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Sample records for mammalian dna methylation

  1. Methylated DNA-binding protein is present in various mammalian cell types

    SciTech Connect

    Supakar, P.C.; Weist, D.; Zhang, D.; Inamdar, N.; Zhang, Xianyang; Khan, R.; Ehrlich, M. ); Ehrlich, K.C. )

    1988-08-25

    A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m{sup 5}C) residues at specific positions. The authors found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

  2. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

    PubMed

    Schroeder, Diane I; Jayashankar, Kartika; Douglas, Kory C; Thirkill, Twanda L; York, Daniel; Dickinson, Pete J; Williams, Lawrence E; Samollow, Paul B; Ross, Pablo J; Bannasch, Danika L; Douglas, Gordon C; LaSalle, Janine M

    2015-08-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo.

  3. [Mammalian DNA methylation and its roles during the induced re-programming of somatic cells].

    PubMed

    Hongwei, Song; Tiezhu, An; Shanhua, Piao; Chunsheng, Wang

    2014-05-01

    The technology of induced pluripotent stem cell (iPS) provides the possibility to reverse the terminal differentiated cells to pluripotent stem cells, and is therefore of great importance in both the theoretical research of stem cells and regenerative medicine. However, the efficiency of current induced reprogramming methods is extremely low, and the incomplete reprogramming often happens. It has been reported that some epigenetic memory of the somatic cells exists in these incomplete reprogrammed iPS cells, and DNA methylation, as a relative long-term and stable epigenetic modification, is one of the important factors that influence the efficiency of reprogramming and differentiative capacity of iPS cells. Mammalian DNA methylation, which normally appears on the CpG sites, occurs on the fifth carbon atom of the cytosine ring. DNA methylation can modulate the expression of somatic cell specific genes, and pluripotent genes; hence, it plays important roles in the processes of mammalian gene regulation, embryonic development and cell reprogramming. In addition, it has also been found that abnormal DNA methylation may lead to the disorder of genetic imprinting and the inactivation of X chromosome in iPS cells. Therefore, in order to provide a concise guidance of DNA methylation studies in iPS, we mainly review the mechanism, the distribution features of DNA methylation, and its roles in induced reprogramming of somatic cells.

  4. Mammalian Brain Development is Accompanied by a Dramatic Increase in Bipolar DNA Methylation

    PubMed Central

    Sun, Ming-an; Sun, Zhixiong; Wu, Xiaowei; Rajaram, Veena; Keimig, David; Lim, Jessica; Zhu, Hongxiao; Xie, Hehuang

    2016-01-01

    DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders. PMID:27585862

  5. Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers.

    PubMed

    Seisenberger, Stefanie; Peat, Julian R; Hore, Timothy A; Santos, Fátima; Dean, Wendy; Reik, Wolf

    2013-01-05

    In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

  6. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA.

    PubMed

    Shen, Li; Gao, Ge; Zhang, Ying; Zhang, He; Ye, Zhiqiang; Huang, Shichao; Huang, Jinyan; Kang, Jiuhong

    2010-10-01

    Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.

  7. Insertion of Foreign DNA into an Established Mammalian Genome Can Alter the Methylation of Cellular DNA Sequences†

    PubMed Central

    Remus, Ralph; Kämmer, Christina; Heller, Hilde; Schmitz, Birgit; Schell, Gudrun; Doerfler, Walter

    1999-01-01

    existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after λ DNA integration might have existed prior to λ DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5′-CG-3′ methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated λ DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion. PMID:9882302

  8. DNA methylation on N6-adenine in mammalian embryonic stem cells

    PubMed Central

    Wu, Tao P.; Wang, Tao; Seetin, Matthew G.; Lai, Yongquan; Zhu, Shijia; Lin, Kaixuan; Liu, Yifei; Byrum, Stephanie D.; Mackintosh, Samuel G.; Zhong, Mei; Tackett, Alan; Wang, Guilin; Hon, Lawrence S.; Fang, Gang; Swenberg, James A.; Xiao, Andrew Z.

    2016-01-01

    It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N6-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N6-methyladenine. An increase of N6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N6-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N6-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N6-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. PMID:27027282

  9. Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

    PubMed Central

    Nyce, J; Liu, L; Jones, P A

    1986-01-01

    Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells. PMID:3086840

  10. Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

    PubMed

    Morano, Annalisa; Angrisano, Tiziana; Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Bartollino, Silvia; Zuchegna, Candida; Babbio, Federica; Bonapace, Ian Marc; Allen, Brittany; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2014-01-01

    We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

  11. 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

  12. 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.

  13. 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

  14. A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases

    PubMed Central

    Benetti, Roberta; Gonzalo, Susana; Jaco, Isabel; Muñoz, Purificación; Gonzalez, Susana; Schoeftner, Stefan; Murchison, Elizabeth; Andl, Thomas; Chen, Taiping; Klatt, Peter; Li, En; Serrano, Manuel; Millar, Sarah; Hannon, Gregory; Blasco, Maria A

    2010-01-01

    Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis. PMID:18311151

  15. Isolation of genomic DNA from mammalian cells.

    PubMed

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Enzymology of Mammalian DNA Methyltransferases.

    PubMed

    Jurkowska, Renata Z; Jeltsch, Albert

    2016-01-01

    DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications.

  17. DNA methylation and its basic function.

    PubMed

    Moore, Lisa D; Le, Thuc; Fan, Guoping

    2013-01-01

    In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. During development, the pattern of DNA methylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation and demethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulates tissue-specific gene transcription. In this chapter, we will review the process of DNA methylation and demethylation in the nervous system. We will describe the DNA (de)methylation machinery and its association with other epigenetic mechanisms such as histone modifications and noncoding RNAs. Intriguingly, postmitotic neurons still express DNA methyltransferases and components involved in DNA demethylation. Moreover, neuronal activity can modulate their pattern of DNA methylation in response to physiological and environmental stimuli. The precise regulation of DNA methylation is essential for normal cognitive function. Indeed, when DNA methylation is altered as a result of developmental mutations or environmental risk factors, such as drug exposure and neural injury, mental impairment is a common side effect. The investigation into DNA methylation continues to show a rich and complex picture about epigenetic gene regulation in the central nervous system and provides possible therapeutic targets for the treatment of neuropsychiatric disorders.

  18. [DNA methylation and epigenetics].

    PubMed

    Vaniushin, B F

    2006-09-01

    In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N6-methyladenine residues in the sequence TGATCA (TGATCA-->TGm6ATCA). Plants possess AdoMet-dependent endonucleases

  19. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  20. DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

    PubMed

    Tran, Robert K; Henikoff, Jorja G; Zilberman, Daniel; Ditt, Renata F; Jacobsen, Steven E; Henikoff, Steven

    2005-01-26

    Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

  1. Mammalian DNA helicase.

    PubMed Central

    Hübscher, U; Stalder, H P

    1985-01-01

    A forked DNA was constructed to serve as a substrate for DNA helicases. It contains features closely resembling a natural replication fork. The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases alpha holoenzyme. One form of DNA polymerase alpha holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P. and Hübscher, U. (1984) Proc. Natl. Acad. Sci. USA 81, 3993-3997). The enzyme can be separated from DNA polymerase alpha by velocity sedimentation in conditions of very low ionic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose. At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable. The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3'hydroxyl end only in the presence of ATP, and this displacement relies on ATP hydrolysis. Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction. By all this token the DNA dependent ATPase appears to be a DNA helicase. It is suggested that this DNA helicase might act in concert with DNA polymerase alpha at the leading strand, possibly pushing the replication fork ahead of the polymerase. Images PMID:3162158

  2. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  3. DNA methylation and differentiation.

    PubMed Central

    Michalowsky, L A; Jones, P A

    1989-01-01

    The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin. Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable. PMID:2466640

  4. 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.

  5. Nutrients and DNA Methylation

    USDA-ARS?s Scientific Manuscript database

    Epigenetics is a new mechanism responsible for development, aging, and disease process such as cancer development. One major epigenetic phenomenon is DNA methylation, which attributes to gene expression and integrity. Deepening the knowledge on one-carbon metabolism is very important to understandin...

  6. DNA Methylation within Transcribed Regions

    PubMed Central

    To, Taiko K.; Saze, Hidetoshi; Kakutani, Tetsuji

    2015-01-01

    DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation. PMID:26143255

  7. DNA Methylation in Mammals

    PubMed Central

    Li, En; Zhang, Yi

    2014-01-01

    DNA methylation is one of the best characterized epigenetic modifications. In mammals it is involved in various biological processes including the silencing of transposable elements, regulation of gene expression, genomic imprinting, and X-chromosome inactivation. This article describes how DNA methylation serves as a cellular memory system and how it is dynamically regulated through the action of the DNA methyltransferase (DNMT) and ten eleven translocation (TET) enzymes. Its role in the regulation of gene expression, through its interplay with histone modifications, is also described, and its implication in human diseases discussed. The exciting areas of investigation that will likely become the focus of research in the coming years are outlined in the summary. PMID:24789823

  8. 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

  9. Methyl nutrients, DNA methylation, and cardiovascular disease.

    PubMed

    Glier, Melissa B; Green, Timothy J; Devlin, Angela M

    2014-01-01

    Diet plays an important role in the development and prevention of cardiovascular disease (CVD), but the molecular mechanisms are not fully understood. DNA methylation has been implicated as an underlying molecular mechanism that may account for the effect of dietary factors on the development and prevention of CVD. DNA methylation is an epigenetic process that provides "marks" in the genome by which genes are set to be transcriptionally activated or silenced. Epigenomic marks are heritable but are also responsive to environmental shifts, such as changes in nutritional status, and are especially vulnerable during development. S-adenosylmethionine is the methyl group donor for DNA methylation and several nutrients are required for the production of S-adenosylmethionine. These methyl nutrients include vitamins (folate, riboflavin, vitamin B12, vitamin B6, choline) and amino acids (methionine, cysteine, serine, glycine). As such, imbalances in the metabolism of these nutrients have the potential to affect DNA methylation. The focus of this review is to provide an overview on the current understanding of the relationship between methyl nutrient status and DNA methylation patterns and the potential role of this interaction in CVD pathology. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. DNA methylation dynamics in neurogenesis.

    PubMed

    Wang, Zhiqin; Tang, Beisha; He, Yuquan; Jin, Peng

    2016-03-01

    Neurogenesis is not limited to the embryonic stage, but continually proceeds in the adult brain throughout life. Epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNA, play important roles in neurogenesis. For decades, DNA methylation was thought to be a stable modification, except for demethylation in the early embryo. In recent years, DNA methylation has proved to be dynamic during development. In this review, we summarize the latest understanding about DNA methylation dynamics in neurogenesis, including the roles of different methylation forms (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine), as well as their 'writers', 'readers' and interactions with histone modifications.

  11. METHYLATION OF SODIUM ARSENITE BY VARIOUS MAMMALIAN CELLS

    EPA Science Inventory


    Methylation of Sodium Arsenite by various Mammalian Cells

    Methylation of arsenite (As 3-1) is thought to play an important role in the carcinogenicity of arsenic. AIM: I. Characterization of methylation of arsenite in primary rodent and transformed human cell lines. ...

  12. METHYLATION OF SODIUM ARSENITE BY VARIOUS MAMMALIAN CELLS

    EPA Science Inventory


    Methylation of Sodium Arsenite by various Mammalian Cells

    Methylation of arsenite (As 3-1) is thought to play an important role in the carcinogenicity of arsenic. AIM: I. Characterization of methylation of arsenite in primary rodent and transformed human cell lines. ...

  13. DNA Methylation in Schizophrenia.

    PubMed

    Pries, Lotta-Katrin; Gülöksüz, Sinan; Kenis, Gunter

    2017-01-01

    Schizophrenia is a highly heritable psychiatric condition that displays a complex phenotype. A multitude of genetic susceptibility loci have now been identified, but these fail to explain the high heritability estimates of schizophrenia. In addition, epidemiologically relevant environmental risk factors for schizophrenia may lead to permanent changes in brain function. In conjunction with genetic liability, these environmental risk factors-likely through epigenetic mechanisms-may give rise to schizophrenia, a clinical syndrome characterized by florid psychotic symptoms and moderate to severe cognitive impairment. These pathophysiological features point to the involvement of epigenetic processes. Recently, a wave of studies examining aberrant DNA modifications in schizophrenia was published. This chapter aims to comprehensively review the current findings, from both candidate gene studies and genome-wide approaches, on DNA methylation changes in schizophrenia.

  14. Event extraction for DNA methylation.

    PubMed

    Ohta, Tomoko; Pyysalo, Sampo; Miwa, Makoto; Tsujii, Jun'ichi

    2011-10-06

    We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  15. Enrichment of methylated DNA by methyl-CpG immunoprecipitation.

    PubMed

    Sonnet, Miriam; Baer, Constance; Rehli, Michael; Weichenhan, Dieter; Plass, Christoph

    2013-01-01

    Normal DNA methylation is an epigenetic modification required for proper development. Aberrant DNA methylation, in contrast, is frequently observed in many different malignancies including leukemias and lymphomas. Global DNA methylation profiling addresses the methylated sequences (methylome) of patient genomes to identify disease-specific methylation patterns. Workload in methylome analyses can be considerably reduced by methylome enrichment using proteins or antibodies with high affinity to methylated DNA. Methyl-CpG Immunoprecipitation (MCIp) employs an immobilized recombinant human methyl-CpG binding domain protein 2, MBD2, which binds methylated CpGs in double-stranded DNA. Elution with increasing salt concentrations allows the fractionated enrichment of different degrees of methylation.

  16. 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.

  17. 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.

  18. 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

  19. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  20. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  1. DNA methylation reprogramming and DNA repair in the mouse zygote.

    PubMed

    Lepikhov, Konstantin; Wossidlo, Mark; Arand, Julia; Walter, Joern

    2010-01-01

    Here, we summarize current knowledge about epigenetic reprogramming during mammalian preimplantation development, as well as the potential mechanisms driving these processes. We will particularly focus on changes taking place in the zygote, where the paternally derived DNA and chromatin undergo the most striking alterations, such as replacement of protamines by histones, histone modifications and active DNA demethylation. The putative mechanisms of active paternal DNA demethylation have been studied for over a decade, accumulating a lot of circumstantial evidence for enzymatic activities provided by the oocyte, protection of the maternal genome against such activities and possible involvement of DNA repair. We will discuss the various facets of dynamic epigenetic changes related to DNA methylation with an emphasis on the putative involvement of DNA repair in DNA demethylation.

  2. Programming of DNA methylation patterns.

    PubMed

    Cedar, Howard; Bergman, Yehudit

    2012-01-01

    DNA methylation represents a form of genome annotation that mediates gene repression by serving as a maintainable mark that can be used to reconstruct silent chromatin following each round of replication. During development, germline DNA methylation is erased in the blastocyst, and a bimodal pattern is established anew at the time of implantation when the entire genome gets methylated while CpG islands are protected. This brings about global repression and allows housekeeping genes to be expressed in all cells of the body. Postimplantation development is characterized by stage- and tissue-specific changes in methylation that ultimately mold the epigenetic patterns that define each individual cell type. This is directed by sequence information in DNA and represents a secondary event that provides long-term expression stability. Abnormal methylation changes play a role in diseases, such as cancer or fragile X syndrome, and may also occur as a function of aging or as a result of environmental influences.

  3. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    PubMed

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

  4. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  5. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  6. Cigarette smoking and DNA methylation

    PubMed Central

    Lee, Ken W. K.; Pausova, Zdenka

    2013-01-01

    DNA methylation is the most studied epigenetic modification, capable of controlling gene expression in the contexts of normal traits or diseases. It is highly dynamic during early embryogenesis and remains relatively stable throughout life, and such patterns are intricately related to human development. DNA methylation is a quantitative trait determined by a complex interplay of genetic and environmental factors. Genetic variants at a specific locus can influence both regional and distant DNA methylation. The environment can have varying effects on DNA methylation depending on when the exposure occurs, such as during prenatal life or during adulthood. In particular, cigarette smoking in the context of both current smoking and prenatal exposure is a strong modifier of DNA methylation. Epigenome-wide association studies have uncovered candidate genes associated with cigarette smoking that have biologically relevant functions in the etiology of smoking-related diseases. As such, DNA methylation is a potential mechanistic link between current smoking and cancer, as well as prenatal cigarette-smoke exposure and the development of adult chronic diseases. PMID:23882278

  7. 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

  8. Methods of DNA methylation analysis.

    USDA-ARS?s Scientific Manuscript database

    The purpose of this review was to provide guidance for investigators who are new to the field of DNA methylation analysis. Epigenetics is the study of mitotically heritable alterations in gene expression potential that are not mediated by changes in DNA sequence. Recently, it has become clear that n...

