prenatal caffeine ingestion can induce DNA hypermethylation of the SR-BI promoter in the rat fetal adrenal. These effects may lead to decreased SR-BI expression and cholesterol uptake, which inhibits steroidogenesis in the fetal adrenal.

  19. Role of TET enzymes in DNA methylation, development, and cancer

    PubMed Central

    Rasmussen, Kasper Dindler

    2016-01-01

    The pattern of DNA methylation at cytosine bases in the genome is tightly linked to gene expression, and DNA methylation abnormalities are often observed in diseases. The ten eleven translocation (TET) enzymes oxidize 5-methylcytosines (5mCs) and promote locus-specific reversal of DNA methylation. TET genes, and especially TET2, are frequently mutated in various cancers, but how the TET proteins contribute to prevent the onset and maintenance of these malignancies is largely unknown. Here, we highlight recent advances in understanding the physiological function of the TET proteins and their role in regulating DNA methylation and transcription. In addition, we discuss some of the key outstanding questions in the field. PMID:27036965

  20. Epigenetic regulation during fetal femur development: DNA methylation matters.

    PubMed

    de Andrés, María C; Kingham, Emmajayne; Imagawa, Kei; Gonzalez, Antonio; Roach, Helmtrud I; Wilson, David I; Oreffo, Richard O C

    2013-01-01

    Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening

  1. Targeting DNA methylation with green tea catechins.

    PubMed

    Yiannakopoulou, Eugenia C

    2015-01-01

    Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate epigenetic processes. This review aims to synthesize evidence on the modulation of DNA methylation by green tea catechins. Green tea catechins have been reported to reverse DNA methylation of tumor suppressor genes and increase transcription of these genes. Green tea catechins and especially epigallocatechin gallate modulate DNA methylation by attenuating the effect of DNA methyltransferase 1 (DNMT1). However, the exact mechanism of DNMT1 inhibition is not delineated. Suggested mechanisms include direct enzymatic inhibition, indirect enzymatic inhibition, reduced DNMT1 expression and translation. The possible effect of green tea catechins on other pathways of DNA methylation, i.e. methyl-CpG binding domain proteins, has not been investigated. Furthermore, the link between redox properties and epigenetic modulation by green tea catechins has not been defined either. Since green tea catechins are natural compounds with a rather acceptable safety profile, further research on their action as inhibitors of DNA methylation seems worthwhile.

  2. DNA Methylation of BDNF Gene in Schizophrenia

    PubMed Central

    Çöpoğlu, Ümit Sertan; İğci, Mehri; Bozgeyik, Esra; Kokaçya, M. Hanifi; İğci, Yusuf Ziya; Dokuyucu, Recep; Arı, Mustafa; Savaş, Haluk A.

    2016-01-01

    Background Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. Material/Methods The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. Results There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. Conclusions Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation. PMID:26851233

  3. Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity.

    PubMed

    Wang, Gang Lin; Zhou, Long Yin; Luo, Hong Qun; Li, Nian Bing

    2013-03-20

    The present work demonstrates a novel signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity using the electroactive complex [Ru(NH3)6](3+) (RuHex) as a signal transducer. The assay exploits the electrostatic interactions between RuHex and DNA strands. Thiolated single strand DNA1 was firstly self-assembled on a gold electrode via Au-S bonding, followed by hybridization with single strand DNA2 to form double strand DNA containing specific recognition sequence of DNA adenine methylation MTase and methylation-responsive restriction endonuclease Dpn I. The double strand DNA may adsorb lots of electrochemical species ([Ru(NH3)6](3+)) via the electrostatic interaction, thus resulting in a high electrochemical signal. In the presence of DNA adenine methylation methyltransferase and S-adenosyl-l-methionine, the formed double strand DNA was methylated by DNA adenine methylation methyltransferase, then the double strand DNA can be cleaved by methylation-responsive restriction endonuclease Dpn I, leading to the dissociation of a large amount of signaling probes from the electrode. As a result, the adsorption amount of RuHex reduced, resulting in a decrease in electrochemical signal. Thus, a sensitive electrochemical method for detection of DNA methylation is proposed. The proposed method yielded a linear response to concentration of Dam MTase ranging from 0.25 to 10UmL(-1) with a detection limit of 0.18UmL(-1) (S/N=3), which might promise this method as a good candidate for monitoring DNA methylation in the future. PMID:23473252

  4. The vegetarian lifestyle and DNA methylation.

    PubMed

    Geisel, Jürgen; Schorr, Heike; Bodis, Marion; Isber, Sonia; Hübner, Ulrich; Knapp, Jean-Pierre; Obeid, Rima; Herrmann, Wolfgang

    2005-01-01

    Vegetarians have a lower intake of vitamin B12 than omnivores do. Vitamin B12 deficiency (holotranscobalamin II <35 pmol/L or methylmalonic acid >271 nmol/L) was found in 58% of 71 vegetarians studied. Higher homocysteine levels (>12 micromol/L) found in 45% indicate disturbed remethylation of homocysteine to methionine. The methylation of DNA is strongly linked to homocysteine metabolism. Since DNA methylation is an important epigenetic factor in the regulation of gene expression, alteration of the methylation pattern has been associated with aging, cancer, atherosclerosis and other diseases. Three observations indicate that DNA methylation could be diminished by a vegetarian lifestyle. The vegetarian diet has a low content of methionine, remethylation of homocysteine is reduced by vitamin B12 deficiency and elevated homocysteine levels can induce the generation of S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases. In our study we observed a significant correlation between SAH and whole-genome methylation (r=-0.36, p<0.01). This observation underlines the role of SAH as a potent inhibitor of methyltransferases. The methylation status was not correlated with homocysteine or S-adenosylemethionine (SAM). These results indicate that the degree of methylation does not depend on the supply of methyl groups and that the reverse generation of SAH has no influence. In addition to whole-genome methylation, the specific promoter methylation of the p66Shc gene was studied. However, the latter did not correlate with SAH, SAM or homocysteine. Obviously, the promoter methylation of the p66Shc gene is controlled in a specific way, without following the general regulating influence of SAH. In conclusion, an inhibitory effect of SAH on whole-genome methylation was found, but from our data no interaction between vegetarian lifestyle and DNA methylation could be determined.

  5. Maternal DNA Methylation Regulates Early Trophoblast Development

    PubMed Central

    Branco, Miguel R.; King, Michelle; Perez-Garcia, Vicente; Bogutz, Aaron B.; Caley, Matthew; Fineberg, Elena; Lefebvre, Louis; Cook, Simon J.; Dean, Wendy; Hemberger, Myriam; Reik, Wolf

    2016-01-01

    Summary Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms. PMID:26812015

  6. Targeting DNA Methylation for Epigenetic Therapy

    PubMed Central

    Yang, Xiaojing; Lay, Fides; Han, Han; Jones, Peter A.

    2010-01-01

    DNA methylation patterns are established during embryonic development and faithfully copied through somatic cell divisions. Based on our understanding of DNA methylation and other interrelated epigenetic modifications, a comprehensive view of the epigenetic landscape and cancer epigenome is evolving. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. During the last few decades, an increasing number of drugs targeting DNA methylation have been developed in an effort to increase efficacy, stability and to decrease toxicity. The earliest and the most successful epigenetic drug to date, 5-Azacytidine, is currently recommended as the first-line treatment for high risk myelodysplastic syndromes (MDS) patients. Encouraging results from clinical trials have prompted further efforts to elucidate epigenetic alterations in cancer and subsequently develop new epigenetic therapies. This review delineates the latest cancer epigenetic models, recent discovery of hypomethylation agents and their application in the clinic. PMID:20846732

  7. Epigenetic DNA Methylation Linked to Social Dominance

    PubMed Central

    Lenkov, Kapa; Lee, Mi H.; Lenkov, Olga D.; Swafford, Andrew; Fernald, Russell D.

    2015-01-01

    Social status hierarchies are ubiquitous in vertebrate social systems, including humans. It is well known that social rank can influence quality of life dramatically among members of social groups. For example, high-ranking individuals have greater access to resources, including food and mating prerogatives that, in turn, have a positive impact on their reproductive success and health. In contrast low ranking individuals typically have limited reproductive success and may experience lasting social and physiological costs. Ultimately, social rank and behavior are regulated by changes in gene expression. However, little is known about mechanisms that transduce social cues into transcriptional changes. Since social behavior is a dynamic process, we hypothesized that a molecular mechanism such as DNA methylation might play a role these changes. To test this hypothesis, we used an African cichlid fish, Astatotilapia burtoni, in which social rank dictates reproductive access. We show that manipulating global DNA methylation state strongly biases the outcomes of social encounters. Injecting DNA methylating and de-methylating agents in low status animals competing for status, we found that animals with chemically increased methylation states were statistically highly likely to ascend in rank. In contrast, those with inhibited methylation processes and thus lower methylation levels were statistically highly unlikely to ascend in rank. This suggests that among its many roles, DNA methylation may be linked to social status and more generally to social behavior. PMID:26717574

  8. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

  9. Covering your bases: inheritance of DNA methylation in plant genomes.

    PubMed

    Niederhuth, Chad E; Schmitz, Robert J

    2014-03-01

    Cytosine methylation is an important base modification that is inherited across mitotic and meiotic cell divisions in plant genomes. Heritable methylation variants can contribute to within-species phenotypic variation. Few methylation variants were known until recently, making it possible to begin to address major unanswered questions: the extent of natural methylation variation within plant genomes, its effects on phenotypic variation, its degree of dependence on genotype, and how it fits into an evolutionary context. Techniques like whole-genome bisulfite sequencing (WGBS) make it possible to determine cytosine methylation states at single-base resolution across entire genomes and populations. Application of this method to natural and novel experimental populations is revealing answers to these long-standing questions about the role of DNA methylation in plant genomes.

  10. Reconfiguration of DNA methylation in aging.

    PubMed

    Zampieri, Michele; Ciccarone, Fabio; Calabrese, Roberta; Franceschi, Claudio; Bürkle, Alexander; Caiafa, Paola

    2015-11-01

    A complex interplay between multiple biological effects shapes the aging process. The advent of genome-wide quantitative approaches in the epigenetic field has highlighted the effective impact of epigenetic deregulation, particularly of DNA methylation, on aging. Age-associated alterations in DNA methylation are commonly grouped in the phenomenon known as "epigenetic drift" which is characterized by gradual extensive demethylation of genome and hypermethylation of a number of promoter-associated CpG islands. Surprisingly, specific DNA regions show directional epigenetic changes in aged individuals suggesting the importance of these events for the aging process. However, the epigenetic information obtained until now in aging needs a re-consideration due to the recent discovery of 5-hydroxymethylcytosine, a new DNA epigenetic mark present on genome. A recapitulation of the factors involved in the regulation of DNA methylation and the changes occurring in aging will be described in this review also considering the data available on 5 hmC.

  11. Eukaryotic DNA methylation as an evolutionary device.

    PubMed

    Colot, V; Rossignol, J L

    1999-05-01

    DNA methylation is catalyzed by a family of conserved DNA methyltransferases and is widespread among protists, plants, fungi and animals. It is however absent in some species and its genomic distribution varies among organisms. Sequence comparisons suggest that known and putative eukaryotic DNA methyltransferases fall into at least five structurally distinct subfamilies. Furthermore, it is now clear that DNA methylation can be involved in several functions, some of which may coexist within the same organism. It can inhibit transcription initiation, arrest transcript elongation, act as an imprinting signal, and suppress homologous recombination. On the basis of these observations, we argue that DNA methylation has been conserved during evolution because it provides unique possibilities for setting up functions of various types.

  12. DNA methylation in inflammatory bowel disease and beyond

    PubMed Central

    Low, Daren; Mizoguchi, Atsushi; Mizoguchi, Emiko

    2013-01-01

    Inflammatory bowel disease (IBD) is a consequence of the complex, dysregulated interplay between genetic predisposition, environmental factors, and microbial composition in the intestine. Despite a great advancement in identifying host-susceptibility genes using genome-wide association studies (GWAS), the majority of IBD cases are still underrepresented. The immediate challenge in post-GWAS era is to identify other causative genetic factors of IBD. DNA methylation has received increasing attention for its mechanistical role in IBD pathogenesis. This stable, yet dynamic DNA modification, can directly affect gene expression that have important implications in IBD development. The alterations in DNA methylation associated with IBD are likely to outset as early as embryogenesis all the way until old-age. In this review, we will discuss the recent advancement in understanding how DNA methylation alterations can contribute to the development of IBD. PMID:23983426

  13. Editing DNA Methylation in the Mammalian Genome.

    PubMed

    Liu, X Shawn; Wu, Hao; Ji, Xiong; Stelzer, Yonatan; Wu, Xuebing; Czauderna, Szymon; Shu, Jian; Dadon, Daniel; Young, Richard A; Jaenisch, Rudolf

    2016-09-22

    Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation. PMID:27662091

  14. Is There a Relationship between DNA Methylation and Phenotypic Plasticity in Invertebrates?

    PubMed Central

    Roberts, Steven B.; Gavery, Mackenzie R.

    2011-01-01

    There is a significant amount of variation in DNA methylation characteristics across organisms. Likewise, the biological role of DNA methylation varies across taxonomic lineages. The complexity of DNA methylation patterns in invertebrates has only recently begun to be characterized in-depth. In some invertebrate species that have been examined to date, methylated DNA is found primarily within coding regions and patterning is closely associated with gene function. Here we provide a perspective on the potential role of DNA methylation in these invertebrates with a focus on how limited methylation may contribute to increased phenotypic plasticity in highly fluctuating environments. Specifically, limited methylation could facilitate a variety of transcriptional opportunities including access to alternative transcription start sites, increasing sequence mutations, exon skipping, and transient methylation. PMID:22232607

  15. Blood-derived DNA methylation markers of cancer risk.

    PubMed

    Marsit, Carmen; Christensen, Brock

    2013-01-01

    The importance of somatic epigenetic alterations in tissues targeted for carcinogenesis is now well recognized and considered a key molecular step in the development of a tumor. Particularly, alteration of gene-specific and genomic DNA methylation has been extensively characterized in tumors, and has become an attractive biomarker of risk due to its specificity and stability in human samples. It also is clear that tumors do not develop as isolated phenomenon in their target tissue, but instead result from altered processes affecting not only the surrounding cells and tissues, but other organ systems, including the immune system. Thus, alterations to DNA methylation profiles detectable in peripheral blood may be useful not only in understanding the carcinogenic process and response to environmental insults, but can also provide critical insights in a systems biological view of tumorigenesis. Research to date has generally focused on how environmental exposures alter genomic DNA methylation content in peripheral blood. More recent work has begun to translate these findings to clinically useful endpoints, by defining the relationship between DNA methylation alterations and cancer risk. This chapter highlights the existing research linking the environment, blood-derived DNA methylation alterations, and cancer risk, and points out how these epigenetic alterations may be contributing fundamentally to carcinogenesis.

  16. DNA methylation and body temperature in fishes.

    PubMed

    Varriale, Annalisa; Bernardi, Giorgio

    2006-12-30

    Previous investigations from our laboratory [Jabbari, K., Cacciò, S., Pais de Barros, J.P., Desgres, J., Bernardi G., 1997. Evolutionary changes in CpG and methylation levels in the genome of vertebrates. Gene 205, 109-118.] led to the discovery of two different methylation levels in the genomes of vertebrates, a higher one exhibited by fishes and amphibians and a lower one shown by mammals and birds. It was also noted that data from the literature indicated a higher CpG level in fishes and amphibians compared to mammals and birds. Such observations led to suggesting the existence of two equilibria and to speculate that the transitions between the two equilibria in DNA methylation and CpG levels were due to a higher deamination rate in warm-blooded vertebrates related to their higher body temperature. Here we used Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis to study methylation levels in a number of fish genomes living at different temperatures. We found that polar fishes exhibit DNA methylation levels that are higher than those of tropical and temperate fishes, the latter being in turn higher than the methylation levels of warm-blooded vertebrates, as expected from previous work. A closer analysis of the data revealed that, among Antarctic fishes, the Channichthyidae (the icefishes, deprived of haemoglobin) had the highest methylation level, and that, among temperate and tropical fishes the latter showed the lowest methylation level. These results confirm the existence of an inverse relationship between DNA methylation and body temperature, when the latter is maintained over evolutionary times.

  17. Native DNA repeats and methylation in Ascobolus.

    PubMed Central

    Goyon, C; Rossignol, J L; Faugeron, G

    1996-01-01

    We identified two classes of native dispersed DNA repeats in the Ascobolus genome. The first class consisted of several kilobase long, methylated repeats. These repeats, named Mars (methylated Ascobolus repeated sequences), fell in one family of LINE-like elements and in three families of LTR-containing retrotransposable elements. The methylation features of Mars elements were those expected if they were natural targets for the MIP (methylation induced premeiotically) previously discovered in Ascobolus. The second class consisted of short repeats, approximately 100 bp long, corresponding to 5S rRNA and tRNA genes. As expected from their size, which was too small to allow MIP to occur, they were unmethylated, as were 26 kb of unique sequences tested. These observations are consistent with the hypothesis that MIP is targeted at natural DNA repeats and constitutes a defensive process against the detrimental consequences of the spreading of mobile elements throughout the genome. The 9 kb tandem repeats harbouring the 28S, 18S and 5.8S rRNA genes displayed methylation features suggesting that rDNA methylation proceeds through a process other than MIP. PMID:8811089

  18. DNA methylation, a hand behind neurodegenerative diseases

    PubMed Central

    Lu, Haoyang; Liu, Xinzhou; Deng, Yulin; Qing, Hong

    2013-01-01

    Epigenetic alterations represent a sort of functional modifications related to the genome that are not responsible for changes in the nucleotide sequence. DNA methylation is one of such epigenetic modifications that have been studied intensively for the past several decades. The transfer of a methyl group to the 5 position of a cytosine is the key feature of DNA methylation. A simple change as such can be caused by a variety of factors, which can be the cause of many serious diseases including several neurodegenerative diseases. In this review, we have reviewed and summarized recent progress regarding DNA methylation in four major neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). The studies of these four major neurodegenerative diseases conclude the strong suggestion of the important role DNA methylation plays in these diseases. However, each of these diseases has not yet been understood completely as details in some areas remain unclear, and will be investigated in future studies. We hope this review can provide new insights into the understanding of neurodegenerative diseases from the epigenetic perspective. PMID:24367332

  19. DNA Methylation and Flavonoids in Genitourinary Cancers

    PubMed Central

    Mukherjee, Neelam; Kumar, Addanki P; Ghosh, Rita

    2015-01-01

    Malignancies of the genitourinary system have some of the highest cancer incidence and mortality rates. For example prostate cancer is the second most common cancer in men and ovarian cancer mortality and incidence are near equal. In addition to genetic changes modulation of the epigenome is critical to cancer development and progression. In this regard epigenetic changes in DNA methylation state and DNA hypermethylation in particular has garnered a great deal of attention. While hypomethylation occurs mostly in repeated sequence such as tandem and interspersed repeats and segment duplications, hypermethylation is associated with CpG islands. Hypomethylation leads to activation of cancer-causing genes with global DNA hypomethylation being commonly associated with metastatic disease. Hypermethylation-mediated silencing of tumor suppressive genes is commonly associated with cancer development. Bioactive phytochemicals such as flavonoids present in fruits, vegetables, beverages etc. have the ability to modulate DNA methylation status and are therefore very valuable agents for cancer prevention. In this review we discuss several commonly methylated genes and flavonoids used to modulate DNA methylation in the prevention of genitourinary cancers. PMID:26005633

  20. Genome-wide quantitative assessment of variation in DNA methylation patterns

    PubMed Central

    Xie, Hehuang; Wang, Min; de Andrade, Alexandre; de F. Bonaldo, Maria; Galat, Vasil; Arndt, Kelly; Rajaram, Veena; Goldman, Stewart; Tomita, Tadanori; Soares, Marcelo B.

    2011-01-01

    Genomic DNA methylation contributes substantively to transcriptional regulations that underlie mammalian development and cellular differentiation. Much effort has been made to decipher the molecular mechanisms governing the establishment and maintenance of DNA methylation patterns. However, little is known about genome-wide variation of DNA methylation patterns. In this study, we introduced the concept of methylation entropy, a measure of the randomness of DNA methylation patterns in a cell population, and exploited it to assess the variability in DNA methylation patterns of Alu repeats and promoters. A few interesting observations were made: (i) within a cell population, methylation entropy varies among genomic loci; (ii) among cell populations, the methylation entropies of most genomic loci remain constant; (iii) compared to normal tissue controls, some tumors exhibit greater methylation entropies; (iv) Alu elements with high methylation entropy are associated with high GC content but depletion of CpG dinucleotides and (v) Alu elements in the intronic regions or far from CpG islands are associated with low methylation entropy. We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters. Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance. PMID:21278160

  1. Global DNA Methylation of Ischemic Stroke Subtypes

    PubMed Central

    Soriano-Tárraga, Carolina; Jiménez-Conde, Jordi; Giralt-Steinhauer, Eva; Mola, Marina; Ois, Ángel; Rodríguez-Campello, Ana; Cuadrado-Godia, Elisa; Fernández-Cadenas, Israel; Carrera, Caty; Montaner, Joan; Elosua, Roberto; Roquer, Jaume

    2014-01-01

    Ischemic stroke (IS), a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA) of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA), small-artery disease (SAD), and cardio-aortic embolism (CE). A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204) were included, distributed across 3 IS subtypes: LAA (78/281, 59/204), SAD (97/281, 53/204), and CE (106/281, 89/204). In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease. PMID:24788121

  2. Information Thermodynamics of Cytosine DNA Methylation.

    PubMed

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    Cytosine DNA methylation (CDM) is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise") induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1) the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2) whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic rules as do current

  3. Epigenomic profiling indicates a role for DNA methylation in early postnatal liver development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The question of whether DNA methylation contributes to the stabilization of gene expression patterns in differentiated mammalian tissues remains controversial. Using genome-wide methylation profiling, we screened 3757 gene promoters for changes in methylation during postnatal liver development to te...

  4. Cytosine methylation of sperm DNA in horse semen after cryopreservation.

    PubMed

    Aurich, Christine; Schreiner, Bettina; Ille, Natascha; Alvarenga, Marco; Scarlet, Dragos

    2016-09-15

    Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure. PMID:27242182

  5. Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA.

    PubMed

    Osakabe, Akihisa; Adachi, Fumiya; Arimura, Yasuhiro; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2015-10-01

    DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

  6. Controlling DNA methylation: many roads to one modification.

    PubMed

    Freitag, Michael; Selker, Eric U

    2005-04-01

    Genetic, biochemical and cytological studies on DNA methylation in several eukaryotic organisms have resulted in leaps of understanding in the past three years. Discoveries of mechanistic links between DNA methylation and histone methylation, and between these processes and RNA interference (RNAi) machineries have reinvigorated the field. The details of the connections between DNA methylation, histone modifications and RNA silencing remain to be elucidated, but it is already clear that no single pathway accounts for all DNA methylation found in eukaryotes. Rather, different taxa use one or more of several general mechanisms to control methylation. Despite recent progress, classic questions remain, including: What are the signals for DNA methylation? Are "de novo" and "maintenance" methylation truly separate processes? How is DNA methylation regulated?

  7. Role of DNA methylation in hybrid vigor in Arabidopsis thaliana

    PubMed Central

    Kawanabe, Takahiro; Ishikura, Sonoko; Miyaji, Naomi; Sasaki, Taku; Wu, Li Min; Itabashi, Etsuko; Takada, Satoko; Shimizu, Motoki; Takasaki-Yasuda, Takeshi; Osabe, Kenji; Peacock, W. James; Dennis, Elizabeth S.; Fujimoto, Ryo

    2016-01-01

    Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis. PMID:27791039

  8. MTHFD1 controls DNA methylation in Arabidopsis.

    PubMed

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J; John, Amala; Smith, Danielle C; Zhai, Jixian; Hale, Christopher J; Long, Jeff A; Hell, Ruediger; Díaz de la Garza, Rocío I; Jacobsen, Steven E

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  9. MTHFD1 controls DNA methylation in Arabidopsis

    PubMed Central

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A.; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J.; John, Amala; Smith, Danielle C.; Zhai, Jixian; Hale, Christopher J.; Long, Jeff A.; Hell, Ruediger; Díaz de la Garza, Rocío I.; Jacobsen, Steven E.

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  10. Dynamic regulation of DNA methylation during mammalian development.

    PubMed

    Guibert, Sylvain; Forné, Thierry; Weber, Michael

    2009-10-01

    DNA methylation occurs on cytosines, is catalyzed by DNA methyltransferases (DNMTs), and is present at high levels in all vertebrates. DNA methylation plays essential roles in maintaining genome integrity, but its implication in orchestrating gene-expression patterns remained a matter of debate for a long time. Recent efforts to map DNA methylation at the genome level helped to get a better picture of the distribution of this mark and revealed that DNA methylation is more dynamic between cell types than previously anticipated. In particular, these datasets showed that DNA methylation is targeted to important developmental genes and might act as a barrier to prevent accidental cellular reprogramming. In this review, we will discuss the distribution and function of DNA methylation in mammalian genomes, with particular emphasis on the waves of global DNA methylation reprogramming occurring in early embryos and primordial germ cells. PMID:22122638

  11. DNA Methylation Analysis: Choosing the Right Method

    PubMed Central

    Kurdyukov, Sergey; Bullock, Martyn

    2016-01-01

    In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful. PMID:26751487

  12. DNA methylation in spermatogenesis and male infertility

    PubMed Central

    Cui, Xiangrong; Jing, Xuan; Wu, Xueqing; Yan, Meiqin; Li, Qiang; Shen, Yan; Wang, Zhenqiang

    2016-01-01

    Infertility is a significant problem for human reproduction, with males and females equally affected. However, the molecular mechanisms underlying male infertility remain unclear. Spermatogenesis is a highly complex process involving mitotic cell division, meiosis cell division and spermiogenesis; during this period, unique and extensive chromatin and epigenetic modifications occur to bring about specific epigenetic profiles in spermatozoa. It has recently been suggested that the dysregulation of epigenetic modifications, in particular the methylation of sperm genomic DNA, may serve an important role in the development of numerous diseases. The present study is a comprehensive review on the topic of male infertility, aiming to elucidate the association between sperm genomic DNA methylation and poor semen quality in male infertility. In addition, the current status of the genetic and epigenetic determinants of spermatogenesis in humans is discussed. PMID:27698683

  13. DNA methylation in spermatogenesis and male infertility

    PubMed Central

    Cui, Xiangrong; Jing, Xuan; Wu, Xueqing; Yan, Meiqin; Li, Qiang; Shen, Yan; Wang, Zhenqiang

    2016-01-01

    Infertility is a significant problem for human reproduction, with males and females equally affected. However, the molecular mechanisms underlying male infertility remain unclear. Spermatogenesis is a highly complex process involving mitotic cell division, meiosis cell division and spermiogenesis; during this period, unique and extensive chromatin and epigenetic modifications occur to bring about specific epigenetic profiles in spermatozoa. It has recently been suggested that the dysregulation of epigenetic modifications, in particular the methylation of sperm genomic DNA, may serve an important role in the development of numerous diseases. The present study is a comprehensive review on the topic of male infertility, aiming to elucidate the association between sperm genomic DNA methylation and poor semen quality in male infertility. In addition, the current status of the genetic and epigenetic determinants of spermatogenesis in humans is discussed.

  14. Dual Functions of the RFTS Domain of Dnmt1 in Replication-Coupled DNA Methylation and in Protection of the Genome from Aberrant Methylation

    PubMed Central

    Kimura, Hironobu; Sharif, Jafar; Muto, Masahiro; Koseki, Haruhiko; Takahashi, Saori; Suetake, Isao; Tajima, Shoji

    2015-01-01

    In mammals, DNA methylation plays important roles in embryogenesis and terminal differentiation via regulation of the transcription-competent chromatin state. The methylation patterns are propagated to the next generation during replication by maintenance DNA methyltransferase, Dnmt1, in co-operation with Uhrf1. In the N-terminal regulatory region, Dnmt1 contains proliferating cell nuclear antigen (PCNA)-binding and replication foci targeting sequence (RFTS) domains, which are thought to contribute to maintenance methylation during replication. To determine the contributions of the N-terminal regulatory domains to the DNA methylation during replication, Dnmt1 lacking the RFTS and/or PCNA-binding domains was ectopically expressed in embryonic stem cells, and then the effects were analyzed. Deletion of both the PCNA-binding and RFTS domains did not significantly affect the global DNA methylation level. However, replication-dependent DNA methylation of the differentially methylated regions of three imprinted genes, Kcnq1ot1/Lit1, Peg3, and Rasgrf1, was impaired in cells expressing the Dnmt1 with not the PCNA-binding domain alone but both the PCNA-binding and RFTS domains deleted. Even in the absence of Uhrf1, which is a prerequisite factor for maintenance DNA methylation, Dnmt1 with both the domains deleted apparently maintained the global DNA methylation level, whilst the wild type and the forms containing the RFTS domain could not perform global DNA methylation under the conditions used. This apparent maintenance of the global DNA methylation level by the Dnmt1 lacking the RFTS domain was dependent on its own DNA methylation activity as well as the presence of de novo-type DNA methyltransferases. We concluded that the RFTS domain, not the PCNA-binding domain, is solely responsible for the replication-coupled DNA methylation. Furthermore, the RFTS domain acts as a safety lock by protecting the genome from replication-independent DNA methylation. PMID:26383849

  15. Dual Functions of the RFTS Domain of Dnmt1 in Replication-Coupled DNA Methylation and in Protection of the Genome from Aberrant Methylation.

    PubMed

    Garvilles, Ronald Garingalao; Hasegawa, Takashi; Kimura, Hironobu; Sharif, Jafar; Muto, Masahiro; Koseki, Haruhiko; Takahashi, Saori; Suetake, Isao; Tajima, Shoji

    2015-01-01

    In mammals, DNA methylation plays important roles in embryogenesis and terminal differentiation via regulation of the transcription-competent chromatin state. The methylation patterns are propagated to the next generation during replication by maintenance DNA methyltransferase, Dnmt1, in co-operation with Uhrf1. In the N-terminal regulatory region, Dnmt1 contains proliferating cell nuclear antigen (PCNA)-binding and replication foci targeting sequence (RFTS) domains, which are thought to contribute to maintenance methylation during replication. To determine the contributions of the N-terminal regulatory domains to the DNA methylation during replication, Dnmt1 lacking the RFTS and/or PCNA-binding domains was ectopically expressed in embryonic stem cells, and then the effects were analyzed. Deletion of both the PCNA-binding and RFTS domains did not significantly affect the global DNA methylation level. However, replication-dependent DNA methylation of the differentially methylated regions of three imprinted genes, Kcnq1ot1/Lit1, Peg3, and Rasgrf1, was impaired in cells expressing the Dnmt1 with not the PCNA-binding domain alone but both the PCNA-binding and RFTS domains deleted. Even in the absence of Uhrf1, which is a prerequisite factor for maintenance DNA methylation, Dnmt1 with both the domains deleted apparently maintained the global DNA methylation level, whilst the wild type and the forms containing the RFTS domain could not perform global DNA methylation under the conditions used. This apparent maintenance of the global DNA methylation level by the Dnmt1 lacking the RFTS domain was dependent on its own DNA methylation activity as well as the presence of de novo-type DNA methyltransferases. We concluded that the RFTS domain, not the PCNA-binding domain, is solely responsible for the replication-coupled DNA methylation. Furthermore, the RFTS domain acts as a safety lock by protecting the genome from replication-independent DNA methylation.

  16. Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment.

    PubMed

    Doherty, Rachael; Couldrey, Christine

    2014-01-01

    Recent advances made in "omics" technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS) and more recently whole genome bisulfite sequencing (WGBS) have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed. PMID:24860595

  17. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

    PubMed Central

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

    2016-01-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  18. Dissolved humic substances initiate DNA-methylation in cladocerans.

    PubMed

    Menzel, Stefanie; Bouchnak, Rihab; Menzel, Ralph; Steinberg, Christian E W

    2011-10-01

    DNA-methylation is one pathway of epigenetic programming of gene expression and can be responsive to environmental challenges such as methylating agents in the food. Here we report on the DNA-methylation in the cladocerans Daphnia magna and Moina macrocopa exposed to humic substances, ubiquitous biogeochemicals. The methylation of DNA can alter the stress response, presumably including exposure to synthetic xenobiotic chemicals. PMID:21963594

  19. Evolution of DNA Methylation Patterns in the Brassicaceae is Driven by Differences in Genome Organization

    PubMed Central

    Hagmann, Jörg; Becker, Claude; Weigel, Detlef

    2014-01-01

    DNA methylation is an ancient molecular modification found in most eukaryotes. In plants, DNA methylation is not only critical for transcriptionally silencing transposons, but can also affect phenotype by altering expression of protein coding genes. The extent of its contribution to phenotypic diversity over evolutionary time is, however, unclear, because of limited stability of epialleles that are not linked to DNA mutations. To dissect the relative contribution of DNA methylation to transposon surveillance and host gene regulation, we leveraged information from three species in the Brassicaceae that vary in genome architecture, Capsella rubella, Arabidopsis lyrata, and Arabidopsis thaliana. We found that the lineage-specific expansion and contraction of transposon and repeat sequences is the main driver of interspecific differences in DNA methylation. The most heavily methylated portions of the genome are thus not conserved at the sequence level. Outside of repeat-associated methylation, there is a surprising degree of conservation in methylation at single nucleotides located in gene bodies. Finally, dynamic DNA methylation is affected more by tissue type than by environmental differences in all species, but these responses are not conserved. The majority of DNA methylation variation between species resides in hypervariable genomic regions, and thus, in the context of macroevolution, is of limited phenotypic consequence. PMID:25393550

  20. Genome-Wide DNA Methylation Profiles Indicate CD8+ T Cell Hypermethylation in Multiple Sclerosis

    PubMed Central

    Bos, Steffan D.; Page, Christian M.; Andreassen, Bettina K.; Elboudwarej, Emon; Gustavsen, Marte W.; Briggs, Farren; Quach, Hong; Leikfoss, Ingvild S.; Bjølgerud, Anja; Berge, Tone; Harbo, Hanne F.; Barcellos, Lisa F.