  9. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  10. Alcohol, DNA Methylation, and Cancer

    PubMed Central

    Varela-Rey, Marta; Woodhoo, Ashwin; Martinez-Chantar, Maria-Luz; Mato, José M.; Lu, Shelly C.

    2013-01-01

    Cancer is one of the most significant diseases associated with chronic alcohol consumption, and chronic drinking is a strong risk factor for cancer, particularly of the upper aerodigestive tract, liver, colorectum, and breast. Several factors contribute to alcohol-induced cancer development (i.e., carcinogenesis), including the actions of acetaldehyde, the first and primary metabolite of ethanol, and oxidative stress. However, increasing evidence suggests that aberrant patterns of DNA methylation, an important epigenetic mechanism of transcriptional control, also could be part of the pathogenetic mechanisms that lead to alcohol-induced cancer development. The effects of alcohol on global and local DNA methylation patterns likely are mediated by its ability to interfere with the availability of the principal biological methyl donor, S-adenosylmethionine (SAMe), as well as pathways related to it. Several mechanisms may mediate the effects of alcohol on DNA methylation, including reduced folate levels and inhibition of key enzymes in one-carbon metabolism that ultimately lead to lower SAMe levels, as well as inhibition of activity and expression of enzymes involved in DNA methylation (i.e., DNA methyltransferases). Finally, variations (i.e., polymorphisms) of several genes involved in one-carbon metabolism also modulate the risk of alcohol-associated carcinogenesis. PMID:24313162

  11. Alcohol, DNA methylation, and cancer.

    PubMed

    Varela-Rey, Marta; Woodhoo, Ashwin; Martinez-Chantar, Maria-Luz; Mato, José M; Lu, Shelly C

    2013-01-01

    Cancer is one of the most significant diseases associated with chronic alcohol consumption, and chronic drinking is a strong risk factor for cancer, particularly of the upper aerodigestive tract, liver, colorectum, and breast. Several factors contribute to alcohol-induced cancer development (i.e., carcinogenesis), including the actions of acetaldehyde, the first and primary metabolite of ethanol, and oxidative stress. However, increasing evidence suggests that aberrant patterns of DNA methylation, an important epigenetic mechanism of transcriptional control, also could be part of the pathogenetic mechanisms that lead to alcohol-induced cancer development. The effects of alcohol on global and local DNA methylation patterns likely are mediated by its ability to interfere with the availability of the principal biological methyl donor, S-adenosylmethionine (SAMe), as well as pathways related to it. Several mechanisms may mediate the effects of alcohol on DNA methylation, including reduced folate levels and inhibition of key enzymes in one-carbon metabolism that ultimately lead to lower SAMe levels, as well as inhibition of activity and expression of enzymes involved in DNA methylation (i.e., DNA methyltransferases). Finally, variations (i.e., polymorphisms) of several genes involved in one-carbon metabolism also modulate the risk of alcohol-associated carcinogenesis.

  12. Stringent programming of DNA methylation in humans.

    PubMed

    Aung, Hnin T; Harrison, Dion K; Findlay, Ian; Mattick, John S; Martin, Nicholas G; Carroll, Bernard J

    2010-10-01

    We describe a PCR-based method called Amplified Methylation Polymorphism (AMP) for scanning genomes for DNA methylation changes. AMP detects tissue-specific DNA methylation signatures often representing junctions between methylated and unmethylated DNA close to intronexon junctions and/or associated with CpG islands. Identical AMP profiles are detected for healthy, young, monozygotic twins.

  13. Primer removal during mammalian mitochondrial DNA replication.

    PubMed

    Uhler, Jay P; Falkenberg, Maria

    2015-10-01

    The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Evolution of DNA Methylation across Insects

    PubMed Central

    Vogel, Kevin J.; Moore, Allen J.; Schmitz, Robert J.

    2017-01-01

    DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. PMID:28025279

  15. DNA methylation in endometriosis (Review)

    PubMed Central

    KOUKOURA, OURANIA; SIFAKIS, STAVROS; SPANDIDOS, DEMETRIOS A.

    2016-01-01

    Endometriosis is defined by the presence and growth of functional endometrial tissue, outside the uterine cavity, primarily in the ovaries, pelvic peritoneum and rectovaginal septum. Although it is a benign disease, it presents with malignant characteristics, such as invasion to surrounding tissues, metastasis to distant locations and recurrence following treatment. Accumulating evidence suggests that various epigenetic aberrations may play an essential role in the pathogenesis of endometriosis. Aberrant DNA methylation represents a possible mechanism repsonsible for this disease, linking gene expression alterations observed in endometriosis with hormonal and environmental factors. Several lines of evidence indicate that endometriosis may partially be due to selective epigenetic deregulations influenced by extrinsic factors. Previous studies have shed light into the epigenetic component of endometriosis, reporting variations in the epigenetic patterns of genes known to be involved in the aberrant hormonal, immunologic and inflammatory status of endometriosis. Although recent studies, utilizing advanced molecular techniques, have allowed us to further elucidate the possible association of DNA methylation with altered gene expression, whether these molecular changes represent the cause or merely the consequence of the disease is a question which remains to be answered. This review provides an overview of the current literature on the role of DNA methylation in the pathophysiology and malignant evolution of endometriosis. We also provide insight into the mechanisms through which DNA methylation-modifying agents may be the next step in the research of the pharmaceutical treatment of endometriosis. PMID:26934855

  16. DNA methylation profiling of human chromosomes 6, 20 and 22

    PubMed Central

    Eckhardt, Florian; Lewin, Joern; Cortese, Rene; Rakyan, Vardhman K.; Attwood, John; Burger, Matthias; Burton, John; Cox, Tony V.; Davies, Rob; Down, Thomas A.; Haefliger, Carolina; Horton, Roger; Howe, Kevin; Jackson, David K.; Kunde, Jan; Koenig, Christoph; Liddle, Jennifer; Niblett, David; Otto, Thomas; Pettett, Roger; Seemann, Stefanie; Thompson, Christian; West, Tony; Rogers, Jane; Olek, Alex; Berlin, Kurt; Beck, Stephan

    2011-01-01

    DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought. PMID:17072317

  17. 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.

  18. Locus-Specific DNA Methylation Reprogramming During Early Porcine Embryogenesis1

    PubMed Central

    Zhao, Ming-Tao; Rivera, Rocio M.; Prather, Randall S.

    2013-01-01

    ABSTRACT During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development. PMID:23303676

  19. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    SciTech Connect

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  20. DNA methylation is critical for Arabidopsis embryogenesis and seed viability.

    PubMed

    Xiao, Wenyan; Custard, Kendra D; Brown, Roy C; Lemmon, Betty E; Harada, John J; Goldberg, Robert B; Fischer, Robert L

    2006-04-01

    DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.

  1. DNA Methylation Is Critical for Arabidopsis Embryogenesis and Seed Viability

    PubMed Central

    Xiao, Wenyan; Custard, Kendra D.; Brown, Roy C.; Lemmon, Betty E.; Harada, John J.; Goldberg, Robert B.; Fischer, Robert L.

    2006-01-01

    DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability. PMID:16531498

  2. Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.

    PubMed

    de la Rica, Lorenzo; Stanley, Jatinder S; Branco, Miguel R

    2016-01-01

    DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.

  3. Methods in DNA methylation profiling

    PubMed Central

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Huey-Jen L; Huang, Tim H-M

    2010-01-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling. PMID:20526417

  4. Methods in DNA methylation profiling.

    PubMed

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Juey-Jen L; Huang, Tim H-M

    2009-12-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.

  5. Dynamics of DNA methylation in aging and Alzheimer's disease.

    PubMed

    Irier, Hasan A; Jin, Peng

    2012-10-01

    Gene expression is modulated by epigenetic factors that come in varying forms, such as DNA methylation, histone modifications, microRNAs, and long noncoding RNAs. Recent studies reveal that these epigenetic marks are important regulatory factors in brain function. In particular, DNA methylation dynamics are found to be essential components of epigenetic regulation in the mammalian central nervous system. In this review, we provide an overview of the literature on DNA methylation in neurodegenerative diseases, with a special focus on methylation of 5-position of cytosine base (5mC) and hydroxymethylation of 5-position of cytosine base (5hmC) in the context of neurodegeneration associated with aging and Alzheimer's disease.

  6. 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.

  7. 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.

  8. DNA methylation profiling of hematopoietic stem cells.

    PubMed

    Begtrup, Amber Hogart

    2014-01-01

    DNA methylation is a key epigenetic mark that is essential for properly functioning hematopoietic stem cells. Determining where functionally relevant DNA methylation marks exist in the genome is crucial to understanding the role that methylation plays in hematopoiesis. This chapter describes a method to profile DNA methylation by selectively enriching methylated DNA sequences that are bound in vitro by methyl-binding domain (MBD) proteins. The MBD-pulldown approach selects for DNA sequences that have the potential to be "read" by the endogenous machinery involved in epigenetic regulation. Furthermore, this approach is feasible with very small quantities of DNA, and is compatible with the use of any downstream high-throughput sequencing approach. This technique offers a reliable, simple, and powerful tool for exploration of the role of DNA methylation in hematopoietic stem cells.

  9. Dynamics and Context-Dependent Roles of DNA Methylation.

    PubMed

    Ambrosi, Christina; Manzo, Massimiliano; Baubec, Tuncay

    2017-05-19

    DNA methylation is one of the most extensively studied epigenetic marks. It is involved in transcriptional gene silencing and plays important roles during mammalian development. Its perturbation is often associated with human diseases. In mammalian genomes, DNA methylation is a prevalent modification that decorates the majority of cytosines. It is found at the promoters and enhancers of inactive genes, at repetitive elements, and within transcribed gene bodies. Its presence at promoters is dynamically linked to gene activity, suggesting that it could directly influence gene expression patterns and cellular identity. The genome-wide distribution and dynamic behaviour of this mark have been studied in great detail in a variety of tissues and cell lines, including early embryonic development and in embryonic stem cells. In combination with functional studies, these genome-wide maps of DNA methylation revealed interesting features of this mark and provided important insights into its dynamic nature and potential functional role in genome regulation. In this review, we discuss how these recent observations, in combination with insights obtained from biochemical and functional genetics studies, have expanded our current knowledge about the regulation and context-dependent roles of DNA methylation in mammalian genomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Molecular and Enzymatic Profiles of Mammalian DNA Methyltransferases: Structures and Targets for Drugs

    PubMed Central

    Xu, F.; Mao, C.; Ding, Y.; Rui, C.; Wu, L.; Shi, A.; Zhang, H.; Zhang, L.; Xu, Z.

    2010-01-01

    DNA methylation is an epigenetic event involved in a variety array of processes that may be the foundation of genetic phenomena and diseases. DNA methyltransferase is a key enzyme for cytosine methylation in DNA, and can be divided into two functional families (Dnmt1 and Dnmt3) in mammals. All mammalian DNA methyltransferases are encoded by their own single gene, and consisted of catalytic and regulatory regions (except Dnmt2). Via interactions between functional domains in the regulatory or catalytic regions and other adaptors or cofactors, DNA methyltransferases can be localized at selective areas (specific DNA/nucleotide sequence) and linked to specific chromosome status (euchromatin/heterochromatin, various histone modification status). With assistance from UHRF1 and Dnmt3L or other factors in Dnmt1 and Dnmt3a/Dnmt3b, mammalian DNA methyltransferases can be recruited, and then specifically bind to hemimethylated and unmethylated double-stranded DNA sequence to maintain and de novo setup patterns for DNA methylation. Complicated enzymatic steps catalyzed by DNA methyltransferases include methyl group transferred from cofactor Ado-Met to C5 position of the flipped-out cytosine in targeted DNA duplex. In the light of the fact that different DNA methyltransferases are divergent in both structures and functions, and use unique reprogrammed or distorted routines in development of diseases, design of new drugs targeting specific mammalian DNA methyltransferases or their adaptors in the control of key steps in either maintenance or de novo DNA methylation processes will contribute to individually treating diseases related to DNA methyltransferases. PMID:20939822

  11. High-Resolution Analysis of Cytosine Methylation in Ancient DNA

    PubMed Central

    Cropley, Jennifer E.; Cooper, Alan; Suter, Catherine M.

    2012-01-01

    Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution. PMID:22276161

  12. The terminal DNA structure of mammalian chromosomes.

    PubMed Central

    McElligott, R; Wellinger, R J

    1997-01-01

    In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated. PMID:9218811

  13. Evolution of DNA Methylation across Insects.

    PubMed

    Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

    2017-03-01

    DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    PubMed

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  15. Interactions within the mammalian DNA methyltransferase family

    PubMed Central

    Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

    2003-01-01

    Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

  16. Identifying DNA methylation in a nanochannel

    PubMed Central

    Sun, Xiaoyin; Yasui, Takao; Yanagida, Takeshi; Kaji, Noritada; Rahong, Sakon; Kanai, Masaki; Nagashima, Kazuki; Kawai, Tomoji; Baba, Yoshinobu

    2016-01-01

    Abstract DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24–96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h. PMID:27877910

  17. Mutation hot spots in mammalian mitochondrial DNA.

    PubMed

    Galtier, Nicolas; Enard, David; Radondy, Yoan; Bazin, Eric; Belkhir, Khalid

    2006-02-01

    Animal mitochondrial DNA is characterized by a remarkably high level of within-species homoplasy, that is, phylogenetic incongruence between sites of the molecule. Several investigators have invoked recombination to explain it, challenging the dogma of maternal, clonal mitochondrial inheritance in animals. Alternatively, a high level of homoplasy could be explained by the existence of mutation hot spots. By using an exhaustive mammalian data set, we test the hot spot hypothesis by comparing patterns of site-specific polymorphism and divergence in several groups of closely related species, including hominids. We detect significant co-occurrence of synonymous polymorphisms among closely related species in various mammalian groups, and a correlation between the site-specific levels of variability within humans (on one hand) and between Hominoidea species (on the other hand), indicating that mutation hot spots actually exist in mammalian mitochondrial coding regions. The whole data, however, cannot be explained by a simple mutation hot spots model. Rather, we show that the site-specific mutation rate quickly varies in time, so that the same sites are not hypermutable in distinct lineages. This study provides a plausible mutation model that potentially accounts for the peculiar distribution of mitochondrial sequence variation in mammals without the need for invoking recombination. It also gives hints about the proximal causes of mitochondrial site-specific hypermutability in humans.

  18. Relationship between nucleosome positioning and DNA methylation

    PubMed Central

    Chodavarapu, Ramakrishna K.; Feng, Suhua; Bernatavichute, Yana V.; Chen, Pao-Yang; Stroud, Hume; Yu, Yanchun; Hetzel, Jonathan; Kuo, Frank; Kim, Jin; Cokus, Shawn J.; Casero, David; Bernal, Maria; Huijser, Peter; Clark, Amander T.; Krämer, Ute; Merchant, Sabeeha S.; Zhang, Xiaoyu; Jacobsen, Steven E.; Pellegrini, Matteo

    2010-01-01

    Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results suggest that nucleosome positioning strongly influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA suggesting that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition. PMID:20512117

  19. 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.

  20. 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.

  1. Whole genome DNA methylation: beyond genes silencing

    PubMed Central

    Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

    2017-01-01

    The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology. PMID:27895318

  2. CG methylation in DNA transcription

    NASA Astrophysics Data System (ADS)

    Chela-Flores, J.; Migoni, R. L.

    1990-08-01

    A simple model of DNA is considered in which the nucleotides cytosine (C) and guanine (G) are not assumed to be identical, and in which macroscopic thermodynamic quantities may be calculated exactly. The H bonds between the C and G nucleotides are assumed to be Morse potentials. We discuss the statistical mechanics of the DNA molecule in the configuration (5'...GGG ...3'; 3'...CCC ...5'), which may be copied by RNA polymerase into a messenger RNA (mRNA) strand (5'...CCC ...3'). This model suggests that replacements of C by 5-methylcytosine (5mC) may be a secondary effect in the inhibition of genetic expression, not interfering directly with the formation of an open state. An experimental test is suggested. The implications of this result are discussed for a related system, in which the enzyme methylase is known to methylate almost exclusively those Cs that are followed by Gs as a regulatory strategy employed by some eukaryotes.

  3. Brain feminization requires active repression of masculinization via DNA methylation

    PubMed Central

    Nugent, Bridget M.; Wright, Christopher L.; Shetty, Amol C.; Hodes, Georgia E.; Lenz, Kathryn M.; Mahurkar, Anup; Russo, Scott J.; Devine, Scott E.; McCarthy, Margaret M.

    2015-01-01

    The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually-dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in females. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation. PMID:25821913

  4. Conventional and nanotechniques for DNA methylation profiling.

    PubMed

    Shanmuganathan, Rajasree; Basheer, Nazeema B; Amirthalingam, Laxmi; Muthukumar, Harshiny; Kaliaperumal, Rajendran; Shanmugam, Kumaran

    2013-01-01

    DNA methylation is critical for gene silencing and is associated with the incidence of many diseases, including cancer. Underlying molecular mechanisms of human diseases and tissue-specific gene expression have been elucidated based on DNA methylation studies. This review highlights the advantages and drawbacks of various methylation screening techniques: blotting, genomic sequencing, bisulfite sequencing, methylation-specific PCR, methylated DNA immunoprecipitation, microarray analysis, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, nanowire transistor detection procedure, quantum dot-based nanoassay, single-molecule real-time detection, fluorimetric assay, electrochemical detection, and atomic force spectroscopy. The review provides insight for selecting a method or a combination of methods for DNA methylation analysis. Convergence of conventional and contemporary nanotechniques to enumerate methylation at specific CpG sites of oncogene would fill the gap in diagnosis of cancer.

  5. Aberrant DNA methylation patterns in diabetic nephropathy

    PubMed Central

    2014-01-01

    Background The aim of this study was to evaluate whether global levels of DNA methylation status were associated with albuminuria and progression of diabetic nephropathy in a case-control study of 123 patients with type 2 diabetes- 53 patients with albuminuria and 70 patients without albuminuria. Methods The 5-methyl cytosine content was assessed by reverse phase high pressure liquid chromatography (RP-HPLC) of peripheral blood mononuclear cells to determine individual global DNA methylation status in two groups. Results Global DNA methylation levels were significantly higher in patients with albuminuria compared with those in normal range of albuminuria (p = 0.01). There were significant differences in global levels of DNA methylation in relation to albuminuria (p = 0.028) and an interesting pattern of increasing global levels of DNA methylation in terms of albuminuria severity. In patients with micro- and macro albuminuria, we found no significant correlations between global DNA methylation levels and duration of diabetes (p > 0.05). In both sub groups, there were not significant differences between global DNA methylation levels with good and poor glycaemic control (p > 0.05). In addition, in patients with albuminuria, no differences in DNA methylation levels were observed between patients with and without other risk factors including age, gender, hypertension, dyslipidaemia and obesity. Conclusions These data may be helpful in further studies to develop novel biomarkers and new strategies for clinical care of patients at risk of diabetic nephropathy. PMID:25028646

  6. Implications of DNA Methylation in Parkinson's Disease.

    PubMed

    Miranda-Morales, Ernesto; Meier, Karin; Sandoval-Carrillo, Ada; Salas-Pacheco, José; Vázquez-Cárdenas, Paola; Arias-Carrión, Oscar

    2017-01-01

    It has been 200 years since Parkinson's disease (PD) was first described, yet many aspects of its etiopathogenesis remain unclear. PD is a progressive and complex neurodegenerative disorder caused by genetic and environmental factors including aging, nutrition, pesticides and exposure to heavy metals. DNA methylation may be altered in response to some of these factors; therefore, it is proposed that epigenetic mechanisms, particularly DNA methylation, can have a fundamental role in gene-environment interactions that are related with PD. Epigenetic changes in PD-associated genes are now widely studied in different populations, to discover the mechanisms that contribute to disease development and identify novel biomarkers for early diagnosis and future pharmacological treatment. While initial studies sought to find associations between promoter DNA methylation and the regulation of associated genes in PD brain tissue, more recent studies have described concordant DNA methylation patterns between blood and brain tissue DNA. These data justify the use of peripheral blood samples instead of brain tissue for epigenetic studies. Here, we summarize the current data about DNA methylation changes in PD and discuss the potential of DNA methylation as a potential biomarker for PD. Additionally, we discuss environmental and nutritional factors that have been implicated in DNA methylation. Although the search for significant DNA methylation changes and gene expression analyses of PD-associated genes have yielded inconsistent and contradictory results, epigenetic modifications remain under investigation for their potential to reveal the link between environmental risk factors and the development of PD.

  7. Altered DNA methylation in PAH deficient phenylketonuria.

    PubMed

    Dobrowolski, Steven F; Lyons-Weiler, James; Spridik, Kayla; Biery, Amy; Breck, Jane; Vockley, Jerry; Yatsenko, Svetlana; Sultana, Tamanna

    2015-01-01

    While phenylalanine (PHE) is the toxic insult in phenylketonuria (PKU), mechanisms underlying PHE toxicity remain ill-defined. Altered DNA methylation in response to toxic exposures is well-recognized. DNA methylation patterns were assessed in blood and brain from PKU patients to determine if PHE toxicity impacts methylation. Methylome assessment, utilizing methylated DNA immunoprecipitation and paired-end sequencing, was performed in DNA obtained from brain tissue of classical PKU patients, leukocytes from poorly controlled PKU patients, leukocytes from well controlled PKU patients, and appropriate control tissues. In PKU brain tissue, expression analysis determined the impact of methylation on gene function. Differential methylation was observed in brain tissue of PKU patients and expression studies identified downstream impact on gene expression. Altered patterns of methylation were observed in leukocytes of well controlled and poorly controlled patients with more extensive methylation in patients with high PHE exposure. Differential methylation of noncoding RNA genes was extensive in patients with high PHE exposure but minimal in well controlled patients. Methylome repatterning leading to altered gene expression was present in brain tissue of PKU patients, suggesting a role in neuropathology. Aberrant methylation is observed in leukocytes of PKU patients and is influenced by PHE exposure. DNA methylation may provide a biomarker relating to historic PHE exposure. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. 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

  9. DNA Methylation and Gene Regulation in Honeybees: From Genome-Wide Analyses to Obligatory Epialleles.

    PubMed

    Wedd, Laura; Maleszka, Ryszard

    2016-01-01

    In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies. Importantly, this gene body methylation is frequently associated with active transcription, and studies in the honeybee have shown that there are strong links between gene body methylation and alternative splicing. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. Here we discuss the current knowledge in this emerging field and highlight both similarities and differences between DNA methylation systems in mammals and invertebrates. Finally, we argue that the relationship between genetic variation, differential DNA methylation, other epigenetic modifications and the transcriptome must be further explored to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.

  10. RNA-directed DNA methylation in Arabidopsis

    PubMed Central

    Aufsatz, Werner; Mette, M. Florian; van der Winden, Johannes; Matzke, Antonius J. M.; Matzke, Marjori

    2002-01-01

    In plants, double-stranded RNA that is processed to short RNAs ≈21–24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA–DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure. PMID:12169664

  11. DNA methylation homeostasis in human and mouse development.

    PubMed

    Iurlaro, Mario; von Meyenn, Ferdinand; Reik, Wolf

    2017-03-02

    The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals. An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species.

  12. DNA DAMAGE BINDING PROTEIN2 Shapes the DNA Methylation Landscape

    PubMed Central

    Schalk, Catherine; Kramdi, Amira; Ahmed, Ikhlak; Cognat, Valérie; Graindorge, Stéfanie; Bergdoll, Marc; Baumberger, Nicolas; Heintz, Dimitri; Bowler, Chris; Genschik, Pascal; Barneche, Fredy; Molinier, Jean

    2016-01-01

    In eukaryotes, DNA repair pathways help to maintain genome integrity and epigenomic patterns. However, the factors at the nexus of DNA repair and chromatin modification/remodeling remain poorly characterized. Here, we uncover a previously unrecognized interplay between the DNA repair factor DNA DAMAGE BINDING PROTEIN2 (DDB2) and the DNA methylation machinery in Arabidopsis thaliana. Loss-of-function mutation in DDB2 leads to genome-wide DNA methylation alterations. Genetic and biochemical evidence indicate that at many repeat loci, DDB2 influences de novo DNA methylation by interacting with ARGONAUTE4 and by controlling the local abundance of 24-nucleotide short interfering RNAs (siRNAs). We also show that DDB2 regulates active DNA demethylation mediated by REPRESSOR OF SILENCING1 and DEMETER LIKE3. Together, these findings reveal a role for the DNA repair factor DDB2 in shaping the Arabidopsis DNA methylation landscape in the absence of applied genotoxic stress. PMID:27531226

  13. Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

    PubMed Central

    Lawley, P. D.; Thatcher, Carolyn J.

    1970-01-01

    1. In neutral aqueous solution N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O6-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed. PMID:5435496

  14. In pursuit of the first recognized epigenetic signal--DNA methylation: a 1976 to 2008 synopsis.

    PubMed

    Doerfler, Walter

    2008-01-01

    A synopsis will be presented of work on DNA methylation, the first epigenetic signal to be recognized. In the author's laboratory, the following problems dealing with DNA methylation have been addressed over the past 32 years: (1) The de novo methylation of foreign DNA integrated into mammalian genomes. (2) Inverse correlations between promoter methylation and activity. (3) The long-term inactivating effect of site-specific promoter methylation. (4) Adenovirus E1 functions in trans and a strong enhancer in cis cancel the silencing effect of promoter methylation. (5) Frog virus 3, an iridovirus with a completely CpG-methylated genome. (6) Mechanisms of de novo methylation. (7) Different segments of the genome possess topical methylation memories. (8) Consequences of foreign DNA insertion into mammalian genomes: alterations of DNA methylation in cis and trans. (9) The epigenetic status of an adenovirus transgenome in Ad12-transformed hamster cells. (10) Cell type-specific patterns of DNA methylation: interindividual concordance in the human genome.

  15. Electrochemical biosensing strategies for DNA methylation analysis.

    PubMed

    Hossain, Tanvir; Mahmudunnabi, Golam; Masud, Mostafa Kamal; Islam, Md Nazmul; Ooi, Lezanne; Konstantinov, Konstantin; Hossain, Md Shahriar Al; Martinac, Boris; Alici, Gursel; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

    2017-02-17

    DNA methylation is one of the key epigenetic modifications of DNA that results from the enzymatic addition of a methyl group at the fifth carbon of the cytosine base. It plays a crucial role in cellular development, genomic stability and gene expression. Aberrant DNA methylation is responsible for the pathogenesis of many diseases including cancers. Over the past several decades, many methodologies have been developed to detect DNA methylation. These methodologies range from classical molecular biology and optical approaches, such as bisulfite sequencing, microarrays, quantitative real-time PCR, colorimetry, Raman spectroscopy to the more recent electrochemical approaches. Among these, electrochemical approaches offer sensitive, simple, specific, rapid, and cost-effective analysis of DNA methylation. Additionally, electrochemical methods are highly amenable to miniaturization and possess the potential to be multiplexed. In recent years, several reviews have provided information on the detection strategies of DNA methylation. However, to date, there is no comprehensive evaluation of electrochemical DNA methylation detection strategies. Herein, we address the recent developments of electrochemical DNA methylation detection approaches. Furthermore, we highlight the major technical and biological challenges involved in these strategies and provide suggestions for the future direction of this important field.

  16. High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions.

    PubMed

    Krebs, Arnaud R; Dessus-Babus, Sophie; Burger, Lukas; Schübeler, Dirk

    2014-09-26

    The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.

  17. Incorporating DNA Methylation Dynamics Into Epigenetic Codes

    PubMed Central

    Szulwach, Keith E.; Jin, Peng

    2014-01-01

    Summary Genomic function is dictated by a combination of DNA sequence and the molecular mechanisms controlling access to genetic information. Access to DNA can be determined by the interpretation of covalent modifications that influence the packaging of DNA into chromatin, including DNA methylation and histone modifications. These modifications are believed to be forms of “epigenetic codes” that exist in discernable combinations that reflect cellular phenotype. Although DNA methylation is known to play important roles in gene regulation and genomic function, its contribution to the encoding of epigenetic information is just beginning to emerge. Here we discuss paradigms associated with the various components of DNA methylation/demethylation and recent advances in the understanding of its dynamic regulation in the genome, integrating these mechanisms into a framework to explain how DNA methylation could contribute to epigenetic codes. PMID:24242211

  18. DNA methylation as a universal biomarker

    PubMed Central

    Levenson, Victor V

    2010-01-01

    Cell-free circulating DNA carries not only tumor-specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation, in certain GC-rich fragments. These fragments are usually located within the promoters and first exons of many genes, comprising CpG islands. Analysis of DNA methylation using cell-free circulating DNA can facilitate development of very accurate biomarkers for detection, diagnosis, prediction of response to therapy and prognosis of outcomes. Recent data suggest that benign and inflammatory diseases have very specific methylation patterns within cell-free circulating DNA, which are different from the pattern of a malignant tumor of the same organ. In addition, specific methylation patterns have been detected for cancers of different organs, so a differential diagnosis of site-specific cancer appears feasible. Currently, cancer-related applications dominate the field, although methylation-based biomarkers may also be possible for other diseases, including neurodegenerative and psychiatric disorders. PMID:20465502

  19. 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

  20. 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

  1. Global DNA methylation in neonatal sepsis.

    PubMed

    Dhas, Benet Bosco; Antony, Hiasindh Ashmi; Bhat, Vishnu; Newton, Banupriya; Parija, Subhash Chandra

    2015-04-01

    To find out whether gDNA methylation can be used as a diagnostic/prognostic method for neonatal sepsis. The study was conducted in the neonatal division of a tertiary care referral hospital. Fifty one newborns as cases and thirty seven newborns as controls were enrolled in the study. Using 5-mC DNA ELISA method, the percentage of genomic DNA methylated in these newborns was established. Highly significant difference in percentage of gDNA methylated was found between the cases and controls (Cases: 2.4 ± 0.39; 2.07 ± 0.35; P < 0.0001). Culture proven and possible cases were also significantly distinguishable (P < 0.05). No significant differences in methylation were observed in terms of gestational age, birth weight and outcomes such shock, thrombocytopenia, except for renal failure. The index results showed that genomic DNA methylation varies significantly among newborns with sepsis (clinical, probable and culture positive) and without sepsis. Although the global DNA methylation was not a highly sensitive diagnostic method, this study reveals that DNA methylation might play a vital role in neonatal sepsis susceptibility. Identification of the specific differentially methylated genes might serve as a promising future diagnostic/prognostic marker for neonatal sepsis.