    2015-01-01

    Objective Determine whether MS-specific DNA methylation profiles can be identified in whole blood or purified immune cells from untreated MS patients. Methods Whole blood, CD4+ and CD8+ T cell DNA from 16 female, treatment naïve MS patients and 14 matched controls was profiled using the HumanMethylation450K BeadChip. Genotype data were used to assess genetic homogeneity of our sample and to exclude potential SNP-induced DNA methylation measurement errors. Results As expected, significant differences between CD4+ T cells, CD8+ T cells and whole blood DNA methylation profiles were observed, regardless of disease status. Strong evidence for hypermethylation of CD8+ T cell, but not CD4+ T cell or whole blood DNA in MS patients compared to controls was observed. Genome-wide significant individual CpG-site DNA methylation differences were not identified. Furthermore, significant differences in gene DNA methylation of 148 established MS-associated risk genes were not observed. Conclusion While genome-wide significant DNA methylation differences were not detected for individual CpG-sites, strong evidence for DNA hypermethylation of CD8+ T cells for MS patients was observed, indicating a role for DNA methylation in MS. Further, our results suggest that large DNA methylation differences for CpG-sites tested here do not contribute to MS susceptibility. In particular, large DNA methylation differences for CpG-sites within 148 established MS candidate genes tested in our study cannot explain missing heritability. Larger studies of homogenous MS patients and matched controls are warranted to further elucidate the impact of CD8+ T cell and more subtle DNA methylation changes in MS development and pathogenesis. PMID:25734800

  1. Global methylation profiles in DNA from different blood cell types

    PubMed Central

    Wu, Hui-Chen; Delgado-Cruzata, Lissette; Flom, Julie D; Kappil, Maya; Ferris, Jennifer S; Liao, Yuyan; Santella, Regina M

    2011-01-01

    DNA methylation measured in white blood cell DNA is increasingly being used in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran) and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources [WBC, Gran, mononuclear (MN) and lymphoblastoid cell lines (LCL)], we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA) and [3H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LCL, MN or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [3H]-methyl acceptance, LINE1 and Alu assays. Methylation in MN was correlated with methylation in WBC for the [3H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant correlations ranging from 0.3–0.7 for each cell type with one exception (Sat2 and Alu in MN). Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39–0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18–0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC. PMID:20890131

  2. CpG methylation patterns of human mitochondrial DNA

    PubMed Central

    Liu, Baojing; Du, Qingqing; Chen, Lu; Fu, Guangping; Li, Shujin; Fu, Lihong; Zhang, Xiaojing; Ma, Chunling; Bin, Cong

    2016-01-01

    The epigenetic modification of mitochondrial DNA (mtDNA) is still in controversy. To clarify this point, we applied the gold standard method for DNA methylation, bisulfite pyrosequencing, to examine human mtDNA methylation status. Before bisulfite conversion, BamHI was used to digest DNA to open the loop of mtDNA. The results demonstrated that the linear mtDNA had significantly higher bisulfite conversion efficiency compared with circular mtDNA. Furthermore, the methylation values obtained from linear mtDNA were significantly lower than that of circular mtDNA, which was verified by SEQUENOM MassARRAY. The above impacts of circular structure were also observed in lung DNA samples but not in saliva DNA samples. Mitochondrial genome methylation of blood samples and saliva samples from 14 unrelated individuals was detected. The detected regions covered 83 CpG sites across mtDNA including D-loop, 12 S rRNA, 16 S rRNA, ND1, COXI, ND3, ND4, ND5, CYTB. We found that the average methylation levels of nine regions were all less than 2% for both sample types. In conclusion, our findings firstly show that the circular structure of mtDNA affects bisulfite conversion efficiency, which leads to overestimation of mtDNA methylation values. CpG methylation in human mtDNA is a very rare event at most DNA regions. PMID:26996456

  3. Global and gene specific DNA methylation changes during zebrafish development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  4. SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®.

    PubMed

    Busato, Florence; Tost, Jörg

    2015-01-01

    The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing(®). Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

  5. Dynamic DNA methylation regulates neuronal intrinsic membrane excitability.

    PubMed

    Meadows, Jarrod P; Guzman-Karlsson, Mikael C; Phillips, Scott; Brown, Jordan A; Strange, Sarah K; Sweatt, J David; Hablitz, John J

    2016-01-01

    Epigenetic modifications, such as DNA cytosine methylation, contribute to the mechanisms underlying learning and memory by coordinating adaptive gene expression and neuronal plasticity. Transcription-dependent plasticity regulated by DNA methylation includes synaptic plasticity and homeostatic synaptic scaling. Memory-related plasticity also includes alterations in intrinsic membrane excitability mediated by changes in the abundance or activity of ion channels in the plasma membrane, which sets the threshold for action potential generation. We found that prolonged inhibition of DNA methyltransferase (DNMT) activity increased intrinsic membrane excitability of cultured cortical pyramidal neurons. Knockdown of the cytosine demethylase TET1 or inhibition of RNA polymerase blocked the increased membrane excitability caused by DNMT inhibition, suggesting that this effect was mediated by subsequent cytosine demethylation and de novo transcription. Prolonged DNMT inhibition blunted the medium component of the after-hyperpolarization potential, an effect that would increase neuronal excitability, and was associated with reduced expression of the genes encoding small-conductance Ca(2+)-activated K(+) (SK) channels. Furthermore, the specific SK channel blocker apamin increased neuronal excitability but was ineffective after DNMT inhibition. Our results suggested that DNMT inhibition enables transcriptional changes that culminate in decreased expression of SK channel-encoding genes and decreased activity of SK channels, thus providing a mechanism for the regulation of neuronal intrinsic membrane excitability by dynamic DNA cytosine methylation. This study has implications for human neurological and psychiatric diseases associated with dysregulated intrinsic excitability. PMID:27555660

  6. Dissecting the role of aberrant DNA methylation in human leukemia

    PubMed Central

    Amabile, Giovanni; Di Ruscio, Annalisa; Müller, Fabian; Welner, Robert S; Yang, Henry; Ebralidze, Alexander K; Zhang, Hong; Levantini, Elena; Qi, Lihua; Martinelli, Giovanni; Brummelkamp, Thijn; Le Beau, Michelle M; Figueroa, Maria E; Bock, Christoph; Tenen, Daniel G

    2015-01-01

    Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL fusion oncogene. However, many molecular mechanisms of the disease progression still remain poorly understood. A growing body of evidence suggests that epigenetic abnormalities are involved in tyrosine kinase resistance in CML, leading to leukemic clone escape and disease propagation. Here we show that, by applying cellular reprogramming to primary CML cells, aberrant DNA methylation contributes to the disease evolution. Importantly, using a BCR-ABL inducible murine model, we demonstrate that a single oncogenic lesion triggers DNA methylation changes which in turn act as a precipitating event in leukemia progression. PMID:25997600

  7. Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes.

    PubMed

    Huff, Jason T; Zilberman, Daniel

    2014-03-13

    Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

  8. DNA Methylation: Insights into Human Evolution

    PubMed Central

    Sharp, Andrew J.; Marques-Bonet, Tomas

    2015-01-01

    A fundamental initiative for evolutionary biologists is to understand the molecular basis underlying phenotypic diversity. A long-standing hypothesis states that species-specific traits may be explained by differences in gene regulation rather than differences at the protein level. Over the past few years, evolutionary studies have shifted from mere sequence comparisons to integrative analyses in which gene regulation is key to understanding species evolution. DNA methylation is an important epigenetic modification involved in the regulation of numerous biological processes. Nevertheless, the evolution of the human methylome and the processes driving such changes are poorly understood. Here, we review the close interplay between Cytosine-phosphate-Guanine (CpG) methylation and the underlying genome sequence, as well as its evolutionary impact. We also summarize the latest advances in the field, revisiting the main literature on human and nonhuman primates. We hope to encourage the scientific community to address the many challenges posed by the field of comparative epigenomics. PMID:26658498

  9. DNA methylation in complex disease: applications in nursing research, practice, and policy.

    PubMed

    Wright, Michelle L; Ralph, Jody L; Ohm, Joyce E; Anderson, Cindy M

    2013-01-01

    DNA methylation is an epigenomic modification that is essential to normal human development and biological processes. DNA methylation patterns are heritable and dynamic throughout the life span. Environmental exposures can alter DNA methylation patterns, contributing to the development of complex disease. Identification and modulation of environmental factors influencing disease susceptibility through alterations in DNA methylation are amenable to nursing intervention and form the basis for individualized patient care. Here we describe the evidence supporting the translation of DNA methylation analyses as a tool for screening, diagnosis, and treatment of complex disease in nursing research and practice. The ethical, legal, social, and economic considerations of advances in genomics are considered as a model for epigenomic policy. We conclude that contemporary and informed nurse scientists and clinicians are uniquely poised to apply innovations in epigenomic research to clinical populations and develop appropriate policies that guide equitable and ethical use of new strategies to improve patient care. PMID:23849553

  10. Epigenetic and genetic influences on DNA methylation variation in maize populations.

    PubMed

    Eichten, Steven R; Briskine, Roman; Song, Jawon; Li, Qing; Swanson-Wagner, Ruth; Hermanson, Peter J; Waters, Amanda J; Starr, Evan; West, Patrick T; Tiffin, Peter; Myers, Chad L; Vaughn, Matthew W; Springer, Nathan M

    2013-08-01

    DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

  11. Infant sex-specific placental cadmium and DNA methylation associations

    SciTech Connect

    Mohanty, April F.; Farin, Fred M.; Bammler, Theo K.; MacDonald, James W.; Afsharinejad, Zahra; Burbacher, Thomas M.; Siscovick, David S.; and others

    2015-04-15

    Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

  12. 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

  13. Genome-Wide DNA Methylation Patterns and Transcription Analysis in Sheep Muscle

    PubMed Central

    Couldrey, Christine; Brauning, Rudiger; Bracegirdle, Jeremy; Maclean, Paul; Henderson, Harold V.; McEwan, John C.

    2014-01-01

    DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species. PMID:25010796

  14. One-pot approach for examining the DNA methylation patterns using an engineered methyl-probe.

    PubMed

    Kim, Seong-Eun; Chang, Matthew; Yuan, Chongli

    2014-08-15

    Aberrant DNA methylation is a common observation in various types of human cancers, i.e., breast and lung cancers. Nevertheless, the current DNA methylation detection approaches require bisulfite treatments and are laborious or costly to perform. To address these challenges, we developed a methyl-probe based on the MBD1 protein. Combined with fluorescence correlation spectroscopy, our probe can sensitively detect the existence of DNA methylation at concentrations above 20nM in a one-pot assay. The probe can quantify the total amount of methylated CG dinucleotides above ~20nM, independent of DNA sequence contexts, concentrations (20-1900nM) and methylation levels (5-100%). Our detection platform offers a simple and cheap alternative DNA methylation detection approach.

  15. De novo methylation, long-term promoter silencing, methylation patterns in the human genome, and consequences of foreign DNA insertion.

    PubMed

    Doerfler, W

    2006-01-01

    This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past 30 years. The text does not attempt to give a complete and meticulous account of the work accomplished in many other laboratories; in that sense it is not a review of the field in a conventional sense. Since the author is also one of the editors of this series of Current Topics in Immunology and Microbiology on DNA methylation, to which contributions by many of our colleagues in this field have been invited, the author's conscience is alleviated that he has not cited many of the relevant and excellent reports by others. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proved their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (1) The de novo methylation of integrated foreign genomes; (2) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (3) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (4) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (5) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; which role has food-ingested DNA played in the elaboration of this mechanism? The interest in problems related to DNA methylation has spread-like the mechanism itself-into many neighboring fields. The nature of the transcriptional programs orchestrating embryonal and fetal development, chromatin structure, genetic imprinting, genetic disease, X

  16. Variable DNA methylation changes during differentiation of human melanoma cells.

    PubMed

    Steigerwald, S D; Pfeifer, G P

    1988-09-01

    The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

  17. DNA methylation epigenotypes in breast cancer molecular subtypes

    PubMed Central

    2010-01-01

    Introduction Identification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes. Methods By using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis. Results The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. Conclusions Our results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer. PMID:20920229

  18. Function and evolution of DNA methylation in Nasonia vitripennis.

    PubMed

    Wang, Xu; Wheeler, David; Avery, Amanda; Rago, Alfredo; Choi, Jeong-Hyeon; Colbourne, John K; Clark, Andrew G; Werren, John H

    2013-01-01

    The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5' regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5' and 3' UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We

  19. Function and Evolution of DNA Methylation in Nasonia vitripennis

    PubMed Central

    Wang, Xu; Wheeler, David; Avery, Amanda; Rago, Alfredo; Choi, Jeong-Hyeon; Colbourne, John K.; Clark, Andrew G.; Werren, John H.

    2013-01-01

    The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression

  20. Competition between DNA methylation and transcription factors determines binding of NRF1.

    PubMed

    Domcke, Silvia; Bardet, Anaïs Flore; Adrian Ginno, Paul; Hartl, Dominik; Burger, Lukas; Schübeler, Dirk

    2015-12-24

    Eukaryotic transcription factors (TFs) are key determinants of gene activity, yet they bind only a fraction of their corresponding DNA sequence motifs in any given cell type. Chromatin has the potential to restrict accessibility of binding sites; however, in which context chromatin states are instructive for TF binding remains mainly unknown. To explore the contribution of DNA methylation to constrained TF binding, we mapped DNase-I-hypersensitive sites in murine stem cells in the presence and absence of DNA methylation. Methylation-restricted sites are enriched for TF motifs containing CpGs, especially for those of NRF1. In fact, the TF NRF1 occupies several thousand additional sites in the unmethylated genome, resulting in increased transcription. Restoring de novo methyltransferase activity initiates remethylation at these sites and outcompetes NRF1 binding. This suggests that binding of DNA-methylation-sensitive TFs relies on additional determinants to induce local hypomethylation. In support of this model, removal of neighbouring motifs in cis or of a TF in trans causes local hypermethylation and subsequent loss of NRF1 binding. This competition between DNA methylation and TFs in vivo reveals a case of cooperativity between TFs that acts indirectly via DNA methylation. Methylation removal by methylation-insensitive factors enables occupancy of methylation-sensitive factors, a principle that rationalizes hypomethylation of regulatory regions. PMID:26675734

  1. Drugging the methylome: DNA methylation and memory.

    PubMed

    Kennedy, Andrew J; Sweatt, J David

    2016-01-01

    Over the past decade, since epigenetic mechanisms were first implicated in memory formation and synaptic plasticity, dynamic DNA methylation reactions have been identified as integral to long-term memory formation, maintenance, and recall. This review incorporates various new findings that DNA methylation mechanisms are important regulators of non-Hebbian plasticity mechanisms, suggesting that these epigenetic mechanisms are a fundamental link between synaptic plasticity and metaplasticity. Because the field of neuroepigenetics is so young and the biochemical tools necessary to probe gene-specific questions are just now being developed and used, this review also speculates about the direction and potential of therapeutics that target epigenetic mechanisms in the central nervous system and the unique pharmacokinetic and pharmacodynamic properties that epigenetic therapies may possess. Mapping the dynamics of the epigenome in response to experiential learning, even a single epigenetic mark in isolation, remains a significant technical and bioinformatic hurdle facing the field, but will be necessary to identify changes to the methylome that govern memory-associated gene expression and effectively drug the epigenome. PMID:26915423

  2. The Influence of DNA Methylation on Bone Cells

    PubMed Central

    Reppe, Sjur; Datta, Harish; Gautvik, Kaare M.

    2015-01-01

    DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain. PMID:27019613

  3. The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation.

    PubMed

    Zakrzewski, Falk; Schubert, Veit; Viehoever, Prisca; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Weisshaar, Bernd; Schmidt, Thomas

    2014-06-01

    Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next-generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome-wide fluorescent in situ hybridization complemented with immunostaining and super-resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44-52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.

  4. Roles of Distal and Genic Methylation in the Development of Prostate Tumorigenesis Revealed by Genome-wide DNA Methylation Analysis

    PubMed Central

    Wang, Yao; Jadhav, Rohit Ramakant; Liu, Joseph; Wilson, Desiree; Chen, Yidong; Thompson, Ian M.; Troyer, Dean A.; Hernandez, Javier; Shi, Huidong; Leach, Robin J.; Huang, Tim H.-M.; Jin, Victor X.

    2016-01-01

    Aberrant DNA methylation at promoters is often linked to tumorigenesis. But many aspects of DNA methylation remain unexplored, including the individual roles of distal and gene body methylation, as well as their collaborative roles with promoter methylation. Here we performed a MBD-seq analysis on prostate specimens classified into low, high, and very high risk group based on Gleason score and TNM stages. We identified gene sets with differential methylation regions (DMRs) in Distal, TSS, gene body and TES. To understand the collaborative roles, TSS was compared with the other three DMRs, resulted in 12 groups of genes with collaborative differential methylation patterns (CDMPs). We found several groups of genes that show opposite methylation patterns in Distal and Genic regions compared to TSS region, and in general they are differentially expressed genes (DEGs) in tumors in TCGA RNA-seq data. IPA (Ingenuity Pathway Analysis) reveals AR/TP53 signaling network to be a major signaling pathway, and survival analysis indicates genes subsets significantly associated with prostate cancer recurrence. Our results suggest that DNA methylation in Distal and Genic regions also plays critical roles in contributing to prostate tumorigenesis, and may act either positively or negatively with TSSs to alter gene regulation in tumors. PMID:26924343

  5. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

    PubMed Central

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  6. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

    PubMed

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  7. Genome-wide analysis of DNA methylation in hepatoblastoma tissues

    PubMed Central

    Cui, Ximao; Liu, Baihui; Zheng, Shan; Dong, Kuiran; Dong, Rui

    2016-01-01

    DNA methylation has a crucial role in cancer biology. In the present study, a genome-wide analysis of DNA methylation in hepatoblastoma (HB) tissues was performed to verify differential methylation levels between HB and normal tissues. As alpha-fetoprotein (AFP) has a critical role in HB, AFP methylation levels were also detected using pyrosequencing. Normal and HB liver tissue samples (frozen tissue) were obtained from patients with HB. Genome-wide analysis of DNA methylation in these tissues was performed using an Infinium HumanMethylation450 BeadChip, and the results were confirmed with reverse transcription-quantitative polymerase chain reaction. The Infinium HumanMethylation450 BeadChip demonstrated distinctively less methylation in HB tissues than in non-tumor tissues. In addition, methylation enrichment was observed in positions near the transcription start site of AFP, which exhibited lower methylation levels in HB tissues than in non-tumor liver tissues. Lastly, a significant negative correlation was observed between AFP messenger RNA expression and DNA methylation percentage, using linear Pearson's R correlation coefficients. The present results demonstrate differential methylation levels between HB and normal tissues, and imply that aberrant methylation of AFP in HB could reflect HB development. Expansion of these findings could provide useful insight into HB biology. PMID:27446465

  8. Classification of Epstein-Barr virus-positive gastric cancers by definition of DNA methylation epigenotypes.

    PubMed

    Matsusaka, Keisuke; Kaneda, Atsushi; Nagae, Genta; Ushiku, Tetsuo; Kikuchi, Yasuko; Hino, Rumi; Uozaki, Hiroshi; Seto, Yasuyuki; Takada, Kenzo; Aburatani, Hiroyuki; Fukayama, Masashi

    2011-12-01

    Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illumina's Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.

  9. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  10. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-10-22

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.

  11. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

    PubMed Central

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A.; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K.; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  12. [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.

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

    PubMed Central

    Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

    2016-01-01

    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. PMID:27056885

  14. Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos

    PubMed Central

    Zhang, Sheng; Chen, Xin; Wang, Fang; An, Xinglan; Tang, Bo; Zhang, Xueming; Sun, Liguang; Li, Ziyi

    2016-01-01

    DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner. PMID:27456302

  15. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  16. Methionine increases BDNF DNA methylation and improves memory in epilepsy

    PubMed Central

    Parrish, R Ryley; Buckingham, Susan C; Mascia, Katherine L; Johnson, Jarvis J; Matyjasik, Michal M; Lockhart, Roxanne M; Lubin, Farah D

    2015-01-01

    Objective Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus. Methods Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model. Results We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade. Interpretation Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE. PMID:25909085

  17. DNA methylation: the future of crime scene investigation?

    PubMed

    Gršković, Branka; Zrnec, Dario; Vicković, Sanja; Popović, Maja; Mršić, Gordan

    2013-07-01

    Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework. PMID:23649761

  18. DNA Methylation in Basal Metazoans: Insights from Ctenophores.

    PubMed

    Dabe, Emily C; Sanford, Rachel S; Kohn, Andrea B; Bobkova, Yelena; Moroz, Leonid L

    2015-12-01

    Epigenetic modifications control gene expression without altering the primary DNA sequence. However, little is known about DNA methylation in invertebrates and its evolution. Here, we characterize two types of genomic DNA methylation in ctenophores, 5-methyl cytosine (5-mC) and the unconventional form of methylation 6-methyl adenine (6-mA). Using both bisulfite sequencing and an ELISA-based colorimetric assay, we experimentally confirmed the presence of 5-mC DNA methylation in ctenophores. In contrast to other invertebrates studied, Mnemiopsis leidyi has lower levels of genome-wide 5-mC methylation, but higher levels of 5-mC methylation in promoters when compared with gene bodies. Phylogenetic analysis showed that ctenophores have distinct forms of DNA methyltransferase 1 (DNMT1); the zf-CXXC domain type, which localized DNMT1 to CpG sites, and is a metazoan specific innovation. We also show that ctenophores encode the full repertoire of putative enzymes for 6-mA DNA methylation, and these genes are expressed in the aboral organ of Mnemiopsis. Using an ELISA-based colorimetric assay, we experimentally confirmed the presence of 6-mA methylation in the genomes of three different species of ctenophores, M. leidyi, Beroe abyssicola, and Pleurobrachia bachei. The functional role of this novel epigenomic mark is currently unknown. In summary, despite their compact genomes, there is a wide variety of epigenomic mechanisms employed by basal metazoans that provide novel insights into the evolutionary origins of biological novelties.

  19. Choline nutrition programs brain development via DNA and histone methylation.

    PubMed

    Blusztajn, Jan Krzysztof; Mellott, Tiffany J

    2012-06-01

    Choline is an essential nutrient for humans. Metabolically choline is used for the synthesis of membrane phospholipids (e.g. phosphatidylcholine), as a precursor of the neurotransmitter acetylcholine, and, following oxidation to betaine, choline functions as a methyl group donor in a pathway that produces S-adenosylmethionine. As a methyl donor choline influences DNA and histone methylation--two central epigenomic processes that regulate gene expression. Because the fetus and neonate have high demands for choline, its dietary intake during pregnancy and lactation is particularly important for normal development of the offspring. Studies in rodents have shown that high choline intake during gestation improves cognitive function in adulthood and prevents memory decline associated with old age. These behavioral changes are accompanied by electrophysiological, neuroanatomical, and neurochemical changes and by altered patterns of expression of multiple cortical and hippocampal genes including those encoding key proteins that contribute to the biochemical mechanisms of learning and memory. These actions of choline are observed long after the exposure to the nutrient ended (months) and correlate with fetal hepatic and cerebral cortical choline-evoked changes in global- and gene-specific DNA cytosine methylation and with dramatic changes of the methylation pattern of lysine residues 4, 9 and 27 of histone H3. Moreover, gestational choline modulates the expression of DNA (Dnmt1, Dnmt3a) and histone (G9a/Ehmt2/Kmt1c, Suv39h1/Kmt1a) methyltransferases. In addition to the central role of DNA and histone methylation in brain development, these processes are highly dynamic in adult brain, modulate the expression of genes critical for synaptic plasticity, and are involved in mechanisms of learning and memory. A recent study documented that in a cohort of normal elderly people, verbal and visual memory function correlated positively with the amount of dietary choline consumption

  20. High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

    PubMed Central

    Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J.; Resch, Eduard

    2016-01-01

    DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50–80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer. PMID:27749902

  1. [Profiles of DNA methylation in normal and cancer cells].

    PubMed

    Weber, Michaël

    2008-01-01

    In eukaryotes, the epigenetic mark DNA methylation is found exclusively at cytosine residues in the CpG islands of genes, transposons and intergenic DNA. Among functional roles, DNA methylation is essential for mammalian embryonic development, and is classically thought to function by stably silencing promoter activity. However, until recently, understanding of the distribution of cytosine methylation in the whole genome - and hence, identification of its targets - was very limited. High-throughput methodologies, including methylated DNA immunoprecipitation, have recently revealed genome-wide mapping of DNA methylation, and provided new and unexpected data. Clearly DNA methylation is selectively associated with some key promoters- and is not a prerequisite for promoter inactivation, since strong CpG island promoters are mostly unmethylated, even when inactive. Most germline-specific genes are methylated and permanently silenced in somatic cells, suggesting a role of this mark in maintaining somatic cellular identity. These large scale studies will also help understanding the deregulation of DNA methylation associated with cancer, among which unmethylation of germinal cells genes, and recent observtion of large hypomethylated regions in tumoral specimens. The next challenge will be to understand if these methylation changes occur randomly, or more likely are specified by oncogenes or linked to environmental pressure. PMID:18789220

  2. DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans

    PubMed Central

    Kesäniemi, Jenni E.; Heikkinen, Liisa; Knott, K. Emily

    2016-01-01

    Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. PMID:27008314

  3. DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans.

    PubMed

    Kesäniemi, Jenni E; Heikkinen, Liisa; Knott, K Emily

    2016-01-01

    Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. PMID:27008314

  4. Structural basis for Klf4 recognition of methylated DNA.

    PubMed

    Liu, Yiwei; Olanrewaju, Yusuf Olatunde; Zheng, Yu; Hashimoto, Hideharu; Blumenthal, Robert M; Zhang, Xing; Cheng, Xiaodong

    2014-04-01

    Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair. PMID:24520114

  5. An atlas of DNA methylation in diverse bovine tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We launched an effort to produce a reference cattle DNA methylation resource to improve animal production. We will employ experimental pipelines built around next generation sequencing technologies to map DNA methylation in cultured cells and primary tissues systems frequently involved in animal pro...

  6. DNA methylation: a permissive mark in memory formation and maintenance.

    PubMed

    Oliveira, Ana M M

    2016-10-01

    DNA methylation was traditionally viewed as a static mechanism required during cell fate determination. This view has been challenged and it is now accepted that DNA methylation is involved in the regulation of genomic responses in mature neurons, particularly in cognitive functions. The evidence for a role of DNA methylation in memory formation and maintenance comes from the increasing number of studies that have assessed the effects of manipulation of DNA methylation modifiers in the ability to form and maintain memories. Moreover, insights from genome-wide analyses of the hippocampal DNA methylation status after neuronal activity show that DNA methylation is dynamically regulated. Despite all the experimental evidence, we are still far from having a clear picture of how DNA methylation regulates long-term adaptations. This review aims on one hand to describe the findings that led to the confirmation of DNA methylation as an important player in memory formation. On the other hand, it tries to integrate these discoveries into the current views of how memories are formed and maintained. PMID:27634149

  7. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  8. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    PubMed

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

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

    PubMed

    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

  10. Effects of DNA Methylation and Chromatin State on Rates of Molecular Evolution in Insects

    PubMed Central

    Glastad, Karl M.; Goodisman, Michael A. D.; Yi, Soojin V.; Hunt, Brendan G.

    2015-01-01

    Epigenetic information is widely appreciated for its role in gene regulation in eukaryotic organisms. However, epigenetic information can also influence genome evolution. Here, we investigate the effects of epigenetic information on gene sequence evolution in two disparate insects: the fly Drosophila melanogaster, which lacks substantial DNA methylation, and the ant Camponotus floridanus, which possesses a functional DNA methylation system. We found that DNA methylation was positively correlated with the synonymous substitution rate in C. floridanus, suggesting a key effect of DNA methylation on patterns of gene evolution. However, our data suggest the link between DNA methylation and elevated rates of synonymous substitution was explained, in large part, by the targeting of DNA methylation to genes with signatures of transcriptionally active chromatin, rather than the mutational effect of DNA methylation itself. This phenomenon may be explained by an elevated mutation rate for genes residing in transcriptionally active chromatin, or by increased structural constraints on genes in inactive chromatin. This result highlights the importance of chromatin structure as the primary epigenetic driver of genome evolution in insects. Overall, our study demonstrates how different epigenetic systems contribute to variation in the rates of coding sequence evolution. PMID:26637432

  11. Effects of DNA Methylation and Chromatin State on Rates of Molecular Evolution in Insects.

    PubMed

    Glastad, Karl M; Goodisman, Michael A D; Yi, Soojin V; Hunt, Brendan G

    2015-12-04

    Epigenetic information is widely appreciated for its role in gene regulation in eukaryotic organisms. However, epigenetic information can also influence genome evolution. Here, we investigate the effects of epigenetic information on gene sequence evolution in two disparate insects: the fly Drosophila melanogaster, which lacks substantial DNA methylation, and the ant Camponotus floridanus, which possesses a functional DNA methylation system. We found that DNA methylation was positively correlated with the synonymous substitution rate in C. floridanus, suggesting a key effect of DNA methylation on patterns of gene evolution. However, our data suggest the link between DNA methylation and elevated rates of synonymous substitution was explained, in large part, by the targeting of DNA methylation to genes with signatures of transcriptionally active chromatin, rather than the mutational effect of DNA methylation itself. This phenomenon may be explained by an elevated mutation rate for genes residing in transcriptionally active chromatin, or by increased structural constraints on genes in inactive chromatin. This result highlights the importance of chromatin structure as the primary epigenetic driver of genome evolution in insects. Overall, our study demonstrates how different epigenetic systems contribute to variation in the rates of coding sequence evolution.

  12. 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

  13. Base-resolution DNA methylation landscape of zebrafish brain and liver.

    PubMed

    Chatterjee, Aniruddha; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

    2014-12-01

    Zebrafish (Danio rerio) is a vertebrate model organism that is widely used for studying a plethora of biological questions, including developmental processes, effects of external cues on phenotype, and human disease modeling. DNA methylation is an important epigenetic mechanism that contributes to gene regulation, and is prevalent in all vertebrates. Reduced representation bisulfite sequencing (RRBS) is a cost-effective technique to generate genome-wide DNA methylation maps and has been used in mammalian genomes (e.g., human, mouse and rat) but not in zebrafish. High-resolution DNA methylation data in zebrafish are limited: increased availability of such data will enable us to model and better understand the roles, causes and consequences of changes in DNA methylation. Here we present five high-resolution DNA methylation maps for wild-type zebrafish brain (two pooled male and two pooled female methylomes) and liver. These data were generated using the RRBS technique (includes 1.43 million CpG sites of zebrafish genome) on the Illumina HiSeq platform. Alignment to the reference genome was performed using the Zv9 genome assembly. To our knowledge, these datasets are the only RRBS datasets and base-resolution DNA methylation data available at this time for zebrafish brain and liver. These datasets could serve as a resource for future studies to document the functional role of DNA methylation in zebrafish. In addition, these datasets could be used as controls while performing analysis on treated samples.

  14. DNA methylation regulates neurophysiological spatial representation in memory formation

    PubMed Central

    Roth, Eric D.; Roth, Tania L.; Money, Kelli M.; SenGupta, Sonda; Eason, Dawn E.; Sweatt, J. David

    2015-01-01

    Epigenetic mechanisms including altered DNA methylation are critical for altered gene transcription subserving synaptic plasticity and the retention of learned behavior. Here we tested the idea that one role for activity-dependent altered DNA methylation is stabilization of cognition-associated hippocampal place cell firing in response to novel place learning. We observed that a behavioral protocol (spatial exploration of a novel environment) known to induce hippocampal place cell remapping resulted in alterations of hippocampal Bdnf DNA methylation. Further studies using neurophysiological in vivo single unit recordings revealed that pharmacological manipulations of DNA methylation decreased long-term but not short-term place field stability. Together our data highlight a role for DNA methylation in regulating neurophysiological spatial representation and memory formation. PMID:25960947

  15. DNA methylation dynamics in muscle development and disease

    PubMed Central

    Carrió, Elvira; Suelves, Mònica

    2015-01-01

    DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis. PMID:25798107

  16. DNA methylation-specific multiplex assays for body fluid identification.

    PubMed

    An, Ja Hyun; Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2013-01-01

    Recent advances in whole-genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to cell or tissue type. Therefore, body fluid-specific differential DNA methylation is a promising indicator for body fluid identification. However, DNA methylation patterns are susceptible to change in response to environmental factors and aging. Therefore, we investigated age-related methylation changes in semen-specific tDMRs using body fluids from young and elderly men. After confirming the stability of the body fluid-specific DNA methylation profile over time, two different multiplex PCR systems were constructed using methylation-sensitive restriction enzyme PCR and methylation SNaPshot, in order to analyze the methylation status of specific CpG sites from the USP49, DACT1, PRMT2, and PFN3 tDMRs. Both multiplex systems could successfully identify semen with spermatozoa and could differentiate menstrual blood and vaginal fluids from blood and saliva. Although including more markers for body fluid identification might be necessary, this study adds to the support that body fluid identification by DNA methylation profiles could be a valuable tool for forensic analysis of body fluids. PMID:22653424

  17. DNA methylation-specific multiplex assays for body fluid identification.

    PubMed

    An, Ja Hyun; Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2013-01-01

    Recent advances in whole-genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to cell or tissue type. Therefore, body fluid-specific differential DNA methylation is a promising indicator for body fluid identification. However, DNA methylation patterns are susceptible to change in response to environmental factors and aging. Therefore, we investigated age-related methylation changes in semen-specific tDMRs using body fluids from young and elderly men. After confirming the stability of the body fluid-specific DNA methylation profile over time, two different multiplex PCR systems were constructed using methylation-sensitive restriction enzyme PCR and methylation SNaPshot, in order to analyze the methylation status of specific CpG sites from the USP49, DACT1, PRMT2, and PFN3 tDMRs. Both multiplex systems could successfully identify semen with spermatozoa and could differentiate menstrual blood and vaginal fluids from blood and saliva. Although including more markers for body fluid identification might be necessary, this study adds to the support that body fluid identification by DNA methylation profiles could be a valuable tool for forensic analysis of body fluids.

  18. Repetitive genomic elements and overall DNA methylation changes in acute myeloid and childhood B-cell lymphoblastic leukemia patients.

    PubMed

    Bujko, Mateusz; Musialik, Ewa; Olbromski, Rafał; Przestrzelska, Marta; Libura, Marta; Pastwińska, Anna; Juszczyński, Przemysław; Zwierzchowski, Lech; Baranowski, Paweł; Siedlecki, Janusz Aleksander

    2014-07-01

    Aberrant epigenetic regulation is a hallmark of neoplastic cells. Increased DNA methylation of individual genes' promoter regions and decreases in overall DNA methylation level are both generally observed in cancer. In solid tumors, this global DNA hypomethylation is related to reduced methylation of repeated DNA elements (REs) and contributes to genome instability. The aim of the present study was to assess methylation level of LINE-1 and ALU REs and total 5-methylcytosine (5metC) content in adult acute myeloid leukemia (AML) (n = 58), childhood B-cell acute lymphoblastic leukemia (ALL) (n = 32), as the most frequent acute leukemias in two age categories and in normal adult bone marrow and children's blood samples. DNA pyrosequencing and ELISA assays were used, respectively. Global DNA hypomethylation was not observed in leukemia patients. Results revealed higher DNA methylation of LINE-1 in AML and ALL samples compared to corresponding normal controls. Elevated methylation of ALU and overall 5metC level were also observed in B-cell ALL patients. Differences of REs and global DNA methylation between AML cytogenetic-risk groups were observed, with the lowest methylation levels in intermediate-risk/cytogenetically normal patients. B-cell ALL is characterized by the highest DNA methylation level compared to AML and controls and overall DNA methylation is correlated with leukocyte count.