  2. Optical biosensing strategies for DNA methylation analysis.

    PubMed

    Nazmul Islam, Md; Yadav, Sharda; Hakimul Haque, Md; Munaz, Ahmed; Islam, Farhadul; Al Hossain, Md Shahriar; Gopalan, Vinod; Lam, Alfred K; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

    2017-06-15

    DNA methylation is an epigenetic modification of DNA, where a methyl group is added at the fifth carbon of the cytosine base to form 5 methyl cytosine (5mC) without altering the DNA sequences. It plays important roles in regulating many cellular processes by modulating key genes expression. Alteration in DNA methylation patterns becomes particularly important in the aetiology of different diseases including cancers. Abnormal methylation pattern could contribute to the pathogenesis of cancer either by silencing key tumor suppressor genes or by activating oncogenes. Thus, DNA methylation biosensing can help in the better understanding of cancer prognosis and diagnosis and aid the development of therapies. Over the last few decades, a plethora of optical detection techniques have been developed for analyzing DNA methylation using fluorescence, Raman spectroscopy, surface plasmon resonance (SPR), electrochemiluminescence and colorimetric readouts. This paper aims to comprehensively review the optical strategies for DNA methylation detection. We also present an overview of the remaining challenges of optical strategies that still need to be focused along with the lesson learnt while working with these techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. 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

  4. Active loss of DNA methylation in two-cell stage goat embryos.

    PubMed

    Park, Jung S; Lee, Doosoo; Cho, Sunwha; Shin, Sang-Tae; Kang, Yong-Kook

    2010-01-01

    Early mammalian embryos are thought to gain nuclear totipotency through DNA methylation reprogramming (DMR). By this process, DNA methylation patterns acquired during gametogenesis that are unnecessary for zygotic development are erased. The DMR patterns of various mammalian species have been studied; however, they do not seem to have a conserved pattern. We examined early goat embryos to find conforming rules underlying mammalian DMR patterns. Immunocytochemical results showed that the overall level of DNA methylation was not greatly changed during the pronucleus stage. At the two-cell stage, active demethylation occurred and simultaneously affected both parental DNAs, resulting in a global loss of 5-methylcytosine. The level of DNA methylation was lowest in the four-cell stage, with increased de novo methylation during the eight-cell stage. Histone H3-lysine 9 was gradually trimethylated in the sperm-derived chromatin, continuing from the pronucleus stage through the two-cell stage. This goat DMR pattern is novel and distinct from the DMRs of other mammalian species. The more mammalian species we included for DMR analysis, the more multifarious patterns we obtained, adding an extra diversity each time to the known mammalian DMR patterns. Nevertheless, the evolutionary significance and developmental consequence of such diverse DMR patterns are currently unknown.

  5. Folate and DNA Methylation: A Review of Molecular Mechanisms and the Evidence for Folate’s Role12

    PubMed Central

    Crider, Krista S.; Yang, Thomas P.; Berry, Robert J; Bailey, Lynn B.

    2012-01-01

    DNA methylation is an epigenetic modification critical to normal genome regulation and development. The vitamin folate is a key source of the one carbon group used to methylate DNA. Because normal mammalian development is dependent on DNA methylation, there is enormous interest in assessing the potential for changes in folate intake to modulate DNA methylation both as a biomarker for folate status and as a mechanistic link to developmental disorders and chronic diseases including cancer. This review highlights the role of DNA methylation in normal genome function, how it can be altered, and the evidence of the role of folate/folic acid in these processes. PMID:22332098

  6. 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

  7. Profiling genome-wide DNA methylation.

    PubMed

    Yong, Wai-Shin; Hsu, Fei-Man; Chen, Pao-Yang

    2016-01-01

    DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

  8. Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

    PubMed Central

    Smith, S S; Kaplan, B E; Sowers, L C; Newman, E M

    1992-01-01

    The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns. Images PMID:1584813

  9. 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. © 2015 S. Karger AG, Basel

  10. 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

  11. DNA Methylation of BDNF Gene in Schizophrenia.

    PubMed

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

    2016-02-06

    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 AND 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.

  12. 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.

  13. DNA methylation in demyelinated multiple sclerosis hippocampus.

    PubMed

    Chomyk, Anthony M; Volsko, Christina; Tripathi, Ajai; Deckard, Sadie A; Trapp, Bruce D; Fox, Robert J; Dutta, Ranjan

    2017-08-18

    Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the human central nervous system (CNS). Memory impairments and hippocampal demyelination are common features in MS patients. Our previous data have shown that demyelination alters neuronal gene expression in the hippocampus. DNA methylation is a common epigenetic modifier of gene expression. In this study, we investigated whether DNA methylation is altered in MS hippocampus following demyelination. Our results show that mRNA levels of DNA methyltransferase were increased in demyelinated MS hippocampus, while de-methylation enzymes were decreased. Comparative methylation profiling identify hypo-methylation within upstream sequences of 6 genes and hyper-methylation of 10 genes in demyelinated MS hippocampus. Genes identified in the current study were also validated in an independent microarray dataset generated from MS hippocampus. Independent validation using RT-PCR revealed that DNA methylation inversely correlated with mRNA levels of the candidate genes. Queries across cell-specific databases revealed that a majority of the candidate genes are expressed by astrocytes and neurons in mouse and human CNS. Taken together, our results expands the list of genes previously identified in MS hippocampus and establish DNA methylation as a mechanism of altered gene expression in MS hippocampus.

  14. Effects of DNA methylation on nucleosome stability.

    PubMed

    Collings, Clayton K; Waddell, Peter J; Anderson, John N

    2013-03-01

    Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

  15. 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

  16. DNA methylation in white blood cells

    PubMed Central

    Delgado-Cruzata, Lissette; Vin-Raviv, Neomi; Wu, Hui Chen; Santella, Regina M

    2011-01-01

    Alterations in DNA methylation patterns, both at specific loci and overall in the genome, have been associated with many different health outcomes. In cancer and other diseases, most of these changes have been observed at the tissue level. Data on whether DNA methylation changes in white blood cells (WBC) can serve as a useful biomarker for different health outcomes are much more limited, but rapidly emerging. Epidemiologic studies have reported associations between global WBC methylation and several different cancers including cancers of the colon, bladder, stomach, breast, and head and neck, as well as schizophrenia and myelodysplastic syndrome. Evidence for WBC methylation at specific loci and disease risk is more limited, but increasing. Differences in WBC DNA methylation by selected risk factors including demographic (age, gender, race), environmental exposures (benzene, persistent organic pollutants, lead, arsenic and air pollution), and other risk factors (cigarette smoke, alcohol drinking, body size, physical activity and diet) have been observed in epidemiologic studies though the patterns are far from consistent. Challenges in inferences from the existing data are primarily due to the cross-sectional fashion and small size of most studies performed to date, as well as to the differences in results across assay type and source of DNA. Large, prospective studies will be needed to understand whether changes in risk factors are associated with changes in DNA methylation patterns, and if changes in DNA methylation patterns are associated with changes in disease endpoints. PMID:21636973

  17. 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

  18. DNA methylation biomarkers: cancer and beyond.

    PubMed

    Mikeska, Thomas; Craig, Jeffrey M

    2014-09-16

    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.

  19. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

    PubMed

    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

  20. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.

    PubMed

    Baubec, Tuncay; Colombo, Daniele F; Wirbelauer, Christiane; Schmidt, Juliane; Burger, Lukas; Krebs, Arnaud R; Akalin, Altuna; Schübeler, Dirk

    2015-04-09

    DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

  1. DNA methylation detection: bisulfite genomic sequencing analysis.

    PubMed

    Li, Yuanyuan; Tollefsbol, Trygve O

    2011-01-01

    DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, plays a pivotal role in many biological procedures such as gene expression, embryonic development, cellular proliferation, differentiation, and chromosome stability. Aberrant DNA methylation is often associated with loss of DNA homeostasis and genomic instability leading to the development of human diseases such as cancer. The importance of DNA methylation creates an urgent demand for effective methods with high sensitivity and reliability to explore innovative diagnostic and therapeutic strategies. Bisulfite genomic sequencing developed by Frommer and colleagues was recognized as a revolution in DNA methylation analysis based on conversion of genomic DNA by using sodium bisulfite. Besides various merits of the bisulfite genomic sequencing method such as being highly qualitative and quantitative, it serves as a fundamental principle to many derived methods to better interpret the mystery of DNA methylation. Here, we present a protocol currently frequently used in our laboratory that has proven to yield optimal outcomes. We also discuss the potential technical problems and troubleshooting notes for a variety of applications in this field.

  2. The evidence for functional non-CpG methylation in mammalian cells

    PubMed Central

    Patil, Vibha; Ward, Robyn L; Hesson, Luke B

    2014-01-01

    In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance. PMID:24717538

  3. DNA methylation abnormalities in congenital heart disease.

    PubMed

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

    2015-01-01

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

  4. Number matters: control of mammalian mitochondrial DNA copy number.

    PubMed

    Clay Montier, Laura L; Deng, Janice J; Bai, Yidong

    2009-03-01

    Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA (mtDNA) depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has advanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.

  5. Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.

    PubMed

    Stewart, Kathleen R; Veselovska, Lenka; Kim, Jeesun; Huang, Jiahao; Saadeh, Heba; Tomizawa, Shin-ichi; Smallwood, Sébastien A; Chen, Taiping; Kelsey, Gavin

    2015-12-01

    Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

  6. Mammalian satellite DNA: a speaking dumb.

    PubMed

    Enukashvily, Natella I; Ponomartsev, Nikita V

    2013-01-01

    The tandemly organized highly repetitive satellite DNA is the main DNA component of centromeric/pericentromeric constitutive heterochromatin. For almost a century, it was considered as "junk DNA," only a small portion of which is used for kinetochore formation. The current review summarizes recent data about satellite DNA transcription. The possible functions of the transcripts are discussed.

  7. DNA Methylation Signatures of the Plant Chromomethyltransferases

    PubMed Central

    Baulcombe, David C.

    2016-01-01

    DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G). By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. In A. thaliana the biased representation of mCCG in heterochromatin is related to specificities of H3K9 methyltransferase SUVH family members. At CHH motifs there is an over-representation of different variant forms of mCHH that, similarly to mCCG hypomethylation, is partitioned into the pericentric regions of the two dicots but dispersed in the monocot chromosomes. The over-represented mCHH motifs in A. thaliana associate with specific types of transposon including both class I and II elements. At mCHH the contextual bias is due to the involvement of various chromomethyltransferases whereas the context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin. This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms associated with maintenance of methylation and the overlying chromatin structure. PMID:27997534

  8. DNA Methylation Signatures of the Plant Chromomethyltransferases.

    PubMed

    Gouil, Quentin; Baulcombe, David C

    2016-12-01

    DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G). By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. In A. thaliana the biased representation of mCCG in heterochromatin is related to specificities of H3K9 methyltransferase SUVH family members. At CHH motifs there is an over-representation of different variant forms of mCHH that, similarly to mCCG hypomethylation, is partitioned into the pericentric regions of the two dicots but dispersed in the monocot chromosomes. The over-represented mCHH motifs in A. thaliana associate with specific types of transposon including both class I and II elements. At mCHH the contextual bias is due to the involvement of various chromomethyltransferases whereas the context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin. This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms associated with maintenance of methylation and the overlying chromatin structure.

  9. Regulation of expression and activity of DNA (cytosine-5) methyltransferases in mammalian cells.

    PubMed

    Kinney, Shannon R Morey; Pradhan, Sriharsa

    2011-01-01

    Three active DNA (cytosine-5) methyltransferases (DNMTs) have been identified in mammalian cells, Dnmt1, Dnmt3a, and Dnmt3b. DNMT1 is primarily a maintenance methyltransferase, as it prefers to methylate hemimethylated DNA during DNA replication and in vitro. DNMT3A and DNMT3B are de novo methyltransferases and show similar activity on unmethylated and hemimethylated DNA. DNMT3L, which lacks the catalytic domain, binds to DNMT3A and DNMT3B variants and facilitates their chromatin targeting, presumably for de novo methylation. There are several mechanisms by which mammalian cells regulate DNMT levels, including varied transcriptional activation of the respective genes and posttranslational modifications of the enzymes that can affect catalytic activity, targeting, and enzyme degradation. In addition, binding of miRNAs or RNA-binding proteins can also alter the expression of DNMTs. These regulatory processes can be disrupted in disease or by environmental factors, resulting in altered DNMT expression and aberrant DNA methylation patterns.

  10. Aberrant DNA Methylation and Prostate Cancer

    PubMed Central

    Majumdar, Sunipa; Buckles, Eric; Estrada, John; Koochekpour, Shahriar

    2011-01-01

    Prostate cancer (PCa) is the most prevalent cancer, a significant contributor to morbidity and a leading cause of cancer-related death in men in Western industrialized countries. In contrast to genetic changes that vary among individual cases, somatic epigenetic alterations are early and highly consistent events. Epigenetics encompasses several different phenomena, such as DNA methylation, histone modifications, RNA interference, and genomic imprinting. Epigenetic processes regulate gene expression and can change malignancy-associated phenotypes such as growth, migration, invasion, or angiogenesis. Methylations of certain genes are associated with PCa progression. Compared to normal prostate tissues, several hypermethylated genes have also been identified in benign prostate hyperplasia, which suggests a role for aberrant methylation in this growth dysfunction. Global and gene-specific DNA methylation could be affected by environmental and dietary factors. Among other epigenetic changes, aberrant DNA methylation might have a great potential as diagnostic or prognostic marker for PCa and could be tested in tumor tissues and various body fluids (e.g., serum, urine). The DNA methylation markers are simple in nature, have high sensitivity, and could be detected either quantitatively or qualitatively. Availability of genome-wide screening methodologies also allows the identification of epigenetic signatures in high throughput population studies. Unlike irreversible genetic changes, epigenetic alterations are reversible and could be used for PCa targeted therapies. PMID:22547956

  11. DNA methylation of Sleeping Beauty with transposition into the mouse genome.

    PubMed

    Park, Chang Won; Kren, Betsy T; Largaespada, David A; Steer, Clifford J

    2005-08-01

    The Sleeping Beauty transposon is a recently developed non-viral vector that can mediate insertion of transgenes into the mammalian genome. Foreign DNA elements that are introduced tend to invoke a host-defense mechanism resulting in epigenetic changes, such as DNA methylation, which may induce transcriptional inactivation of mammalian genes. To assess potential epigenetic modifications associated with Sleeping Beauty transposition, we investigated the DNA methylation pattern of transgenes inserted into the mouse genome as well as genomic regions flanking the insertion sites with bisulfite-mediated genomic sequencing. Transgenic mouse lines were created with two different Sleeping Beauty transposons carrying either the Agouti or eGFP transgene. Our results showed that DNA methylation in the keratin-14 promoter and Agouti transgene were negligible. In addition, two different genomic loci flanking the Agouti insertion site exhibited patterns of DNA methylation similar to wild-type mice. In contrast, high levels of DNA methylation were observed in the eGFP transgene and its ROSA26 promoter. These results indicate that transposition via Sleeping Beauty into the mouse genome may result in a significant level of de novo DNA methylation. This may depend on a number of different factors including the cargo DNA sequence, chromosomal context of the insertion site, and/or host genetic background.

  12. An endoparasitoid wasp influences host DNA methylation

    PubMed Central

    Kumar, Sunil; Kim, Yonggyun

    2017-01-01

    Parasitism by endoparasitoid wasps changes the expression of various host genes, and alters host immune and developmental processes. However, it is not clearly understood how parasitism changes host gene expression in a whole genome scale. This study focused on an epigenetic control of Cotesia plutellae, an endoparasitoid wasp, against its host, Plutella xylostella. Two DNA methyltransferases (DNMT-1 and DNMT-2) are encoded in the genome of P. xylostella. In addition, methyl-binding domain proteins (MBDs) and DNA demethylation factor, ten-eleven translation protein (TET) are encoded. DNA methylation of P. xylostella genomic DNA was confirmed by restriction digestion with Gla I specific to 5-methylcytosine. DNA methylation intensity in parasitized (P) larvae was decreased compared to that in nonparasitized (NP) larvae, especially at late parasitic stage, at which expression levels of both DNMT-1 and DNMT-2 were also decreased. DNA demethylation of P. xylostella was confirmed in both NP and P larvae by restriction digestion with PvuRts1I recognizing 5-hydroxymethyl cytosine. Parasitism also suppressed expression levels of TET and MBDs. Treatment of 5-aza-2′-deoxycytidine (AZA) reduced DNA methylation intensity of NP larvae, causing suppression of hemocyte-spreading behavior and delay of immature development. RNA interference of DNMT-1 or DNMT-2 mimicked the adverse effects of AZA. PMID:28230192

  13. Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug.

    PubMed

    Wang, Tieli; Pickard, Amanda J; Gallo, James M

    2016-07-01

    The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas -known as glioblastoma multiforme (GBM)- an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  14. Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

    PubMed Central

    Pickard, Amanda J.; Diaz, Anthony Joseph; Mura, Hugo; Nyuwen, Lila; Coello, Daniel; Sheva, Saif; Maria, Nava; Gallo, James M.; Wang, Tieli

    2017-01-01

    Background/Aim The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas –known as glioblastoma multiforme (GBM)– an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Materials and Methods Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Results Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. Conclusion The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment. PMID:27354585

  15. DNA methylation in memory formation: Emerging insights

    PubMed Central

    Heyward, Frankie D.; Sweatt, J. David

    2016-01-01

    The establishment of synaptic plasticity and long-term memory requires lasting cellular and molecular modifications that, as a whole, must endure despite the rapid turnover of their constituent parts. Such a molecular feat must be mediated by a stable, self-perpetuating, cellular information storage mechanism. DNA methylation, being the archetypal cellular information storage mechanism, has been heavily implicated as being necessary for stable activity-dependent transcriptional alterations within the central nervous system (CNS). This review details the foundational discoveries from both gene-targeted, as well as whole-genome sequencing, studies that have successfully brought DNA methylation to our attention as a chief regulator of activity- and experience-dependent transcriptional alterations within the CNS. We present a hypothetical framework with which the disparate experimental findings dealing with distinct manipulations of the DNA methylation, and their effect on memory, might be resolved while taking into account the unique impact activity-dependent alterations in DNA methylation potentially have on both memory promoting and memory-suppressing gene expression. And last, we discuss potential avenues for future inquiry into the role of DNA methylation during remote memory formation. PMID:25832671

  16. DNA Methylation in Memory Formation: Emerging Insights.

    PubMed

    Heyward, Frankie D; Sweatt, J David

    2015-10-01

    The establishment of synaptic plasticity and long-term memory requires lasting cellular and molecular modifications that, as a whole, must endure despite the rapid turnover of their constituent parts. Such a molecular feat must be mediated by a stable, self-perpetuating, cellular information storage mechanism. DNA methylation, being the archetypal cellular information storage mechanism, has been heavily implicated as being necessary for stable activity-dependent transcriptional alterations within the CNS. This review details the foundational discoveries from both gene-targeted and whole-genome sequencing studies that have brought DNA methylation to our attention as a chief regulator of activity- and experience-dependent transcriptional alterations within the CNS. We present a hypothetical framework to resolve disparate experimental findings regarding distinct manipulations of DNA methylation and their effect on memory, taking into account the unique impact activity-dependent alterations in DNA methylation potentially have on both memory-promoting and memory-suppressing gene expression. And last, we discuss potential avenues for future inquiry into the role of DNA methylation during remote memory formation. © The Author(s) 2015.

  17. Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®.

    PubMed

    Huntriss, John; Woodfine, Kathryn; Huddleston, Joanna E; Murrell, Adele; Picton, Helen M

    2015-01-01

    Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression.

  18. Epigenetic consequences of foreign DNA insertions: de novo methylation and global alterations of methylation patterns in recipient genomes.

    PubMed

    Doerfler, Walter

    2011-11-01

    The insertion of foreign DNA into mammalian or plant genomes is a frequent event in biology. My laboratory has pursued a long-standing interest in the structure of integrated adenovirus genomes and in the mechanism of foreign DNA insertions in mammalian cells. The long-term consequences of the integration of alien DNA are only partly known, and even less well understood are the mechanisms that bring them about. Evidence from viral systems has contributed to the realization that foreign DNA insertions entail a complex of sequelae that have also become apparent in non-viral systems: (i) The de novo methylation of integrated foreign DNA sequences has frequently been observed. (ii) Alterations of DNA methylation patterns in the recipient genome at and remote from the site of foreign DNA insertion have been demonstrated but it remains to be investigated how generally this phenomenon occurs. Many viral genomes find and have found entry into the genomes of present-day organisms. A major portion of mammalian genomes represents incomplete retroviral genomes that frequently have become permanently silenced by DNA methylation. It is still unknown how and to what extent the insertion of retroviral or retrotransposon sequences into established genomes has altered and shaped the methylation and transcription profiles of present day genomes. An additional reason for concern about the effects of foreign DNA integration is the fact that in all fields of molecular biology and medicine, the generation of transgenic or transgenomic cells and organisms has become a ubiquitously applied experimental technique. Copyright © 2011 John Wiley & Sons, Ltd.

  19. DNA Methylation-Targeted Drugs.

    PubMed

    Da Costa, Elodie M; McInnes, Gabrielle; Beaudry, Annie; Raynal, Noël J-M

    Targeting DNA hypermethylation, using nucleoside analogs, is an efficient approach to reprogram cancer cell epigenome leading to reduced proliferation, increased differentiation, recognition by the immune system, and ultimately cancer cell death. DNA methyltransferase inhibitors have been approved for the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myelogenous leukemia. To improve clinical efficacy and overcome mechanisms of drug resistance, a second generation of DNA methyltransferase inhibitors has been designed and is currently in clinical trials. Although efficient in monotherapy against hematologic malignancies, the potential of DNA methyltransferase inhibitors to synergize with small molecules targeting chromatin or immunotherapy will provide additional opportunities for their future clinical application against leukemia and solid tumors.

  20. DNA methylation during differentiation of a lower eukaryote, Physarum polycephalum.

    PubMed

    Fronk, J; Magiera, R

    1994-11-15

    Starvation-induced differentiation of the slime mould Physarum polycephalum is accompanied by continuous methylation of DNA. No stable changes in the overall level of DNA methylation are evident, but a gene known to be transcribed specifically during differentiation is subject to increased methylation. Inhibitors of DNA methylation preclude differentiation of P. polycephalum, although they are only marginally inhibitory to normal growth. Taken together these results indicate that methylation of DNA is involved in differentiation of this lower eukaryote.

  1. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements.

    PubMed

    Prior, Sara; Miousse, Isabelle R; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R; Allen, Antiño R; Raber, Jacob; Tackett, Alan J; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2016-10-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2'-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5'-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Is the Fungus Magnaporthe Losing DNA Methylation?

    PubMed Central

    Ikeda, Ken-ichi; Van Vu, Ba; Kadotani, Naoki; Tanaka, Masaki; Murata, Toshiki; Shiina, Kohta; Chuma, Izumi; Tosa, Yukio; Nakayashiki, Hitoshi

    2013-01-01

    The long terminal repeat retrotransposon, Magnaporthe gypsy-like element (MAGGY), has been shown to be targeted for cytosine methylation in a subset of Magnaporthe oryzae field isolates. Analysis of the F1 progeny from a genetic cross between methylation-proficient (Br48) and methylation-deficient (GFSI1-7-2) isolates revealed that methylation of the MAGGY element was governed by a single dominant gene. Positional cloning followed by gene disruption and complementation experiments revealed that the responsible gene was the DNA methyltransferase, MoDMT1, an ortholog of Neurospora crassa Dim-2. A survey of MAGGY methylation in 60 Magnaporthe field isolates revealed that 42 isolates from rice, common millet, wheat, finger millet, and buffelgrass were methylation proficient while 18 isolates from foxtail millet, green bristlegrass, Japanese panicgrass, torpedo grass, Guinea grass, and crabgrass were methylation deficient. Phenotypic analyses showed that MoDMT1 plays no major role in development and pathogenicity of the fungus. Quantitative polymerase chain reaction analysis showed that the average copy number of genomic MAGGY elements was not significantly different between methylation-deficient and -proficient field isolates even though the levels of MAGGY transcript were generally higher in the former group. MoDMT1 gene sequences in the methylation-deficient isolates suggested that at least three independent mutations were responsible for the loss of MoDMT1 function. Overall, our data suggest that MoDMT1 is not essential for the natural life cycle of the fungus and raise the possibility that the genus Magnaporthe may be losing the mechanism of DNA methylation on the evolutionary time scale. PMID:23979580

  3. 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

  4. Prognostic DNA Methylation Markers for Prostate Cancer

    PubMed Central

    Strand, Siri H.; Orntoft, Torben F.; Sorensen, Karina D.

    2014-01-01

    Prostate cancer (PC) is the most commonly diagnosed neoplasm and the third most common cause of cancer-related death amongst men in the Western world. PC is a clinically highly heterogeneous disease, and distinction between aggressive and indolent disease is a major challenge for the management of PC. Currently, no biomarkers or prognostic tools are able to accurately predict tumor progression at the time of diagnosis. Thus, improved biomarkers for PC prognosis are urgently needed. This review focuses on the prognostic potential of DNA methylation biomarkers for PC. Epigenetic changes are hallmarks of PC and associated with malignant initiation as well as tumor progression. Moreover, DNA methylation is the most frequently studied epigenetic alteration in PC, and the prognostic potential of DNA methylation markers for PC has been demonstrated in multiple studies. The most promising methylation marker candidates identified so far include PITX2, C1orf114 (CCDC181) and the GABRE~miR-452~miR-224 locus, in addition to the three-gene signature AOX1/C1orf114/HAPLN3. Several other biomarker candidates have also been investigated, but with less stringent clinical validation and/or conflicting evidence regarding their possible prognostic value available at this time. Here, we review the current evidence for the prognostic potential of DNA methylation markers in PC. PMID:25238417

  5. Intragenic DNA methylation prevents spurious transcription initiation.

    PubMed

    Neri, Francesco; Rapelli, Stefania; Krepelova, Anna; Incarnato, Danny; Parlato, Caterina; Basile, Giulia; Maldotti, Mara; Anselmi, Francesca; Oliviero, Salvatore

    2017-03-02

    In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

  6. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  7. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  8. Hydroxymethyluracil DNA glycosylase in mammalian cells

    SciTech Connect

    Hollstein, M.C.; Brooks, P.; Linn, S.; Ames, B.N.

    1984-07-01

    An activity has been purified 350-fold from extracts of mouse plasmacytoma cells that forms 5-hydroxymethyluracil (..cap alpha..-hydroxythymine) and apyrimidinic sites with phage SPO1 DNA, which contains this base in place of thymine. This DNA glycosylase presumably functions to eliminate hydroxymethyluracil, a major thymine-derived DNA lesion produced by ionizing radiation and oxidative damage. The enzyme has no cofactor requirement and is active in EDTA. Neither intermediate formation nor hydrolysis of hydroxymethyldeoxyuridine or hydroxymethyldeoxyuridine monophosphate was detected. The enzyme does not cleave apyrimidinic sites in DNA. It does release uracil from the uracil-containing DNA of phage PBS2, but this acitivity is less than 2% of the predominant uracil DNA glycosytase activity of the cell, which is separated by phosphocellulose chromatography. The major uracil DNA glycosylase does not release hydroxymethyluracil from SPO1 DNA. The hydroxymethyluracil glycosylase is also separated upon phosphocelluose chromatography from a thymine glycol DNA glycosylase activity that is accompanied by an apyrimidinic endonuclease activity. 33 references, 2 figures, 2 tables.

  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. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  10. In silico design of the first DNA-independent mechanism-based inhibitor of mammalian DNA methyltransferase Dnmt1

    PubMed Central

    Nikolić, Patrik

    2017-01-01

    Background We use our earlier experimental studies of the catalytic mechanism of DNA methyltransferases to prepare in silico a family of novel mechanism-based inhibitors of human Dnmt1. Highly specific inhibitors of DNA methylation can be used for analysis of human epigenome and for the creation of iPS cells. Results We describe a set of adenosyl-1-methyl-pyrimidin-2-one derivatives as novel mechanism-based inhibitors of mammalian DNA methyltransferase Dnmt1. The inhibitors have been designed to bind simultaneously in the active site and the cofactor site and thus act as transition-state analogues. Molecular dynamics studies showed that the lead compound can form between 6 to 9 binding interactions with Dnmt1. QM/MM analysis showed that the upon binding to Dnmt1 the inhibitor can form a covalent adduct with active site Cys1226 and thus act as a mechanism-based suicide-inhibitor. The inhibitor can target DNA-bond and DNA-free form of Dnmt1, however the suicide-inhibition step is more likely to happen when DNA is bound to Dnmt1. The validity of presented analysis is described in detail using 69 modifications in the lead compound structure. In total 18 of the presented 69 modifications can be used to prepare a family of highly specific inhibitors that can differentiate even between closely related enzymes such as Dnmt1 and Dnmt3a DNA methyltransferases. Conclusions Presented results can be used for preparation of some highly specific and potent inhibitors of mammalian DNA methylation with specific pharmacological properties. PMID:28399172

  11. Natural history of eukaryotic DNA methylation systems.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2011-01-01

    Methylation of cytosines and adenines in DNA is a widespread epigenetic mark in both prokaryotes and eukaryotes. In eukaryotes, it has a profound influence on chromatin structure and dynamics. Recent advances in genomics and biochemistry have considerably elucidated the functions and provenance of these DNA modifications. DNA methylases appear to have emerged first in bacterial restriction-modification (R-M) systems from ancient RNA-modifying enzymes, in transitions that involved acquisition of novel catalytic residues and DNA-recognition features. DNA adenine methylases appear to have been acquired by ciliates, heterolobosean amoeboflagellates, and certain chlorophyte algae. Six distinct clades of cytosine methylases, including the DNMT1, DNMT2, and DNMT3 clades, were acquired by eukaryotes through independent lateral transfer of their precursors from bacteria or bacteriophages. In addition to these, multiple adenine and cytosine methylases were acquired by several families of eukaryotic transposons. In eukaryotes, the DNA-methylase module was often combined with distinct modified and unmodified peptide recognition domains and other modules mediating specialized interactions, for example, the RFD module of DNMT1 which contains a permuted Sm domain linked to a helix-turn-helix domain. In eukaryotes, the evolution of DNA methylases appears to have proceeded in parallel to the elaboration of histone-modifying enzymes and the RNAi system, with functions related to counter-viral and counter-transposon defense, and regulation of DNA repair and differential gene expression being their primary ancestral functions. Diverse DNA demethylation systems that utilize base-excision repair via DNA glycosylases and cytosine deaminases appear to have emerged in multiple eukaryotic lineages. Comparative genomics suggests that the link between cytosine methylation and DNA glycosylases probably emerged first in a novel R-M system in bacteria. Recent studies suggest that the 5mC is not

  12. Regulation and function of DNA methylation in plants and animals

    PubMed Central

    He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

    2011-01-01

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

  13. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

    USDA-ARS?s Scientific Manuscript database

    DNA methylation is an epigenetic mechanism central to the development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. We used whole genome bisulfite sequencing, and found that differentiation of mouse colonic intestinal stem cell...

  14. 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

  15. Information Thermodynamics of Cytosine DNA Methylation

    PubMed Central

    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

  16. Topoisomerase II regulates the maintenance of DNA methylation.

    PubMed

    Lu, Lin-Yu; Kuang, Henry; Korakavi, Gautam; Yu, Xiaochun

    2015-01-09

    The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. 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.

  18. Regulation of maintenance DNA methylation via histone ubiquitylation

    PubMed Central

    Nishiyama, Atsuya; Yamaguchi, Luna; Nakanishi, Makoto

    2016-01-01

    DNA methylation is one of the most stable but dynamically regulated epigenetic marks that act as determinants of cell fates during embryonic development through regulation of various forms of gene expression. DNA methylation patterns must be faithfully propagated throughout successive cell divisions in order to maintain cell-specific function. We have recently demonstrated that Uhrf1-dependent ubiquitylation of histone H3 at lysine 23 is critical for Dnmt1 recruitment to DNA replication sites, which catalyzes the conversion of hemi-methylated DNA to fully methylated DNA. In this review, we provide an overview of recent progress in understanding the mechanism underlying maintenance DNA methylation. PMID:26590302

  19. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:26484126

  1. DNA ligase I is not essential for mammalian cell viability.

    PubMed

    Han, Li; Masani, Shahnaz; Hsieh, Chih-lin; Yu, Kefei

    2014-04-24

    Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  2. Distinctive Klf4 mutants determine preference for DNA methylation status

    SciTech Connect

    Hashimoto, Hideharu; Wang, Dongxue; Steves, Alyse N.; Jin, Peng; Blumenthal, Robert M.; Zhang, Xing; Cheng, Xiaodong

    2016-09-04

    Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

  3. Distinctive Klf4 mutants determine preference for DNA methylation status

    PubMed Central

    Hashimoto, Hideharu; Wang, Dongxue; Steves, Alyse N.; Jin, Peng; Blumenthal, Robert M.; Zhang, Xing; Cheng, Xiaodong

    2016-01-01

    Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay. PMID:27596594

  4. 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.