  19. Sequences Sufficient for Programming Imprinted Germline DNA Methylation Defined

    PubMed Central

    Park, Yoon Jung; Herman, Herry; Gao, Ying; Lindroth, Anders M.; Hu, Benjamin Y.; Murphy, Patrick J.; Putnam, James R.; Soloway, Paul D.

    2012-01-01

    Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice. PMID:22403732

  20. DNA methylation-mediated control of learning and memory.

    PubMed

    Yu, Nam-Kyung; Baek, Sung Hee; Kaang, Bong-Kiun

    2011-01-19

    Animals constantly receive and respond to external or internal stimuli, and these experiences are learned and memorized in their brains. In animals, this is a crucial feature for survival, by making it possible for them to adapt their behavioral patterns to the ever-changing environment. For this learning and memory process, nerve cells in the brain undergo enormous molecular and cellular changes, not only in the input-output-related local subcellular compartments but also in the central nucleus. Interestingly, the DNA methylation pattern, which is normally stable in a terminally differentiated cell and defines the cell type identity, is emerging as an important regulatory mechanism of behavioral plasticity. The elucidation of how this covalent modification of DNA, which is known to be the most stable epigenetic mark, contributes to the complex orchestration of animal behavior is a fascinating new research area. We will overview the current understanding of the mechanism of modifying the methyl code on DNA and its impact on learning and memory.

  1. Uncovering Driver DNA Methylation Events in Nonsmoking Early Stage Lung Adenocarcinoma.

    PubMed

    Zhang, Xindong; Gao, Lin; Liu, Zhi-Ping; Jia, Songwei; Chen, Luonan

    2016-01-01

    As smoking rates decrease, proportionally more cases with lung adenocarcinoma occur in never-smokers, while aberrant DNA methylation has been suggested to contribute to the tumorigenesis of lung adenocarcinoma. It is extremely difficult to distinguish which genes play key roles in tumorigenic processes via DNA methylation-mediated gene silencing from a large number of differentially methylated genes. By integrating gene expression and DNA methylation data, a pipeline combined with the differential network analysis is designed to uncover driver methylation genes and responsive modules, which demonstrate distinctive expressions and network topology in tumors with aberrant DNA methylation. Totally, 135 genes are recognized as candidate driver genes in early stage lung adenocarcinoma and top ranked 30 genes are recognized as driver methylation genes. Functional annotation and the differential network analysis indicate the roles of identified driver genes in tumorigenesis, while literature study reveals significant correlations of the top 30 genes with early stage lung adenocarcinoma in never-smokers. The analysis pipeline can also be employed in identification of driver epigenetic events for other cancers characterized by matched gene expression data and DNA methylation data. PMID:27610367

  2. Uncovering Driver DNA Methylation Events in Nonsmoking Early Stage Lung Adenocarcinoma

    PubMed Central

    Jia, Songwei; Chen, Luonan

    2016-01-01

    As smoking rates decrease, proportionally more cases with lung adenocarcinoma occur in never-smokers, while aberrant DNA methylation has been suggested to contribute to the tumorigenesis of lung adenocarcinoma. It is extremely difficult to distinguish which genes play key roles in tumorigenic processes via DNA methylation-mediated gene silencing from a large number of differentially methylated genes. By integrating gene expression and DNA methylation data, a pipeline combined with the differential network analysis is designed to uncover driver methylation genes and responsive modules, which demonstrate distinctive expressions and network topology in tumors with aberrant DNA methylation. Totally, 135 genes are recognized as candidate driver genes in early stage lung adenocarcinoma and top ranked 30 genes are recognized as driver methylation genes. Functional annotation and the differential network analysis indicate the roles of identified driver genes in tumorigenesis, while literature study reveals significant correlations of the top 30 genes with early stage lung adenocarcinoma in never-smokers. The analysis pipeline can also be employed in identification of driver epigenetic events for other cancers characterized by matched gene expression data and DNA methylation data. PMID:27610367

  3. Inhibition of DNA Methylation Impairs Synaptic Plasticity during an Early Time Window in Rats

    PubMed Central

    Díaz, Paula; Ardiles, Álvaro O.

    2016-01-01

    Although the importance of DNA methylation-dependent gene expression to neuronal plasticity is well established, the dynamics of methylation and demethylation during the induction and expression of synaptic plasticity have not been explored. Here, we combined electrophysiological, pharmacological, molecular, and immunohistochemical approaches to examine the contribution of DNA methylation and the phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) to synaptic plasticity. We found that, at twenty minutes after theta burst stimulation (TBS), the DNA methylation inhibitor 5-aza-2-deoxycytidine (5AZA) impaired hippocampal long-term potentiation (LTP). Surprisingly, after two hours of TBS, when LTP had become a transcription-dependent process, 5AZA treatment had no effect. By comparing these results to those in naive slices, we found that, at two hours after TBS, an intergenic region of the RLN gene was hypomethylated and that the phosphorylation of residue S80 of MeCP2 was decreased, while the phosphorylation of residue S421 was increased. As expected, 5AZA affected only the methylation of the RLN gene and exerted no effect on MeCP2 phosphorylation patterns. In summary, our data suggest that tetanic stimulation induces critical changes in synaptic plasticity that affects both DNA methylation and the phosphorylation of MeCP2. These data also suggest that early alterations in DNA methylation are sufficient to impair the full expression of LTP. PMID:27493805

  4. Uncovering Driver DNA Methylation Events in Nonsmoking Early Stage Lung Adenocarcinoma

    PubMed Central

    Jia, Songwei; Chen, Luonan

    2016-01-01

    As smoking rates decrease, proportionally more cases with lung adenocarcinoma occur in never-smokers, while aberrant DNA methylation has been suggested to contribute to the tumorigenesis of lung adenocarcinoma. It is extremely difficult to distinguish which genes play key roles in tumorigenic processes via DNA methylation-mediated gene silencing from a large number of differentially methylated genes. By integrating gene expression and DNA methylation data, a pipeline combined with the differential network analysis is designed to uncover driver methylation genes and responsive modules, which demonstrate distinctive expressions and network topology in tumors with aberrant DNA methylation. Totally, 135 genes are recognized as candidate driver genes in early stage lung adenocarcinoma and top ranked 30 genes are recognized as driver methylation genes. Functional annotation and the differential network analysis indicate the roles of identified driver genes in tumorigenesis, while literature study reveals significant correlations of the top 30 genes with early stage lung adenocarcinoma in never-smokers. The analysis pipeline can also be employed in identification of driver epigenetic events for other cancers characterized by matched gene expression data and DNA methylation data.

  5. Ras regulation of DNA-methylation and cancer

    SciTech Connect

    Patra, Samir Kumar

    2008-04-01

    Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Ras family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.

  6. DNA Methylation: A Timeline of Methods and Applications

    PubMed Central

    Harrison, Alan; Parle-McDermott, Anne

    2011-01-01

    DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation is of paramount importance to biological health and disease. Recently, the quest to unravel the Human Epigenome commenced, calling for a modernization of previous DNA methylation profiling techniques. Here, we describe the major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome). The earliest techniques were based on the separation of methylated and unmethylated cytosines via chromatography. The following years would see molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context, notably immunoprecipitation via anti-5′methylcytosine and selective digestion with methylation-sensitive restriction endonucleases. With the advent of sodium bisulfite treatment of DNA, a deamination reaction that converts cytosine to uracil only when unmethylated, the epigenetic modification can now be identified in the same manner as a DNA base-pair change. More recently, these three techniques have been applied to more technically advanced systems such as DNA microarrays and next-generation sequencing platforms, bringing us closer to unveiling a complete human epigenetic profile. PMID:22303369

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats

    PubMed Central

    Li, Xiumei; Gao, Xiaoxiao; Zhang, Kaifa; Luo, Lei; Ding, Jianping; Zhang, Yunhai; Li, Yunsheng; Cao, Hongguo; Ling, Yinghui; Zhang, Xiaorong; Liu, Ya; Fang, Fugui

    2016-01-01

    Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3′-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5′-UTRs and increased from 5′-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5′-UTRs and 3′-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset. PMID:27788248

  10. DNA methylation of SPARC and chronic low back pain

    PubMed Central

    2011-01-01

    Background The extracellular matrix protein SPARC (Secreted Protein, Acidic, Rich in Cysteine) has been linked to degeneration of the intervertebral discs and chronic low back pain (LBP). In humans, SPARC protein expression is decreased as a function of age and disc degeneration. In mice, inactivation of the SPARC gene results in the development of accelerated age-dependent disc degeneration concurrent with age-dependent behavioral signs of chronic LBP. DNA methylation is the covalent modification of DNA by addition of methyl moieties to cytosines in DNA. DNA methylation plays an important role in programming of gene expression, including in the dynamic regulation of changes in gene expression in response to aging and environmental signals. We tested the hypothesis that DNA methylation down-regulates SPARC expression in chronic LBP in pre-clinical models and in patients with chronic LBP. Results Our data shows that aging mice develop anatomical and behavioral signs of disc degeneration and back pain, decreased SPARC expression and increased methylation of the SPARC promoter. In parallel, we show that human subjects with back pain exhibit signs of disc degeneration and increased methylation of the SPARC promoter. Methylation of either the human or mouse SPARC promoter silences its activity in transient transfection assays. Conclusions This study provides the first evidence that DNA methylation of a single gene plays a role in chronic pain in humans and animal models. This has important implications for understanding the mechanisms involved in chronic pain and for pain therapy. PMID:21867537

  11. DNA Methylation Profile at the DNMT3L Promoter

    PubMed Central

    Gokul, Gopinathan; Gautami, Bhimana; Malathi, Surapaneni; Sowjanya, A. Pavani; Poli, Usha Rani; Jain, Meenakshi; Ramakrishna, Gayatri; Khosla, Sanjeev

    2007-01-01

    Epigenetic events play a prominent role during cancer development. This is evident from the fact that almost all cancer types show aberrant DNA methylation. These abnormal DNA methylation levels are not restricted to just a few genes but affect the whole genome. Previous studies have shown genome-wide DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most cancers. Molecules like DNA methyltransferase act as effectors of epigenetic reprogramming. In the present study we have examined the possibility that the reprogramming genes themselves undergo epigenetic modifications reflecting their changed transcriptional status during cancer development. Comparison of DNA methylation status between the normal and cervical cancer samples was carried out at the promoters of a few reprogramming molecules. Our study revealed statistically significant DNA methylation differences within the promoter of DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L promoter was found to have lost DNA methylation to varying levels in 14 out of 15 cancer cervix samples analysed. The present study highlights the importance of DNA methylation profile at DNMT3L promoter not only as a promising biomarker for cervical cancer, which is the second most common cancer among women worldwide, but also provides insight into the possible role of DNMT3L in cancer development. PMID:17965599

  12. DNA Methylation Variation Trends during the Embryonic Development of Chicken

    PubMed Central

    Li, Shizhao; Zhu, Yufei; Zhi, Lihui; Han, Xiaoying; Shen, Jing; Liu, Yanli; Yao, Junhu; Yang, Xiaojun

    2016-01-01

    The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation

  13. [DNA methylation and demethylation: current status and future perspective].

    PubMed

    Dajun, Deng

    2014-05-01

    DNA methylation plays important roles in cell differentiation, embryonic development, host adaptations to environmental factors, and pathogenesis through regulation of gene transcription and imprinting, X-inactivation, and defense of foreign genetic material invasion, is currently one of the hottest research fields on epigenetics. In the past few years, a number of important findings on DNA methylation have been achieved. These findings include discovery of TETs-catalyzed cytosine hydroxymethylation and its functions in the early embryonic development; the relationship between active and passive DNA demethylation; establishment and maintenance of DNA methylation patterns and their associations with histone modifications, chromatin configuration, polycomb group proteins and non-coding RNA bindings. DNA methylation has become a new potential biomarker and therapy target.

  14. Structural and Functional Coordination of DNA and Histone Methylation

    PubMed Central

    Cheng, Xiaodong

    2014-01-01

    One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on structural aspects of enzymatic activities of histone (arginine and lysine) methylation and demethylation and functional links between the methylation status of the DNA and histones. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. PMID:25085914

  15. Epigenome-Wide Association Analysis Identified Nine Skin DNA Methylation Loci for Psoriasis.

    PubMed

    Zhou, Fusheng; Wang, Wenjun; Shen, Changbing; Li, Hui; Zuo, Xianbo; Zheng, Xiaodong; Yue, Min; Zhang, Cuicui; Yu, Liang; Chen, Mengyun; Zhu, Caihong; Yin, Xianyong; Tang, Mingjun; Li, Yongjiang; Chen, Gang; Wang, Zaixing; Liu, Shengxiu; Zhou, Yi; Zhang, Fengyu; Zhang, Weijia; Li, Caihua; Yang, Sen; Sun, Liangdan; Zhang, Xuejun

    2016-04-01

    Psoriasis is a chronic hyperproliferative and inflammatory skin disease caused by the interplay of genetic and environmental factors. DNA methylation has been linked to psoriasis, but the manner in which this process contributes to the disease is not fully understood. In this study, we carried out a three-stage epigenome-wide association study to identify disease-associated differentially methylated sites using a combination of 262 skin and 48 peripheral blood mononuclear cell samples. We not only revealed genome-wide methylation patterns for psoriasis but also identified strong associations between the skin-specific DNA methylation of nine disease-associated differentially methylated sites and psoriasis (Wilcoxon ranked PBonferroni < 0.01; methylation level difference > 0.10). Further analysis revealed that these nine disease-associated differentially methylated sites were not significantly affected by genetic variations, supporting their remarkable contributions to disease status. The expression of CYP2S1, ECE1, EIF2C2, MAN1C1, and DLGAP4 was negatively correlated with DNA methylation. These findings will help us to better understand the molecular mechanism of psoriasis. PMID:26743604

  16. Epigenome-Wide Association Analysis Identified Nine Skin DNA Methylation Loci for Psoriasis.

    PubMed

    Zhou, Fusheng; Wang, Wenjun; Shen, Changbing; Li, Hui; Zuo, Xianbo; Zheng, Xiaodong; Yue, Min; Zhang, Cuicui; Yu, Liang; Chen, Mengyun; Zhu, Caihong; Yin, Xianyong; Tang, Mingjun; Li, Yongjiang; Chen, Gang; Wang, Zaixing; Liu, Shengxiu; Zhou, Yi; Zhang, Fengyu; Zhang, Weijia; Li, Caihua; Yang, Sen; Sun, Liangdan; Zhang, Xuejun

    2016-04-01

    Psoriasis is a chronic hyperproliferative and inflammatory skin disease caused by the interplay of genetic and environmental factors. DNA methylation has been linked to psoriasis, but the manner in which this process contributes to the disease is not fully understood. In this study, we carried out a three-stage epigenome-wide association study to identify disease-associated differentially methylated sites using a combination of 262 skin and 48 peripheral blood mononuclear cell samples. We not only revealed genome-wide methylation patterns for psoriasis but also identified strong associations between the skin-specific DNA methylation of nine disease-associated differentially methylated sites and psoriasis (Wilcoxon ranked PBonferroni < 0.01; methylation level difference > 0.10). Further analysis revealed that these nine disease-associated differentially methylated sites were not significantly affected by genetic variations, supporting their remarkable contributions to disease status. The expression of CYP2S1, ECE1, EIF2C2, MAN1C1, and DLGAP4 was negatively correlated with DNA methylation. These findings will help us to better understand the molecular mechanism of psoriasis.

  17. DNA Methylation Reduces Binding and Cleavage by Bleomycin

    PubMed Central

    2015-01-01

    In a recent study, we described the enhanced double-strand cleavage of hairpin DNAs by Fe·bleomycin (Fe·BLM) that accompanies increasingly strong binding of this antitumor agent and suggested that this effect may be relevant to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM-induced cleavage at the site of methylation and to result in enhanced cleavage at adjacent nonmethylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe·BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed. PMID:25187079

  18. Quantitative reconstruction of leukocyte subsets using DNA methylation

    PubMed Central

    2014-01-01

    Background Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach. Results Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods. Conclusions Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells. PMID:24598480

  19. DNA methylation and histone modification in onion chromosomes.

    PubMed

    Suzuki, Go; Shiomi, Maho; Morihana, Sayuri; Yamamoto, Maki; Mukai, Yasuhiko

    2010-01-01

    Onion, Allium cepa, is a model plant for experimental observation of somatic cell division, whose mitotic chromosome is extremely large, and contains the characteristic terminal heterochromatin. Epigenetic status of the onion chromosome is a matter of deep interest from a molecular cytogenetic point of view, because epigenetic marks regulate chromatin structure and gene expression. Here we examined chromosomal distribution of DNA methylation and histone modification in A. cepa in order to reveal the chromatin structure in detail. Immunodetection of 5-methylcytosine (5mC) and in situ nick-translation analysis showed that onion genomic DNA was highly methylated, and the methylated CG dinucleotides were distributed in entire chromosomes. In addition, distributions of histone methylation codes, which occur in close association with DNA methylation, were similar to those of other large genome species. From these results, a highly heterochromatic and less euchromatic state of large onion chromosomes were demonstrated at an epigenetic level.

  20. Allele-Specific DNA Methylation Detection by Pyrosequencing®.

    PubMed

    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide information on the methylation status of individual alleles of genes. This information may be of importance in many situations. In particular, in cancer both alleles of tumour suppressor genes generally need to be inactivated for a phenotypic effect to be observed. Here, we present a simple and cost-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon. PMID:26103906

  1. DNA methylation mediates genetic variation for adaptive transgenerational plasticity.

    PubMed

    Herman, Jacob J; Sultan, Sonia E

    2016-09-14

    Environmental stresses experienced by individual parents can influence offspring phenotypes in ways that enhance survival under similar conditions. Although such adaptive transgenerational plasticity is well documented, its transmission mechanisms are generally unknown. One possible mechanism is environmentally induced DNA methylation changes. We tested this hypothesis in the annual plant Polygonum persicaria, a species known to express adaptive transgenerational plasticity in response to parental drought stress. Replicate plants of 12 genetic lines (sampled from natural populations) were grown in dry versus moist soil. Their offspring were exposed to the demethylating agent zebularine or to control conditions during germination and then grown in dry soil. Under control germination conditions, the offspring of drought-stressed parents grew longer root systems and attained greater biomass compared with offspring of well-watered parents of the same genetic lines. Demethylation removed these adaptive developmental effects of parental drought, but did not significantly alter phenotypic expression in offspring of well-watered parents. The effect of demethylation on the expression of the parental drought effect varied among genetic lines. Differential seed provisioning did not contribute to the effect of parental drought on offspring phenotypes. These results demonstrate that DNA methylation can mediate adaptive, genotype-specific effects of parental stress on offspring phenotypes. PMID:27629032

  2. Horizontal transfer of DNA methylation patterns into bacterial chromosomes

    PubMed Central

    Shin, Jung-Eun; Lin, Chris; Lim, Han N.

    2016-01-01

    Horizontal gene transfer (HGT) is the non-inherited acquisition of novel DNA sequences. HGT is common and important in bacteria because it enables the rapid generation of new phenotypes such as antibiotic resistance. Here we show that in vivo and in vitro DNA methylation patterns can be horizontally transferred into bacterial chromosomes to program cell phenotypes. The experiments were performed using a synthetic system in Escherichia coli where different DNA methylation patterns within the cis-regulatory sequence of the agn43 gene turn on or off a fluorescent reporter (CFP). With this system we demonstrated that DNA methylation patterns not only accompany the horizontal transfer of genes into the bacterial cytoplasm but can be transferred into chromosomes by: (i) bacteriophage P1 transduction; and (ii) transformation of extracellular synthetic DNA. We also modified the experimental system by replacing CFP with the SgrS small RNA, which regulates glucose and methyl α-D-glucoside uptake, and showed that horizontally acquired DNA methylation patterns can increase or decrease cell fitness. That is, horizontally acquired DNA methylation patterns can result in the selection for and against cells that have HGT. Findings from these proof-of-concept experiments have applications in synthetic biology and potentially broad implications for bacterial adaptation and evolution. PMID:27084942

  3. Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome.

    PubMed

    Wong, Yan-Fung; Micklem, Chris N; Taguchi, Masataka; Itonaga, Hidehiro; Sawayama, Yasushi; Imanishi, Daisuke; Nishikawa, Shinichi; Miyazaki, Yasushi; Jakt, Lars Martin

    2014-10-01

    Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression.

  4. Research Advances in Pituitary Adenoma and DNA Methylation.

    PubMed

    Wei, Zhen-Qing; Li, Yang; Li, Wei-Hua; Lou, Jia-Cheng; Zhang, Bo

    2016-08-01

    DNA methylation is closely related to the genesis and development of pituitary adenoma. Studies have shown that high methylation in the promoter region of potassium voltage-gated chanel,shaker related subfamily,beta member 2,O-6-methylguanine-DNA methyltransferase,echinoderm microtubule associated protein like 2 ,ras homolog family member D ,homeobox B1 ,NNAT, and P16 inhibits the expression of these genes and regulates of the proliferation of pituitary adenoma. DNA methylation is also closely related to invasive pituitary adenoma. Therefore,further study on molecular mechanism of DNA methylation of pituitary adenoma will offer a new strategy for the diagnosis and treatment of pituitary adenoma. PMID:27594164

  5. DNA methylation studies using twins: what are they telling us?

    PubMed

    Bell, Jordana T; Spector, Tim D

    2012-01-01

    Recent studies have identified both heritable DNA methylation effects and differential methylation in disease-discordant identical twins. Larger sample sizes, replication, genetic-epigenetic analyses and longitudinal assays are now needed to establish the role of epigenetic variants in disease. PMID:23078798

  6. Integration of DNA sequence and DNA methylation changes in monozygotic twin pairs discordant for schizophrenia.

    PubMed

    Castellani, C A; Melka, M G; Gui, J L; O'Reilly, R L; Singh, S M

    2015-12-01

    Schizophrenia is a complex mental disorder with high heritability (80%), extensive genetic heterogeneity, environmental contributions and only 50% concordance in discordant monozygotic (MZ) twins. Discordant MZ twins provide an exceptional opportunity to assess patient specific genome-wide genetic and epigenetic changes that may account for the disease phenotype. A combined analysis of genetic and epigenetic changes on the same twin pairs is expected to provide a more effective approach for two reasons. First, it is now possible to generate relatively reliable complete genome sequences as well as promoter methylation states on an individual level and second, the unaffected twin that originated from the same zygote provides a near perfect genetic match for contrast and comparison. This report deals with the combined analysis of DNA sequence data and methylation data on two pairs of discordant MZ twins that have been clinically followed for over 20 years. Results on Family 1 show that 58 genes differ in DNA sequence as well as promoter methylation in a schizophrenia-affected twin as compared to her healthy co-twin. The corresponding number for family 2 was 13. The two lists are over represented by neuronal genes and include a number of known schizophrenia candidate genes and drug targets. The results argue that changes in multiple genes via co-localized genetic and epigenetic alteration contribute to a liability threshold that is necessary for development of schizophrenia. This novel hypothesis, although logical, remains to be validated.

  7. Advancements in the Underlying Pathogenesis of Schizophrenia: Implications of DNA Methylation in Glial Cells

    PubMed Central

    Chen, Xing-Shu; Huang, Nanxin; Michael, Namaka; Xiao, Lan

    2015-01-01

    Schizophrenia (SZ) is a chronic and severe mental illness for which currently there is no cure. At present, the exact molecular mechanism involved in the underlying pathogenesis of SZ is unknown. The disease is thought to be caused by a combination of genetic, biological, psychological, and environmental factors. Recent studies have shown that epigenetic regulation is involved in SZ pathology. Specifically, DNA methylation, one of the earliest found epigenetic modifications, has been extensively linked to modulation of neuronal function, leading to psychiatric disorders such as SZ. However, increasing evidence indicates that glial cells, especially dysfunctional oligodendrocytes undergo DNA methylation changes that contribute to the pathogenesis of SZ. This review primarily focuses on DNA methylation involved in glial dysfunctions in SZ. Clarifying this mechanism may lead to the development of new therapeutic interventional strategies for the treatment of SZ and other illnesses by correcting abnormal methylation in glial cells. PMID:26696822

  8. Expression of DNA methylation genes in secondary progressive multiple sclerosis.

    PubMed

    Fagone, Paolo; Mangano, Katia; Di Marco, Roberto; Touil-Boukoffa, Chafia; Chikovan, Tinatin; Signorelli, Santo; Lombardo, Giuseppe A G; Patti, Francesco; Mammana, Santa; Nicoletti, Ferdinando

    2016-01-15

    Multiple sclerosis (MS) is an immunoinflammatory disease of the central nervous system that seems to be influenced by DNA methylation. We sought to explore the expression pattern of genes involved in the control of DNA methylation in Secondary Progressive (SP) MS patients' PBMCs. We have found that SP MS is characterized by a significant upregulation of two genes belonging to the MBD family genes, MBD2 and MBD4, and by a downregulation of TDG and TET3. PMID:26711572

  9. Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation.

    PubMed

    Zhang, Qingzhu; Wang, Dong; Lang, Zhaobo; He, Li; Yang, Lan; Zeng, Liang; Li, Yanqiang; Zhao, Cheng; Huang, Huan; Zhang, Heng; Zhang, Huiming; Zhu, Jian-Kang

    2016-07-19

    DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. PMID:27382183

  10. DNA Methylation: An Epigenetic Insight into Type 2 Diabetes Mellitus.

    PubMed

    Alam, Fahmida; Islam, Md Asiful; Gan, Siew Hua; Mohamed, Mafauzy; Sasongko, Teguh Haryo

    2016-01-01

    DNA methylation, a major regulator of epigenetic modifications has been shown to alter the expression of genes that are involved in aspects of glucose metabolism such as glucose intolerance, insulin resistance, β-cell dysfunction and other conditions, and it ultimately leads to the pathogenesis of type 2 diabetes mellitus (T2DM). Current evidences indicate an association of DNA methylation with T2DM. This review provides an overview of how various factors play crucial roles in T2DM pathogenesis and how DNA methylation interacts with these factors. Additionally, an update on current techniques of DNA methylation analysis with their pros and cons is provided as a basis for the adoption of suitable techniques in future DNA methylation research towards better management of T2DM. To elucidate the mechanistic relationship between vital environmental factors and the development of T2DM, a better understanding of the changes in gene expression associated with DNA methylation at the molecular level is still needed. PMID:27229720

  11. Oxidative Stress and DNA Methylation in Prostate Cancer

    PubMed Central

    Donkena, Krishna Vanaja; Young, Charles Y. F.; Tindall, Donald J.

    2010-01-01

    The protective effects of fruits, vegetables, and other foods on prostate cancer may be due to their antioxidant properties. An imbalance in the oxidative stress/antioxidant status is observed in prostate cancer patients. Genome oxidative damage in prostate cancer patients is associated with higher lipid peroxidation and lower antioxidant levels. Oxygen radicals are associated with different steps of carcinogenesis, including structural DNA damage, epigenetic changes, and protein and lipid alterations. Epigenetics affects genetic regulation, cellular differentiation, embryology, aging, cancer, and other diseases. DNA methylation is perhaps the most extensively studied epigenetic modification, which plays an important role in the regulation of gene expression and chromatin architecture, in association with histone modification and other chromatin-associated proteins. This review will provide a broad overview of the interplay of oxidative stress and DNA methylation, DNA methylation changes in regulation of gene expression, lifestyle changes for prostate cancer prevention, DNA methylation as biomarkers for prostate cancer, methods for detection of methylation, and clinical application of DNA methylation inhibitors for epigenetic therapy. PMID:20671914

  12. Dicer-independent RNA-directed DNA methylation in Arabidopsis.

    PubMed

    Yang, Dong-Lei; Zhang, Guiping; Tang, Kai; Li, Jingwen; Yang, Lan; Huang, Huan; Zhang, Heng; Zhu, Jian-Kang

    2016-01-01

    RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci. PMID:26642813

  13. DNA Methylation in the Medial Prefrontal Cortex Regulates Alcohol-Induced Behavior and Plasticity

    PubMed Central

    Tapocik, Jenica D.; Juergens, Nathan; Pitcairn, Caleb; Borich, Abbey; Schank, Jesse R.; Sun, Hui; Schuebel, Kornel; Zhou, Zhifeng; Yuan, Qiaoping; Vendruscolo, Leandro F.; Goldman, David; Heilig, Markus

    2015-01-01

    Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism. PMID:25878287

  14. DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity.

    PubMed

    Barbier, Estelle; Tapocik, Jenica D; Juergens, Nathan; Pitcairn, Caleb; Borich, Abbey; Schank, Jesse R; Sun, Hui; Schuebel, Kornel; Zhou, Zhifeng; Yuan, Qiaoping; Vendruscolo, Leandro F; Goldman, David; Heilig, Markus

    2015-04-15

    Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

  15. Alternation of histone and DNA methylation in human atherosclerotic carotid plaques.

    PubMed

    Greißel, A; Culmes, M; Napieralski, R; Wagner, E; Gebhard, H; Schmitt, M; Zimmermann, A; Eckstein, H-H; Zernecke, A; Pelisek, J

    2015-08-01

    Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development. PMID:25993995

  16. Genome-wide profiling of DNA methylation and gene expression in Crassostrea gigas male gametes

    PubMed Central

    Olson, Claire E.; Roberts, Steven B.

    2014-01-01

    DNA methylation patterns and functions are variable across invertebrate taxa. In order to provide a better understanding of DNA methylation in the Pacific oyster (Crassostrea gigas), we characterized the genome-wide DNA methylation profile in male gamete cells using whole-genome bisulfite sequencing. RNA-Seq analysis was performed to examine the relationship between DNA methylation and transcript expression. Methylation status of over 7.6 million CpG dinucleotides was described with a majority of methylated regions occurring among intragenic regions. Overall, 15% of the CpG dinucleotides were determined to be methylated and the mitochondrial genome lacked DNA methylation. Integrative analysis of DNA methylation and RNA-Seq data revealed a positive association between methylation status, both in gene bodies and putative promoter regions, and expression. This study provides a comprehensive characterization of the distribution of DNA methylation in the oyster male gamete tissue and suggests that DNA methylation is involved in gene regulatory activity. PMID:24987376

  17. Sensing DNA methylation in the protozoan parasite Entamoeba histolytica.

    PubMed

    Lavi, Tal; Isakov, Elada; Harony, Hala; Fisher, Ohad; Siman-Tov, Rama; Ankri, Serge

    2006-12-01

    In the protozoan parasite Entamoeba histolytica, 5-methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non-long-terminal repeat retrotransposon (RT LINE) compared with the non-methylated RT LINE. This protein, named E. histolytica-methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non-Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C-terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA-methylated binding activity in protozoa.

  18. Adiposity is associated with DNA methylation profile in adipose tissue

    PubMed Central

    Agha, Golareh; Houseman, E Andres; Kelsey, Karl T; Eaton, Charles B; Buka, Stephen L; Loucks, Eric B

    2015-01-01

    Background: Adiposity is a risk factor for type 2 diabetes and cardiovascular disease, suggesting an important role for adipose tissue in the development of these conditions. The epigenetic underpinnings of adiposity are not well understood, and studies of DNA methylation in relation to adiposity have rarely focused on target adipose tissue. Objectives were to evaluate whether genome-wide DNA methylation profiles in subcutaneous adipose tissue and peripheral blood leukocytes are associated with measures of adiposity, including central fat mass, body fat distribution and body mass index. Methods: Participants were 106 men and women (mean age 47 years) from the New England Family Study. DNA methylation was evaluated using the Infinium HumanMethylation450K BeadChip. Adiposity phenotypes included dual-energy X-ray absorptiometry-assessed android fat mass, android:gynoid fat ratio and trunk:limb fat ratio, as well as body mass index. Results: Adipose tissue genome-wide DNA methylation profiles were associated with all four adiposity phenotypes, after adjusting for race, sex and current smoking (omnibus p-values <0.001). After further adjustment for adipose cell-mixture effects, associations with android fat mass, android:gynoid fat ratio, and trunk:limb fat ratio remained. In gene-specific analyses, adiposity phenotypes were associated with adipose tissue DNA methylation in several genes that are biologically relevant to the development of adiposity, such as AOC3, LIPE, SOD3, AQP7 and CETP. Blood DNA methylation profiles were not associated with adiposity, before or after adjustment for blood leukocyte cell mixture effects. Conclusion: Findings show that DNA methylation patterns in adipose tissue are associated with adiposity. PMID:25541553

  19. Principles Governing DNA Methylation during Neuronal Lineage and Subtype Specification

    PubMed Central

    Sharma, Ali; Klein, Shifra S.; Barboza, Luendreo; Lohdi, Niraj

    2016-01-01

    Although comprehensively described during early neuronal development, the role of DNA methylation/demethylation in neuronal lineage and subtype specification is not well understood. By studying two distinct neuronal progenitors as they differentiate to principal neurons in mouse hippocampus and striatum, we uncovered several principles governing neuronal DNA methylation during brain development. (1) The program consists of three stages: an initial genome-wide methylation during progenitor proliferation is followed by loss of methylation during the transition of regional progenitors to “young” hippocampal/striatal neurons, which is then reversed by gain in methylation during maturation to subtype-specific neurons. (2) At the first two stages, gain and loss of methylation are limited to CpGs, whereas during the third maturation stage, methylation also occurs at non-CpG sites in both lineages. (3) Methylation/demethylation, similar to transcription, are initially highly similar in the two lineages, whereas diversification in methylation and transcription during maturation creates subtype-specific methylation differences. (4) Initially, methylation targets all genomic locations, whereas later, during early and late differentiation, the preferred targets are intronic/intergenic sequences with enhancer-like activity. (5) Differentially methylated genes are enriched in sequential neurodevelopmental functions (such as progenitor proliferation, migration, neuritogenesis, and synaptic transmission); upregulated genes represent current and consecutive stage-specific functions, and downregulated genes represent preceding functions that are no longer required. The main conclusion of our work is that the neuronal methylation/demethylation program is predominantly developmental with minimal lineage specificity, except in the final stage of development when neuron subtype-specific differences also emerge. SIGNIFICANCE STATEMENT Our work is the first to describe a set of

  20. Characterization of DNA Methylation in Circulating Tumor Cells

    PubMed Central

    Pixberg, Constantin F.; Schulz, Wolfgang A.; Stoecklein, Nikolas H.; Neves, Rui P. L.