  5. DNA repair genes of mammalian cells

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Salazar, E.P.; Fuscoe, J.C.; Weber, C.A.

    1985-09-27

    In the CHO cell line various mutations affecting DNA repair have been obtained. Mutants that belong to five genetic complementation groups for UV sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. A sixth genetic complementation group for UV sensitivity has now been identified with UV27-1. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutants analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has very high SCE, are both corrected by chromosome 19. 46 refs., 3 figs.

  6. 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

  7. Identification of genes required for de novo DNA methylation in Arabidopsis

    PubMed Central

    Greenberg, Maxim VC; Ausin, Israel; Chan, Simon WL; Cokus, Shawn J; Cuperus, Josh T; Feng, Suhua; Law, Julie A; Chu, Carolyn; Pellegrini, Matteo; Carrington, James C

    2011-01-01

    De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation. PMID:21150311

  8. Whole-genome DNA methylation profile of the jewel wasp (Nasonia vitripennis).

    PubMed

    Beeler, Suzannah M; Wong, Garrett T; Zheng, Jennifer M; Bush, Eliot C; Remnant, Emily J; Oldroyd, Benjamin P; Drewell, Robert A

    2014-03-20

    The epigenetic mark of DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3' direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.

  9. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation

    PubMed Central

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-01-01

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  10. 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.

  11. DNA methylation as a biomarker for preeclampsia.

    PubMed

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

    2014-10-01

    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. 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. 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). 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. 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. © The Author(s) 2013.

  12. A DNA methylation biomarker of alcohol consumption.

    PubMed

    Liu, C; Marioni, R E; Hedman, Å K; Pfeiffer, L; Tsai, P-C; Reynolds, L M; Just, A C; Duan, Q; Boer, C G; Tanaka, T; Elks, C E; Aslibekyan, S; Brody, J A; Kühnel, B; Herder, C; Almli, L M; Zhi, D; Wang, Y; Huan, T; Yao, C; Mendelson, M M; Joehanes, R; Liang, L; Love, S-A; Guan, W; Shah, S; McRae, A F; Kretschmer, A; Prokisch, H; Strauch, K; Peters, A; Visscher, P M; Wray, N R; Guo, X; Wiggins, K L; Smith, A K; Binder, E B; Ressler, K J; Irvin, M R; Absher, D M; Hernandez, D; Ferrucci, L; Bandinelli, S; Lohman, K; Ding, J; Trevisi, L; Gustafsson, S; Sandling, J H; Stolk, L; Uitterlinden, A G; Yet, I; Castillo-Fernandez, J E; Spector, T D; Schwartz, J D; Vokonas, P; Lind, L; Li, Y; Fornage, M; Arnett, D K; Wareham, N J; Sotoodehnia, N; Ong, K K; van Meurs, J B J; Conneely, K N; Baccarelli, A A; Deary, I J; Bell, J T; North, K E; Liu, Y; Waldenberger, M; London, S J; Ingelsson, E; Levy, D

    2016-11-15

    The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10(-7). Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10(-7). In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.Molecular Psychiatry advance online publication, 15 November 2016; doi:10.1038/mp.2016.192.

  13. 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

  14. DNA Methylation Analysis: Choosing the Right Method.

    PubMed

    Kurdyukov, Sergey; Bullock, Martyn

    2016-01-06

    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.

  15. 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

  16. Transcription factors as readers and effectors of DNA methylation.

    PubMed

    Zhu, Heng; Wang, Guohua; Qian, Jiang

    2016-08-01

    Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease.

  17. Transcription factors as readers and effectors of DNA methylation

    PubMed Central

    Zhu, Heng; Wang, Guohua; Qian, Jiang

    2017-01-01

    Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease. PMID:27479905

  18. 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. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA

    PubMed Central

    Stewart, James Bruce; Freyer, Christoph; Elson, Joanna L; Wredenberg, Anna; Cansu, Zekiye; Trifunovic, Aleksandra; Larsson, Nils-Göran

    2008-01-01

    There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity. PMID:18232733

  20. Induced DNA repair pathway in mammalian cells

    SciTech Connect

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-..beta..-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells.

  1. DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus

    PubMed Central

    2013-01-01

    Background DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. Results We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. Conclusions Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo. PMID:23714178

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

    PubMed

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

    2011-01-01

    DNA methylation measured in white blood cell DNA is increasingly being used as 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 [(3)H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [(3)H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [(3)H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive 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.

  3. Importin-7 Mediates Nuclear Trafficking of DNA in Mammalian Cells

    PubMed Central

    Dhanoya, Arjun; Wang, Tse; Keshavarz-Moore, Eli; Fassati, Ariberto; Chain, Benjamin M

    2013-01-01

    Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response. PMID:23067392

  4. 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. Copyright © 2014 Elsevier GmbH. All rights reserved.

  5. In situ analysis of DNA methylation in plants.

    PubMed

    Kathiria, Palak; Kovalchuk, Igor

    2010-01-01

    Epigenetic changes in the plant genome are associated with differential genome methylation, histone modifications, and the binding of various chromatin-binding factors. Methylation of cytosine residues is one of the most versatile mechanisms of epigenetic regulation. The analysis of DNA methylation can be performed in different ways. However, most of these procedures involve the extraction of chromatin from cells with further isolation and analysis of DNA. Modest success has been achieved in DNA methylation analysis in plant tissues in situ. Here, we present an in situ method for DNA methylation analysis, which has high sensitivity and good reproducibility.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. DNA Methylation Biomarkers for Nasopharyngeal Carcinoma: Diagnostic and Prognostic Tools.

    PubMed

    Jiang, Wei; Cai, Rui; Chen, Qiu-Qiu

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is a common tumor in southern China and south-eastern Asia. Effective strategies for the prevention or screening of NPC are limited. Exploring effective biomarkers for the early diagnosis and prognosis of NPC continues to be a rigorous challenge. Evidence is accumulating that DNA methylation alterations are involved in the initiation and progression of NPC. Over the past few decades, aberrant DNA methylation in single or multiple tumor suppressor genes (TSGs) in various biologic samples have been described in NPC, which potentially represents useful biomarkers. Recently, large-scale DNA methylation analysis by genome-wide methylation platform provides a new way to identify candidate DNA methylated markers of NPC. This review summarizes the published research on the diagnostic and prognostic potential biomarkers of DNA methylation for NPC and discusses the current knowledge on DNA methylation as a biomarker for the early detection and monitoring of progression of NPC.

  8. 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

  9. Temporal regulation of DNA replication in mammalian cells.

    PubMed

    Méndez, Juan

    2009-01-01

    Eukaryotic cells follow a temporal program to duplicate their genomes. Chromosomes are divided into domains with a specific DNA replication timing (RT), not dictated by DNA sequence alone, which is conserved from one cell cycle to the next. Timing of replication correlates with gene density, transcriptional activity, chromatin structure and nuclear position, making it an intriguing epigenetic mark. The differentiation from embryonic stem cells to specialized cell types is accompanied by global changes in the RT program. This review covers our current understanding of the mechanisms that determine RT in mammalian cells, its possible biological significance and how unscheduled alterations of the RT program may predispose to human disease.

  10. Collaborations between CpG sites in DNA methylation

    NASA Astrophysics Data System (ADS)

    Song, You; Ren, Honglei; Lei, Jinzhi

    2017-08-01

    DNA methylation patterns have profound impacts on genome stability, gene expression and development. The molecular base of DNA methylation patterns has long been focused at single CpG sites level. Here, we construct a kinetic model of DNA methylation with collaborations between CpG sites, from which a correlation function was established based on experimental data. The function consists of three parts that suggest three possible sources of the correlation: movement of enzymes along DNA, collaboration between DNA methylation and nucleosome modification, and global enzyme concentrations within a cell. Moreover, the collaboration strength between DNA methylation and nucleosome modification is universal for mouse early embryo cells. The obtained correlation function provides insightful understanding for the mechanisms of inheritance of DNA methylation patterns.

  11. Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain.

    PubMed

    Guo, Junjie U; Su, Yijing; Shin, Joo Heon; Shin, Jaehoon; Li, Hongda; Xie, Bin; Zhong, Chun; Hu, Shaohui; Le, Thuc; Fan, Guoping; Zhu, Heng; Chang, Qiang; Gao, Yuan; Ming, Guo-li; Song, Hongjun

    2014-02-01

    DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base-resolution DNA methylome from adult mouse dentate neurons consists of both CpG (~75%) and CpH (~25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

  12. CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing.

    PubMed

    Hetzl, Jennifer; Foerster, Andrea M; Raidl, Günther; Mittelsten Scheid, Ortrun

    2007-08-01

    Cytosine methylation is a hallmark of epigenetic information in the DNA of many fungi, vertebrates and plants. The technique of bisulphite genomic sequencing reveals the methylation state of every individual cytosine in a sequence, and thereby provides high-resolution data on epigenetic diversity; however, the manual evaluation and documentation of large amounts of data is laborious and error-prone. While some software is available for facilitating the analysis of mammalian DNA methylation, which is found nearly exclusively at CG sites, there is no software optimally suited for data from DNA with significant non-CG methylation. We describe CyMATE (Cytosine Methylation Analysis Tool for Everyone) for in silico analysis of DNA sequences after bisulphite conversion of plant DNA, in which methylation is more divergent with respect to sequence context and biological relevance. From aligned sequences, CyMATE includes and distinguishes methylation at CG, CHG and CHH (where H = A, C or T), and can extract both quantitative and qualitative data regarding general and pattern-specific methylation per sequence and per position, i.e. data for individual sites in a sequence and the epigenetic divergence within a sample. In addition, it can provide graphical output from alignments in either an overview or a 'zoom-in' view as pdf files. Detailed information, including a quality control of the sequencing data, is provided in text format. We applied CyMATE to the analysis of DNA methylation at transcriptionally silenced promoters in diploid and polyploid Arabidopsis and found significant hypermethylation, high stability of the methylated state independent of chromosome number, and non-redundant patterns of mC distribution. CyMATE is freely available for non-commercial use at http://www.gmi.oeaw.ac.at/CyMATE.

  13. 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

  14. The role of DNA methylation on Octopus vulgaris development and their perspectives

    PubMed Central

    Díaz-Freije, Eva; Gestal, Camino; Castellanos-Martínez, Sheila; Morán, Paloma

    2014-01-01

    DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for

  15. Genome-wide gene expression and DNA methylation differences in abnormally cloned and normally natural mating piglets.

    PubMed

    Zou, C; Fu, Y; Li, C; Liu, H; Li, G; Li, J; Zhang, H; Wu, Y; Li, C

    2016-08-01

    Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer.

  16. 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

  17. Analysis of Mammalian rDNA Internal Transcribed Spacers

    PubMed Central

    Coleman, Annette W.

    2013-01-01

    Nuclear rDNA Internal Transcribed Spacers, ITS1 and ITS2, are widely used for eukaryote phylogenetic studies from the ordinal level to the species level, and there is even a database for ITS2 sequences. However, ITS regions have been ignored in mammalian phylogenetic studies, and only a few rodent and ape sequences are represented in GenBank. The reasons for this dearth, and the remedies, are described here. We have recovered these sequences, mostly >1 kb in length, for 36 mammalian species. Sequence alignment and transcript folding comparisons reveal the rRNA transcript secondary structure. Mammalian ITS regions, though quite long, still fold into the recognizable secondary structure of other eukaryotes. The ITS2 in particular bears the four standard helix loops, and loops II and III have the hallmark characters universal to eukaryotes. Both sequence and insertions/deletions of transcript secondary structure helices observed here support the four superorder taxonomy of Placentalia. On the family level, major unique indels, neatly excising entire helices, will be useful when additional species are represented, resulting in significant further understanding of the details of mammalian evolutionary history. Furthermore, the identification of a highly conserved element of ITS1 common to warm-blooded vertebrates may aid in deciphering the complex mechanism of RNA transcript processing. This is the last major group of terrestrial vertebrates for which rRNA ITS secondary structure has been resolved. PMID:24260162

  18. Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

    PubMed Central

    Liang, Gangning; Chan, Matilda F.; Tomigahara, Yoshitaka; Tsai, Yvonne C.; Gonzales, Felicidad A.; Li, En; Laird, Peter W.; Jones, Peter A.

    2002-01-01

    We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells. PMID:11756544

  19. Phase resetting of the mammalian circadian clock by DNA damage.

    PubMed

    Oklejewicz, Małgorzata; Destici, Eugin; Tamanini, Filippo; Hut, Roelof A; Janssens, Roel; van der Horst, Gijsbertus T J

    2008-02-26

    To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

  20. Whole-genome DNA methylation profiling using MethylCap-seq.

    PubMed

    Brinkman, Arie B; Simmer, Femke; Ma, Kelong; Kaan, Anita; Zhu, Jingde; Stunnenberg, Hendrik G

    2010-11-01

    MethylCap-seq is a robust procedure for genome-wide profiling of DNA methylation. The approach consists of the capture of methylated DNA using the MBD domain of MeCP2, and subsequent next-generation sequencing of eluted DNA. Elution of the captured methylated DNA is done in steps using a salt gradient, which stratifies the genome into fractions with different CpG density. The enrichment reached within the individual eluates allows for cost-effective deep sequence coverage. The profiles together yield a detailed genome-wide map of methylated regions and readily allows detection of DNA methylation in known and novel regions. Here, we describe principles and details of the MethylCap-seq procedure using different sources of starting material.

  1. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA).

    PubMed

    Rauch, Tibor A; Pfeifer, Gerd P

    2010-11-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

  2. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA)

    PubMed Central

    Rauch, Tibor A.; Pfeifer, Gerd P.

    2010-01-01

    The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences. PMID:20304072

  3. Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

    PubMed

    Li, Qing; Hermanson, Peter J; Springer, Nathan M

    2018-01-01

    DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

  4. Age-associated changes in DNA methylation across multiple tissues in an inbred mouse model

    PubMed Central

    Spiers, Helen; Hannon, Eilis; Wells, Sara; Williams, Brenda; Fernandes, Cathy; Mill, Jonathan

    2016-01-01

    Epigenetic disruption has been implicated in many diseases of aging, and age-associated DNA methylation changes at specific genomic loci in humans are strongly correlated with chronological age. The aim of this study was to explore the specificity of selected age-associated differentially methylated positions (aDMPs) identified in human epidemiological studies by quantifying DNA methylation across multiple tissues in homologous regions of the murine genome. We selected four high-confidence aDMPs (located in the vicinity of the ELOVL2, GLRA1, MYOD1 and PDE4C genes) and quantified DNA methylation across these regions in four tissues (blood, lung, cerebellum and hippocampus) from male and female C57BL/6J mice, ranging in age from fetal (embryonic day 17) to 630 days. We observed tissue-specific age-associated changes in DNA methylation that was directionally consistent with those observed in humans. These findings lend further support to the notion that changes in DNA methylation are associated with chronological age and suggest that these processes are often conserved across tissues and between mammalian species. Our data highlight the relevance of utilizing model systems, in which environmental and genetic influences can be carefully controlled, for the further study of these phenomena. PMID:26861500

  5. Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities

    DTIC Science & Technology

    2016-10-01

    1 AD______________ AWARD NUMBER: W81XWH-14-1-0529 TITLE: Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities...Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-0529 5c. PROGRAM...American (EA), and after diagnosis, AA men are more likely to die from prostate cancer than EA men. We hypothesize that differences in DNA methylation

  6. Genome-wide DNA methylation profile in mungbean

    PubMed Central

    Kang, Yang Jae; Bae, Ahra; Shim, Sangrea; Lee, Taeyoung; Lee, Jayern; Satyawan, Dani; Kim, Moon Young; Lee, Suk-Ha

    2017-01-01

    DNA methylation on cytosine residues is known to affect gene expression and is potentially responsible for the phenotypic variations among different crop cultivars. Here, we present the whole-genome DNA methylation profiles and assess the potential effects of single nucleotide polymorphisms (SNPs) for two mungbean cultivars, Sunhwanogdu (VC1973A) and Kyunggijaerae#5 (V2984). By measuring the DNA methylation levels in leaf tissue with the bisulfite sequencing (BSseq) approach, we show both the frequencies of the various types of DNA methylation and the distribution of weighted gene methylation levels. SNPs that cause nucleotide changes from/to CHH – where C is cytosine and H is any other nucleotide – were found to affect DNA methylation status in VC1973A and V2984. In order to better understand the correlation between gene expression and DNA methylation levels, we surveyed gene expression in leaf tissues of VC1973A and V2984 using RNAseq. Transcript expressions of paralogous genes were controlled by DNA methylation within the VC1973A genome. Moreover, genes that were differentially expressed between the two cultivars showed distinct DNA methylation patterns. Our mungbean genome-wide methylation profiles will be valuable resources for understanding the phenotypic variations between different cultivars, as well as for molecular breeding. PMID:28084412

  7. 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

  8. DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.

    PubMed

    Stenzig, Justus; Hirt, Marc N; Löser, Alexandra; Bartholdt, Lena M; Hensel, Jan-Tobias; Werner, Tessa R; Riemenschneider, Mona; Indenbirken, Daniela; Guenther, Thomas; Müller, Christian; Hübner, Norbert; Stoll, Monika; Eschenhagen, Thomas

    2016-01-01

    DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 µM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (α-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 µM, nucleosidic inhibitor), RG108 (60 µM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 µM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy.

  9. Tissue-specific DNA methylation is conserved across human, mouse, and rat, and driven by primary sequence conservation.

    PubMed

    Zhou, Jia; Sears, Renee L; Xing, Xiaoyun; Zhang, Bo; Li, Daofeng; Rockweiler, Nicole B; Jang, Hyo Sik; Choudhary, Mayank N K; Lee, Hyung Joo; Lowdon, Rebecca F; Arand, Jason; Tabers, Brianne; Gu, C Charles; Cicero, Theodore J; Wang, Ting

    2017-09-12

    Uncovering mechanisms of epigenome evolution is an essential step towards understanding the evolution of different cellular phenotypes. While studies have confirmed DNA methylation as a conserved epigenetic mechanism in mammalian development, little is known about the conservation of tissue-specific genome-wide DNA methylation patterns. Using a comparative epigenomics approach, we identified and compared the tissue-specific DNA methylation patterns of rat against those of mouse and human across three shared tissue types. We confirmed that tissue-specific differentially methylated regions are strongly associated with tissue-specific regulatory elements. Comparisons between species revealed that at a minimum 11-37% of tissue-specific DNA methylation patterns are conserved, a phenomenon that we define as epigenetic conservation. Conserved DNA methylation is accompanied by conservation of other epigenetic marks including histone modifications. Although a significant amount of locus-specific methylation is epigenetically conserved, the majority of tissue-specific DNA methylation is not conserved across the species and tissue types that we investigated. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, evolutionary dynamics of tissue-specific DNA methylation are best explained by the maintenance or turnover of binding sites for important transcription factors. Our study extends the limited literature of comparative epigenomics and suggests a new paradigm for epigenetic conservation without genetic conservation through analysis of transcription factor binding sites.

  10. 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].

  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. 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

  13. Mammalian non-CG methylations are conserved and cell-type specific and may have been involved in the evolution of transposon elements

    PubMed Central

    Guo, Weilong; Zhang, Michael Q.; Wu, Hong

    2016-01-01

    Although non-CG methylations are abundant in several mammalian cell types, their biological significance is sparsely characterized. We gathered 51 human and mouse DNA methylomes from brain neurons, embryonic stem cells and induced pluripotent stem cells, primordial germ cells and oocytes. We utilized an unbiased sub-motif prediction method and reported CW as the representative non-CG methylation context, which is distinct from CC methylation in terms of sequence context and genomic distribution. A two-dimensional comparison of non-CG methylations across cell types and species was performed. Unambiguous studies of sequence preferences and genomic region enrichment showed that CW methylation is cell-type specific and is also conserved between humans and mice. In brain neurons, it was found that active long interspersed nuclear element-1 (LINE-1) lacked CW methylations but not CG methylations. Coincidentally, both human Alu and mouse B1 elements preferred high CW methylations at specific loci during their respective evolutionary development. Last, the strand-specific distributions of CW methylations in introns and long interspersed nuclear elements are also cell-type specific and conserved. In summary, our results illustrate that CW methylations are highly conserved among species, are dynamically regulated in each cell type, and are potentially involved in the evolution of transposon elements. PMID:27573482

  14. Mammalian non-CG methylations are conserved and cell-type specific and may have been involved in the evolution of transposon elements.

    PubMed

    Guo, Weilong; Zhang, Michael Q; Wu, Hong

    2016-08-30

    Although non-CG methylations are abundant in several mammalian cell types, their biological significance is sparsely characterized. We gathered 51 human and mouse DNA methylomes from brain neurons, embryonic stem cells and induced pluripotent stem cells, primordial germ cells and oocytes. We utilized an unbiased sub-motif prediction method and reported CW as the representative non-CG methylation context, which is distinct from CC methylation in terms of sequence context and genomic distribution. A two-dimensional comparison of non-CG methylations across cell types and species was performed. Unambiguous studies of sequence preferences and genomic region enrichment showed that CW methylation is cell-type specific and is also conserved between humans and mice. In brain neurons, it was found that active long interspersed nuclear element-1 (LINE-1) lacked CW methylations but not CG methylations. Coincidentally, both human Alu and mouse B1 elements preferred high CW methylations at specific loci during their respective evolutionary development. Last, the strand-specific distributions of CW methylations in introns and long interspersed nuclear elements are also cell-type specific and conserved. In summary, our results illustrate that CW methylations are highly conserved among species, are dynamically regulated in each cell type, and are potentially involved in the evolution of transposon elements.

  15. 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

  16. [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.

  17. 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.

  18. DNA methylation of the LIN28 pseudogene family.

    PubMed

    Davis, Aaron P; Benninghoff, Abby D; Thomas, Aaron J; Sessions, Benjamin R; White, Kenneth L

    2015-04-11

    DNA methylation directs the epigenetic silencing of selected regions of DNA, including the regulation of pseudogenes, and is widespread throughout the genome. Pseudogenes are decayed copies of duplicated genes that have spread throughout the genome by transposition. Pseudogenes are transcriptionally silenced by DNA methylation, but little is known about how pseudogenes are targeted for methylation or how methylation levels are maintained in different tissues. We employed bisulfite next generation sequencing to examine the methylation status of the LIN28 gene and four processed pseudogenes derived from LIN28. The objective was to determine whether LIN28 pseudogenes maintain the same pattern of methylation as the parental gene or acquire a methylation pattern independent of the gene of origin. In this study, we determined that the methylation status of LIN28 pseudogenes does not resemble the pattern evident for the LIN28 gene, but rather these pseudogenes appear to acquire methylation patterns independent of the parental gene. Furthermore, we observed that methylation levels of the examined pseudogenes correlate to the location of insertion within the genome. LIN28 pseudogenes inserted into gene bodies were highly methylated in all tissues examined. In contrast, pseudogenes inserted into genomic regions that are not proximal to genes were differentially methylated in various tissue types. Our analysis suggests that Lin28 pseudogenes do not acquire patterns of tissue-specific methylation as for the parental gene, but rather are methylated in patterns specific to the local genomic environment into which they were inserted.

  19. The implications of DNA methylation for toxicology: toward toxicomethylomics, the toxicology of DNA methylation.

    PubMed

    Szyf, Moshe

    2011-04-01

    Identifying agents that have long-term deleterious impact on health but exhibit no immediate toxicity is of prime importance. It is well established that long-term toxicity of chemicals could be caused by their ability to generate changes in the DNA sequence through the process of mutagenesis. Several assays including the Ames test and its different modifications were developed to assess the mutagenic potential of chemicals (Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. (1973a). Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 70, 2281-2285; Ames, B. N., Lee, F. D., and Durston, W. E. (1973b). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70, 782-786). These tests have also been employed for assessing the carcinogenic potential of compounds. However, the DNA molecule contains within its chemical structure two layers of information. The DNA sequence that bears the ancestral genetic information and the pattern of distribution of covalently bound methyl groups on cytosines in DNA. DNA methylation patterns are generated by an innate program during gestation but are attuned to the environment in utero and throughout life including physical and social exposures. DNA function and health could be stably altered by exposure to environmental agents without changing the sequence, just by changing the state of DNA methylation. Our current screening tests do not detect agents that have long-range impact on the phenotype without altering the genotype. The realization that long-range damage could be caused without changing the DNA sequence has important implications on the way we assess the safety of chemicals, drugs, and food and broadens the scope of definition of toxic agents.

  20. DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition.

    PubMed

    Ivanova, Elena; Chen, Jian-Hua; Segonds-Pichon, Anne; Ozanne, Susan E; Kelsey, Gavin

    2012-10-01

    The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming-isocaloric protein restriction to female mice during gestation or lactation-to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.

  1. The Ciona intestinalis cleavage clock is independent of DNA methylation.

    PubMed

    Suzuki, Miho M; Mori, Tomoko; Satoh, Noriyuki

    2016-10-01

    The initiation of embryonic gene expression in ascidian embryos appears to be tightly regulated by the number of DNA replication cycles. DNA methylation is thought to contribute to the clock mechanism that counts the rounds of DNA replication. We used mass spectrometry and whole genome bisulfite sequencing to characterize DNA methylation changes that occur in early developmental stages of the ascidian, Ciona intestinalis. We found that global DNA methylation in early Ciona development was static, and a base-wise comparison between the genomes of consecutive developmental stages found no DNA demethylation that was related to zygotic gene activation. Additionally, 5hmC was hardly detected by mass spectrometry in the developmental samples, suggesting a lack of demethylation mediated by ten eleven translocation (TET) methylcytosine dioxygenase in C. intestinalis. We conclude that DNA methylation is not involved in regulating DNA replication-dependent transcriptional activation.

  2. Nucleosomes protect DNA from DNA methylation in vivo and in vitro

    PubMed Central

    Felle, Max; Hoffmeister, Helen; Rothammer, Julia; Fuchs, Andreas; Exler, Josef H.; Längst, Gernot

    2011-01-01

    Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo. PMID:21622955

  3. 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

  4. Variable patterns of total DNA and rDNA methylation in animals.

    PubMed Central

    Bird, A P; Taggart, M H

    1980-01-01

    Restriction endonucleases were used to determine the degree of methylation at the sequences CCGG and GCGC in a wide range of animal DNAs. Both total DNA methylation and ribosomal DNA methylation were studied. Whole DNA methylation was indetectable in arthropods, fractional in other invertebrate phyla, and high in the vertebrates. Ribosomal DNA was predominantly unmethylated in all animals except fish and amphibia, where it was heavily methylated. We discuss the evolutionary and functional implications of these results, and suggest that the large differences between genome types are the result of evolutionary changes in the relative size of heavily methylated and unmethylated compartments. Images PMID:6253937

  5. DNA methylation profiles at birth and child ADHD symptoms.

    PubMed

    van Mil, Nina H; Steegers-Theunissen, Régine P M; Bouwland-Both, Marieke I; Verbiest, Michael M P J; Rijlaarsdam, Jolien; Hofman, Albert; Steegers, Eric A P; Heijmans, Bastiaan T; Jaddoe, Vincent W V; Verhulst, Frank C; Stolk, Lisette; Eilers, Paul H C; Uitterlinden, André G; Tiemeier, Henning

    2014-02-01

    Attention deficit/hyperactivity disorder (ADHD) is a common and highly heritable psychiatric disorder. In addition, early life environmental factors contribute to the occurrence of ADHD. Recently, DNA methylation has emerged as a mechanism potentially mediating genetic and environmental effects. Here, we investigated whether newborn DNA methylation patterns of selected candidate genes involved in psychiatric disorders or fetal growth are associated with ADHD symptoms in childhood. Participants were 426 children from a large population based cohort of Dutch national origin. Behavioral data were obtained at age 6 years with the Child Behavior Checklist. For the current study, 11 regions at 7 different genes were selected. DNA methylation levels of cord blood DNA were measured for the 11 regions combined and for each region separately. We examined the association between DNA methylation levels at different regions and ADHD symptoms with linear mixed models. DNA methylation levels were negatively associated with ADHD symptom score in the overall analysis of all 11 regions. This association was largely explained by associations of DRD4 and 5-HTT regions. Other candidate genes showed no association between DNA methylation levels and ADHD symptom score. Associations between DNA methylation levels and ADHD symptom score were attenuated by co-occurring Oppositional defiant disorder and total symptoms. Lower DNA methylation levels of the 7 genes assessed at birth, were associated with more ADHD symptoms of the child at 6 years of age. Further studies are needed to confirm our results and to investigate the possible underlying mechanism.

  6. 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.

  7. Implications of DNA Methylation in Parkinson’s Disease

    PubMed Central

    Miranda-Morales, Ernesto; Meier, Karin; Sandoval-Carrillo, Ada; Salas-Pacheco, José; Vázquez-Cárdenas, Paola; Arias-Carrión, Oscar

    2017-01-01

    It has been 200 years since Parkinson’s disease (PD) was first described, yet many aspects of its etiopathogenesis remain unclear. PD is a progressive and complex neurodegenerative disorder caused by genetic and environmental factors including aging, nutrition, pesticides and exposure to heavy metals. DNA methylation may be altered in response to some of these factors; therefore, it is proposed that epigenetic mechanisms, particularly DNA methylation, can have a fundamental role in gene–environment interactions that are related with PD. Epigenetic changes in PD-associated genes are now widely studied in different populations, to discover the mechanisms that contribute to disease development and identify novel biomarkers for early diagnosis and future pharmacological treatment. While initial studies sought to find associations between promoter DNA methylation and the regulation of associated genes in PD brain tissue, more recent studies have described concordant DNA methylation patterns between blood and brain tissue DNA. These data justify the use of peripheral blood samples instead of brain tissue for epigenetic studies. Here, we summarize the current data about DNA methylation changes in PD and discuss the potential of DNA methylation as a potential biomarker for PD. Additionally, we discuss environmental and nutritional factors that have been implicated in DNA methylation. Although the search for significant DNA methylation changes and gene expression analyses of PD-associated genes have yielded inconsistent and contradictory results, epigenetic modifications remain under investigation for their potential to reveal the link between environmental risk factors and the development of PD. PMID:28769760

  8. DNA Methylation Profiling Reveals Correlation of Differential Methylation Patterns with Gene Expression in Human Epilepsy.

    PubMed

    Wang, Liang; Fu, Xinwei; Peng, Xi; Xiao, Zheng; Li, Zhonggui; Chen, Guojun; Wang, Xuefeng

    2016-05-01

    DNA methylation plays important roles in regulating gene expression and has been reported to be related with epilepsy. This study aimed to define differential DNA methylation patterns in drug-refractory epilepsy patients and to investigate the role of DNA methylation in human epilepsy. We performed DNA methylation profiling in brain tissues from epileptic and control patients via methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing PCR, and the messenger RNA (mRNA) levels of candidate genes were evaluated by reverse transcriptase PCR. We found 224 genes that showed differential DNA methylation between epileptic patients and controls. Among the seven candidate genes, three genes (TUBB2B, ATPGD1, and HTR6) showed relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited hypermethylation and decreased mRNA levels, whereas HTR6 displayed hypomethylation and increased mRNA levels in the epileptic samples. Our findings suggest that certain genes become differentially regulated by DNA methylation in human epilepsy.