    2015-01-01

    Epigenetics contributes to molecular mechanisms leading to tumor cell transformation and systemic progression of cancer. However, the dynamics of epigenetic remodeling during metastasis remains unexplored. In this context, circulating tumor cells (CTCs) might enable a direct insight into epigenetic mechanisms relevant for metastasis by providing direct access to systemic cancer. CTCs can be used as prognostic markers in cancer patients and are regarded as potential metastatic precursor cells. However, despite substantial technical progress, the detection and molecular characterization of CTCs remain challenging, in particular the analysis of DNA methylation. As recent studies have started to address the epigenetic state of CTCs, we discuss here the potential of such investigations to elucidate mechanisms of metastasis and to develop tumor biomarkers. PMID:26506390

  1. Expression and DNA methylation analysis of SNRPN in Prader-Willi patients

    SciTech Connect

    Glenn, C.C.; Jong, M.T.C.; Driscoll, D.J.

    1994-09-01

    The human SNRPN gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the Prader-Willi syndrome critical region in 15q11-q13. We have previously demonstrated functional imprinting of SNRPN, with absent expression in PWS skin fibroblasts and lymphoblasts. We now show a similar lack of expression in blood of PWS patients, which appear to correlate with DNA methylation of NotI sites in the 5{prime} region of the gene. RNA and DNA was extracted from peripheral blood of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) deletion patients to be used for RT-PCR with SNRPN gene-specific primers and DNA methylation analysis. Either no or highly reduced levels of SNRPN RT-PCR product were detected in nine PWS samples but was present in normals, AS patients, and one clinically typical PWS patient. Parent-of-origin DNA methylation imprints are also present within the SNRPN gene. PWS patients having only a maternal contribution of SNRPN have several NotI restriction sites near the transcription start site which are methylated, while these same sites are unmethylated on the paternal chromosome (i.e., AS samples). Several CpG sites approximately 22 kb downstream of the transcription start site are methylated preferentially on the paternal allele. These observations for human SNRPN are similar to those of the mouse imprinted gene Igf2r, which exhibits DNA methylation of a CpG island 27 kb from the transcription start site on the expressed allele, and DNA methylation in the promoter region of the repressed allele. We suggest that RT-PCR and/or DNA methylation analysis from blood of PWS patients may be the most accurate means of diagnosing classical PWS. These results further indicate a role for SNRPN in the pathogenesis of PWS, and may serve as a model to study other human imprinted genes.

  2. Parent-of-Origin DNA Methylation Dynamics during Mouse Development.

    PubMed

    Stelzer, Yonatan; Wu, Hao; Song, Yuelin; Shivalila, Chikdu S; Markoulaki, Styliani; Jaenisch, Rudolf

    2016-09-20

    Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development, suggesting that imprints are faithfully maintained in embryos and adults. To test this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single-cell resolution. Biallelic gain or loss of IG-DMR methylation occurred in a small fraction of mouse embryonic stem cells, significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation, causing substantial and consistent tissue- and cell-type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis, resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance. PMID:27653683

  3. DNA methylation detection based on difference of base content

    NASA Astrophysics Data System (ADS)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  4. Quantification of 5-methyl-2'-deoxycytidine in the DNA.

    PubMed

    Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

    2015-01-01

    Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues. PMID:26098716

  5. Dynamic Changes in DNA Methylation in Ischemic Tolerance

    PubMed Central

    Meller, Robert; Pearson, Andrea; Simon, Roger P.

    2015-01-01

    Epigenetic mediators of gene expression are hypothesized to regulate transcriptomic responses to preconditioning ischemia and ischemic tolerance. Here, we utilized a methyl-DNA enrichment protocol and sequencing (ChIP-seq) to identify patterns of DNA methylation in an established model of ischemic tolerance in neuronal cultures (oxygen and glucose deprivation: OGD). We observed an overall decrease in global DNA methylation at 4 h following preconditioning ischemia (30 min OGD), harmful ischemia (120 min OGD), and in ischemic tolerant neuronal cultures (30 min OGD, 24 h recovery, 120 min OGD). We detected a smaller cohort of hypermethylated regions following ischemic conditions, which were further analyzed revealing differential chromosomal localization of methylation, and a differential concentration of methylation on genomic regions. Together, these data show that the temporal profiles of DNA methylation with respect to chromatin hyper- and hypo-methylation following various ischemic conditions are highly dynamic, and may reveal novel targets for neuroprotection. PMID:26029158

  6. The Methylated DNA Immunoprecipitation [MeDIP] to Investigate the Epigenetic Remodeling in Cell Fate Determination and Cancer Development.

    PubMed

    Masciarelli, Silvia; Bellissimo, Teresa; Iosue, Ilaria; Fazi, Francesco

    2016-01-01

    Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and the expression of noncoding RNAs contribute to the regulation of gene expression for the cell fate determination and tissue development. The disruption of these epigenetic mechanisms, in conjunction with genetic alterations, is a decisive element for cancer development and progression. The cancer phenotype is characterized by global DNA hypomethylation and gene-specific hypermethylation. The methylated DNA immunoprecipitation [MeDIP] is a useful approach currently used to clarify the functional consequences of DNA methylation on cell fate determination and cancer development.

  7. Examining the Causes and Consequences of Context-Specific Differential DNA Methylation in Maize.

    PubMed

    Li, Qing; Song, Jawon; West, Patrick T; Zynda, Greg; Eichten, Steven R; Vaughn, Matthew W; Springer, Nathan M

    2015-08-01

    DNA methylation is a stable modification of chromatin that can contribute to epigenetic variation through the regulation of genes or transposons. Profiling of DNA methylation in five maize (Zea mays) inbred lines found that while DNA methylation levels for more than 99% of the analyzed genomic regions are similar, there are still 5,000 to 20,000 context-specific differentially methylated regions (DMRs) between any two genotypes. The analysis of identical-by-state genomic regions that have limited genetic variation provided evidence that DMRs can occur without local sequence variation, but they are less common than in regions with genetic variation. Characterization of the sequence specificity of DMRs, location of DMRs relative to genes and transposons, and patterns of DNA methylation in regions flanking DMRs reveals a distinct subset of DMRs. Transcriptome profiling of the same tissue revealed that only approximately 20% of genes with qualitative (on-off) differences in gene expression are associated with DMRs, and there is little evidence for association of DMRs with genes that show quantitative differences in gene expression. We also identify a set of genes that may represent cryptic information that is silenced by DNA methylation in the reference B73 genome. Many of these genes exhibit natural variation in other genotypes, suggesting the potential for selection to act upon existing epigenetic natural variation. This study provides insights into the origin and influences of DMRs in a crop species with a complex genome organization.

  8. Mobile small RNAs regulate genome-wide DNA methylation

    PubMed Central

    Lewsey, Mathew G.; Hardcastle, Thomas J.; Melnyk, Charles W.; Molnar, Attila; Valli, Adrián; Urich, Mark A.; Nery, Joseph R.; Baulcombe, David C.; Ecker, Joseph R.

    2016-01-01

    RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21–24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession. PMID:26787884

  9. Mobile small RNAs regulate genome-wide DNA methylation.

    PubMed

    Lewsey, Mathew G; Hardcastle, Thomas J; Melnyk, Charles W; Molnar, Attila; Valli, Adrián; Urich, Mark A; Nery, Joseph R; Baulcombe, David C; Ecker, Joseph R

    2016-02-01

    RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession. PMID:26787884

  10. Methylsorb: a simple method for quantifying DNA methylation using DNA-gold affinity interactions.

    PubMed

    Sina, Abu Ali Ibn; Carrascosa, Laura G; Palanisamy, Ramkumar; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-10-21

    The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

  11. Evolutionary patterns of genic DNA methylation vary across land plants.

    PubMed

    Takuno, Shohei; Ran, Jin-Hua; Gaut, Brandon S

    2016-01-01

    Little is known about patterns of genic DNA methylation across the plant kingdom or about the evolutionary processes that shape them. To characterize gene-body methylation (gbM) within exons, we have gathered single-base resolution methylome data that span the phylogenetic breadth of land plants. We find that a basal land plant, Marchantia polymorpha, lacks any evident signal of gbM within exons, but conifers have high levels of both CG and CHG (where H is A, C or T) methylation in expressed genes. To begin to understand the evolutionary forces that shape gbM, we first tested for correlations in methylation levels across orthologues(1,2). Genic CG methylation levels, but not CHG or CHH levels, are correlated across orthologues for species as distantly related as ferns and angiosperms. Hence, relative levels of CG methylation are a consistent property across genes, even for species that diverged ∼400 million years ago(3,4). In contrast, genic CHG methylation correlates with genome size, suggesting that the host epigenetic response to transposable elements also affects genes. Altogether, our data indicate that the evolutionary forces acting on DNA methylation vary substantially across species, genes and methylation contexts. PMID:27249194

  12. Use of capillary electrophoresis to study methylation patterns in DNA

    NASA Astrophysics Data System (ADS)

    Voss, Karl; Roos, Pieter; Zhang, Jian Z.; Dovichi, Norman J.

    1996-04-01

    A four-color multiple capillary DNA sequencer is used to determine the methylation pattern of double stranded DNA. The DNA sample is treated with bisulfite under conditions that convert cytosine to uracil. Methyl-cytosine is inert under these reaction conditions. After PCR amplification, the reaction products are subjected to a four-color fluorescent Sanger sequencing reaction. The sequence is then determined by use of capillary electrophoresis. Comparison of the sequence obtained after bisulfite treatment with the original sequence reveals that certain of the Cs in the original sequence are converted to Ts. This conversion occurs only if the original C was not methylated. Those Cs that are common to both sequences were methylated in the original sequence. Methylation patterns have been implicated in aging, developmental biology, and cancer; however, there has been no simple and rapid method for determining the methylation pattern in genomic DNA. The method described in this paper is quick, simple, and accurate, and demonstrates an exciting application of capillary electrophoresis DNA sequencing.

  13. Analysis of DNA Methylation in Various Swine Tissues

    PubMed Central

    Niu, Weiping; Yang, Runjun; Zhang, Yonghong; Qiu, Zhengyan; Sun, Boxing; Zhao, Zhihui

    2011-01-01

    DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively. In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome. PMID:21283691

  14. DNA Methylation Profiling Identifies Global Methylation Differences and Markers of Adrenocortical Tumors

    PubMed Central

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

    2012-01-01

    Context: It is not known whether there are any DNA methylation alterations in adrenocortical tumors. Objective: The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. Methods: Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. Results: Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. Conclusions: Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors. PMID:22472567

  15. Methylation matters? Decreased methylation status of genomic DNA in the blood of schizophrenic twins.

    PubMed

    Bönsch, Dominikus; Wunschel, Michael; Lenz, Bernd; Janssen, Gesa; Weisbrod, Matthias; Sauer, Heinrich

    2012-08-15

    Studies of schizophrenia inheritance in identical twins show a concordance of about 50%, which supports an epigenetic model. In our present study we investigated methylation of genomic DNA and promoter methylation of Reelin and SOX10 genes in peripheral blood of twins suffering from schizophrenia. Global DNA methylation was reduced (52.3%) in schizophrenic twins if compared with healthy control twins (65.7%). The reduced methylation was significant in males only. We also found a similar hypomethylation in the non-affected twins of discordant pairs and a mixed group of psychiatric controls. In discordant twins there was a relative hypermethylation of the SOX10 promoter. Within-pair-difference of methylation of Reelin promoter was significantly lower in monozygotic twins than in dizygotic twins. PMID:23102571

  16. Third trimester phthalate exposure is associated with DNA methylation of growth-related genes in human placenta

    PubMed Central

    Zhao, Yan; Chen, Jiao; Wang, Xiu; Song, Qi; Xu, Hui-Hui; Zhang, Yun-Hui

    2016-01-01

    Strong evidence implicates maternal phthalate exposure during pregnancy in contributing to adverse birth outcomes. Recent research suggests these effects might be mediated through the improper regulation of DNA methylation in offspring tissue. In this study, we examined associations between prenatal phthalate exposure and DNA methylation in human placenta. We recruited 181 mother-newborn pairs (80 fetal growth restriction newborns, 101 normal newborns) in Wenzhou, China and measured third trimester urinary phthalate metabolite concentrations and placental DNA methylation levels of IGF2 and AHRR. We found urinary concentrations of mono (2-ethyl-5- hydroxyhexyl) phthalate (MEHHP), and mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) were significantly inversely associated with placental IGF2 DNA methylation. The associations were much more evident in fetal growth restriction (FGR) newborns than those in normal newborns. These findings suggest that changes in placental DNA methylation might be part of the underlying biological pathway between prenatal phthalate exposure and adverse fetal growth. PMID:27653773

  17. Third trimester phthalate exposure is associated with DNA methylation of growth-related genes in human placenta

    NASA Astrophysics Data System (ADS)

    Zhao, Yan; Chen, Jiao; Wang, Xiu; Song, Qi; Xu, Hui-Hui; Zhang, Yun-Hui

    2016-09-01

    Strong evidence implicates maternal phthalate exposure during pregnancy in contributing to adverse birth outcomes. Recent research suggests these effects might be mediated through the improper regulation of DNA methylation in offspring tissue. In this study, we examined associations between prenatal phthalate exposure and DNA methylation in human placenta. We recruited 181 mother-newborn pairs (80 fetal growth restriction newborns, 101 normal newborns) in Wenzhou, China and measured third trimester urinary phthalate metabolite concentrations and placental DNA methylation levels of IGF2 and AHRR. We found urinary concentrations of mono (2-ethyl-5- hydroxyhexyl) phthalate (MEHHP), and mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) were significantly inversely associated with placental IGF2 DNA methylation. The associations were much more evident in fetal growth restriction (FGR) newborns than those in normal newborns. These findings suggest that changes in placental DNA methylation might be part of the underlying biological pathway between prenatal phthalate exposure and adverse fetal growth.

  18. 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

  19. Dynamics of nucleosome assembly and effects of DNA methylation.

    PubMed

    Lee, Ju Yeon; Lee, Jaehyoun; Yue, Hongjun; Lee, Tae-Hee

    2015-02-13

    The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)2 tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)2 tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA. PMID:25550164

  20. Dynamics of nucleosome assembly and effects of DNA methylation.

    PubMed

    Lee, Ju Yeon; Lee, Jaehyoun; Yue, Hongjun; Lee, Tae-Hee

    2015-02-13

    The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)2 tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)2 tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.

  1. Effects of cytosine methylation on DNA charge transport

    NASA Astrophysics Data System (ADS)

    Hihath, Joshua; Guo, Shaoyin; Zhang, Peiming; Tao, Nongjian

    2012-04-01

    The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 Å, its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.

  2. Genetic and environmental impacts on DNA methylation levels in twins.

    PubMed

    Yet, Idil; Tsai, Pei-Chien; Castillo-Fernandez, Juan E; Carnero-Montoro, Elena; Bell, Jordana T

    2016-01-01

    Epigenetics describes the study of cellular modifications that can modify the expression of genes without changing the DNA sequence. DNA methylation is one of the most stable and prevalent epigenetic mechanisms. Twin studies have been a valuable model for unraveling the genetic and epigenetic epidemiology of complex traits, and now offer a potential to dissect the factors that impact DNA methylation variability and its biomedical significance. The twin design specifically allows for the study of genetic, environmental and lifestyle factors, and their potential interactions, on epigenetic profiles. Furthermore, genetically identical twins offer a unique opportunity to assess nongenetic impacts on epigenetic profiles. Here, we summarize recent findings from twin studies of DNA methylation profiles across tissues, to define current knowledge regarding the genetic and nongenetic factors that influence epigenetic variation. PMID:26678685

  3. EPIGENETIC EFFECTS OF SHIFTWORK ON BLOOD DNA METHYLATION

    PubMed Central

    Bollati, Valentina; Baccarelli, Andrea; Sartori, Samantha; Tarantini, Letizia; Motta, Valeria; Rota, Federica; Costa, Giovanni

    2012-01-01

    In the present study, the authors investigated the effects of shiftwork exposure on DNA methylation using peripheral blood DNA from subjects working in two chemical plants in Northern Italy. The investigation was designed to evaluate (a) DNA methyl- ation changes in Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements as a surrogate of global methylation and (b) promoter methylation of gluco- corticoid receptor (GCR), tumor necrosis factor alpha (TNF-α), and interferon- gamma (IFN-γ). One hundred and fifty white male workers (mean ± SD: 41.0 ± 9 yrs of age) were examined: 100 3 × 8 rotating shiftworkers (40.4 ± 8.7 yrs of age) and 50 day workers (42.2 ± 9.4 yrs of age). The authors used bisulfite-pyrosequencing to esti- mate repetitive elements and gene-specific methylation. Multiple regression analysis, adjusted for age, body mass index (BMI), and job seniority, did not show any signifi- cant association between the five DNA methylation markers and shiftwork. However, job seniority, in all subjects, was significantly associated with Alu (β = −0.019, p = .033) and IFN-γ (β = −0.224, p < .001) methylation, whereas TNF-α methylation was inversely correlated with age (β = −0.093, p < .001). Considering only shiftworkers, multiple regression analysis, adjusted for age, BMI, and job seniority, showed a sig- nificant difference between morning and evening types in TNF-α methylation (mean morning type [MT] 11.425 %5mC versus evening type [ET] 12.975 %5mC; β = 1.33, p = .022). No difference was observed between good and poor tolerance to shiftwork. Increasing job seniority (<5, 5–15, >15 yrs) was associated with significantly lower Alu (β = −0.86, p = .006) and IFN-γ methylation (β = −6.50, p = .007) after adjust- ment for age, BMI, and morningness/eveningness. In addition, GCR significantly increased with length of shiftwork (β = 3.33, p = .05). The data showed alterations in blood DNA methylation in a group of

  4. DNA methylation: conducting the orchestra from exposure to phenotype?

    PubMed

    Leenen, Fleur A D; Muller, Claude P; Turner, Jonathan D

    2016-01-01

    DNA methylation, through 5-methyl- and 5-hydroxymethylcytosine (5mC and 5hmC), is considered to be one of the principal interfaces between the genome and our environment, and it helps explain phenotypic variations in human populations. Initial reports of large differences in methylation level in genomic regulatory regions, coupled with clear gene expression data in both imprinted genes and malignant diseases, provided easily dissected molecular mechanisms for switching genes on or off. However, a more subtle process is becoming evident, where small (<10 %) changes to intermediate methylation levels are associated with complex disease phenotypes. This has resulted in two clear methylation paradigms. The latter "subtle change" paradigm is rapidly becoming the epigenetic hallmark of complex disease phenotypes, although we are currently hampered by a lack of data addressing the true biological significance and meaning of these small differences. Our initial expectation of rapidly identifying mechanisms linking environmental exposure to a disease phenotype led to numerous observational/association studies being performed. Although this expectation remains unmet, there is now a growing body of literature on specific genes, suggesting wide ranging transcriptional and translational consequences of such subtle methylation changes. Data from the glucocorticoid receptor (NR3C1) has shown that a complex interplay between DNA methylation, extensive 5'UTR splicing, and microvariability gives rise to the overall level and relative distribution of total and N-terminal protein isoforms generated. Additionally, the presence of multiple AUG translation initiation codons throughout the complete, processed mRNA enables translation variability, hereby enhancing the translational isoforms and the resulting protein isoform diversity, providing a clear link between small changes in DNA methylation and significant changes in protein isoforms and cellular locations. Methylation changes in

  5. A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.

    PubMed

    Pélissier, T; Wassenegger, M

    2000-01-01

    In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed. PMID:10668798

  6. Experimental factors affecting the robustness of DNA methylation analysis

    PubMed Central

    Pharo, Heidi D.; Honne, Hilde; Vedeld, Hege M.; Dahl, Christina; Andresen, Kim; Liestøl, Knut; Jeanmougin, Marine; Guldberg, Per; Lind, Guro E.

    2016-01-01

    Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data. PMID:27671843

  7. N6-methyladenine: the other methylated base of DNA

    PubMed Central

    Ratel, David; Ravanat, Jean-Luc; Berger, François; Wion, Didier

    2006-01-01

    DNA methylation is an epigenetic mechanism involved in many biological functions in prokaryotes and eukaryotes. Contrary to mammalian DNA, which is thought to contain only 5-methylcytosine (m5C), bacterial DNA contains two additional methylated bases, namely N6-methyladenine (m6A), and a more recently discovered minor base N4-methylcytosine (m4C). These modified bases are involved in the protection of bacterial DNA from the action of specific endonucleases via the host-specific restriction-modification system which is regarded as a defense mechanism against bacteriophage infection. However, if the main function of m5C and m4C in bacteria is the protection against restriction enzymes, the roles of m6A are multiple and include for example the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA. Hence, in regard to the multiple roles of m6A in bacteria, and to the well known tendency for m5C to deaminate in thymine, the selection of the mutagenic m5C instead of m6A in mammals as the only methylated base may seem surprising. However, even if adenine methylation is usually considered as a bacterial DNA feature, the presence of m6A is not restricted to prokaryotic DNA since this methylated base has been found in protist and plant DNAs. Furthermore, indirect evidence suggests the presence of m6A in mammal DNA, raising the possibility that this base has remained undetected due to the low sensitivity of the analytical methods used. This points to the importance to consider m6A as the sixth element of DNA. PMID:16479578

  8. The potential role of DNA methylation in abdominal aortic aneurysms.

    PubMed

    Ryer, Evan J; Ronning, Kaitryn E; Erdman, Robert; Schworer, Charles M; Elmore, James R; Peeler, Thomas C; Nevius, Christopher D; Lillvis, John H; Garvin, Robert P; Franklin, David P; Kuivaniemi, Helena; Tromp, Gerard

    2015-01-01

    Abdominal aortic aneurysm (AAA) is a complex disorder that has a significant impact on the aging population. While both genetic and environmental risk factors have been implicated in AAA formation, the precise genetic markers involved and the factors influencing their expression remain an area of ongoing investigation. DNA methylation has been previously used to study gene silencing in other inflammatory disorders and since AAA has an extensive inflammatory component, we sought to examine the genome-wide DNA methylation profiles in mononuclear blood cells of AAA cases and matched non-AAA controls. To this end, we collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers (n = 11), AAA non-smokers (n = 9), control smokers (n = 10) and control non-smokers (n = 11). Methylation data were obtained using the Illumina 450k Human Methylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9), and adenylate cyclase 10 pseudogene 1 (ADCY10P1). Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9. These findings are novel and suggest DNA methylation may play a role in AAA pathobiology. PMID:25993294

  9. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  10. Accounting for population stratification in DNA methylation studies.

    PubMed

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable.

  11. Accounting for population stratification in DNA methylation studies.

    PubMed

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable. PMID:24478250

  12. Associations between Serum Perfluoroalkyl Acids and LINE-1 DNA Methylation

    PubMed Central

    Watkins, Deborah J.; Wellenius, Gregory A.; Butler, Rondi A.; Bartell, Scott M.; Fletcher, Tony; Kelsey, Karl T.

    2014-01-01

    Perfluoroalkyl acids (PFAAs) are persistent, synthetic compounds that are used in a number of consumer products. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been associated with cardiovascular risk factors, and changes in gene expression and DNA methylation in animals and cellular systems. However, whether PFAA exposure is associated with LINE-1 DNA methylation, a potential marker of cardiovascular risk, in humans remains unknown. We sought to evaluate the cross-sectional associations between serum PFAAs and LINE-1 DNA methylation in a population highly exposed to PFOA. We measured serum PFAAs twice four to five years apart in 685 adult participants (47% male, mean age ± SD=42 ± 11 years). We measured percent LINE-1 DNA methylation in peripheral blood leukocytes at the second time point (follow-up), and estimated absolute differences in LINE-1 methylation associated with an interquartile (IQR) shift in mean PFAA serum levels. IQR increases in mean serum PFOA, PFOS, perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS) were associated with differences of −0.04 (p=0.16), 0.20 (p=0.001), 0.06 (p=0.19), and 0.02 (p=0.57), respectively, in % LINE-1 methylation at follow-up after adjustment for potential confounders. We observed a monotonic increase in LINE-1 DNA methylation across tertiles of PFOS and PFNA (ptrend=0.02 for both associations), but not across tertiles of PFOA or PFHxS (ptrend=0.71 and 0.44, respectively). In summary, serum PFOS was associated with LINE-1 methylation, while serum PFOA, PFHxS, and PFNA were not. Additional research is needed to more precisely determine whether these compounds are epigenetically active. PMID:24263140

  13. Human body epigenome maps reveal noncanonical DNA methylation variation.

    PubMed

    Schultz, Matthew D; He, Yupeng; Whitaker, John W; Hariharan, Manoj; Mukamel, Eran A; Leung, Danny; Rajagopal, Nisha; Nery, Joseph R; Urich, Mark A; Chen, Huaming; Lin, Shin; Lin, Yiing; Jung, Inkyung; Schmitt, Anthony D; Selvaraj, Siddarth; Ren, Bing; Sejnowski, Terrence J; Wang, Wei; Ecker, Joseph R

    2015-07-01

    Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual's cells, with epigenetic mechanisms that could have tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals' phased genome, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in several genomic contexts varies substantially among human tissues.

  14. DNA methylation on N6-adenine in C. elegans

    PubMed Central

    Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei; Sendinc, Erdem; Liu, Jianzhao; Aristizábal-Corrales, David; Hsu, Chih-Hung; Aravind, L.; He, Chuan; Shi, Yang

    2015-01-01

    Summary In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. PMID:25936839

  15. Aberrant DNA methylation is a dominant mechanism in MDS progression to AML

    PubMed Central

    Jiang, Ying; Dunbar, Andrew; Gondek, Lukasz P.; Mohan, Sanjay; Rataul, Manjot; O'Keefe, Christine; Sekeres, Mikkael

    2009-01-01

    Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). As such, study of MDS/AML can provide insight into the mechanisms of neoplastic evolution. In 184 patients with MDS and AML, DNA methylation microarray and high-density single nucleotide polymorphism array (SNP-A) karyotyping were used to assess the relative contributions of aberrant DNA methylation and chromosomal deletions to tumor-suppressor gene (TSG) silencing during disease progression. Aberrant methylation was seen in every sample, on average affecting 91 of 1505 CpG loci in early MDS and 179 of 1505 loci after blast transformation (refractory anemia with excess blasts [RAEB]/AML). In contrast, chromosome aberrations were seen in 79% of early MDS samples and 90% of RAEB/AML samples, and were not as widely distributed over the genome. Analysis of the most frequently aberrantly methylated genes identified FZD9 as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML. PMID:18832655

  16. DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts.

    PubMed

    Karouzakis, E; Rengel, Y; Jüngel, A; Kolling, C; Gay, R E; Michel, B A; Tak, P P; Gay, S; Neidhart, M; Ospelt, C

    2011-12-01

    In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction. PMID:21753787

  17. Extra-coding RNAs regulate neuronal DNA methylation dynamics

    PubMed Central

    Savell, Katherine E.; Gallus, Nancy V. N.; Simon, Rhiana C.; Brown, Jordan A.; Revanna, Jasmin S.; Osborn, Mary Katherine; Song, Esther Y.; O'Malley, John J.; Stackhouse, Christian T.; Norvil, Allison; Gowher, Humaira; Sweatt, J. David; Day, Jeremy J.

    2016-01-01

    Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states. PMID:27384705

  18. Discordance of DNA methylation variance between two accessible human tissues.

    PubMed

    Jiang, Ruiwei; Jones, Meaghan J; Chen, Edith; Neumann, Sarah M; Fraser, Hunter B; Miller, Gregory E; Kobor, Michael S

    2015-01-01

    Population epigenetic studies have been seeking to identify differences in DNA methylation between specific exposures, demographic factors, or diseases in accessible tissues, but relatively little is known about how inter-individual variability differs between these tissues. This study presents an analysis of DNA methylation differences between matched peripheral blood mononuclear cells (PMBCs) and buccal epithelial cells (BECs), the two most accessible tissues for population studies, in 998 promoter-located CpG sites. Specifically we compared probe-wise DNA methylation variance, and how this variance related to demographic factors across the two tissues. PBMCs had overall higher DNA methylation than BECs, and the two tissues tended to differ most at genomic regions of low CpG density. Furthermore, although both tissues showed appreciable probe-wise variability, the specific regions and magnitude of variability differed strongly between tissues. Lastly, through exploratory association analysis, we found indication of differential association of BEC and PBMC with demographic variables. The work presented here offers insight into variability of DNA methylation between individuals and across tissues and helps guide decisions on the suitability of buccal epithelial or peripheral mononuclear cells for the biological questions explored by epigenetic studies in human populations.

  19. Predicting DNA methylation level across human tissues.

    PubMed

    Ma, Baoshan; Wilker, Elissa H; Willis-Owen, Saffron A G; Byun, Hyang-Min; Wong, Kenny C C; Motta, Valeria; Baccarelli, Andrea A; Schwartz, Joel; Cookson, William O C M; Khabbaz, Kamal; Mittleman, Murray A; Moffatt, Miriam F; Liang, Liming

    2014-04-01

    Differences in methylation across tissues are critical to cell differentiation and are key to understanding the role of epigenetics in complex diseases. In this investigation, we found that locus-specific methylation differences between tissues are highly consistent across individuals. We developed a novel statistical model to predict locus-specific methylation in target tissue based on methylation in surrogate tissue. The method was evaluated in publicly available data and in two studies using the latest IlluminaBeadChips: a childhood asthma study with methylation measured in both peripheral blood leukocytes (PBL) and lymphoblastoid cell lines; and a study of postoperative atrial fibrillation with methylation in PBL, atrium and artery. We found that our method can greatly improve accuracy of cross-tissue prediction at CpG sites that are variable in the target tissue [R(2) increases from 0.38 (original R(2) between tissues) to 0.89 for PBL-to-artery prediction; from 0.39 to 0.95 for PBL-to-atrium; and from 0.81 to 0.98 for lymphoblastoid cell line-to-PBL based on cross-validation, and confirmed using cross-study prediction]. An extended model with multiple CpGs further improved performance. Our results suggest that large-scale epidemiology studies using easy-to-access surrogate tissues (e.g. blood) could be recalibrated to improve understanding of epigenetics in hard-to-access tissues (e.g. atrium) and might enable non-invasive disease screening using epigenetic profiles. PMID:24445802

  20. Non-symmetrical cytosine methylation in tobacco pollen DNA.

    PubMed

    Oakeley, E J; Jost, J P

    1996-07-01

    We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants. PMID:8806424

  1. DNA methylation: A source of random variation in natural populations.

    PubMed

    Massicotte, Rachel; Whitelaw, Emma; Angers, Bernard

    2011-04-01

    Epigenetic processes (e.g., DNA methylation) have been proposed as potentially important evolutionary mechanisms. However, before drawing conclusions about their evolutionary relevance, we need to evaluate the independence of epigenetic variation from genetic variation, as well as the extent of methylation polymorphism in nature. We evaluated these in natural populations of a clonal fish, Chrosomus eos-neogaeus, for which genetically identical individuals may be found in distinct environments. A genomic survey confirms the genetic uniformity of individuals, whereas a substantial level of inter-individual variation results in DNA methylation. Survey of the methylation status of the CpG dinucleotides of a fragment of a retrotransposon confirmed a marked difference in epiallelic composition among tissues, as well as among individuals. This study provides further evidence of epigenetic variation in the absence of genetic variation and demonstrates that this process can be a source of random variation in natural populations. PMID:21266851

  2. DNA methylation perspectives in the pathogenesis of autoimmune diseases.

    PubMed

    Sun, Bao; Hu, Lei; Luo, Zhi-Ying; Chen, Xiao-Ping; Zhou, Hong-Hao; Zhang, Wei

    2016-03-01

    DNA methylation is now widely recognized as being critical to maintain the function of immune cells. Recent studies suggest that aberrant DNA methylation levels not only can result in immune cells autoreactivity in vitro, but also are related to autoimmunity in vivo. Environmental factors and genetic polymorphisms cause abnormal methylation, which affects the expression of certain immune-related genes, being becoming hot spot of explaining the mechanism of autoimmune diseases. This paper reviews the importance of abnormal methylation during the development of common autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and type 1 diabetes, aiming at a better understanding of the pathogenesis of autoimmune diseases and providing new ideas for the treatment of these diseases. PMID:26821302

  3. Developmental modulation of DNA methylation in the fungus Phycomyces blakesleeanus.

    PubMed Central

    Antequera, F; Tamame, M; Vilanueva, J R; Santos, T

    1985-01-01

    DNA methylation is a rather sparse event among fungi. Phycomyces blakesleeanus seems to be one of the few exceptions in this context. 5-Methylcytosine represents 2.9% of the total cytosine in spore DNA and is located in approximately the same amount at any of the four CA, CT, CC or CG dinucleotides. A progressive and gradual drop in total 5-methylcytosine parallels the development of the fungus. This demethylation is non random but sequence specific and is not accounted for equally by the four different methylated dinucleotides, CG being much less affected (20% demethylated) than CA, CT and CC (more than 90% demethylated at the same time). "De novo" methylation to restore the initial pattern probably takes place during spore maturation. By using specific hybridization probes we have been able to show that the rRNA genes are not significantly methylated at any stage of development, regardless of their transcription status. Images PMID:2997714

  4. High-frequency aberrantly methylated targets in pancreatic adenocarcinoma identified via global DNA methylation analysis using methylCap-seq

    PubMed Central

    2014-01-01

    Background Extensive reprogramming and dysregulation of DNA methylation is an important characteristic of pancreatic cancer (PC). Our study aimed to characterize the genomic methylation patterns in various genomic contexts of PC. The methyl capture sequencing (methylCap-seq) method was used to map differently methylated regions (DMRs) in pooled samples from ten PC tissues and ten adjacent non-tumor (PN) tissues. A selection of DMRs was validated in an independent set of PC and PN samples using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and methylation sensitive restriction enzyme-based qPCR (MSRE-qPCR). The mRNA and expressed sequence tag (EST) expression of the corresponding genes was investigated using RT-qPCR. Results A total of 1,131 PC-specific and 727 PN-specific hypermethylated DMRs were identified in association with CpG islands (CGIs), including gene-associated CGIs and orphan CGIs; 2,955 PC-specific and 2,386 PN-specific hypermethylated DMRs were associated with gene promoters, including promoters containing or lacking CGIs. Moreover, 1,744 PC-specific and 1,488 PN-specific hypermethylated DMRs were found to be associated with CGIs or CGI shores. These results suggested that aberrant hypermethylation in PC typically occurs in regions surrounding the transcription start site (TSS). The BSP, MSP, MSRE-qPCR, and RT-qPCR data indicated that the aberrant DNA methylation in PC tissue and in PC cell lines was associated with gene (or corresponding EST) expression. Conclusions Our study characterized the genome-wide DNA methylation patterns in PC and identified DMRs that were distributed among various genomic contexts that might influence the expression of corresponding genes or transcripts to promote PC. These DMRs might serve as diagnostic biomarkers or therapeutic targets for PC. PMID:25276247

  5. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders?

    PubMed Central

    Keil, Kimberly P.; Lein, Pamela J.

    2016-01-01

    There is now compelling evidence that gene by environment interactions are important in the etiology of autism spectrum disorders (ASDs). However, the mechanisms by which environmental factors interact with genetic susceptibilities to confer individual risk for ASD remain a significant knowledge gap in the field. The epigenome, and in particular DNA methylation, is a critical gene expression regulatory mechanism in normal and pathogenic brain development. DNA methylation can be influenced by environmental factors such as diet, hormones, stress, drugs, or exposure to environmental chemicals, suggesting that environmental factors may contribute to adverse neurodevelopmental outcomes of relevance to ASD via effects on DNA methylation in the developing brain. In this review, we describe epidemiological and experimental evidence implicating altered DNA methylation as a potential mechanism by which environmental chemicals confer risk for ASD, using polychlorinated biphenyls (PCBs), lead, and bisphenol A (BPA) as examples. Understanding how environmental chemical exposures influence DNA methylation and how these epigenetic changes modulate the risk and/or severity of ASD will not only provide mechanistic insight regarding gene-environment interactions of relevance to ASD but may also suggest potential intervention strategies for these and potentially other neurodevelopmental disorders. PMID:27158529

  6. Mitochondrial regulation of cancer associated nuclear DNA methylation

    SciTech Connect

    Xie Chenghui; Naito, Akihiro; Mizumachi, Takatsugu; Evans, Teresa T.; Douglas, Michael G.; Cooney, Craig A.; Fan Chunyang; Higuchi, Masahiro

    2007-12-21

    The onset and progression of cancer is associated with the methylation-dependent silencing of specific genes, however, the mechanism and its regulation have not been established. We previously demonstrated that reduction of mitochondrial DNA content induces cancer progression. Here we found that mitochondrial DNA-deficient LN{rho}0-8 activates the hypermethylation of the nuclear DNA promoters including the promoter CpG islands of the endothelin B receptor, O{sup 6}-methylguanine-DNA methyltransferase, and E-cadherin. These are unmethylated and the corresponding gene products are expressed in the parental LNCaP containing mitochondrial DNA. The absence of mitochondrial DNA induced DNA methyltransferase 1 expression which was responsible for the methylation patterns observed. Inhibition of DNA methyltransferase eliminated hypermethylation and expressed gene products in LN{rho}0-8. These studies demonstrate loss or reduction of mitochondrial DNA resulted in the induction of DNA methyltransferase 1, hypermethylation of the promoters of endothelin B receptor, O{sup 6}-methylguanine-DNA methyltransferase, and E-cadherin, and reduction of the corresponding gene products.

  7. High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

    PubMed

    Naydenov, Mladen; Baev, Vesselin; Apostolova, Elena; Gospodinova, Nadezhda; Sablok, Gaurav; Gozmanova, Mariyana; Yahubyan, Galina

    2015-02-01

    Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

  8. DNA Methylation Machinery in the Endometrium and Endometrial Cancer.

    PubMed

    Caplakova, Veronika; Babusikova, Eva; Blahovcova, Eva; Balharek, Tomas; Zelieskova, Maria; Hatok, Jozef

    2016-09-01

    During the normal menstrual cycle, endometrial tissue undergoes many biochemical and morphological changes which are under the control of steroid hormone levels. DNA methylation plays a key role in gene expression regulation and influences functional changes in endometrial tissue. Eliminating senescent cells from the functional layer of the endometrium is mediated by apoptotic cell death, which helps maintain cellular homeostasis. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and thus apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients with advanced disease. Despite intensive study of the cancer epigenome, there is missing information about disrupted apoptotic gene regulation in DNA methylation levels. Therefore, it is necessary to spread our knowledge in the field of epigenetics to help us differentiate normal and cancer tissues and detect the early stages of cancer disease. PMID:27630276

  9. 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

    2016-05-01

    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. PMID:26967308

  10. Estimating DNA methylation levels by joint modeling of multiple methylation profiles from microarray data

    PubMed Central

    Wang, Tao; Chen, Mengjie; Zhao, Hongyu

    2015-01-01

    Summary DNA methylation studies have been revolutionized by the recent development of high throughput array-based platforms. Most of the existing methods analyze microarray methylation data on a probe-by-probe basis, ignoring probe-specific effects and correlations among methylation levels at neighboring genomic locations. These methods can potentially miss functionally relevant findings associated with genomic regions. In this paper, we propose a statistical model that allows us to pool information on the same probe across multiple samples to estimate the probe affinity effect, and to borrow strength from the neighboring probe sites to better estimate the methylation values. Using a simulation study we demonstrate that our method can provide accurate model-based estimates. We further use the proposed method to develop a new procedure for detecting differentially methylated regions, and compare it with a state-of-the-art approach via a data application. PMID:26433612

  11. Estimating DNA methylation levels by joint modeling of multiple methylation profiles from microarray data.

    PubMed

    Wang, Tao; Chen, Mengjie; Zhao, Hongyu

    2016-06-01

    DNA methylation studies have been revolutionized by the recent development of high throughput array-based platforms. Most of the existing methods analyze microarray methylation data on a probe-by-probe basis, ignoring probe-specific effects and correlations among methylation levels at neighboring genomic locations. These methods can potentially miss functionally relevant findings associated with genomic regions. In this article, we propose a statistical model that allows us to pool information on the same probe across multiple samples to estimate the probe affinity effect, and to borrow strength from the neighboring probe sites to better estimate the methylation values. Using a simulation study, we demonstrate that our method can provide accurate model-based estimates. We further use the proposed method to develop a new procedure for detecting differentially methylated regions, and compare it with a state-of-the-art approach via a data application.

  12. Differential DNA Methylation Analysis without a Reference Genome.

    PubMed

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  13. Differential DNA Methylation Analysis without a Reference Genome.

    PubMed

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. PMID:26673328

  14. Differential DNA Methylation Analysis without a Reference Genome

    PubMed Central

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C.; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-01-01

    Summary Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. PMID:26673328

  15. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    NASA Astrophysics Data System (ADS)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-06-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  16. Genome-wide Mapping of Nucleosome Positioning and DNA Methylation Within Individual DNA Molecules

    PubMed Central

    Liu, Yaping; Lay, Fides D.; Liang, Gangning; Berman, Benjamin P.; Jones, Peter A.; Kelly, Terry

    2012-01-01

    DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to footprint nucleosome positioning genome-wide using less than 1 million cells, which does not suffer from sequence based biases associated with MNase digestion and retains endogenous DNA methylation information. Using a novel bioinformatics pipeline we identify chromatin configurations associated with a variety of functional genomic loci including distinct promoter types, enhancers, insulators, X-inactivated and imprinted genes. Importantly, DNA methylation and nucleosome positioning information are obtained from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule level that can be used to monitor disease progression and response to therapy.

  17. Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2.

    PubMed

    Kinde, Benyam; Gabel, Harrison W; Gilbert, Caitlin S; Griffith, Eric C; Greenberg, Michael E

    2015-06-01

    DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system. PMID:25739960

  18. Acetylsalicylic acid, aging and coronary artery disease are associated with ABCA1 DNA methylation in men

    PubMed Central

    2014-01-01

    Background Previous studies have suggested that DNA methylation contributes to coronary artery disease (CAD) risk variability. DNA hypermethylation at the ATP-binding cassette transporter A1 (ABCA1) gene, an important modulator of high-density lipoprotein cholesterol and reverse cholesterol transport, has been previously associated with plasma lipid levels, aging and CAD, but the association with CAD has yet to be replicated. Results ABCA1 DNA methylation levels were measured in leucocytes of 88 men using bis-pyrosequencing. We first showed that DNA methylation at the ABCA1 gene promoter locus is associated with aging and CAD occurrence in men (P < 0.05). The latter association is stronger among older men with CAD (≥61 years old; n = 19), who showed at least 4.7% higher ABCA1 DNA methylation levels as compared to younger men with CAD (<61 years old; n = 19) or men without CAD (n = 50; P < 0.001). Higher ABCA1 DNA methylation levels in older men were also associated with higher total cholesterol (r = 0.34, P = 0.03), low-density lipoprotein cholesterol (r = 0.32, P = 0.04) and triglyceride levels (r = 0.26, P = 0.09). Furthermore, we showed that acetylsalicylic acid therapy is associated with 3.6% lower ABCA1 DNA methylation levels (P = 0.006), independent of aging and CAD status of patients. Conclusions This study provides new evidence that the ABCA1 epigenetic profile is associated with CAD and aging, and highlights that epigenetic modifications might be a significant molecular mechanism involved in the pathophysiological processes associated with CAD. Acetylsalicylic acid treatment for CAD prevention might involve epigenetic mechanisms. PMID:25093045

  19. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    NASA Astrophysics Data System (ADS)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  20. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    PubMed Central

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/. PMID:26797014

  1. Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice.

    PubMed

    Kanzleiter, Timo; Jähnert, Markus; Schulze, Gunnar; Selbig, Joachim; Hallahan, Nicole; Schwenk, Robert Wolfgang; Schürmann, Annette

    2015-05-15

    The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice (n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs (P < 0.05, meth diff >5%, coverage >10) in their putative promoter regions. Alignment with gene expression data (n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors (Plexin A2) as well as genes that participate in muscle hypertrophy (Igfbp4) and motor neuron innervation (Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.

  2. Genome-Wide Analysis of DNA Methylation and Cigarette Smoking in a Chinese Population

    PubMed Central

    Zhu, Xiaoyan; Li, Jun; Deng, Siyun; Yu, Kuai; Liu, Xuezhen; Deng, Qifei; Sun, Huizhen; Zhang, Xiaomin; He, Meian; Guo, Huan; Chen, Weihong; Yuan, Jing; Zhang, Bing; Kuang, Dan; He, Xiaosheng; Bai, Yansen; Han, Xu; Liu, Bing; Li, Xiaoliang; Yang, Liangle; Jiang, Haijing; Zhang, Yizhi; Hu, Jie; Cheng, Longxian; Luo, Xiaoting; Mei, Wenhua; Zhou, Zhiming; Sun, Shunchang; Zhang, Liyun; Liu, Chuanyao; Guo, Yanjun; Zhang, Zhihong; Hu, Frank B.; Liang, Liming; Wu, Tangchun

    2016-01-01

    Background: Smoking is a risk factor for many human diseases. DNA methylation has been related to smoking, but genome-wide methylation data for smoking in Chinese populations is limited. Objectives: We aimed to investigate epigenome-wide methylation in relation to smoking in a Chinese population. Methods: We measured the methylation levels at > 485,000 CpG sites (CpGs) in DNA from leukocytes using a methylation array and conducted a genome-wide meta-analysis of DNA methylation and smoking in a total of 596 Chinese participants. We further evaluated the associations of smoking-related CpGs with internal polycyclic aromatic hydrocarbon (PAH) biomarkers and their correlations with the expression of corresponding genes. Results: We identified 318 CpGs whose methylation levels were associated with smoking at a genome-wide significance level (false discovery rate < 0.05), among which 161 CpGs annotated to 123 genes were not associated with smoking in recent studies of Europeans and African Americans. Of these smoking-related CpGs, methylation levels at 80 CpGs showed significant correlations with the expression of corresponding genes (including RUNX3, IL6R, PTAFR, ANKRD11, CEP135 and CDH23), and methylation at 15 CpGs was significantly associated with urinary 2-hydroxynaphthalene, the most representative internal monohydroxy-PAH biomarker for smoking. Conclusion: We identified DNA methylation markers associated with smoking in a Chinese population, including some markers that were also correlated with gene expression. Exposure to naphthalene, a byproduct of tobacco smoke, may contribute to smoking-related methylation. Citation: Zhu X, Li J, Deng S, Yu K, Liu X, Deng Q, Sun H, Zhang X, He M, Guo H, Chen W, Yuan J, Zhang B, Kuang D, He X, Bai Y, Han X, Liu B, Li X, Yang L, Jiang H, Zhang Y, Hu J, Cheng L, Luo X, Mei W, Zhou Z, Sun S, Zhang L, Liu C, Guo Y, Zhang Z, Hu FB, Liang L, Wu T. 2016. Genome-wide analysis of DNA methylation and cigarette smoking in Chinese. Environ

  3. Forensic DNA methylation profiling--potential opportunities and challenges.

    PubMed

    Vidaki, Athina; Daniel, Barbara; Court, Denise Syndercombe

    2013-09-01

    Investigating the DNA sequence is the most powerful tool that can be employed in forensic genetics for the identification of an individual, or to determine specific ethnic and phenotypic characteristics. However, there are also other heritable changes in gene function or cellular phenotype which are caused by mechanisms other than differences in the DNA sequence itself. Over the last decade it has become evident that epigenetic markers can be of substantial forensic significance. The determination of possible alterations in DNA methylation patterns could aid various forensic investigations, such as differentiating monozygotic twins, identifying the tissue source or determining the age of tissue donors. This review aims to give a brief overview of the possible advantages of forensic DNA methylation profiling and sheds light on the limitations of this approach.

  4. DNA methylation provides insight into intergenerational risk for preterm birth in African Americans

    PubMed Central

    Parets, Sasha E; Conneely, Karen N; Kilaru, Varun; Menon, Ramkumar; Smith, Alicia K

    2015-01-01

    African Americans are at increased risk for spontaneous preterm birth (PTB). Though PTB is heritable, genetic studies have not identified variants that account for its intergenerational risk, prompting the hypothesis that epigenetic factors may also contribute. The objective of this study was to evaluate DNA methylation from maternal leukocytes to identify patterns specific to PTB and its intergenerational risk. DNA from peripheral leukocytes from African American women that delivered preterm (24–34 weeks; N = 16) or at term (39–41 weeks; N = 24) was assessed for DNA methylation using the HumanMethylation450 BeadChip. In maternal samples, 17,829 CpG sites associated with PTB, but no CpG site remained associated after correction for multiple comparisons. Examination of paired maternal-fetal samples identified 5,171 CpG sites in which methylation of maternal samples correlated with methylation of her respective fetus (FDR < 0.05). These correlated sites were enriched for association with PTB in maternal leukocytes. The majority of correlated CpG sites could be attributed to one or more genetic variants. They were also significantly more likely to be in genes involved in metabolic, cardiovascular, and immune pathways, suggesting a role for genetic and environmental contributions to PTB risk and chronic disease. The results of this study may provide insight into the factors underlying intergenerational risk for PTB and its consequences. PMID:26090903

  5. Genome-wide analysis of DNA methylation in the sexual stage of the insect pathogenic fungus Cordyceps militaris.

    PubMed

    Wang, Yu-long; Wang, Zhang-xun; Liu, Chun; Wang, Si-bao; Huang, Bo

    2015-12-01

    DNA methylation is a basic epigenetic mechanism found in eukaryotes, but its patterns and roles vary significantly among diverse taxa. In fungi, DNA methylation has various effects on diverse biological processes. However, its function in the sexual development of fungi remains unclear. Cordyceps militaris, readily performs sexual reproduction and thus provides a remarkably rich model for understanding epigenetic processes in sexual development. Here, we surveyed the methylome of C. militaris at single-base resolution to assess DNA methylation patterns during sexual development using genomic bisulfite sequencing (BS-Seq). The results showed that approximately 0.4 % of cytosines are methylated, similar to the DNA methylation level (0.39 %) during asexual development. Importantly, we found that DNA methylation in the fungi undergoes global reprogramming during fungal development. Moreover, RNA-Seq analysis indicated that the differentially methylated regions (DMRs) have no correlation with the genes that have roles during fungal sexual development in C. militaris. These results provide a comprehensive characterization of DNA methylation in the sexual development of C. militaris, which will contribute to future investigations of epigenetics in fungi. PMID:26615747

  6. The key culprit in the pathogenesis of systemic lupus erythematosus: Aberrant DNA methylation.

    PubMed

    Wu, Haijing; Zhao, Ming; Tan, Lina; Lu, Qianjin

    2016-07-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple organ involvement. It is characterized by abundant autoantibodies that form immune complex with autoantigens and deposit in organs and cause tissue damage by inducing inflammation. The pathogenesis of SLE has been intensively studied but remains unclear. B and T lymphocyte abnormalities, dysregulation of apoptosis, defects in the clearance of apoptotic materials, and various genetic and epigenetic factors are believed to contribute to the initiation and development of SLE. The up-to-date research findings point to the relationship between abnormal DNA methylation and SLE, which has attracted considerable interest worldwide. Besides the global hypomethylation on lupus T and B cells, the gene specific and site-specific methylation has been identified and documented to be responsible for SLE. The purpose of this review was to present and summarize the association between aberrant DNA methylation of immune cells and SLE, the possible mechanisms of immune dysfunction caused by DNA methylation, and to better understand the roles of aberrant DNA methylation in the initiation and development of SLE and to provide an insight into the related diagnosis biomarkers and therapeutic options in SLE.

  7. DNA Methylation as Surrogate Marker For Gastric Cancer

    PubMed Central

    Oh, Jung-Hwan; Jung, Sung-Hoon; Hong, Seung-Jin; Rhyu, Mun-Gan

    2015-01-01

    Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern. PMID:26473155

  8. DNA methylation presents distinct binding sites for human transcription factors.

    PubMed

    Hu, Shaohui; Wan, Jun; Su, Yijing; Song, Qifeng; Zeng, Yaxue; Nguyen, Ha Nam; Shin, Jaehoon; Cox, Eric; Rho, Hee Sool; Woodard, Crystal; Xia, Shuli; Liu, Shuang; Lyu, Huibin; Ming, Guo-Li; Wade, Herschel; Song, Hongjun; Qian, Jiang; Zhu, Heng

    2013-01-01

    DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001. PMID:24015356

  9. DNA methylation status predicts cell type-specific enhancer activity

    PubMed Central

    Wiench, Malgorzata; John, Sam; Baek, Songjoon; Johnson, Thomas A; Sung, Myong-Hee; Escobar, Thelma; Simmons, Catherine A; Pearce, Kenneth H; Biddie, Simon C; Sabo, Pete J; Thurman, Robert E; Stamatoyannopoulos, John A; Hager, Gordon L

    2011-01-01

    Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR–DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors. PMID:21701563

  10. siRNA-directed DNA Methylation in Plants.

    PubMed

    Xie, Meng; Yu, Bin

    2015-02-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  11. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Ponts, Nadia; Fu, Lijuan; Harris, Elena Y.; Zhang, Jing; Chung, Duk-Won D.; Cervantes, Michael C.; Prudhomme, Jacques; Atanasova-Penichon, Vessela; Zehraoui, Enric; Bunnik, Evelien; Rodrigues, Elisandra M.; Lonardi, Stefano; Hicks, Glenn R.; Wang, Yinsheng; Le Roch, Karine G.

    2014-01-01

    SUMMARY Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase, PfDNMT, that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated, in which only one DNA strand is methylated, and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated and transcript levels correlate with intra-exonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and uniqueness of PfDNMT suggest that the methylation pathway is a potential target for anti-malarial strategies. PMID:24331467

  12. Analysis of DNA methylation in pancreatic cancer: an update.

    PubMed

    Pilarsky, Christian; Grützmann, Robert

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor and the fourth common cause of cancer death in the Western world. The lack of effective therapeutic strategies is due to the late diagnosis of this disease. Methylation markers could improve early detection and help in the surveillance of PDAC after treatment. Analysis of hypermethylation in the tumor tissue might help to identify new therapeutic strategies and aid in the understanding of the pathophysiological changes occurring in pancreatic cancer. There are several methods for the detection of methylated events, but methylation-specific PCR (MSP-PCR) is the method of choice if a small number of genes will be tested in a larger set of patients samples. After isolation of the DNA by standard procedure, the DNA is then modified using sodium bisulfide. PMID:25421660

  13. The interplay between DNA methylation, folate and neurocognitive development.

    PubMed

    Irwin, Rachelle E; Pentieva, Kristina; Cassidy, Tony; Lees-Murdock, Diane J; McLaughlin, Marian; Prasad, Girijesh; McNulty, Helene; Walsh, Colum P

    2016-06-01

    DNA methylation provides an attractive possible means for propagating the effects of environmental inputs during fetal life and impacting subsequent adult mental health, which is leading to increasing collaboration between molecular biologists, nutritionists and psychiatrists. An area of interest is the potential role of folate, not just in neural tube closure in early pregnancy, but in later major neurodevelopmental events, with consequences for later sociocognitive maturation. Here, we set the scene for recent discoveries by reviewing the major events of neural development during fetal life, with an emphasis on tissues and structures where dynamic methylation changes are known to occur. Following this, we give an indication of some of the major classes of genes targeted by methylation and important for neurological and behavioral development. Finally, we highlight some cognitive disorders where methylation changes are implicated as playing an important role. PMID:27319574

  14. Differences of DNA methylation profiles between monozygotic twins' blood samples.

    PubMed

    Li, Chengtao; Zhao, Shumin; Zhang, Na; Zhang, Suhua; Hou, Yiping

    2013-09-01

    Monozygotic twins (MZs) share an identical genomic sequence, which makes it impossible to discriminate one another with conventional genetic markers like STRs. On the other hand, phenotypic discordance between MZs implies the existence of different epigenetic characteristics. DNA methylation, an essential epigenetic modification, however, might be a potential biomarker to solve the forensic puzzle. In this study, we examined 22 pairs of MZs with a methylation BeadChip including 27,578 CpG sites. The results suggested that MZs exhibited remarkable differences of genome-wide 5-methylcytosine. According to a set of criteria of selection, 92 CpG sites with significant differences of methylation status within MZs were identified from the global epigenome. In conclusion, this pilot study suggested that CpG methylation profile could be a useful biomarker in individual identification of MZs. PMID:23649773

  15. Dnmt2-dependent methylomes lack defined DNA methylation patterns

    PubMed Central

    Raddatz, Günter; Guzzardo, Paloma M.; Olova, Nelly; Fantappié, Marcelo Rosado; Rampp, Markus; Schaefer, Matthias; Reik, Wolf; Hannon, Gregory J.; Lyko, Frank

    2013-01-01

    Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms. PMID:23641003

  16. Reversible DNA methylation regulates seasonal photoperiodic time measurement

    PubMed Central

    Stevenson, Tyler J.; Prendergast, Brian J.

    2013-01-01

    In seasonally breeding vertebrates, changes in day length induce categorically distinct behavioral and reproductive phenotypes via thyroid hormone-dependent mechanisms. Winter photoperiods inhibit reproductive neuroendocrine function but cannot sustain this inhibition beyond 6 mo, ensuring vernal reproductive recrudescence. This genomic plasticity suggests a role for epigenetics in the establishment of seasonal reproductive phenotypes. Here, we report that DNA methylation of the proximal promoter for the type III deiodinase (dio3) gene in the hamster hypothalamus is reversible and critical for photoperiodic time measurement. Short photoperiods and winter-like melatonin inhibited hypothalamic DNA methyltransferase expression and reduced dio3 promoter DNA methylation, which up-regulated dio3 expression and induced gonadal regression. Hypermethylation attenuated reproductive responses to short photoperiods. Vernal refractoriness to short photoperiods reestablished summer-like methylation of the dio3 promoter, dio3 expression, and reproductive competence, revealing a dynamic and reversible mechanism of DNA methylation in the mammalian brain that plays a central role in physiological orientation in time. PMID:24067648

  17. Ancestry dependent DNA methylation and influence of maternal nutrition.

    PubMed

    Mozhui, Khyobeni; Smith, Alicia K; Tylavsky, Frances A

    2015-01-01

    There is extensive variation in DNA methylation between individuals and ethnic groups. These differences arise from a combination of genetic and non-genetic influences and potential modifiers include nutritional cues, early life experience, and social and physical environments. Here we compare genome-wide DNA methylation in neonatal cord blood from African American (AA; N = 112) and European American (EA; N = 91) participants of the CANDLE Study (Conditions Affecting Neurocognitive Development and Learning in Early Childhood). Our goal is to determine if there are replicable ancestry-specific methylation patterns that may implicate risk factors for diseases that have differential prevalence between populations. To identify the most robust ancestry-specific CpG sites, we replicate our results in lymphoblastoid cell lines from Yoruba African and CEPH European panels of HapMap. We also evaluate the influence of maternal nutrition--specifically, plasma levels of vitamin D and folate during pregnancy--on methylation in newborns. We define stable ancestry-dependent methylation of genes that include tumor suppressors and cell cycle regulators (e.g., APC, BRCA1, MCC). Overall, there is lower global methylation in African ancestral groups. Plasma levels of 25-hydroxy vitamin D are also considerably lower among AA mothers and about 60% of AA and 40% of EA mothers have concentrations below 20 ng/ml. Using a weighted correlation analysis, we define a network of CpG sites that is jointly modulated by ancestry and maternal vitamin D. Our results show that differences in DNA methylation patterns are remarkably stable and maternal micronutrients can exert an influence on the child epigenome. PMID:25742137

  18. DNA methylation and childhood asthma in the inner city

    PubMed Central

    Yang, Ivana V.; Pedersen, Brent S.; Liu, Andrew; O’Connor, George T.; Teach, Stephen J.; Kattan, Meyer; Misiak, Rana Tawil; Gruchalla, Rebecca; Steinbach, Suzanne F.; Szefler, Stanley J.; Gill, Michelle A.; Calatroni, Agustin; David, Gloria; Hennessy, Corinne E.; Davidson, Elizabeth J.; Zhang, Weiming; Gergen, Peter; Togias, Alkis; Busse, William W.; Schwartz, David A.

    2015-01-01

    Background Epigenetic marks are heritable, influenced by the environment, direct the maturation of T lymphocytes, and in mice enhance the development of allergic airway disease. Thus it is important to define epigenetic alterations in asthmatic populations. Objective We hypothesize that epigenetic alterations in circulating PBMCs are associated with allergic asthma. Methods We compared DNA methylation patterns and gene expression in inner-city children with persistent atopic asthma versus healthy control subjects by using DNA and RNA from PBMCs. Results were validated in an independent population of asthmatic patients. Results Comparing asthmatic patients (n = 97) with control subjects (n = 97), we identified 81 regions that were differentially methylated. Several immune genes were hypomethylated in asthma, including IL13, RUNX3, and specific genes relevant to T lymphocytes (TIGIT). Among asthmatic patients, 11 differentially methylated regions were associated with higher serum IgE concentrations, and 16 were associated with percent predicted FEV1. Hypomethylated and hypermethylated regions were associated with increased and decreased gene expression, respectively (P < .6 × 10−11 for asthma and P < .01 for IgE). We further explored the relationship between DNA methylation and gene expression using an integrative analysis and identified additional candidates relevant to asthma (IL4 and ST2). Methylation marks involved in T-cell maturation (RUNX3), TH2 immunity (IL4), and oxidative stress (catalase) were validated in an independent asthmatic cohort of children living in the inner city. Conclusions Our results demonstrate that DNA methylation marks in specific gene loci are associated with asthma and suggest that epigenetic changes might play a role in establishing the immune phenotype associated with asthma. PMID:25769910

  19. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

    PubMed Central

    Maresca, Alessandra; Zaffagnini, Mirko; Caporali, Leonardo; Carelli, Valerio; Zanna, Claudia

    2015-01-01

    Autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E) are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5)-methyltransferase 1 (DNMT1). DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy, and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA) suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however, mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is regulated and

  20. Aberrant DNA Methylation of rDNA and PRIMA1 in Borderline Personality Disorder

    PubMed Central

    Teschler, Stefanie; Gotthardt, Julia; Dammann, Gerhard; Dammann, Reinhard H.

    2016-01-01

    Borderline personality disorder (BPD) is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5′-external transcribed spacer/5′ETS) and the promoter of the proline rich membrane anchor 1 gene (PRIMA1) in peripheral blood samples of 24 female patients (mean age (33 ± 11) years) diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7) years). A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5′ETS were significantly lower (0.9-fold) in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5′ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD. PMID:26742039

  1. Aberrant DNA Methylation of rDNA and PRIMA1 in Borderline Personality Disorder.

    PubMed

    Teschler, Stefanie; Gotthardt, Julia; Dammann, Gerhard; Dammann, Reinhard H

    2016-01-01

    Borderline personality disorder (BPD) is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5'-external transcribed spacer/5'ETS) and the promoter of the proline rich membrane anchor 1 gene (PRIMA1) in peripheral blood samples of 24 female patients (mean age (33 ± 11) years) diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7) years). A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5'ETS were significantly lower (0.9-fold) in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5'ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD. PMID:26742039

  2. Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity.

    PubMed

    Wang, Gang Lin; Luo, Hong Qun; Li, Nian Bing

    2014-09-21

    A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit of 0.25 U mL(-1), which might make this method a good candidate for monitoring DNA methylation in the future. PMID:25028809

  3. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  4. DNA methylation and differential gene regulation in photoreceptor cell death

    PubMed Central

    Farinelli, P; Perera, A; Arango-Gonzalez, B; Trifunovic, D; Wagner, M; Carell, T; Biel, M; Zrenner, E; Michalakis, S; Paquet-Durand, F; Ekström, P A R

    2014-01-01

    Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP. PMID:25476906

  5. Arsenic exposure and DNA methylation among elderly men

    PubMed Central

    Lambrou, Angeliki; Baccarelli, Andrea; Wright, Robert O.; Weisskopf, Marc; Bollati, Valentina; Amarasiriwardena, Chitra; Vokonas, Pantel; Schwartz, Joel

    2012-01-01

    BACKGROUND Arsenic exposure has been linked to epigenetic modifications such as DNA methylation in in vitro and animal studies. This association has also been explored in highly exposed human populations, but studies among populations environmentally exposed to low arsenic levels are lacking. METHODS We evaluated the association between exposure to arsenic, measured in toenails, and blood DNA methylation in Alu and Long Interspersed Nucleotide Element-1 (LINE-1) repetitive elements in elderly men environmentally exposed to low levels of arsenic. We also explored potential effect modification by plasma folate, cobalamin (vitamin B12), and pyridoxine (vitamin B6). The study population was 581 participants from the Normative Aging Study in Boston, of whom 434, 140, and 7 had 1, 2, and 3 visits, respectively, between 1999-2002 and 2006-2007. We used mixed-effects models and included interaction terms to assess potential effect modification by nutritional factors. RESULTS There was a trend of increasing Alu and decreasing LINE-1 DNA methylation as arsenic exposure increased. In subjects with plasma folate below the median (< 14.1 ng/ml), arsenic was positively associated with Alu DNA methylation (β=0.08 [95% confidence interval = 0.03 to 0.13] for one interquartile range [0.06μg/g] increase in arsenic) while a negative association was observed in subjects with plasma folate above the median (β=-0.08 [-0.17 to 0.01]). CONCLUSIONS We found an association between arsenic exposure and DNA methylation in Alu repetitive elements that varied by folate level. This suggests a potential role for nutritional factors in arsenic toxicity. PMID:22833016

  6. Electrostatic contribution to the bending of DNA.

    PubMed

    Sivolob, A; Khrapunov, S N

    1997-09-01

    A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.

  7. methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles.

    PubMed

    Akalin, Altuna; Kormaksson, Matthias; Li, Sheng; Garrett-Bakelman, Francine E; Figueroa, Maria E; Melnick, Ari; Mason, Christopher E

    2012-01-01

    DNA methylation is a chemical modification of cytosine bases that is pivotal for gene regulation, cellular specification and cancer development. Here, we describe an R package, methylKit, that rapidly analyzes genome-wide cytosine epigenetic profiles from high-throughput methylation and hydroxymethylation sequencing experiments. methylKit includes functions for clustering, sample quality visualization, differential methylation analysis and annotation features, thus automating and simplifying many of the steps for discerning statistically significant bases or regions of DNA methylation. Finally, we demonstrate methylKit on breast cancer data, in which we find statistically significant regions of differential methylation and stratify tumor subtypes. methylKit is available at http://code.google.com/p/methylkit. PMID:23034086

  8. Heritability and Reversibility of DNA Methylation Induced by in vitro Grafting between Brassica juncea and B. oleracea

    PubMed Central

    Cao, Liwen; Yu, Ningning; Li, Junxing; Qi, Zhenyu; Wang, Dan; Chen, Liping

    2016-01-01

    Grafting between tuber mustard and red cabbage produced a chimeric shoot apical meristem (SAM) of TTC, consisting of Layers I and II from Tuber mustard and Layer III from red Cabbage. Phenotypic variations, which mainly showed in leaf shape and SAM, were observed in selfed progenies GSn (GS = grafting-selfing, n = generations) of TTC. Here the heritability of phenotypic variation and its association with DNA methylation changes in GSn were investigated. Variation in leaf shape was found to be stably inherited to GS5, but SAM variation reverted over generations. Subsequent measurement of DNA methylation in GS1 revealed 5.29–6.59% methylation changes compared with tuber mustard (TTT), and 31.58% of these changes were stably transmitted to GS5, but the remainder reverted to the original status over generations, suggesting grafting-induced DNA methylation changes could be both heritable and reversible. Sequence analysis of differentially methylated fragments (DMFs) revealed methylation mainly changed within transposons and exon regions, which further affected the expression of genes, including flowering time- and gibberellin response-related genes. Interestingly, DMFs could match differentially expressed siRNA of GS1, GS3 and GS5, indicating that grafting-induced DNA methylation could be directed by siRNA changes. These results suggest grafting-induced DNA methylation may contribute to phenotypic variations induced by grafting. PMID:27257143

  9. Heritability and Reversibility of DNA Methylation Induced by in vitro Grafting between Brassica juncea and B. oleracea.

    PubMed

    Cao, Liwen; Yu, Ningning; Li, Junxing; Qi, Zhenyu; Wang, Dan; Chen, Liping

    2016-01-01

    Grafting between tuber mustard and red cabbage produced a chimeric shoot apical meristem (SAM) of TTC, consisting of Layers I and II from Tuber mustard and Layer III from red Cabbage. Phenotypic variations, which mainly showed in leaf shape and SAM, were observed in selfed progenies GSn (GS = grafting-selfing, n = generations) of TTC. Here the heritability of phenotypic variation and its association with DNA methylation changes in GSn were investigated. Variation in leaf shape was found to be stably inherited to GS5, but SAM variation reverted over generations. Subsequent measurement of DNA methylation in GS1 revealed 5.29-6.59% methylation changes compared with tuber mustard (TTT), and 31.58% of these changes were stably transmitted to GS5, but the remainder reverted to the original status over generations, suggesting grafting-induced DNA methylation changes could be both heritable and reversible. Sequence analysis of differentially methylated fragments (DMFs) revealed methylation mainly changed within transposons and exon regions, which further affected the expression of genes, including flowering time- and gibberellin response-related genes. Interestingly, DMFs could match differentially expressed siRNA of GS1, GS3 and GS5, indicating that grafting-induced DNA methylation could be directed by siRNA changes. These results suggest grafting-induced DNA methylation may contribute to phenotypic variations induced by grafting. PMID:27257143

  10. RNA Splicing Factors and RNA-Directed DNA Methylation

    PubMed Central

    Huang, Chao-Feng; Zhu, Jian-Kang

    2014-01-01

    RNA-directed histone and/or DNA modification is a conserved mechanism for the establishment of epigenetic marks from yeasts and plants to mammals. The heterochromation formation in yeast is mediated by RNAi-directed silencing mechanism, while the establishment of DNA methylation in plants is through the RNA-directed DNA methylation (RdDM) pathway. Recently, splicing factors are reported to be involved in both RNAi-directed heterochromatin formation in yeast and the RdDM pathway in plants. In yeast, splicing factors may provide a platform for facilitating the siRNA generation through an interaction with RDRC and thereby affect the heterochromatin formation, whereas in plants, various splicing factors seem to act at different steps in the RdDM pathway. PMID:24833507

  11. Transition of LINE-1 DNA methylation status and altered expression in first and third trimester placentas.

    PubMed

    He, Zhi-ming; Li, Jinping; Hwa, Yi Lisa; Brost, Brian; Fang, Qun; Jiang, Shi-Wen

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions.

  12. Transition of LINE-1 DNA Methylation Status and Altered Expression in First and Third Trimester Placentas

    PubMed Central

    Hwa, Yi Lisa; Brost, Brian; Fang, Qun; Jiang, Shi-Wen

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression, genomic DNA stability, cell proliferation, and malignant transformation. Common cellular features including fast tissue expansion, invasive growth, and active angiogenesis, have been noticed between placental development and tumorigenesis by many investigators. While the DNA hypomethylation and transcriptional activation of LINE-1 has been found to be a feature of tumorigenesis, it is not clear if similar changes could be involved in placental development. In this study, we assessed LINE-1 methylation in human placentas from different gestational ages and observed a significant decrease of LINE-1 methylation levels in third trimester placentas compared to first trimester placentas. Accompanying with this change is the significantly increased LINE-1 mRNA levels in third trimester placentas. Since no global DNA methylation change was detected between first and third trimesters, LINE-1 methylation changes appeared to be a specific epigenetic entity contributing to placental development. Indeed, further analyses showed that LINE-1 upregulation was correlated with higher levels of PCNA, suggesting a link between LINE-1 activation and fast proliferation of certain cellular components in third trimester placentas. Measurement of the DNMT1, DNMT3A, and DNMT3B expression found a significant reduction of DNMT3B between third and first trimesters, pointing to the possible involvement of this enzyme in the regulation of LINE-1 methylation. Taken together these results provided evidence for a dynamic temporal regulation of LINE-1 methylation and activation during placental development. These studies have laid a foundation for future investigation on the function of LINE-1 expression in human placenta under different patho-physiological conditions. PMID:24821186

  13. Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

    PubMed

    Jenkins, Timothy G; Aston, Kenneth I; Pflueger, Christian; Cairns, Bradley R; Carrell, Douglas T

    2014-07-01

    Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists. PMID:25010591

  14. DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels

    PubMed Central

    Pfeiffer, Liliane; Wahl, Simone; Pilling, Luke C.; Reischl, Eva; Sandling, Johanna K.; Kunze, Sonja; Holdt, Lesca M.; Kretschmer, Anja; Schramm, Katharina; Adamski, Jerzy; Klopp, Norman; Illig, Thomas; Hedman, Åsa K.; Roden, Michael; Hernandez, Dena G.; Singleton, Andrew B.; Thasler, Wolfgang E.; Grallert, Harald; Gieger, Christian; Herder, Christian; Teupser, Daniel; Meisinger, Christa; Spector, Timothy D.; Kronenberg, Florian; Prokisch, Holger; Melzer, David; Peters, Annette; Deloukas, Panos; Ferrucci, Luigi; Waldenberger, Melanie

    2016-01-01

    Background Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases. PMID:25583993

  15. N6-methyladenine: the other methylated base of DNA.

    PubMed

    Ratel, David; Ravanat, Jean-Luc; Berger, François; Wion, Didier

    2006-03-01

    Contrary to mammalian DNA, which is thought to contain only 5-methylcytosine (m5C), bacterial DNA contains two additional methylated bases, namely N6-methyladenine (m6A), and N4-methylcytosine (m4C). However, if the main function of m5C and m4C in bacteria is protection against restriction enzymes, the roles of m6A are multiple and include, for example, the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA. Interestingly, even if adenine methylation is usually considered a bacterial DNA feature, the presence of m6A has been found in protist and plant DNAs. Furthermore, indirect evidence suggests the presence of m6A in mammal DNA, raising the possibility that this base has remained undetected due to the low sensitivity of the analytical methods used. This highlights the importance of considering m6A as the sixth element of DNA. PMID:16479578

  16. Alterations in cardiac DNA methylation in human dilated cardiomyopathy

    PubMed Central

    Haas, Jan; Frese, Karen S; Park, Yoon Jung; Keller, Andreas; Vogel, Britta; Lindroth, Anders M; Weichenhan, Dieter; Franke, Jennifer; Fischer, Simon; Bauer, Andrea; Marquart, Sabine; Sedaghat-Hamedani, Farbod; Kayvanpour, Elham; Köhler, Doreen; Wolf, Nadine M; Hassel, Sarah; Nietsch, Rouven; Wieland, Thomas; Ehlermann, Philipp; Schultz, Jobst-Hendrik; Dösch, Andreas; Mereles, Derliz; Hardt, Stefan; Backs, Johannes; Hoheisel, Jörg D; Plass, Christoph; Katus, Hugo A; Meder, Benjamin

    2013-01-01

    Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome-wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass-spectrometric analysis and bisulphite-sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure. PMID:23341106

  17. Histone H1 Limits DNA Methylation in Neurospora crassa

    PubMed Central

    Seymour, Michael; Ji, Lexiang; Santos, Alex M.; Kamei, Masayuki; Sasaki, Takahiko; Basenko, Evelina Y.; Schmitz, Robert J.; Zhang, Xiaoyu; Lewis, Zachary A.

    2016-01-01

    Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the ΔhH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the ΔhH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa. PMID:27172195

  18. Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules

    PubMed Central

    Kelly, Theresa K.; Liu, Yaping; Lay, Fides D.; Liang, Gangning; Berman, Benjamin P.; Jones, Peter A.

    2012-01-01

    DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy. PMID:22960375

  19. DNA methylation profiles in type 1 diabetes twins point to strong epigenetic effects on etiology.

    PubMed

    Stefan, Mihaela; Zhang, Weijia; Concepcion, Erlinda; Yi, Zhengzi; Tomer, Yaron

    2014-05-01

    Type 1 diabetes (T1D) shows ∼40% concordance rate in monozygotic twins (MZ) suggesting a role for environmental factors and/or epigenetic modifications in the etiology of the disease. The aim of our study was to dissect the contribution of epigenetic factors, particularly, DNA methylation (DNAm), to the incomplete penetrance of T1D. We performed DNAm profiling in lymphocyte cell lines from 3 monozygotic (MZ) twin pairs discordant for T1D and 6 MZ twin pairs concordant for the disease using HumanMethylation27 BeadChip. This assay assesses the methylation state of 27,578 CpG sites, mostly located within proximal promoter regions. We identified 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. Functional annotation of the genes with distinct CpG methylation profiles in T1D samples showed differential DNAm of immune response and defense response pathways between affected and unaffected twins. Integration of DNAm data with GWAS data mapped several known T1D associated genes, HLA, INS, IL-2RB, CD226, which showed significant differences in DNAm between affected and unaffected of twins. Our findings suggest that abnormalities of DNA methylation patterns, known to regulate gene transcription, may be involved in the pathogenesis of T1D. PMID:24210274

  20. Investigating the effects of the presence of foreign DNA on DNA methylation and DNA repair events in cultured eukaryotic cells.

    PubMed

    Du Toit, J; van der Westhuizen, F H; Pretorius, P J

    2013-01-01

    Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.

  1. Gamma Irradiation Does Not Induce Detectable Changes in DNA Methylation Directly following Exposure of Human Cells

    PubMed Central

    Lahtz, Christoph; Bates, Steven E.; Jiang, Yong; Li, Arthur X.; Wu, Xiwei; Hahn, Maria A.; Pfeifer, Gerd P.

    2012-01-01

    Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 (137Cs) radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis) or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells. PMID:23024770

  2. Abnormalities of the DNA methylation mark and its machinery: an emerging cause of neurologic dysfunction.

    PubMed

    Weissman, Jacqueline; Naidu, Sakkubai; Bjornsson, Hans T

    2014-07-01

    Recently, Mendelian disorders of the DNA methylation machinery have been described which demonstrate the complex roles of epigenetics in neurodevelopment and disease. For example, defects of DNMT1, the maintenance methyltransferase, lead to adult-onset progressive neurologic disorders, whereas defects of the de novo methyltransferases DNMT3A and DNMT3B lead to nonprogressive neurodevelopmental conditions. Furthermore, patients with DNMT3A deficiency demonstrate overgrowth, a feature common to disorders of histone machinery and imprinting disorders, highlighting the interconnectedness of the many epigenetic layers. Disorders of the DNA methylation machinery include both the aforementioned "writers" and also the "readers" of the methyl mark, such as MeCP2, the cause of Rett syndrome. Any dosage disruption, either haploinsufficiency or overexpression of DNA methylation machinery leads to widespread gene expression changes in trans, disrupting expression of a subset of target genes that contribute to individual disease phenotypes. In contrast, classical imprinting disorders such as Angelman syndrome have been thought generally to cause epigenetic dysregulation in cis. However, the recent description of multilocus methylation disorders challenges this generalization. Here, in addition to summarizing recent developments in identifying the pathogenesis of these diseases, we highlight clinical considerations and some unexpected therapeutic opportunities, such as topoisomerase inhibitors for classical imprinting disorders. PMID:25192503

  3. DNA Methylation Affects the SP1-regulated Transcription of FOXF2 in Breast Cancer Cells.

    PubMed

    Tian, Hong-Pan; Lun, Shu-Min; Huang, Huan-Jing; He, Rui; Kong, Peng-Zhou; Wang, Qing-Shan; Li, Xiao-Qing; Feng, Yu-Mei

    2015-07-31

    FOXF2 (forkhead box F2) is a mesenchyme-specific transcription factor that plays a critical role in tissue homeostasis through the maintenance of epithelial polarity. In a previous study, we demonstrated that FOXF2 is specifically expressed in basal-like breast cancer (BLBC) cells and functions as an epithelial-mesenchymal transition suppressor. FOXF2 deficiency enhances the metastatic ability of BLBC cells through activation of the epithelial-mesenchymal transition program, but reduces cell proliferation. In this study, we demonstrate that CpG island methylation of the FOXF2 proximal promoter region is involved in the regulatory mechanism of the subtype-specific expression of FOXF2 in breast cancer cells. DNMT1, DNMT3A, and DNMT3B commonly or individually contributed to this DNA methylation in different breast cancer cells. SP1 regulated the transcriptional activity of FOXF2 through direct binding to the proximal promoter region, whereas this binding was abrogated through DNA methylation. FOXF2 mediated the SP1-regulated suppression of progression and promotion of proliferation of non-methylated BLBC cells. Thus, we conclude that the subtype-specific expression and function of FOXF2 in breast cancer cells are regulated through the combined effects of DNA methylation and SP1 transcriptional regulation.

  4. Abnormalities of the DNA Methylation Mark and Its Machinery: An Emerging Cause of Neurologic Dysfunction

    PubMed Central

    Weissman, Jacqueline; Naidu, Sakkubai; Bjornsson, Hans T.

    2015-01-01

    Recently, Mendelian disorders of the DNA methylation machinery have been described which demonstrate the complex roles of epigenetics in neurodevelopment and disease. For example, defects of DNMT1, the maintenance methyltransferase, lead to adult-onset progressive neurologic disorders, whereas defects of the de novo methyltransferases DNMT3A and DNMT3B lead to nonprogressive neurodevelopmental conditions. Furthermore, patients with DNMT3A deficiency demonstrate overgrowth, a feature common to disorders of histone machinery and imprinting disorders, highlighting the interconnectedness of the many epigenetic layers. Disorders of the DNA methylation machinery include both the aforementioned “writers” and also the “readers” of the methyl mark, such as MeCP2, the cause of Rett syndrome. Any dosage disruption, either haploinsufficiency or overexpression of DNA methylation machinery leads to wide-spread gene expression changes in trans, disrupting expression of a subset of target genes that contribute to individual disease phenotypes. In contrast, classical imprinting disorders such as Angelman syndrome have been thought generally to cause epigenetic dysregulation in cis. However, the recent description of multilocus methylation disorders challenges this generalization. Here, in addition to summarizing recent developments in identifying the pathogenesis of these diseases, we highlight clinical considerations and some unexpected therapeutic opportunities, such as to poisomerase inhibitors for classical imprinting disorders. PMID:25192503

  5. Dissecting the precise role of H3K9 methylation in crosstalk with DNA maintenance methylation in mammals.

    PubMed

    Zhao, Qian; Zhang, Jiqin; Chen, Ruoyu; Wang, Lina; Li, Bo; Cheng, Hao; Duan, Xiaoya; Zhu, Haijun; Wei, Wei; Li, Jiwen; Wu, Qihan; Han, Jing-Dong J; Yu, Wenqiang; Gao, Shaorong; Li, Guohong; Wong, Jiemin

    2016-01-01

    In mammals it is unclear if UHRF1-mediated DNA maintenance methylation by DNMT1 is strictly dependent on histone H3K9 methylation. Here we have generated an Uhrf1 knockin (KI) mouse model that specifically abolishes the H3K9me2/3-binding activity of Uhrf1. The homozygous Uhrf1 KI mice are viable and fertile, and exhibit ∼10% reduction of DNA methylation in various tissues. The reduced DNA methylation occurs globally in the genome and does not restrict only to the H3K9me2/3 enriched repetitive sequences. In vitro UHRF1 binds with higher affinity to reconstituted nucleosome with hemi-methylated CpGs than that with H3K9me2/3, although it binds cooperatively to nucleosome with both modifications. We also show that the nucleosome positioning affects the binding of methylated DNA by UHRF1. Thus, while our study supports a role for H3K9 methylation in promoting DNA methylation, it demonstrates for the first time that DNA maintenance methylation in mammals is largely independent of H3K9 methylation. PMID:27554592

  6. Genome-wide analysis of methylation in bovine clones by methylated DNA immunoprecipitation (MeDIP).

    PubMed

    Kiefer, Hélène

    2015-01-01

    Methylated DNA immunoprecipitation (MeDIP), when coupled to high-throughput sequencing or microarray hybridization, allows for the identification of methylated loci at a genome-wide scale. Genomic regions affected by incomplete reprogramming after nuclear transfer can potentially be delineated by comparing the MeDIP profiles of bovine clones and non-clones. This chapter presents a MeDIP protocol largely inspired from Mohn and colleagues (Mohn et al., Methods Mol Biol 507:55-64, 2009), with PCR primers specific for cattle, and when possible, overviews of experimental designs adapted to the comparison between clones and non-clones.

  7. Compendium of aberrant DNA methylation and histone modifications in cancer.

    PubMed

    Hattori, Naoko; Ushijima, Toshikazu

    2014-12-01

    Epigenetics now refers to the study or research field related to DNA methylation and histone modifications. Historically, global DNA hypomethylation was first revealed in 1983, and, after a decade, silencing of a tumor suppressor gene by regional DNA hypermethylation was reported. After the proposal of the histone code in the 2000s, alterations of histone methylation were also identified in cancers. Now, it is established that aberrant epigenetic alterations are involved in cancer development and progression, along with mutations and chromosomal losses. Recent cancer genome analyses have revealed a large number of mutations of epigenetic modifiers, supporting their important roles in cancer pathogenesis. Taking advantage of the reversibility of epigenetic alterations, drugs targeting epigenetic regulators and readers have been developed for restoration of normal pattern of the epigenome, and some have already demonstrated clinical benefits. In addition, DNA methylation of specific marker genes can be used as a biomarker for cancer diagnosis, including risk diagnosis, detection of cancers, and pathophysiological diagnosis. In this paper, we will summarize the major concepts of cancer epigenetics, placing emphasis on history.

  8. The role of DNA methylation in aging, rejuvenation, and age-related disease.

    PubMed

    Johnson, Adiv A; Akman, Kemal; Calimport, Stuart R G; Wuttke, Daniel; Stolzing, Alexandra; de Magalhães, João Pedro

    2012-10-01

    DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-L-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation.

  9. The Role of DNA Methylation in Aging, Rejuvenation, and Age-Related Disease

    PubMed Central

    Johnson, Adiv A.; Akman, Kemal; Calimport, Stuart R.G.; Wuttke, Daniel; de Magalhães, João Pedro

    2012-01-01

    Abstract DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-l-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation. PMID:23098078

  10. DNA methylation map of mouse and human brain identifies target genes in Alzheimer's disease.

    PubMed

    Sanchez-Mut, Jose V; Aso, Ester; Panayotis, Nicolas; Lott, Ira; Dierssen, Mara; Rabano, Alberto; Urdinguio, Rocio G; Fernandez, Agustin F; Astudillo, Aurora; Martin-Subero, Jose I; Balint, Balazs; Fraga, Mario F; Gomez, Antonio; Gurnot, Cecile; Roux, Jean-Christophe; Avila, Jesus; Hensch, Takao K; Ferrer, Isidre; Esteller, Manel

    2013-10-01

    The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimer's disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5'-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimer's disease. We were able to translate these findings to patients with Alzheimer's disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease.

  11. Aberrant DNA Methylation: Implications in Racial Health Disparity

    PubMed Central

    Wang, Xuefeng; Ji, Ping; Zhang, Yuanhao; LaComb, Joseph F.; Tian, Xinyu; Li, Ellen; Williams, Jennie L.

    2016-01-01

    Background Incidence and mortality rates of colorectal carcinoma (CRC) are higher in African Americans (AAs) than in Caucasian Americans (CAs). Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations. Materials and Methods Total DNA and RNA samples of paired tumor and adjacent normal colon tissues were prepared from AA and CA CRC specimens. Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing were employed to evaluate total genome methylation of 5’-regulatory regions and dysregulation of gene expression, respectively. Robust analysis was conducted using a trimming-and-retrieving scheme for RRBS library mapping in conjunction with the BStool toolkit. Results DNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs). Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4), and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC) were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins) and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p]) in AA patients with CRC versus CA patients. Conclusion DNA methylation profile and/or products of its downstream targets could serve as biomarker(s) addressing racial health disparity. PMID:27111221

  12. DNA methylation as a target of epigenetic therapeutics in cancer.

    PubMed

    Li, Keqin K; Li, Fangcheng; Li, Qiushi S; Yang, Kun; Jin, Bilian

    2013-02-01

    Epigenetic alterations have been implicated in the development and progression of human cancer. It is noteworthy that epigenetic modifications, in contrast to genetic mutations, are intrinsically reversible. This triggers an impressive interest of researchers in treatment of cancer patients via targeting epigenetic mechanisms, leading to subsequent intensive investigations of epigenetic drugs as a novel therapeutic intervention. DNA methylation, the major form of epigenetic modifications, is catalyzed by the maintenance DNA methyltransferase (DNMT) 1 and/or the de novo methyltransferases DNMT3A and DNMT3B. Aberrant expression of DNMTs and disruption of DNA methylation are closely associated with multiple forms of cancer, although the exact mechanisms underlying this link remain elusive. An array of tumor suppressor genes (TSGs) frequently sustain promoter hypermethylation, which results in epigenetic silencing of these genes and makes cancer cells acquire growth advantages. DNA demethylating agents, re-activating TSGs via inhibiting hypermethylation of their promoter regions, are currently being tested in clinical trials, and several of them are already applied in clinics. DNA demethylating agents, used either alone or in combination with other agents, such as chemotherapeutic drugs and the histone deacetylase inhibitors, have shown to be effective in treatment of cancer, although only in a small set of patients. In this review, we examine and discuss the most recent advances in epigenetic therapy of cancer, with a focus on DNA demethylating agents.

  13. Inheritance and Variation of Genomic DNA Methylation in Diploid and Triploid Pacific Oyster (Crassostrea gigas).

    PubMed

    Jiang, Qun; Li, Qi; Yu, Hong; Kong, Lingfeng

    2016-02-01

    DNA methylation is an important epigenetic mechanism that could be responsive to environmental changes indicating a potential role in natural selection and adaption. In order to evaluate an evolutionary role of DNA methylation, it is essential to first gain a better insight into inheritability. To address this question, this study investigated DNA methylation variation from parents to offspring in the Pacific oyster Crassostrea gigas using fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis. Most of parental methylated loci were stably transmitted to offspring segregating following Medelian expectation. However, methylated loci deviated more often than non-methylated loci and offspring showed a few de novo methylated loci indicating DNA methylation changes from parents to offspring. Interestingly, some male-specific methylated loci were found in this study which might help to explore sex determination in oyster. Despite environmental stimuli, genomic stresses such as polyploidization also can induce methylation changes. This study also compared global DNA methylation level and individual methylated loci between diploid and triploid oysters. Results showed no difference in global methylation state but a few ploidy-specific loci were detected. DNA methylation variation during polyploidization was less than autonomous methylation variation from parents to offspring.

  14. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    SciTech Connect

    Song, Jikui; Rechkoblit, Olga; Bestor, Timothy H.; Patel, Dinshaw J.

    2011-09-06

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  15. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    SciTech Connect

    J Song; O Rechkoblit; T Bestor; D Patel

    2011-12-31

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  16. Regulation of the transcriptional program by DNA methylation during human αβ T-cell development

    PubMed Central

    Rodriguez, Ramon M.; Suarez-Alvarez, Beatriz; Mosén-Ansorena, David; García-Peydró, Marina; Fuentes, Patricia; García-León, María J.; Gonzalez-Lahera, Aintzane; Macias-Camara, Nuria; Toribio, María L.; Aransay, Ana M.; Lopez-Larrea, Carlos

    2015-01-01

    Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis. PMID:25539926

  17. Targeted DNA methylation analysis by next-generation sequencing.

    PubMed

    Masser, Dustin R; Stanford, David R; Freeman, Willard M

    2015-02-24

    The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

  18. eMethylsorb: electrochemical quantification of DNA methylation at CpG resolution using DNA-gold affinity interactions.

    PubMed

    Sina, Abu Ali Ibn; Howell, Sidney; Carrascosa, Laura G; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-01

    We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

  19. Microarray-based resonance light scattering assay for detecting DNA methylation and human DNA methyltransferase simultaneously with high sensitivity.

    PubMed

    Ma, Lan; Su, Min; Li, Tao; Wang, Zhenxin

    2014-07-21

    A microarray-based resonance light scattering assay, with the combination of methylation-sensitive endonuclease and gold nanoparticle (GNP) probes, has been proposed to sensitively distinguish the DNA methylation level as low as 0.01% (10 pM methylated DNA in 100 nM total DNA) and detect human DNA methyltransferase 1 (Dnmt1) down to 0.1 U mL(-1).

  20. Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

    PubMed

    Steegenga, Wilma T; Boekschoten, Mark V; Lute, Carolien; Hooiveld, Guido J; de Groot, Philip J; Morris, Tiffany J; Teschendorff, Andrew E; Butcher, Lee M; Beck, Stephan; Müller, Michael

    2014-06-01

    Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.

  1. Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

    PubMed

    Steegenga, Wilma T; Boekschoten, Mark V; Lute, Carolien; Hooiveld, Guido J; de Groot, Philip J; Morris, Tiffany J; Teschendorff, Andrew E; Butcher, Lee M; Beck, Stephan; Müller, Michael

    2014-06-01

    Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression. PMID:24789080

  2. Genome-wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice.

    PubMed

    Yan, Huihuang; Kikuchi, Shinji; Neumann, Pavel; Zhang, Wenli; Wu, Yufeng; Chen, Feng; Jiang, Jiming

    2010-08-01

    We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.

  3. The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

    PubMed Central

    Laget, Sophie; Joulie, Michael; Le Masson, Florent; Sasai, Nobuhiro; Christians, Elisabeth; Pradhan, Sriharsa; Roberts, Richard J.; Defossez, Pierre-Antoine

    2010-01-01

    Background MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental retardation. Principal Findings Here we report the first functional characterization of MBD5 and MBD6. We have observed that the proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD. However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro, neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested. Conclusions Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a possible role in epigenetic reprogramming after fertilization. PMID:20700456

  4. FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

    PubMed

    Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

    2014-01-01

    Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

  5. DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

    PubMed Central

    Sailani, M. Reza; Santoni, Federico A.; Letourneau, Audrey; Borel, Christelle; Makrythanasis, Periklis; Hibaoui, Youssef; Popadin, Konstantin; Bonilla, Ximena; Guipponi, Michel; Gehrig, Corinne; Vannier, Anne; Carre-Pigeon, Frederique; Feki, Anis; Nizetic, Dean; Antonarakis, Stylianos E.

    2015-01-01

    DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes. PMID:26317209

  6. Lsh Is Essential for Maintaining Global DNA Methylation Levels in Amphibia and Fish and Interacts Directly with Dnmt1

    PubMed Central

    Dunican, Donncha S.; Pennings, Sari; Meehan, Richard R.

    2015-01-01

    Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase), predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a) show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b) support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance. PMID:26491684

  7. Interleukin-6 promotes tumorigenesis by altering DNA methylation in oral cancer cells.

    PubMed

    Gasche, Jacqueline A; Hoffmann, Jürgen; Boland, C Richard; Goel, Ajay

    2011-09-01

    Worldwide oral squamous cell carcinoma (OSCC) accounts for more than 100,000 deaths each year. Chronic inflammation constitutes one of the key risk factors for OSCC. Accumulating evidence suggests that aberrant DNA methylation may contribute to OSCC tumorigenesis. This study investigated whether chronic inflammation alters DNA methylation and expression of cancer-associated genes in OSCC. We established an in vitro model of interleukin (IL)-6 mediating chronic inflammation in OSCC cell lines. Thereafter, we measured the ability of IL-6 to induce global hypomethylation of long interspersed nuclear element-1 (LINE-1) sequences, as well as CpG methylation changes using multiple methodologies including quantitative pyrosequencing, methylation-specific multiplex ligation-dependent probe amplification and sensitive melting analysis after real-time-methylation-specific polymerase chain reaction (PCR). Gene expression was investigated by quantitative reverse transcriptase-PCR. IL-6 induced significant global LINE-1 hypomethylation (p=0.016) in our in vitro model of inflammatory stress in OSCC cell lines. Simultaneously, IL-6 induced CpG promoter methylation changes in several important putative tumor suppressor genes including CHFR, GATA5 and PAX6. Methylation changes correlated inversely with the changes in the expression of corresponding genes. Our results indicate that IL-6-induced inflammation promotes tumorigenesis in the oral cavity by altering global LINE-1 hypomethylation. In addition, concurrent hypermethylation of multiple tumor suppressor genes by IL-6 suggests that epigenetic gene silencing may be an important consequence of chronic inflammation in the oral cavity. These findings have clinical relevance, as both methylation and inflammation are suitable targets for developing novel preventive and therapeutic measures.

  8. Mercury biomarkers and DNA methylation among Michigan dental professionals.

    PubMed

    Goodrich, Jaclyn M; Basu, Niladri; Franzblau, Alfred; Dolinoy, Dana C

    2013-04-01

    Modification of the epigenome may be a mechanism underlying toxicity and disease following chemical exposure. Animal and human data suggest that mercury (Hg) impacts DNA methylation. We hypothesize that methylmercury and inorganic Hg exposures from fish consumption and dental amalgams, respectively, may be associated with altered DNA methylation at global repetitive elements (long interspersed elements, LINE-1) and candidate genes related to epigenetic processes (DNMT1) and protection against Hg toxicity (SEPW1, SEPP1). Dental professionals were recruited at Michigan Dental Association (MDA) meetings in 2009 and 2010. Subjects (n=131) provided survey data (e.g. exposure sources, demographics) and biological samples for Hg measurement and epigenetic analysis. Total Hg was quantified via atomic absorption spectrophotometry in hair and urine, indicative of methylmercury and inorganic Hg exposures, respectively. Global repetitive and candidate gene methylation was quantified via pyrosequencing of bisulfite converted DNA isolated from buccal mucosa. Hair Hg (geometric mean (95% CI): 0.37 (0.31-0.44) µg/g) and urine Hg (0.70 (0.60-0.83) µg/L) were associated with sources of exposure (fish consumption and dental amalgams, respectively). Multivariable linear regression revealed a trend of SEPP1 hypomethylation with increasing hair Hg levels, and this was significant (P<0.05) among males. The trend remained when excluding non-dentists. No significant relationships between urine Hg and DNA methylation were observed. Thus, in a limited cohort, we identified an association between methylmercury exposure and hypomethylation of a potentially labile region of the genome (SEPP1 promoter), and this relationship was gender specific. PMID:23444121

  9. Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

    PubMed

    Vaes, Bart L T; Lute, Carolien; van der Woning, Sebastian P; Piek, Ester; Vermeer, Jenny; Blom, Henk J; Mathers, John C; Müller, Michael; de Groot, Lisette C P G M; Steegenga, Wilma T

    2010-02-01

    S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

  10. DNA nucleoside composition and methylation in several species of microalgae

    SciTech Connect

    Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. )

    1992-06-01

    Total DNA was isolated from 10 species of microalgae, including representatives of the Chlorophyceae (Chlorella ellipsoidea, Chlamydomonas reinhardtii, and Monoraphidium minutum), Bacillariophyceae (Cyclotella cryptica, Navicula saprophila, Nitzschia pusilla, and Phaeodactylum tricornutum), Charophyceae (Stichococcus sp.), Dinophyceae (Crypthecodinium cohnii), and Prasinophyceae (Tetraselmis suecica). Control samples of Escherichia coli and calf thymus DNA were also analyzed. The nucleoside base composition of each DNA sample was determined by reversed-phase high performance liquid chromatography. All samples contained 5-methyldeoxycytidine, although at widely varying levels. In M. minutum, about one-third of the cytidine residues were methylated. Restriction analysis supported this high degree of methylation in M. minutum and suggested that methylation is biased toward 5[prime]-CG dinucleotides. The guanosine + cytosine (GC) contents of the green algae were, with the exception of Stichococcus sp., consistently higher than those of the diatoms. Monoraphidium minutum exhibited an extremely high GC content of 71%. Such a value is rare among eukaryotic organisms and might indicate an unusual codon usage. This work is important for developing strategies for transformation and gene cloning in these algae. 46 refs., 1 fig., 2 tabs.

  11. DNA methylation profiling of primary neuroblastoma tumors using methyl-CpG-binding domain sequencing

    PubMed Central

    Decock, Anneleen; Ongenaert, Maté; Van Criekinge, Wim; Speleman, Frank; Vandesompele, Jo

    2016-01-01

    Comprehensive genome-wide DNA methylation studies in neuroblastoma (NB), a childhood tumor that originates from precursor cells of the sympathetic nervous system, are scarce. Recently, we profiled the DNA methylome of 102 well-annotated primary NB tumors by methyl-CpG-binding domain (MBD) sequencing, in order to identify prognostic biomarker candidates. In this data descriptor, we give details on how this data set was generated and which bioinformatics analyses were applied during data processing. Through a series of technical validations, we illustrate that the data are of high quality and that the sequenced fragments represent methylated genomic regions. Furthermore, genes previously described to be methylated in NB are confirmed. As such, these MBD sequencing data are a valuable resource to further study the association of NB risk factors with the NB methylome, and offer the opportunity to integrate methylome data with other -omic data sets on the same tumor samples such as gene copy number and gene expression, also publically available. PMID:26836295

  12. DNA methylation profiling of primary neuroblastoma tumors using methyl-CpG-binding domain sequencing.

    PubMed

    Decock, Anneleen; Ongenaert, Maté; Van Criekinge, Wim; Speleman, Frank; Vandesompele, Jo

    2016-01-01

    Comprehensive genome-wide DNA methylation studies in neuroblastoma (NB), a childhood tumor that originates from precursor cells of the sympathetic nervous system, are scarce. Recently, we profiled the DNA methylome of 102 well-annotated primary NB tumors by methyl-CpG-binding domain (MBD) sequencing, in order to identify prognostic biomarker candidates. In this data descriptor, we give details on how this data set was generated and which bioinformatics analyses were applied during data processing. Through a series of technical validations, we illustrate that the data are of high quality and that the sequenced fragments represent methylated genomic regions. Furthermore, genes previously described to be methylated in NB are confirmed. As such, these MBD sequencing data are a valuable resource to further study the association of NB risk factors with the NB methylome, and offer the opportunity to integrate methylome data with other -omic data sets on the same tumor samples such as gene copy number and gene expression, also publically available. PMID:26836295

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

    PubMed

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

    2016-09-01

    DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. PMID:27474941

  14. Review of the alterations in DNA methylation in esophageal squamous cell carcinoma.

    PubMed

    Baba, Yoshifumi; Watanabe, Masayuki; Baba, Hideo

    2013-12-01

    Epigenetic changes such as DNA methylation, histone modification, and loss of genome imprinting play a crucial role in esophageal squamous cell carcinogenesis, along with genomic and genetic alterations. DNA methylation is a fundamental epigenetic process that modulates gene expression. Cancer cells exhibit two types of alterations of DNA methylation: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. In several types of human cancers, the methods of detecting an aberrant methylation status have been applied to clinical fields to stratify high-risk groups, detect early cancer, and predict clinical outcomes. Importantly, epigenetic changes, including alterations in DNA methylation, are reversible and can thus be targets for cancer therapy or chemoprevention. Therefore, a better understanding of the DNA methylation in esophageal squamous cell carcinoma (ESCC) is important for optimizing cancer therapy and chemoprevention. We herein summarize the current knowledge regarding alterations in DNA methylation and the clinical implications in ESCC.

  15. Emerging technologies for studying DNA methylation for the molecular diagnosis of cancer

    PubMed Central

    Marzese, Diego M.; Hoon, Dave S.B.

    2015-01-01

    DNA methylation is an epigenetic mechanism that plays a key role in regulating gene expression and other functions. Although this modification is seen in different sequence contexts, the most frequently detected DNA methylation in mammals involves cytosine-guanine dinucleotides. Pathological alterations in DNA methylation patterns are described in a variety of human diseases, including cancer. Unlike genetic changes, DNA methylation is heavily influenced by subtle modifications in the cellular microenvironment. In all cancers, aberrant DNA methylation is involved in the alteration of a large number of oncological pathways with relevant theranostic utility. Several technologies for DNA methylation mapping were recently developed and successfully applied in cancer studies. The scope of these technologies varies from assessing a single cytosine-guanine locus to genome-wide distribution of DNA methylation. Here, we review the strengths and weaknesses of these approaches in the context of clinical utility for the molecular diagnosis of human cancers. PMID:25797072

  16. DNA methylation patterns of protein coding genes and long noncoding RNAs in female schizophrenic patients.

    PubMed

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Gao, Shugui; Duan, Shiwei

    2015-02-01

    Schizophrenia (SCZ) is a complex mental disorder contributed by both genetic and epigenetic factors. Long noncoding RNAs (lncRNAs) was recently found playing an important regulatory role in mental disorders. However, little was known about the DNA methylation of lncRNAs, although numerous SCZ studies have been performed on genetic polymorphisms or epigenetic marks in protein coding genes. We presented a comprehensive genome wide DNA methylation study of both protein coding genes and lncRNAs in female patients with paranoid and undifferentiated SCZ. Using the methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), 8,163 and 764 peaks were identified in paranoid and undifferentiated SCZ, respectively (p < 1 × 10-5). Gene ontology analysis showed that the hypermethylated regions were enriched in the genes related to neuron system and brain for both paranoid and undifferentiated SCZ (p < 0.05). Among these peaks, 121 peaks were located in gene promoter regions that might affect gene expression and influence the SCZ related pathways. Interestingly, DNA methylation of 136 and 23 known lncRNAs in Refseq database were identified in paranoid and undifferentiated SCZ, respectively. In addition, ∼20% of intergenic peaks annotated based on Refseq genes were overlapped with lncRNAs in UCSC and gencode databases. In order to show the results well for most biological researchers, we created an online database to display and visualize the information of DNA methyation peaks in both types of SCZ (http://www.bioinfo.org/scz/scz.htm). Our results showed that the aberrant DNA methylation of lncRNAs might be another important epigenetic factor for SCZ.

  17. Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains

    PubMed Central

    Stephens, Dominique C.; Poon, Gregory M. K.

    2016-01-01

    Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited. PMID:27270080

  18. The DNA methylation landscape of human early embryos.

    PubMed

    Guo, Hongshan; Zhu, Ping; Yan, Liying; Li, Rong; Hu, Boqiang; Lian, Ying; Yan, Jie; Ren, Xiulian; Lin, Shengli; Li, Junsheng; Jin, Xiaohu; Shi, Xiaodan; Liu, Ping; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Li, Xianlong; Guo, Fan; Wu, Xinglong; Fan, Xiaoying; Yong, Jun; Wen, Lu; Xie, Sunney X; Tang, Fuchou; Qiao, Jie

    2014-07-31

    DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

  19. Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus

    PubMed Central

    McLean, Chelsea M.; Dokshin, Gregoriy A.; Persson, Jenna M.; Herman, Herry; Pasini, Diego; Miró, Xavier; Donohoe, Mary E.; Lee, Jeannie T.; Helin, Kristian; Soloway, Paul D.

    2008-01-01

    At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development. PMID:18670629

  20. An osmium-DNA interstrand complex: application to facile DNA methylation analysis.

    PubMed

    Tanaka, Kazuo; Tainaka, Kazuki; Umemoto, Tadashi; Nomura, Akiko; Okamoto, Akimitsu

    2007-11-21

    Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

  1. Genomic patterns of DNA methylation: targets and function of an epigenetic mark.

    PubMed

    Weber, Michael; Schübeler, Dirk

    2007-06-01

    Methylation of cytosines can mediate epigenetic gene silencing and is the only known DNA modification in eukaryotes. Recent efforts to map DNA methylation across mammalian genomes revealed limited DNA methylation at regulatory regions but widespread methylation in intergenic regions and repeats. This is consistent with the idea that hypermethylation is the default epigenetic state and serves in maintaining genome integrity. DNA methylation patterns at regulatory regions are generally stable, but a minor subset of regulatory regions show variable DNA methylation between cell types, suggesting an additional dynamic component. Such promoter de novo methylation might be involved in the maintenance rather than the initiation of silencing of defined genes during development. How frequently such dynamic methylation occurs, its biological relevance and the pathways involved deserve investigation. PMID:17466503

  2. Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

    PubMed Central

    Song, Min-Ae; Tiirikainen, Maarit; Kwee, Sandi; Okimoto, Gordon; Yu, Herbert; Wong, Linda L.

    2013-01-01

    Background Hepatocellular carcinoma (HCC) is one of the most common cancers and frequently presents with an advanced disease at diagnosis. There is only limited knowledge of genome-scale methylation changes in HCC. Methods and Findings We performed genome-wide methylation profiling in a total of 47 samples including 27 HCC and 20 adjacent normal liver tissues using the Illumina HumanMethylation450 BeadChip. We focused on differential methylation patterns in the promoter CpG islands as well as in various less studied genomic regions such as those surrounding the CpG islands, i.e. shores and shelves. Of the 485,577 loci studied, significant differential methylation (DM) was observed between HCC and adjacent normal tissues at 62,692 loci or 13% (p<1.03e-07). Of them, 61,058 loci (97%) were hypomethylated and most of these loci were located in the intergenic regions (43%) or gene bodies (33%). Our analysis also identified 10,775 differentially methylated (DM) loci (17% out of 62,692 loci) located in or surrounding the gene promoters, 4% of which reside in known Differentially Methylated Regions (DMRs) including reprogramming specific DMRs and cancer specific DMRs, while the rest (10,315) involving 4,106 genes could be potential new HCC DMR loci. Interestingly, the promoter-related DM loci occurred twice as frequently in the shores than in the actual CpG islands. We further characterized 982 DM loci in the promoter CpG islands to evaluate their potential biological function and found that the methylation changes could have effect on the signaling networks of Cellular development, Gene expression and Cell death (p = 1.0e-38), with BMP4, CDKN2A, GSTP1, and NFATC1 on the top of the gene list. Conclusion Substantial changes of DNA methylation at a genome-wide level were observed in HCC. Understanding epigenetic changes in HCC will help to elucidate the pathogenesis and may eventually lead to identification of molecular markers for liver cancer diagnosis, treatment and

  3. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    PubMed

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  4. Role of DNA methylation and the DNA methyltransferases in learning and memory.

    PubMed

    Morris, Michael J; Monteggia, Lisa M

    2014-09-01

    Dynamic regulation of chromatin structure in postmitotic neurons plays an important role in learning and memory. Methylation of cytosine nucleotides has historically been considered the strongest and least modifiable of epigenetic marks. Accumulating recent data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning, memory formation, and behavioral plasticity. The current review focuses on the emergence of data that support the role of DNA methylation and demethylation, and its molecular mediators in memory formation.

  5. Role of DNA methylation and the DNA methyltransferases in learning and memory

    PubMed Central

    Morris, Michael J.; Monteggia, Lisa M.

    2014-01-01

    Dynamic regulation of chromatin structure in postmitotic neurons plays an important role in learning and memory. Methylation of cytosine nucleotides has historically been considered the strongest and least modifiable of epigenetic marks. Accumulating recent data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning, memory formation, and behavioral plasticity. The current review focuses on the emergence of data that support the role of DNA methylation and demethylation, and its molecular mediators in memory formation. PMID:25364286

  6. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    PubMed

    Schrader, Anna; Gross, Thomas; Thalhammer, Verena; Längst, Gernot

    2015-01-01

    The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  7. Functions of DNA methylation and hydroxymethylation in mammalian development.

    PubMed

    Guibert, Sylvain; Weber, Michael

    2013-01-01

    DNA methylation occurs at cytosines, predominantly in the CpG dinucleotide context and is a key epigenetic regulator of embryogenesis and stem-cell differentiation in mammals. The genomic patterns of 5-methylcytosine are extensively reprogrammed during early embryonic development as well as in the germ-cell lineage. Thanks to improvements in high-throughput mapping technologies, it is now possible to characterize the dynamics of this epigenetic mark at the genome scale. DNA methylation plays multiple roles during development and serves to establish long-term gene silencing. In 2009, it was revealed that 5-hydroxymethylcytosine (5hmC) is another prominent cytosine modification catalyzed by the enzymes of the TET family and abundant in certain cell types. 5hmC has been thought to serve as an intermediate in the reaction of DNA demethylation or act as a signal for chromatin factors. Here, we review the current knowledge on the roles of these DNA epigenetic marks in development, epigenetic reprogramming, and pluripotency. PMID:23587238

  8. A Pilot Genome-Scale Profiling of DNA Methylation in Sporadic Pituitary Macroadenomas: Association with Tumor Invasion and Histopathological Subtype

    PubMed Central

    Ling, Chao; Pease, Matthew; Shi, Lingling; Punj, Vasu; Shiroishi, Mark S.; Commins, Deborah; Weisenberger, Daniel J.; Wang, Kai; Zada, Gabriel

    2014-01-01

    Pituitary adenomas (PAs) are neoplasms that may cause a variety of neurological and endocrine effects. Although known causal contributors include heredity, hormonal influence and somatic mutations, the pathophysiologic mechanisms driving tumorigenesis and invasion of sporadic PAs remain unknown. We hypothesized that alterations in DNA methylation are associated with PA invasion and histopathology subtype, and that genome-scale methylation analysis may complement current classification methods for sporadic PAs. Twenty-four surgically-resected sporadic PAs with varying histopathological subtypes were assigned dichotomized Knosp invasion scores and examined using genome-wide DNA methylation profiling and RNA sequencing. PA samples clustered into subgroups according to functional status. Compared with hormonally-active PAs, nonfunctional PAs exhibited global DNA hypermethylation (mean beta-value 0.47 versus 0.42, P = 0.005); the most significant site of differential DNA methylation was within the promoter region of the potassium voltage-gated channel KCNAB2 (FDR = 5.11×10−10). Pathway analysis of promoter-associated CpGs showed that nonfunctional PAs are potentially associated with the ion-channel activity signal pathway. DNA hypermethylation tended to be negatively correlated with gene expression. DNA methylation analysis may be used to identify candidate genes involved in PA function and may potentially complement current standard immunostaining classification in sporadic PAs. DNA hypermethylation of KCNAB2 and downstream ion-channel activity signal pathways may contribute to the endocrine-inactive status of nonfunctional PAs. PMID:24781529

  9. Novel features of telomere biology revealed by the absence of telomeric DNA methylation.

    PubMed

    Vega-Vaquero, Alejandro; Bonora, Giancarlo; Morselli, Marco; Vaquero-Sedas, María I; Rubbi, Liudmilla; Pellegrini, Matteo; Vega-Palas, Miguel A

    2016-08-01

    Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length. PMID:27405804

  10. Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors.

    PubMed

    Planello, Aline C; Ji, Junfeng; Sharma, Vivek; Singhania, Rajat; Mbabaali, Faridah; Müller, Fabian; Alfaro, Javier A; Bock, Christoph; De Carvalho, Daniel D; Batada, Nizar N

    2014-01-01

    The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities. PMID:25408883

  11. Symmetrical Dose-Dependent DNA-Methylation Profiles in Children with Deletion or Duplication of 7q11.23

    PubMed Central

    Strong, Emma; Butcher, Darci T.; Singhania, Rajat; Mervis, Carolyn B.; Morris, Colleen A.; De Carvalho, Daniel; Weksberg, Rosanna; Osborne, Lucy R.

    2015-01-01

    Epigenetic dysfunction has been implicated in a growing list of disorders that include cancer, neurodevelopmental disorders, and neurodegeneration. Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders with broad phenotypic spectra caused by deletion and duplication, respectively, of a 1.5-Mb region that includes several genes with a role in epigenetic regulation. We have identified striking differences in DNA methylation across the genome between blood cells from children with WS or Dup7 and blood cells from typically developing (TD) children. Notably, regions that were differentially methylated in both WS and Dup7 displayed a significant and symmetrical gene-dose-dependent effect, such that WS typically showed increased and Dup7 showed decreased DNA methylation. Differentially methylated genes were significantly enriched with genes in pathways involved in neurodevelopment, autism spectrum disorder (ASD) candidate genes, and imprinted genes. Using alignment with ENCODE data, we also found the differentially methylated regions to be enriched with CCCTC-binding factor (CTCF) binding sites. These findings suggest that gene(s) within 7q11.23 alter DNA methylation at specific sites across the genome and result in dose-dependent DNA-methylation profiles in WS and Dup7. Given the extent of DNA-methylation changes and the potential impact on CTCF binding and chromatin regulation, epigenetic mechanisms most likely contribute to the complex neurological phenotypes of WS and Dup7. Our findings highlight the importance of DNA methylation in the pathogenesis of WS and Dup7 and provide molecular mechanisms that are potentially shared by WS, Dup7, and ASD. PMID:26166478

  12. Prostaglandin E₂ increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression.

    PubMed

    Huang, Steven K; Scruggs, Anne M; Donaghy, Jake; McEachin, Richard C; Fisher, Aaron S; Richardson, Bruce C; Peters-Golden, Marc

    2012-09-01

    Although alterations in DNA methylation patterns have been associated with specific diseases and environmental exposures, the mediators and signaling pathways that direct these changes remain understudied. The bioactive lipid mediator prostaglandin E(2) (PGE(2)) has been shown to exert a myriad of effects on cell survival, proliferation, and differentiation. Here, we report that PGE(2) also signals to increase global DNA methylation and DNA methylation machinery in fibroblasts. HumanMethylation27 BeadChip array analysis of primary fetal (IMR-90) and adult lung fibroblasts identified multiple genes that were hypermethylated in response to PGE(2). PGE(2), compared with nontreated controls, increased expression and activity (EC(50)∼10(7) M) of one specific isoform of DNA methyltransferase, DNMT3a. Silencing of DNMT3a negated the ability of PGE(2) to increase DNMT activity. The increase in DNMT3a expression was mediated by PGE(2) signaling via its E prostanoid 2 receptor and the second messenger cAMP. PGE(2), compared with the untreated control, increased the expression and activity of Sp1 and Sp3 (EC(50)∼3×10(7) M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE(2) increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE(2) decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is regulated by a ubiquitous bioactive endogenous mediator. Given that PGE(2) biosynthesis is modulated by environmental toxins, various disease states, and commonly used pharmacological agents, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.

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

    PubMed Central

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

    2015-01-01

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

  14. Real time in vitro regulation of DNA methylation using a 5-fluorouracil conjugated DNA-based stimuli-responsive platform.

    PubMed

    Mao, Xiuhai; Wei, Ming; Zhu, Chengfeng; Lu, Jianxin; Gao, Jimin; Simon, Anna J; Shi, Jiye; Huang, Qing; Fan, Chunhai

    2013-04-10

    DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and many methylases have been regarded as promising targets for antimicrobial drugs. In this work, we report a stimulus responsive, self-regulating anticancer drug release platform, comprising a multifunctional DNA that upon methylation by methyltransferase (MTase) releases 5-fluorouracil (5-Fu) and in turn inhibits subsequent expression of MTase. The multifunctional DNA with anticancer drug are first methylated by DNA adenine methylation (DAM) methyltransferase (MTase) and then cut by the methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the functional DNA by the methylation/cleavage process will release the anticancer drug, resulting in inhibition of the activity of DAM in turn. Consequently, the enzyme activity of DAM MTase can be self-regulated. Furthermore, we found that the inhibition efficiency of 5-Fu significantly increase as it is functionalized with DNA.

  15. Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks

    PubMed Central

    Zilberman, Daniel; Coleman-Derr, Devin; Ballinger, Tracy; Henikoff, Steven

    2010-01-01

    Eukaryotic chromatin is separated into functional domains differentiated by posttranslational histone modifications, histone variants, and DNA methylation1–6. Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are critical for eukaryotic development4,5, and aberrant methylation-induced silencing of tumor suppressor genes is a common feature of human cancer7. In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5′ ends of genes where it promotes transcriptional competence8–20. How DNA methylation and H2A.Z influence transcription remains largely unknown. Here we show that in the plant Arabidopsis thaliana, regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation4,5, engenders opposite changes in H2A.Z deposition, while mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z17 leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation. PMID:18815594

  16. Novel method to detect DNA methylation using gold nanoparticles coupled with enzyme-linkage reactions.

    PubMed

    Liu, Tao; Zhao, Jing; Zhang, Dongmei; Li, Genxi

    2010-01-01

    DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as promising targets for antimicrobial drugs. In this paper, we propose a simple and sensitive colorimetric assay method to detect the activity of methylases so as to monitor DNA methylation using DNA-modified gold nanoparticles (AuNPs) coupled with enzyme-linkage reactions. The duplex DNA molecules modified on the surface of AuNPs are first methylated by DNA adenine methylation (Dam) methyltransferase (MTase) and then cut by methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the AuNP surfaces by the methylation/cleavage process will destabilize the nanoparticles, resulting in aggregation of AuNPs and a red-to-blue color change. Consequently, the enzyme activity of Dam MTase can be assayed and DNA methylation can be detected. Furthermore, this study may provide a sensitive platform to screen inhibitors for Dam MTase.

  17. Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as measured by bisulfite sequencing

    PubMed Central

    Hao, Yue; Cui, Yanjun; Gu, Xianhong

    2016-01-01

    Heat stress affects muscle development and meat quality in food animals; however, little is known regarding its regulatory mechanisms at the epigenetic level, such as via DNA methylation. In this study, we aimed to compare the DNA methylation profiles between control and heat-stressed pigs to identify candidate genes for skeletal muscle development and meat quality. Whole-genome bisulfite sequencing was used to investigate the genome-wide DNA methylation patterns in the longissimus dorsi muscles of the pigs. Both groups showed similar proportions of methylation at CpG sites but exhibited different proportions at non-CpG sites. A total of 57,147 differentially methylated regions were identified between the two groups, which corresponded to 1,422 differentially methylated genes. Gene ontogeny and KEGG pathway analyses indicated that these were mainly involved in energy and lipid metabolism, cellular defense and stress responses, and calcium signaling pathways. This study revealed the global DNA methylation pattern of pig muscle between normal and heat stress conditions. The result of this study might contribute to a better understanding of epigenetic regulation in pig muscle development and meat quality. PMID:27264107

  18. CHST11 gene expression and DNA methylation in breast cancer

    PubMed Central

    HERMAN, DAMIR; LEAKEY, TATIANA I.; BEHRENS, ALICE; YAO-BORENGASSER, AIWEI; COONEY, CRAIG A.; JOUSHEGHANY, FARIBA; PHANAVANH, BOUNLEUT; SIEGEL, ERIC R.; SAFAR, A. MAZIN; KOROURIAN, SOHEILA; KIEBER-EMMONS, THOMAS; MONZAVI-KARBASSI, BEHJATOLAH

    2015-01-01

    Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2′-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the

  19. Genomic landscape of human allele-specific DNA methylation.

    PubMed

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J; Smith, Andrew D

    2012-05-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  20. Genomic landscape of human allele-specific DNA methylation

    PubMed Central

    Fang, Fang; Hodges, Emily; Molaro, Antoine; Dean, Matthew; Hannon, Gregory J.; Smith, Andrew D.

    2012-01-01

    DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species. PMID:22523239

  1. Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

    PubMed

    Kraiczy, J; Nayak, K; Ross, A; Raine, T; Mak, T N; Gasparetto, M; Cario, E; Rakyan, V; Heuschkel, R; Zilbauer, M

    2016-05-01

    DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.

  2. Unmasking risk loci: DNA methylation illuminates the biology of cancer predisposition: analyzing DNA methylation of transcriptional enhancers reveals missed regulatory links between cancer risk loci and genes.

    PubMed

    Aran, Dvir; Hellman, Asaf

    2014-02-01

    Paradoxically, DNA sequence polymorphisms in cancer risk loci rarely correlate with the expression of cancer genes. Therefore, the molecular mechanism underlying an individual's susceptibility to cancer has remained largely unknown. However, recent evaluations of the correlations between DNA methylation and gene expression levels across healthy and cancerous genomes have revealed enrichment of disease-related DNA methylation variations within disease-associated risk loci. Moreover, it appears that transcriptional enhancers embedded in cancer risk loci often contain DNA methylation sites that closely define the expression of prominent cancer genes, despite the lack of significant correlations between gene expression levels and the surrounding disease-associated polymorphic sequences. We suggest that DNA methylation variations may obscure the effect of co-residing risk sequence alleles. Analysis of enhancer methylation data may help to reveal the regulatory circuits underlying predisposition to cancers and other common diseases.

  3. Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

    PubMed

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B

    2016-03-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects. PMID:26581878

  4. Dysregulation of DNA Methylation Induced by Past Arsenic Treatment Causes Persistent Genomic Instability in Mammalian Cells

    PubMed Central

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B.

    2016-01-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects. PMID:26581878

  5. Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

    PubMed

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B

    2016-03-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects.

  6. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae.

    PubMed

    Jeon, Junhyun; Choi, Jaeyoung; Lee, Gir-Won; Park, Sook-Young; Huh, Aram; Dean, Ralph A; Lee, Yong-Hwan

    2015-02-24

    DNA methylation is an important epigenetic modification that regulates development of plants and mammals. To investigate the roles of DNA methylation in fungal development, we profiled genome-wide methylation patterns at single-nucleotide resolution during vegetative growth, asexual reproduction, and infection-related morphogenesis in a model plant pathogenic fungus, Magnaporthe oryzae. We found that DNA methylation occurs in and around genes as well as transposable elements and undergoes global reprogramming during fungal development. Such reprogramming of DNA methylation suggests that it may have acquired new roles other than controlling the proliferation of TEs. Genetic analysis of DNA methyltransferase deletion mutants also indicated that proper reprogramming in methylomes is required for asexual reproduction in the fungus. Furthermore, RNA-seq analysis showed that DNA methylation is associated with transcriptional silencing of transposable elements and transcript abundance of genes in context-dependent manner, reinforcing the role of DNA methylation as a genome defense mechanism. This comprehensive approach suggests that DNA methylation in fungi can be a dynamic epigenetic entity contributing to fungal development and genome defense. Furthermore, our DNA methylomes provide a foundation for future studies exploring this key epigenetic modification in fungal development and pathogenesis.

  7. A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

    PubMed

    Wei, Wei; Gao, Chunyan; Xiong, Yanxiang; Zhang, Yuanjian; Liu, Songqin; Pu, Yuepu

    2015-01-01

    DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate.

  8. Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

    PubMed Central

    Zhou, Feng C.; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan

    2016-01-01

    Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology. PMID:27583369

  9. Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum.

    PubMed

    Zhou, Feng C; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan

    2016-01-01

    Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology. PMID:27583369

  10. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    PubMed

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  11. DNA methylation, an epigenetic mechanism connecting folate to healthy embryonic development and aging

    PubMed Central

    Kim, Kyong-chol; Friso, Simonetta; Choi, Sang-Woon

    2009-01-01

    Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our life time by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation. PMID:19733471

  12. DNA Methylation and Chromatin Organization in Insects: Insights from the Ant Camponotus floridanus

    PubMed Central

    Glastad, Karl M.; Hunt, Brendan G.; Goodisman, Michael A. D.

    2015-01-01

    Epigenetic information regulates gene function and has important effects on development in eukaryotic organisms. DNA methylation, one such form of epigenetic information, has been implicated in the regulation of gene function in diverse metazoan taxa. In insects, DNA methylation has been shown to play a role in the regulation of gene expression and splicing. However, the functional basis for this role remains relatively poorly understood, and other epigenetic systems likely interact with DNA methylation to affect gene expression. We investigated associations between DNA methylation and histone modifications in the genome of the ant Camponotus floridanus in order to provide insight into how different epigenetic systems interact to affect gene function. We found that many histone modifications are strongly predictive of DNA methylation levels in genes, and that these epigenetic signals are more predictive of gene expression when considered together than when considered independently. We also found that peaks of DNA methylation are associated with the spatial organization of chromatin within active genes. Finally, we compared patterns of differential histone modification enrichment to patterns of differential DNA methylation to reveal that several histone modifications significantly covary with DNA methylation between C. floridanus phenotypes. As the first genomic comparison of DNA methylation to histone modifications within a single insect taxon, our investigation provides new insight into the regulatory significance of DNA methylation. PMID:25724207

  13. Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype

    PubMed Central

    Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

    2016-01-01

    Study Objectives: We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Methods: Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Results: Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (−114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (−531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (−608/−618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (−194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. Conclusions: IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. Commentary: A

  14. Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

    PubMed

    Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2010-11-01

    Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

  15. [Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

    PubMed

    Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

    2007-06-01

    The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

  16. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates

    PubMed Central

    Rivière, Guillaume

    2014-01-01

    DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing, and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position, and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster's developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5′-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment. PMID:24778620

  17. Sex-dichotomous effects of NOS1AP promoter DNA methylation on intracranial aneurysm and brain arteriovenous malformation.

    PubMed

    Wang, Zhepei; Zhao, Jikuang; Sun, Jie; Nie, Sheng; Li, Keqing; Gao, Feng; Zhang, Tiefeng; Duan, Shiwei; Di, Yazhen; Huang, Yi; Gao, Xiang

    2016-05-16

    The goal of this study was to investigate the contribution of NOS1AP-promoter DNA methylation to the risk of intracranial aneurysm (IA) and brain arteriovenous malformation (BAVM) in a Han Chinese population. A total of 48 patients with IAs, 22 patients with BAVMs, and 26 control individuals were enrolled in the study. DNA methylation was tested using bisulfite pyrosequencing technology. We detected significantly higher DNA methylation levels in BAVM patients than in IA patients based on the multiple testing correction (CpG4-5 methylation: 5.86±1.04% vs. 4.37±2.64%, P=0.006). In women, CpG4-5 methylation levels were much lower in IA patients (3.64±1.97%) than in BAVM patients (6.11±1.20%, P<0.0001). However, in men, CpG1-3 methylation levels were much higher in the controls (6.92±0.78%) than in BAVM patients (5.99±0.70%, P=0.008). Additionally, there was a gender-based difference in CpG1 methylation within the controls (men vs. women: 5.75±0.50% vs. 4.99±0.53%, P=0.003) and BAVM patients (men vs. women: 4.70±0.74% vs. 5.50±0.87%, P=0.026). A subgroup analysis revealed significantly higher CpG3 methylation in patients who smoked than in those who did not (P=0.041). Our results suggested that gender modulated the interaction between NOS1AP promoter DNA methylation in IA and BAVM patients. Our results also confirmed that regular tobacco smoking was associated with increased NOS1AP methylation in humans. Additional studies with larger sample sizes are required to replicate and extend these findings. PMID:27080431

  18. Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate–Vertebrate Boundary

    PubMed Central

    Keller, Thomas E.; Han, Priscilla; Yi, Soojin V.

    2016-01-01

    Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate–vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate–vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression. PMID:26715626

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

    PubMed Central

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

    2011-01-01

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

  20. Analysis of DNA methylation level by methylation-sensitive amplification polymorphism in half smooth tongue sole (Cynoglossus semilaevis) subjected to salinity stress

    NASA Astrophysics Data System (ADS)

    Li, Siping; He, Feng; Wen, Haishen; Li, Jifang; Si, Yufeng; Liu, Mingyuan; He, Huiwen; Huang, Zhengju

    2016-09-01

    Increasingly arisen environmental constraints may contribute to heritable phenotypic variation including methylation changes, which can help the animals with development, growth and survival. In this study, we assessed the DNA methylation levels in three tissues (gonad, kidney and gill) of half smooth tongue sole under the salinity stress. The methylation-sensitive amplification polymorphism (MSAP) technique was applied to illustrate the regulation of epigenetic mechanism in environmental stimuli. Fish were subjected to 15 salinity treatment for 7 and 60 days, respectively. A total of 11259 fragments were amplified with 8 pairs of selective primers. The levels of methylated DNA in different tissues of females and males without salinity stress were analyzed, which were 32.76% and 47.32% in gonad; 38.13% and 37.69% in kidney; 37.58% and 34.96% in gill, respectively. In addition, the significant difference was observed in gonad between females and males, indicating that discrepant regulation in gonadal development and differentiation may involve sex-related genes. Further analysis showed that total and hemi-methylation were significantly decreased under 15 salinity for 7 days, probably resulting in up-regulating salt-tolerance genes expression to adjust salt changing. With the adjustment for 60 days, total and hemi-methylation prominently went back to its normal levels to obtain equilibrium. Particularly, full methylation levels were steady along with salinity stress to maintain the stability of gene expression. Additionally, the data showed that gonads in females and gills in males were superior in adaptability. As a result, DNA methylation regulates tissue- specific epiloci, and may respond to salinity stress by regulating gene expression to maintain animal survival and activity.

  1. Human age estimation from blood using mRNA, DNA methylation, DNA rearrangement, and telomere length.

    PubMed

    Zubakov, Dmitry; Liu, Fan; Kokmeijer, Iris; Choi, Ying; van Meurs, Joyce B J; van IJcken, Wilfred F J; Uitterlinden, André G; Hofman, Albert; Broer, Linda; van Duijn, Cornelia M; Lewin, Jörn; Kayser, Manfred

    2016-09-01

    Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DNA methylation markers in blood from the same young and old individuals using microarray technologies. The obtained candidate markers were validated in independent samples covering a wide age range using alternative technologies together with previously proposed DNA methylation, sjTREC, and telomere length markers. Cross-validated multiple regression analysis was applied for estimating and validating the age predictive power of various sets of biomarkers within and across different marker types. We found that DNA methylation markers outperformed mRNA, sjTREC, and telomere length in age predictive power. The best performing model included 8 DNA methylation markers derived from 3 CpG islands reaching a high level of accuracy (cross-validated R(2)=0.88, SE±6.97 years, mean absolute deviation 5.07 years). However, our data also suggest that mRNA markers can provide independent age information: a model using a combined set of 5 DNA methylation markers and one mRNA marker could provide

  2. Human age estimation from blood using mRNA, DNA methylation, DNA rearrangement, and telomere length.

    PubMed

    Zubakov, Dmitry; Liu, Fan; Kokmeijer, Iris; Choi, Ying; van Meurs, Joyce B J; van IJcken, Wilfred F J; Uitterlinden, André G; Hofman, Albert; Broer, Linda; van Duijn, Cornelia M; Lewin, Jörn; Kayser, Manfred

    2016-09-01

    Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DNA methylation markers in blood from the same young and old individuals using microarray technologies. The obtained candidate markers were validated in independent samples covering a wide age range using alternative technologies together with previously proposed DNA methylation, sjTREC, and telomere length markers. Cross-validated multiple regression analysis was applied for estimating and validating the age predictive power of various sets of biomarkers within and across different marker types. We found that DNA methylation markers outperformed mRNA, sjTREC, and telomere length in age predictive power. The best performing model included 8 DNA methylation markers derived from 3 CpG islands reaching a high level of accuracy (cross-validated R(2)=0.88, SE±6.97 years, mean absolute deviation 5.07 years). However, our data also suggest that mRNA markers can provide independent age information: a model using a combined set of 5 DNA methylation markers and one mRNA marker could provide

  3. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    PubMed Central

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R.; Pradhan, Sriharsa

    2015-01-01

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

  4. DNA methylation and leukemia susceptibility in China: Evidence from an updated meta-analysis

    PubMed Central

    Jiang, Danjie; Li, Yirun; Hong, Qingxiao; Shen, Yusheng; Xu, Chunjing; Xu, Yan; Zhu, Huangkai; Dai, Dongjun; Ouyang, Guifang; Duan, Shiwei

    2016-01-01

    Mounting evidence supports a role for DNA methylation in the pathogenesis of leukemia; however, there no overview of these results in the Chinese population. The present study performed a comprehensive meta-analysis to establish candidate genes with an altered methylation status in Chinese leukemia patients. Eligible studies were identified through searching the National Center of Biotechnology Information PubMed and Wanfang databases. Studies were pooled and overall odds ratios with corresponding confidence intervals were calculated. A total of 4,325 leukemia patients and 2,010 controls from 94 studies on 53 genes were included in this meta-analysis, and 47 genes were found to be aberrantly methylated in leukemia patients. A further subgroup meta-analysis by leukemia subtype demonstrated that hypermethylation of 5 genes, namely cyclin-dependent kinase (CDKN)2A, DNA-binding protein inhibitor-4, CDKN2B, glioma pathogenesis-related protein 1 and p73, contributed to the risk of various subtypes of leukemia. In addition, a strong association between CDKN2A and leukemia was identified in Chinese (P<0.00001) but not in European patients. The aberrantly methylated genes identified in the present meta-analysis may help elucidate the mechanisms underlying the development of leukemia in Chinese patients. PMID:27588182

  5. Gene-set Analysis with CGI Information for Differential DNA Methylation Profiling

    PubMed Central

    Chang, Chia-Wei; Lu, Tzu-Pin; She, Chang-Xian; Feng, Yen-Chen; Hsiao, Chuhsing Kate

    2016-01-01

    DNA methylation is a well-established epigenetic biomarker for many diseases. Studying the relationships among a group of genes and their methylations may help to unravel the etiology of diseases. Since CpG-islands (CGIs) play a crucial role in the regulation of transcription during methylation, including them in the analysis may provide further information in understanding the pathogenesis of cancers. Such CGI information, however, has usually been overlooked in existing gene-set analyses. Here we aimed to include both pathway information and CGI status to rank competing gene-sets and identify among them the genes most likely contributing to DNA methylation changes. To accomplish this, we devised a Bayesian model for matched case-control studies with parameters for CGI status and pathway associations, while incorporating intra-gene-set information. Three cancer studies with candidate pathways were analyzed to illustrate this approach. The strength of association for each candidate pathway and the influence of each gene were evaluated. Results show that, based on probabilities, the importance of pathways and genes can be determined. The findings confirm that some of these genes are cancer-related and may hold the potential to be targeted in drug development. PMID:27090937

  6. Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes.

    PubMed

    Fisher, Marisa M; Watkins, Renecia A; Blum, Janice; Evans-Molina, Carmella; Chalasani, Naga; DiMeglio, Linda A; Mather, Kieren J; Tersey, Sarah A; Mirmira, Raghavendra G

    2015-11-01

    Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease. PMID:26216854

  7. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    PubMed

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  8. Nutritional control of reproductive status in honeybees via DNA methylation.

    PubMed

    Kucharski, R; Maleszka, J; Foret, S; Maleszka, R

    2008-03-28

    Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. We show that silencing the expression of DNA methyltransferase Dnmt3, a key driver of epigenetic global reprogramming, in newly hatched larvae led to a royal jelly-like effect on the larval developmental trajectory; the majority of Dnmt3 small interfering RNA-treated individuals emerged as queens with fully developed ovaries. Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.

  9. Optimized analysis of DNA methylation and gene expression from small, anatomically-defined areas of the brain.

    PubMed

    Bettscheider, Marc; Kuczynska, Arleta; Almeida, Osborne; Spengler, Dietmar

    2012-07-12

    Exposure to diet, drugs and early life adversity during sensitive windows of life can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases. DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides. CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample. Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

  10. Pancreatic Cancer Patient Survival Correlates with DNA Methylation of Pancreas Development Genes

    PubMed Central

    Thompson, Michael J.; Rubbi, Liudmilla; Dawson, David W.; Donahue, Timothy R.; Pellegrini, Matteo

    2015-01-01

    DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival. PMID:26039411

  11. Pancreatic cancer patient survival correlates with DNA methylation of pancreas development genes.

    PubMed

    Thompson, Michael J; Rubbi, Liudmilla; Dawson, David W; Donahue, Timothy R; Pellegrini, Matteo

    2015-01-01

    DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival.

  12. Combined analysis of DNA methylation and cell cycle in cancer cells

    PubMed Central

    Desjobert, Cécile; El Maï, Mounir; Gérard-Hirne, Tom; Guianvarc'h, Dominique; Carrier, Arnaud; Pottier, Cyrielle; Arimondo, Paola B; Riond, Joëlle

    2015-01-01

    DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase. PMID:25531272

  13. Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study.

    PubMed

    Wu, Hui-Chen; Wang, Qiao; Chung, Wendy K; Andrulis, Irene L; Daly, Mary B; John, Esther M; Keegan, Theresa H M; Knight, Julia; Bradbury, Angela R; Kappil, Maya A; Gurvich, Irina; Santella, Regina M; Terry, Mary Beth

    2014-07-01

    Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6-15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.

  14. Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract.

    PubMed

    Keil, Kimberly P; Altmann, Helene M; Mehta, Vatsal; Abler, Lisa L; Elton, Erik A; Vezina, Chad M

    2013-12-01

    The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.

  15. DNA hypermethylation in hyperhomocysteinemia contributes to abnormal extracellular matrix metabolism in the kidney

    PubMed Central

    Pushpakumar, Sathnur; Kundu, Sourav; Narayanan, Nithya; Sen, Utpal

    2015-01-01

    Hyperhomocysteinemia (HHcy) is prevalent in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Emerging studies suggest that epigenetic mechanisms contribute to the development and progression of fibrosis in CKD. HHcy and its intermediates are known to alter the DNA methylation pattern, which is a critical regulator of epigenetic information. In this study, we hypothesized that HHcy causes renovascular remodeling by DNA hypermethylation, leading to glomerulosclerosis. We also evaluated whether the DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5-Aza) could modulate extracellular matrix (ECM) metabolism and reduce renovascular fibrosis. C57BL/6J (wild-type) and cystathionine-β-synthase (CBS+/−) mice, treated without or with 5-Aza (0.5 mg/kg body weight, i.p.), were used. CBS+/− mice showed high plasma Hcy levels, hypertension, and significant glomerular and arteriolar injury. 5-Aza treatment normalized blood pressure and reversed renal injury. CBS+/− mice showed global hypermethylation and up-regulation of DNA methyltransferase-1 and -3a. Methylation-specific PCR showed an imbalance between matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and also increased collagen and galectin-3 expression. 5-Aza reduced abnormal DNA methylation and restored the MMP-9/TIMP-1, -2 balance. In conclusion, our data suggest that during HHcy, abnormal DNA methylation and an imbalance between MMP-9 and TIMP-1 and -2 lead to ECM remodeling and renal fibrosis.—Pushpakumar, S., Kundu, S., Narayanan, N., Sen, U. DNA hypermethylation in hyperhomocysteinemia contributes to abnormal extracellular matrix metabolism in the kidney. PMID:26224753

  16. The contribution of oceanic methyl iodide to stratospheric iodine

    NASA Astrophysics Data System (ADS)

    Tegtmeier, S.; Krüger, K.; Quack, B.; Atlas, E.; Blake, D. R.; Boenisch, H.; Engel, A.; Hepach, H.; Hossaini, R.; Navarro, M. A.; Raimund, S.; Sala, S.; Shi, Q.; Ziska, F.

    2013-12-01

    We investigate the contribution of oceanic methyl iodide (CH3I) to the stratospheric iodine budget. Based on CH3I measurements from three tropical ship campaigns and the Lagrangian transport model FLEXPART, we provide a detailed analysis of CH3I transport from the ocean surface to the cold point in the upper tropical tropopause layer (TTL). While average oceanic emissions differ by less than 50% from campaign to campaign, the measurements show much stronger variations within each campaign. A positive correlation between the oceanic CH3I emissions and the efficiency of CH3I troposphere-stratosphere transport has been identified for some cruise sections. The mechanism of strong horizontal surface winds triggering large emissions on the one hand and being associated with tropical convective systems, such as developing typhoons, on the other hand, could explain the identified correlations. As a result of the simultaneous occurrence of large CH3I emissions and strong vertical uplift, localized maximum mixing ratios of 0.6 ppt CH3I at the cold point have been determined for observed peak emissions during the SHIVA (Stratospheric Ozone: Halogen Impacts in a Varying Atmosphere)-Sonne research vessel campaign in the coastal western Pacific. The other two campaigns give considerably smaller maxima of 0.1 ppt CH3I in the open western Pacific and 0.03 ppt in the coastal eastern Atlantic. In order to assess the representativeness of the large local mixing ratios, we use climatological emission scenarios to derive global upper air estimates of CH3I abundances. The model results are compared with available upper air measurements, including data from the recent ATTREX and HIPPO2 aircraft campaigns. In the eastern Pacific region, the location of the available measurement campaigns in the upper TTL, the comparisons give a good agreement, indicating that around 0.01 to 0.02 ppt of CH3I enter the stratosphere. However, other tropical regions that are subject to stronger convective

  17. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation

    PubMed Central

    Sanchez, Robersy; Mackenzie, Sally A.

    2016-01-01

    Cytosine DNA methylation (CDM) is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1) the amount of information gained/lost with the CDM changes IR and (2) the uncertainty of not observing a SNP LCR. We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI) patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on IR and on LCR, respectively. A statistical-physical relationship between IR and LCR was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment. PMID:27322251

  18. Environmental pollution and DNA methylation: carcinogenesis, clinical significance, and practical applications.

    PubMed

    Cao, Yi

    2015-09-01

    Environmental pollution is one of the main causes of human cancer. Exposures to environmental carcinogens result in genetic and epigenetic alterations which induce cell transformation. Epigenetic changes caused by environmental pollution play important roles in the development and progression of environmental pollution-related cancers. Studies on DNA methylation are among the earliest and most conducted epigenetic research linked to cancer. In this review, the roles of DNA methylation in carcinogenesis and their significance in clinical medicine were summarized, and the effects of environmental pollutants, particularly air pollutants, on DNA methylation were introduced. Furthermore, prospective applications of DNA methylation to environmental pollution detection and cancer prevention were discussed.

  19. DNA methylation is crucial for the early development in the Oyster C. gigas.

    PubMed

    Riviere, Guillaume; Wu, Guan-Chung; Fellous, Alexandre; Goux, Didier; Sourdaine, Pascal; Favrel, Pascal

    2013-12-01

    In vertebrates, epigenetic modifications influence gene transcription, and an appropriate DNA methylation is critical in development. Indeed, a precise temporal and spatial pattern of early gene expression is mandatory for a normal embryogenesis. However, such a regulation and its underlying mechanisms remain poorly understood in more distant organisms such as Lophotrochozoa. Thus, despite DNA in the oyster genome being methylated, the role of DNA methylation in development is unknown. To clarify this point, oyster genomic DNA was examined during early embryogenesis and found differentially methylated. Reverse transcriptase quantitative polymerase chain reaction indicated stage-specific levels of transcripts encoding DNA-methyltransferase (DNMT) and methyl-binding domain proteins. In addition, as highlighted by electronic microscopy and immunohistochemistry, the DNMT inhibitor 5-aza-cytidine induced alterations in the quantity and the localisation of methylated DNA and severe dose-dependent development alterations and was lethal after zygotic genome reinitiation. Furthermore, methyl-DNA-immunoprecipitation-quantitative polymerase chain reaction revealed that the transcription level of most of the homeobox gene orthologues examined, but not of the other early genes investigated, was inversely correlated with their specific DNA methylation. Altogether, our results demonstrate that DNA methylation influences gene expression in Crassostrea gigas and is critical for oyster development, possibly by specifically controlling the transcription level of homeobox orthologues. These findings provide evidence for the importance of epigenetic regulation of development in Lophotrochozoans and bring new insights into the early life of C. gigas, one of the most important aquaculture resources worldwide.

  20. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

    PubMed

    Wion, Didier; Casadesús, Josep

    2006-03-01

    N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

  1. Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

    PubMed

    Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

    2015-01-01

    Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements. PMID:26368560

  2. De novo DNA methylation of the paternal genome in 2-cell mouse embryos.

    PubMed

    Ma, X S; Wang, X G; Qin, L; Song, C L; Lin, F; Song, J M; Zhu, C C; Liu, H L

    2014-10-27

    The developmental dynamics of DNA methylation events have been well studied. Active demethylation of the paternal genome occurs in the zygote, passive demethylation occurs during cleavage stages, and de novo methylation occurs by the blastocyst stage. It is believed that the paternal genome has lower levels of methylation during early development than the maternal genome. However, in this study, we provide direct and indirect evidence of genome-wide de novo DNA methylation of the paternal genome after the first cell cycle in mouse embryos. Although very little methylation was detected within the male pronucleus in zygotes, an intense methylation signal was clearly visible within the androgenetic 2-cell embryos. Moreover, the DNA methylation level of the paternal genome in the post-zygotic metaphase embryos was similar to that of the maternal genome. Using indirect immunofluorescence with an antibody to methylated lysine 9 in histone H3, we provided new evidence to support the concept of spatial compartmentalization of parental genomes in 2-cell mouse embryos. Nevertheless, the transient segregation of parental genomes was not observed by determining the DNA methylation distribution in the 2-cell embryos even though DNA methylation asymmetry between the maternal and paternal pronucleus existed in the 1-cell stage. The disappearance of separate immunofluorescence signals of 5-methyl cytosine in the 2-cell embryos might be attributed to the de novo methylation of the paternal genome during the first mitotic cycle.

  3. A Pre-mRNA-Splicing Factor Is Required for RNA-Directed DNA Methylation in Arabidopsis

    PubMed Central

    Huang, Chao-Feng; Miki, Daisuke; Tang, Kai; Zhou, Hao-Ran; Zheng, Zhimin; Chen, Wei; Ma, Ze-Yang; Yang, Lan; Zhang, Heng; Liu, Renyi; He, Xin-Jian; Zhu, Jian-Kang

    2013-01-01

    Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation. PMID:24068953

  4. Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

    PubMed Central

    Roberts, C J; Selker, E U

    1995-01-01

    A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora. Images PMID:8532524

  5. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

    PubMed

    Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

    2015-01-01

    Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. PMID:25848745

  6. A Comparative Study of Tests for Homogeneity of Variances with Application to DNA Methylation Data

    PubMed Central

    Li, Xuan; Qiu, Weiliang; Morrow, Jarrett; DeMeo, Dawn L.; Weiss, Scott T.; Fu, Yuejiao; Wang, Xiaogang

    2015-01-01

    Variable DNA methylation has been associated with cancers and complex diseases. Researchers have identified many DNA methylation markers that have different mean methylation levels between diseased subjects and normal subjects. Recently, researchers found that DNA methylation markers with different variabilities between subject groups could also have biological meaning. In this article, we aimed to help researchers choose the right test of equal variance in DNA methylation data analysis. We performed systematic simulation studies and a real data analysis to compare the performances of 7 equal-variance tests, including 2 tests recently proposed in the DNA methylation analysis literature. Our results showed that the Brown-Forsythe test and trimmed-mean-based Levene's test had good performance in testing for equality of variance in our simulation studies and real data analyses. Our results also showed that outlier profiles could be biologically very important. PMID:26683022

  7. Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State

    PubMed Central

    Boby, Céline; Leroux, Christine; Declerck, Ken; Szarc vel Szic, Katarzyna; Heyninck, Karen; Laukens, Kris; Bizet, Martin; Defrance, Matthieu; Dedeurwaerder, Sarah; Calonne, Emilie; Fuks, Francois; Haegeman, Guy; Haenen, Guido R. M. M.; Bast, Aalt; Weseler, Antje R.

    2014-01-01

    Background In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. Conclusion Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans. PMID:24763279

  8. The contribution of oceanic methyl iodide to stratospheric iodine

    NASA Astrophysics Data System (ADS)

    Tegtmeier, S.; Krüger, K.; Quack, B.; Atlas, E.; Blake, D. R.; Boenisch, H.; Engel, A.; Hepach, H.; Hossaini, R.; Navarro, M. A.; Raimund, S.; Sala, S.; Shi, Q.; Ziska, F.

    2013-04-01

    We investigate the contribution of oceanic methyl iodide (CH3I) to the stratospheric iodine budget. Based on CH3I measurements during three tropical ship campaigns and the Lagrangian transport model FLEXPART we provide a detailed analysis of CH3I transport from the ocean surface to the cold point in the upper tropical tropopause layer (TTL). While average oceanic emissions differ by less than 50% from campaign to campaign, the measurements show much stronger variations within each campaign. A positive correlation between the oceanic CH3I emissions and the efficiency of CH3I troposphere-stratosphere transport has been identified for some cruise sections. The mechanism of strong horizontal surface winds triggering large emissions on the one hand and being associated with tropical convective systems, such as developing typhoons, on the other hand, could explain the identified correlations. As a result of the simultaneous occurrence of large CH3I emissions and strong vertical uplift, localized maximum mixing ratios of 0.6 ppt CH3I at the cold point have been determined for observed peak emissions during the SHIVA-Sonne campaign in the coastal West Pacific. The other two campaigns give considerable smaller maxima of 0.1 ppt CH3I for the TransBrom campaign in the open West Pacific and 0.03 ppt for emissions from the coastal East Atlantic during the DRIVE campaign. In order to assess the representativeness of the large local mixing ratios we use climatological emission scenarios to derive global upper air estimates of CH3I abundances. The model results are compared to available upper air measurements including data from the recent ATTREX and HIPPO2 aircraft campaigns. In the East Pacific region, the location of the available measurement campaigns in the upper TTL, the comparisons give a good agreement indicating that around 0.01 to 0.02 ppt of CH3I enter the stratosphere. However, other tropical regions, which are subject to stronger convective activity show larger CH3I

  9. Forest harvest contribution to Boreal freshwater methyl mercury load

    NASA Astrophysics Data System (ADS)

    Kronberg, Rose-Marie; Drott, Andreas; Jiskra, Martin; Wiederhold, Jan G.; Björn, Erik; Skyllberg, Ulf

    2016-06-01

    Effects of Boreal forest harvest on mercury (Hg) and methyl mercury (MeHg) soil pools and export by stream runoff were quantified by comparing 10 reference watersheds (REFs) covered by >80 year old Norway spruce (Picea abies Karst.) forests with 10 similar watersheds subjected to clear-cutting (CCs). While total Hg soil storage did not change, MeHg pools increased seven times (p = 0.006) in the organic topsoil 2 years after clear-cutting. In undulating terrain, situated above the postglacial marine limit (ML) of the ancient Baltic Sea, the mass ratio between flux-weighted MeHg and dissolved organic carbon (MeHg/DOC) in stream runoff increased 1.8 times (p < 0.004) as a consequence of forest harvest. When recalculated to 100% clear-cutting of the watershed, the annual MeHg stream export increased 3.8 times (p = 0.047). Below the ML, where the terrain was flatter, neither the MeHg/DOC ratio nor the annual export of MeHg differed between REFs and CCs, likely because of the larger contribution of MeHg exported from peaty soils and small wetlands. The most robust measure, MeHg/DOC, was used to calculate MeHg loadings to Boreal headwaters. If the forest harvest effect lasts 10 years, clear-cutting increases MeHg runoff by 12-20% in Sweden and 2% in the Boreal zone as a whole. In Sweden, having intensely managed forests, 37% and 56% of MeHg are exported from peatlands and forest soils, respectively, and forest clear-cutting is adding another 6.6%. In the Boreal zone as a whole peatlands and forests soils contribute with 53% and 46%, respectively, and clear-cutting is estimated to add another 1.0%. An expected rapid increase in Boreal forest harvest and disturbance urge for inclusion of land use effects in mercury biogeochemical cycling models at different scales.

  10. A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling.

    PubMed

    Kneip, Christoph; Schmidt, Bernd; Fleischhacker, Michael; Seegebarth, Anke; Lewin, Jörn; Flemming, Nadja; Seemann, Stefanie; Schlegel, Thomas; Witt, Christian; Liebenberg, Volker; Dietrich, Dimo

    2009-09-01

    DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.

  11. Genome-wide DNA methylation detection by MethylCap-seq and Infinium HumanMethylation450 BeadChips: an independent large-scale comparison

    PubMed Central

    De Meyer, Tim; Bady, Pierre; Trooskens, Geert; Kurscheid, Sebastian; Bloch, Jocelyne; Kros, Johan M.; Hainfellner, Johannes A.; Stupp, Roger; Delorenzi, Mauro; Hegi, Monika E.; Van Criekinge, Wim

    2015-01-01

    Two cost-efficient genome-scale methodologies to assess DNA-methylation are MethylCap-seq and Illumina’s Infinium HumanMethylation450 BeadChips (HM450). Objective information regarding the best-suited methodology for a specific research question is scant. Therefore, we performed a large-scale evaluation on a set of 70 brain tissue samples, i.e. 65 glioblastoma and 5 non-tumoral tissues. As MethylCap-seq coverages were limited, we focused on the inherent capacity of the methodology to detect methylated loci rather than a quantitative analysis. MethylCap-seq and HM450 data were dichotomized and performances were compared using a gold standard free Bayesian modelling procedure. While conditional specificity was adequate for both approaches, conditional sensitivity was systematically higher for HM450. In addition, genome-wide characteristics were compared, revealing that HM450 probes identified substantially fewer regions compared to MethylCap-seq. Although results indicated that the latter method can detect more potentially relevant DNA-methylation, this did not translate into the discovery of more differentially methylated loci between tumours and controls compared to HM450. Our results therefore indicate that both methodologies are complementary, with a higher sensitivity for HM450 and a far larger genome-wide coverage for MethylCap-seq, but also that a more comprehensive character does not automatically imply more significant results in biomarker studies. PMID:26482909

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

    PubMed Central

    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

    2014-01-01

    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

  13. Blast induced neurotrauma causes overpressure dependent changes to the DNA methylation equilibrium.

    PubMed

    Bailey, Zachary S; Grinter, Michael B; De La Torre Campos, Diego; VandeVord, Pamela J

    2015-09-14

    Traumatic brain injury (TBI) has a high prevalence in our society and often leads to morbidity and mortality. TBI also occurs frequently in a military setting where exposure to blast waves is common. Abnormal gene expression involved with oxidative stress, inflammation and neuronal apoptosis has been well documented following blast induced neurotrauma (BINT). Altered epigenetic transcriptional regulation through DNA methylation has been implicated in the pathology of the injury. Imbalance between DNA methylation and DNA demethylation may lead to altered methylation patterns and subsequent changes in gene transcription. DNA methyltransferase enzymes (DNMT1, DNMT3a, and DNMT3b) are responsible for the addition of methyl groups to DNA, DNA methylation. Whereas the combined function of ten-eleven translocation enzymes (TET1, TET2, and TET3) and thymine-DNA glycosylase (TDG) result in the removal of methyl groups from DNA, DNA demethylation. We used an established rodent model of BINT to assess changes in DNA methylation and demethylation enzymes following injury. Three different blast overpressures were investigated (10, 17 and 23psi). Gene expression was investigated in the prefrontal cortex and hippocampus two weeks following injury. We observed DNMT, TET and TDG expression changes between pressure groups and brain regions. The hippocampus was more vulnerable to enzyme expression changes than the prefrontal cortex, which correlated with aberrant DNA methylation. A significant negative correlation was found between global DNA methylation and the magnitude of blast overpressure exposure. Through transcriptional regulation, altered DNA methylation patterns may offer insight into the characteristic outcomes associated with the injury pathology including inflammation, oxidative stress and apoptosis. As such, these enzymes may be important targets to future therapeutic intervention strategies. PMID:26232681

  14. Mood Stabilizers and the Influence on Global Leukocyte DNA Methylation in Bipolar Disorder

    PubMed Central

    Backlund, Lena; Wei, Ya Bin; Martinsson, Lina; Melas, Philippe A.; Liu, Jia Jia; Mu, Ninni; Östenson, Claes-Göran; Ekström, Tomas J.; Schalling, Martin; Lavebratt, Catharina

    2015-01-01

    Little is known about the relationship between treatments for bipolar disorder (BD), their therapeutic responses and the DNA methylation status. We investigated whether global DNA methylation levels differ between healthy controls and bipolar patients under different treatments. Global DNA methylation was measured in leukocyte DNA from bipolar patients under lithium monotherapy (n = 29) or combination therapy (n = 32) and from healthy controls (n = 26). Lithium response was assessed using the Alda scale. Lithium in monotherapy was associated with hypomethylation (F = 4.63, p = 0.036). Lithium + valproate showed a hypermethylated pattern compared to lithium alone (F = 7.27, p = 0.011). Lithium response was not associated with DNA methylation levels. These data suggest that the choice of treatment in BD may lead to different levels of global DNA methylation. However, further research is needed to understand its clinical significance.

  15. Genome-wide Mapping Reveals Conservation of Promoter DNA Methylation Following Chicken Domestication

    PubMed Central

    Li, Qinghe; Wang, Yuanyuan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Ning

    2015-01-01

    It is well-known that environment influences DNA methylation, however, the extent of heritable DNA methylation variation following animal domestication remains largely unknown. Using meDIP-chip we mapped the promoter methylomes for 23,316 genes in muscle tissues of ancestral and domestic chickens. We systematically examined the variation of promoter DNA methylation in terms of different breeds, differentially expressed genes, SNPs and genes undergo genetic selection sweeps. While considerable changes in DNA sequence and gene expression programs were prevalent, we found that the inter-strain DNA methylation patterns were highly conserved in promoter region between the wild and domestic chicken breeds. Our data suggests a global preservation of DNA methylation between the wild and domestic chicken breeds in either a genome-wide or locus-specific scale in chick muscle tissues. PMID:25735894

  16. Genome-wide mapping reveals conservation of promoter DNA methylation following chicken domestication.

    PubMed

    Li, Qinghe; Wang, Yuanyuan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Ning

    2015-01-01

    It is well-known that environment influences DNA methylation, however, the extent of heritable DNA methylation variation following animal domestication remains largely unknown. Using meDIP-chip we mapped the promoter methylomes for 23,316 genes in muscle tissues of ancestral and domestic chickens. We systematically examined the variation of promoter DNA methylation in terms of different breeds, differentially expressed genes, SNPs and genes undergo genetic selection sweeps. While considerable changes in DNA sequence and gene expression programs were prevalent, we found that the inter-strain DNA methylation patterns were highly conserved in promoter region between the wild and domestic chicken breeds. Our data suggests a global preservation of DNA methylation between the wild and domestic chicken breeds in either a genome-wide or locus-specific scale in chick muscle tissues.

  17. Effects of cytosine methylation on DNA morphology: An atomic force microscopy study.

    PubMed

    Cassina, V; Manghi, M; Salerno, D; Tempestini, A; Iadarola, V; Nardo, L; Brioschi, S; Mantegazza, F

    2016-01-01

    Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.

  18. Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.

    PubMed

    Heimer, Brandon W; Tam, Brooke E; Sikes, Hadley D

    2015-12-01

    Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application. PMID:26384511

  19. Analysis of Chromatin Regulators Reveals Specific Features of Rice DNA Methylation Pathways.

    PubMed

    Tan, Feng; Zhou, Chao; Zhou, Qiangwei; Zhou, Shaoli; Yang, Wenjing; Zhao, Yu; Li, Guoliang; Zhou, Dao-Xiu

    2016-07-01

    Plant DNA methylation that occurs at CG, CHG, and CHH sites (H = A, C, or T) is a hallmark of the repression of repetitive sequences and transposable elements (TEs). The rice (Oryza sativa) genome contains about 40% repetitive sequence and TEs and displays specific patterns of genome-wide DNA methylation. The mechanism responsible for the specific methylation patterns is unclear. Here, we analyzed the function of OsDDM1 (Deficient in DNA Methylation 1) and OsDRM2 (Deficient in DNA Methylation 1) in genome-wide DNA methylation, TE repression, small RNA accumulation, and gene expression. We show that OsDDM1 is essential for high levels of methylation at CHG and, to a lesser extent, CG sites in heterochromatic regions and also is required for CHH methylation that mainly locates in the genic regions of the genome. In addition to a large member of TEs, loss of OsDDM1 leads to hypomethylation and up-regulation of many protein-coding genes, producing very severe growth phenotypes at the initial generation. Importantly, we show that OsDRM2 mutation results in a nearly complete loss of CHH methylation and derepression of mainly small TE-associated genes and that OsDDM1 is involved in facilitating OsDRM2-mediated CHH methylation. Thus, the function of OsDDM1 and OsDRM2 defines distinct DNA methylation pathways in the bulk of DNA methylation of the genome, which is possibly related to the dispersed heterochromatin across chromosomes in rice and suggests that DNA methylation mechanisms may vary among different plant species. PMID:27208249

  20. Divergence of Gene Body DNA Methylation and Evolution of Plant Duplicate Genes

    PubMed Central

    Wang, Jun; Marowsky, Nicholas C.; Fan, Chuanzhu

    2014-01-01

    It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica) genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences) of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes. PMID:25310342

  1. Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome

    PubMed Central

    Brebi-Mieville, Priscilla; Ili-Gangas, Carmen; Leal-Rojas, Pamela; Noordhuis, Maartje; Soudry, Ethan; Perez, Jimena; Roa, Juan Carlos; Sidransky, David; Guerrero-Preston, Rafael

    2012-01-01

    The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies. PMID:22207357

  2. DNA methylation directs functional maturation of pancreatic β cells

    PubMed Central

    Dhawan, Sangeeta; Tschen, Shuen-Ing; Zeng, Chun; Guo, Tingxia; Hebrok, Matthias; Matveyenko, Aleksey; Bhushan, Anil

    2015-01-01

    Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specific Dnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function. PMID:26098213

  3. Deciphering a methylome: what can we read into patterns of DNA methylation?

    PubMed

    Flores, Kevin B; Amdam, Gro V

    2011-10-01

    The methylation of cytosines within cytosine-guanine (CG) dinucleotides is an epigenetic mark that can modify gene transcription. With the advent of high-throughput sequencing, it is possible to map methylomes, i.e. detect methylated CGs on a genome-wide scale. The methylomes sequenced to date reveal a divergence in prevalence and targeting of CG methylation between taxa, despite the conservation of the DNA methyltransferase enzymes that cause DNA methylation. Therefore, interspecific methylation usage is predicted to diverge. In various taxa, this tenet gains support from patterns of CG depletion that can be traced in DNA before methylomes are explicitly mapped. Depletion of CGs in methylated genomic regions is expected because methylated cytosines are subject to increased mutability caused by nucleotide deamination. However, the basis of diverging interspecific methylation usage is less clear. We use insights from the methylome of honeybees (Apis mellifera) to emphasize the possible importance of organismal life histories in explaining methylation usage and the accuracy of methylation prediction based on CG depletion. Interestingly, methylated genes in honeybees are more conserved across taxa than non-methylated genes despite the divergence in utilization of methylation and the increased mutability caused by deamination.

  4. Identification and Functional Relevance of de novo DNA Methylation in Cancerous B-Cell Populations

    PubMed Central

    Wang, Xiao-Ming; Greiner, Timothy C.; Bibikova, Marina; Pike, Brian L.; Siegmund, Kimberly D.; Sinha, Uttam K.; Müschen, Markus; Jaeger, Erich B.; Weisenburger, Dennis J.; Chan, Wing C.; Shibata, Darryl; Fan, Jian-Bing; Hacia, Joseph G.

    2011-01-01

    Epigenetic remodeling is a hallmark of cancer, with the frequent acquisition of de novo DNA methylation in CpG islands. However, the functional relevance of de novo DNA methylation in cancer is less well-defined. To begin to address this issue in B-cells, we used BeadArray assays to survey the methylation status of 1,500 cancer-related CpG loci in two molecular subtypes of diffuse large B-cell lymphoma (ABC-DLBCL and GCB-DLBCL) and cognate normal B-cell populations. We identified 81 loci that showed frequent de novo DNA methylation in GCB-DLBCL and 67 loci that showed frequent de novo DNA methylation in ABC-DLBCL. These de novo methylated CpG loci included reported targets of polycomb repressive complexes (PRC) in stem cells. All candidate loci in GCB-DLBCL are proximal to genes that are poorly expressed or silent in purified normal germinal center (GC) B-cells. This is consistent with the hypothesis that de novo DNA methylation in cancer is more frequently involved in the maintenance rather than the initiation of gene silencing (de novo repression). This suggests that epigenetic switching occurs during tumorigenesis with de novo DNA methylation locking in gene silencing normally mediated by transcriptional repressors. Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis. PMID:20069569

  5. Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice.

    PubMed Central

    Lettmann, C; Schmitz, B; Doerfler, W

    1991-01-01

    In cultured mammalian cells, foreign DNA can be integrated into the host genome. Foreign DNA is frequently de novo methylated in specific patterns with successive cell generations. The sequence-specific methylation of promoter sequences in integrated foreign DNA is associated with the long-term inactivation of eukaryotic genes. We have now extended these experiments to studies on transgenic mice. As in previous work, a construct (pAd2E2AL-CAT) has been used which consists of the late E2A promoter of adenovirus type 2 (Ad2) DNA fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT). This construct has been integrated in the non-methylated in the 5'-CCGG-3' premethylated form in the genomes of transgenic mice. DNA from various organs was analyzed by HpaII/MspI cleavage to assess the state of methylation in 5'-CCGG-3' sequences. The results demonstrate that the transgenic construct is in general stable. Non-methylated constructs have remained partly non-methylated for four generations or can become de novo methylated at all or most 5'-CCGG-3' sequences in the founder animal. Preimposed patterns of 5'-CCGG-3' methylation have been preserved for up to four generations beyond the founder animal. In the testes of two different founder animals and two F1 males, the transgenic DNA has become demethylated by an unknown mechanism. In all other organs, the transgenic DNA preserves the preimposed 5'-CCGG-3' methylation pattern. In the experiments performed so far we have not observed differences in the transmission of methylation patterns depending on whether the transgene has been maternally or paternally inherited. The 5'-CCGG-3' premethylated transgene does not catalyze CAT activity in several organs, except in one example of the testes of an animal in which the transgenic construct has become demethylated. In contrast, when the nonmethylated construct has been integrated and remained largely non-methylated, CAT activity has been detected in extracts from

  6. Position-dependent correlations between DNA methylation and the evolutionary rates of mammalian coding exons

    PubMed Central

    Chuang, Trees-Juen; Chen, Feng-Chi; Chen, Yen-Zho

    2012-01-01

    DNA cytosine methylation is a central epigenetic marker that is usually mutagenic and may increase the level of sequence divergence. However, methylated genes have been reported to evolve more slowly than unmethylated genes. Hence, there is a controversy on whether DNA methylation is correlated with increased or decreased protein evolutionary rates. We hypothesize that this controversy has resulted from the differential correlations between DNA methylation and the evolutionary rates of coding exons in different genic positions. To test this hypothesis, we compare human–mouse and human–macaque exonic evolutionary rates against experimentally determined single-base resolution DNA methylation data derived from multiple human cell types. We show that DNA methylation is significantly related to within-gene variations in evolutionary rates. First, DNA methylation level is more strongly correlated with C-to-T mutations at CpG dinucleotides in the first coding exons than in the internal and last exons, although it is positively correlated with the synonymous substitution rate in all exon positions. Second, for the first exons, DNA methylation level is negatively correlated with exonic expression level, but positively correlated with both nonsynonymous substitution rate and the sample specificity of DNA methylation level. For the internal and last exons, however, we observe the opposite correlations. Our results imply that DNA methylation level is differentially correlated with the biological (and evolutionary) features of coding exons in different genic positions. The first exons appear more prone to the mutagenic effects, whereas the other exons are more influenced by the regulatory effects of DNA methylation. PMID:23019368

  7. DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

    PubMed Central

    Sater, Mohamad R. Abdul; Lamelas, Araceli; Wang, Guilin; Clark, Tyson A.; Röltgen, Katharina; Mane, Shrikant; Korlach, Jonas; Pluschke, Gerd; Schmid, Christoph D.

    2015-01-01

    The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of

  8. DNA methylation of the LEP gene is associated with craving during alcohol withdrawal.

    PubMed

    Hillemacher, Thomas; Weinland, Christian; Lenz, Bernd; Kraus, Thomas; Heberlein, Annemarie; Glahn, Alexander; Muschler, Marc A N; Bleich, Stefan; Kornhuber, Johannes; Frieling, Helge

    2015-01-01

    Different studies have described evidence for an association between leptin serum levels and craving in alcohol dependent patients. As leptin expression is regulated by DNA methylation we investigated changes of DNA methylation of the LEP gene promoter region in alcohol dependent patients undergoing withdrawal. Results show that low methylation status is associated with increasing serum leptin levels and elevation of craving for alcohol in the referring patients group. These findings point towards a pathophysiological relevance of changes in DNA methylation of the LEP gene promoter region in alcohol dependence.

  9. MethMarker: user-friendly design and optimization of gene-specific DNA methylation assays

    PubMed Central

    2009-01-01

    DNA methylation is a key mechanism of epigenetic regulation that is frequently altered in diseases such as cancer. To confirm the biological or clinical relevance of such changes, gene-specific DNA methylation changes need to be validated in multiple samples. We have developed the MethMarker http://methmarker.mpi-inf.mpg.de/ software to help design robust and cost-efficient DNA methylation assays for six widely used methods. Furthermore, MethMarker implements a bioinformatic workflow for transforming disease-specific differentially methylated genomic regions into robust clinical biomarkers. PMID:19804638

  10. Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons

    PubMed Central

    Iwamoto, Kazuya; Bundo, Miki; Ueda, Junko; Oldham, Michael C.; Ukai, Wataru; Hashimoto, Eri; Saito, Toshikazu; Geschwind, Daniel H.; Kato, Tadafumi

    2011-01-01

    Epigenome information in mammalian brain cells reflects their developmental history, neuronal activity, and environmental exposures. Studying the epigenetic modifications present in neuronal cells is critical to a more complete understanding of the role of the genome in brain functions. We performed comprehensive DNA methylation analysis in neuronal and non-neuronal nuclei obtained from the human prefrontal cortex. Neuronal nuclei manifest qualitatively and quantitatively distinctive DNA methylation patterns, including relative global hypomethylation, differential enrichment of transcription-factor binding sites, and higher methylation of genes expressed in astrocytes. Non-neuronal nuclei showed indistinguishable DNA methylation patterns from bulk cortex and higher methylation of synaptic transmission-related genes compared wit