  9. Controlling for conservation in genome-wide DNA methylation studies.

    PubMed

    Singer, Meromit; Pachter, Lior

    2015-05-30

    A commonplace analysis in high-throughput DNA methylation studies is the comparison of methylation extent between different functional regions, computed by averaging methylation states within region types and then comparing averages between regions. For example, it has been reported that methylation is more prevalent in coding regions as compared to their neighboring introns or UTRs, leading to hypotheses about novel forms of epigenetic regulation. We have identified and characterized a bias present in these seemingly straightforward comparisons that results in the false detection of differences in methylation intensities across region types. This bias arises due to differences in conservation rates, rather than methylation rates, and is broadly present in the published literature. When controlling for conservation at coding start sites the differences in DNA methylation rates disappear. Moreover, a re-evaluation of methylation rates at intronexon junctions reveals that the magnitude of previously reported differences is greatly exaggerated. We introduce two correction methods to address this bias, an inferencebased matrix completion algorithm and an averaging approach, tailored to address different underlying biological questions. We evaluate how analysis using these corrections affects the detection of differences in DNA methylation across functional boundaries. We report here on a bias in DNA methylation comparative studies that originates in conservation rate differences and manifests itself in the false discovery of differences in DNA methylation intensities and their extents. We have characterized this bias and its broad implications, and show how to control for it so as to enable the study of a variety of biological questions.

  10. Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing.

    PubMed

    Bialic, Marta; Coulon, Vincent; Drac, Marjorie; Gostan, Thierry; Schwob, Etienne

    2015-01-01

    How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.

  11. 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.

  12. DNA Methylation in Basal Metazoans: Insights from Ctenophores

    PubMed Central

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

    2015-01-01

    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. PMID:26173712

  13. Effects of DNA methylation inhibitors and conventional antidepressants on mice behaviour and brain DNA methylation levels.

    PubMed

    Sales, Amanda Juliana; Joca, Sâmia Regiane Lourenço

    2016-02-01

    Stress increases DNA methylation and decreases the expression of genes involved in neural plasticity, while treatment with DNA methyltransferase inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in preclinical models. Therefore, the aim of the present work was to further investigate the potential antidepressant-like effect induced by DNMTi by evaluating the behavioural effects induced by associating DNMTi treatment with conventional antidepressant drugs in mice submitted to the forced swimming test (FST). In addition, brain levels of DNA methylation were also investigated. Mice received systemic injections of 5-aza-2'-deoxycytidine (5-AzaD, 0.1, 0.2 mg/kg), RG108 (0.1, 0.2, 0.4 mg/kg), desipramine (DES, 2.5, 5, 10 mg/kg) or fluoxetine (FLX, 5, 10, 20, 30 mg/kg) and were submitted to the FST or to the open field test (OFT). Additional groups received a combination of subeffective doses of 5-AzaD or RG108 (DNMTi) with subeffective doses of DES or FLX (antidepressants). Subeffective doses of RG108 (0.1 mg/kg) or 5-AzaD (0.1 mg/kg) in association with subeffective doses of DES (2.5 mg/kg) or FLX (10 mg/kg) induced significant antidepressant-like effects. Effective doses of RG108 (0.2 mg/kg), 5-AzaD (0.2 mg/kg), DES (10 mg/kg) and FLX (20 mg/kg) atenuated stress-induced changes in DNA methylation levels in the hippocampus and prefrontal cortex. None of the treatments induced locomotor effects in the OFT. These results suggest that DNMTi potentiate the behavioural effects of antidepressant drugs in the FST and that antidepressants, as well as DNMTi, are able to modulate stress-induced changes in DNA methylation in brain regions closely associated with the neurobiology of depression.

  14. 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

  15. 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

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

    PubMed

    Shiratori, Hiromi; 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.

  17. 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.

  18. 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

  19. DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination

    PubMed Central

    Zamudio, Natasha; Barau, Joan; Teissandier, Aurélie; Walter, Marius; Borsos, Maté; Servant, Nicolas; Bourc'his, Déborah

    2015-01-01

    DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L−/− meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events. PMID:26109049

  20. DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination.

    PubMed

    Zamudio, Natasha; Barau, Joan; Teissandier, Aurélie; Walter, Marius; Borsos, Maté; Servant, Nicolas; Bourc'his, Déborah

    2015-06-15

    DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L(-/-) meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events.

  1. Intermediate DNA methylation is a conserved signature of genome regulation

    PubMed Central

    Elliott, GiNell; Hong, Chibo; Xing, Xiaoyun; Zhou, Xin; Li, Daofeng; Coarfa, Cristian; Bell, Robert J.A.; Maire, Cecile L.; Ligon, Keith L.; Sigaroudinia, Mahvash; Gascard, Philippe; Tlsty, Thea D.; Harris, R. Alan; Schalkwyk, Leonard C.; Bilenky, Misha; Mill, Jonathan; Farnham, Peggy J.; Kellis, Manolis; Marra, Marco A.; Milosavljevic, Aleksandar; Hirst, Martin; Stormo, Gary D.; Wang, Ting; Costello, Joseph F.

    2015-01-01

    The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. PMID:25691127

  2. Allele-specific DNA methylation: beyond imprinting.

    PubMed

    Tycko, Benjamin

    2010-10-15

    Allele-specific DNA methylation (ASM) and allele-specific gene expression (ASE) have long been studied in genomic imprinting and X chromosome inactivation. But these types of allelic asymmetries, along with allele-specific transcription factor binding (ASTF), have turned out to be far more pervasive-affecting many non-imprinted autosomal genes in normal human tissues. ASM, ASE and ASTF have now been mapped genome-wide by microarray-based methods and NextGen sequencing. Multiple studies agree that all three types of allelic asymmetries, as well as the related phenomena of expression and methylation quantitative trait loci, are mostly accounted for by cis-acting regulatory polymorphisms. The precise mechanisms by which this occurs are not yet understood, but there are some testable hypotheses and already a few direct clues. Future challenges include achieving higher resolution maps to locate the epicenters of cis-regulated ASM, using this information to test mechanistic models, and applying genome-wide maps of ASE/ASM/ASTF to pinpoint functional regulatory polymorphisms influencing disease susceptibility.

  3. Detection of regional DNA methylation using DNA-graphene affinity interactions.

    PubMed

    Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

    2017-01-15

    We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Folate, colorectal cancer and the involvement of DNA methylation.

    PubMed

    Williams, Elizabeth A

    2012-11-01

    Diet is a major factor in the aetiology of colorectal cancer (CRC). Epidemiological evidence suggests that folate confers a modest protection against CRC risk. However, the relationship is complex, and evidence from human intervention trials and animal studies suggests that a high-dose of folic acid supplementation may enhance the risk of colorectal carcinogenesis in certain circumstances. The molecular mechanisms underlying the apparent dual modulatory effect of folate on colorectal carcinogenesis are not fully understood. Folate is central to C1 metabolism and is needed for both DNA synthesis and DNA methylation, providing plausible biological mechanisms through which folate could modulate cancer risk. Aberrant DNA methylation is an early event in colorectal carcinogenesis and is typically associated with the transcriptional silencing of tumour suppressor genes. Folate is required for the production of S-adenosyl methionine, which serves as a methyl donor for DNA methylation events; thereby folate availability is proposed to modulate DNA methylation status. The evidence for an effect of folate on DNA methylation in the human colon is limited, but a modulation of DNA methylation in response to folate has been demonstrated. More research is required to clarify the optimum intake of folate for CRC prevention and to elucidate the effect of folate availability on DNA methylation and the associated impact on CRC biology.

  5. An atlas of DNA methylation in diverse bovine tissues

    USDA-ARS?s Scientific Manuscript database

    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. Analyses of cattle DNA methylation patterns in diverse tissues

    USDA-ARS?s Scientific Manuscript database

    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 culture cells and primary tissues systems frequently involved in animal prod...

  7. DNA methylation profiling using bisulfite-based epityping of pooled genomic DNA.

    PubMed

    Docherty, Sophia J; Davis, Oliver S P; Haworth, Claire M A; Plomin, Robert; Mill, Jonathan

    2010-11-01

    DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however they require large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. DNA pooling approaches are already widely used in large-scale studies of DNA sequence and gene expression. In this paper, we describe the application of this economical DNA pooling technique to the study of DNA methylation profiles. This method generates accurate quantitative assessments of group DNA methylation averages, reducing the time, cost and amount of DNA starting material required for large-scale epigenetic investigation of disease phenotypes.

  8. 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

  9. Comparison of DNA methylation profiles in human fetal and adult red blood cell progenitors.

    PubMed

    Lessard, Samuel; Beaudoin, Mélissa; Benkirane, Karim; Lettre, Guillaume

    2015-01-01

    DNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts. We used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors. We identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts. Our DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of

  10. Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities

    DTIC Science & Technology

    2015-10-01

    after diagnosis , AA men are more likely to die from prostate cancer than EA men. We hypothesize that differences in DNA methylation patterns across...AD AWARD NUMBER: W81XWH-14-1-0529 TITLE: Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities PRINCIPAL INVESTIGATOR...Methylation Features that Underlie Prostate Cancer Disparities 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0529 5c. PROGRAM ELEMENT NUMBER 6

  11. DNA methylation and gene expression in Mimulus guttatus.

    PubMed

    Colicchio, Jack M; Miura, Fumihito; Kelly, John K; Ito, Takashi; Hileman, Lena C

    2015-07-07

    The presence of methyl groups on cytosine nucleotides across an organism's genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R(2) = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression.

  12. The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2015-04-10

    Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

  13. Biomarkers of lead exposure and DNA methylation within retrotransposons.

    PubMed

    Wright, Robert O; Schwartz, Joel; Wright, Rosalind J; Bollati, Valentina; Tarantini, Letizia; Park, Sung Kyun; Hu, Howard; Sparrow, David; Vokonas, Pantel; Baccarelli, Andrea

    2010-06-01

    DNA methylation is an epigenetic mark that regulates gene expression. Changes in DNA methylation within white blood cells may result from cumulative exposure to environmental metals such as lead. Bone lead, a marker of cumulative exposure, may therefore better predict DNA methylation than does blood lead. In this study we compared associations between lead biomarkers and DNA methylation. We measured global methylation in participants of the Normative Aging Study (all men) who had archived DNA samples. We measured patella and tibia lead levels by K-X-Ray fluorescence and blood lead by atomic absorption spectrophotometry. DNA samples from blood were used to determine global methylation averages within CpG islands of long interspersed nuclear elements-1 (LINE-1) and Alu retrotransposons. A mixed-effects model using repeated measures of Alu or LINE-1 as the dependent variable and blood/bone lead (tibia or patella in separate models) as the primary exposure marker was fit to the data. Overall mean global methylation (+/- SD) was 26.3 +/- 1.0 as measured by Alu and 76.8 +/- 1.9 as measured by LINE-1. In the mixed-effects model, patella lead levels were inversely associated with LINE-1 (beta = -0.25; p < 0.01) but not Alu (beta = -0.03; p = 0.4). Tibia lead and blood lead did not predict global methylation for either Alu or LINE-1. Patella lead levels predicted reduced global DNA methylation within LINE-1 elements. The association between lead exposure and LINE-1 DNA methylation may have implications for the mechanisms of action of lead on health outcomes, and also suggests that changes in DNA methylation may represent a biomarker of past lead exposure.

  14. A DNA methylation fingerprint of 1628 human samples

    PubMed Central

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

    2012-01-01

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

  15. Comprehensive DNA methylation analysis of the Aedes aegypti genome.

    PubMed

    Falckenhayn, Cassandra; Carneiro, Vitor Coutinho; de Mendonça Amarante, Anderson; Schmid, Katharina; Hanna, Katharina; Kang, Seokyoung; Helm, Mark; Dimopoulos, George; Fantappié, Marcelo Rosado; Lyko, Frank

    2016-11-02

    Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.

  16. Comprehensive DNA methylation analysis of the Aedes aegypti genome

    PubMed Central

    Falckenhayn, Cassandra; Carneiro, Vitor Coutinho; de Mendonça Amarante, Anderson; Schmid, Katharina; Hanna, Katharina; Kang, Seokyoung; Helm, Mark; Dimopoulos, George; Fantappié, Marcelo Rosado; Lyko, Frank

    2016-01-01

    Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation. PMID:27805064

  17. Sequences sufficient for programming imprinted germline DNA methylation defined.

    PubMed

    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.

  18. DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro.

    PubMed

    Duan, Li; Liang, Yujie; Ma, Bin; Wang, Daming; Liu, Wei; Huang, Jianghong; Xiong, Jianyi; Peng, Liangquan; Chen, Jielin; Zhu, Weimin; Wang, Daping

    2017-07-01

    DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

  19. DNA Replication Origins and Fork Progression at Mammalian Telomeres

    PubMed Central

    Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

    2017-01-01

    Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

  20. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  1. DNA methylation-based variation between human populations.

    PubMed

    Kader, Farzeen; Ghai, Meenu

    2017-02-01

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

  2. A collaborative exercise on DNA methylation based body fluid typing.

    PubMed

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

    2016-10-01

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

  3. 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.

  4. Epigenetics in Alzheimer's Disease: Perspective of DNA Methylation.

    PubMed

    Qazi, Talal Jamil; Quan, Zhenzhen; Mir, Asif; Qing, Hong

    2017-01-14

    Research over the years has shown that causes of Alzheimer's disease are not well understood, but over the past years, the involvement of epigenetic mechanisms in the developing memory formation either under pathological or physiological conditions has become clear. The term epigenetics represents the heredity of changes in phenotype that are independent of altered DNA sequences. Different studies validated that cytosine methylation of genomic DNA decreases with age in different tissues of mammals, and therefore, the role of epigenetic factors in developing neurological disorders in aging has been under focus. In this review, we summarized and reviewed the involvement of different epigenetic mechanisms especially the DNA methylation in Alzheimer's disease (AD), late-onset Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and autosomal dominant Alzheimer's disease (ADAD). Down to the minutest of details, we tried to discuss the methylation patterns like mitochondrial DNA methylation and ribosomal DNA (rDNA) methylation. Additionally, we mentioned some therapeutic approaches related to epigenetics, which could provide a potential cure for AD. Moreover, we reviewed some recent studies that validate DNA methylation as a potential biomarker and its role in AD. We hope that this review will provide new insights into the understanding of AD pathogenesis from the epigenetic perspective especially from the perspective of DNA methylation.

  5. 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

  6. A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

    PubMed Central

    Claus, Rainer; Wilop, Stefan; Hielscher, Thomas; Sonnet, Miriam; Dahl, Edgar; Galm, Oliver; Jost, Edgar; Plass, Christoph

    2012-01-01

    Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly. PMID:22647397

  7. Curcumin modulates DNA methylation in colorectal cancer cells.

    PubMed

    Link, Alexander; Balaguer, Francesc; Shen, Yan; Lozano, Juan Jose; Leung, Hon-Chiu E; Boland, C Richard; Goel, Ajay

    2013-01-01

    Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza

  8. 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

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

    PubMed

    Yang, Chen; Ye, Jing; 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.

  10. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks

    PubMed Central

    Wyatt, Michael D.; Pittman, Douglas L.

    2008-01-01

    The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a ‘radiomimetic,’ i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired. PMID:17173371

  11. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation.

    PubMed

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J; Cowling, Victoria H; Cairns, Bradley R; White, Robert J

    2015-03-23

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription.

  12. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    PubMed Central

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  13. 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

  14. Methylation-induced blocks to in vitro DNA replication.

    PubMed

    Larson, K; Sahm, J; Shenkar, R; Strauss, B

    1985-01-01

    Single-stranded primed M13mp2 templates and double-stranded templates were treated with either dimethyl sulfate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine and used for DNA synthesis in vitro. Methylation inhibits the ability of the molecules to serve as templates. When either E. coli DNA polymerase I or AMV reverse transcriptase were used as polymerases, DNA synthesis terminated one nucleotide 3' to the site of adenine residues in the template. Heating of the templates resulted in the appearance of additional termination bands one nucleotide before the site of G's in the template. We assume that methylated A's but not methylated G's are blocks to in vitro DNA synthesis and that heating converts a portion of the sites of methylated G to AP sites which are blocks to synthesis.

  15. DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP

    PubMed Central

    Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

    2009-01-01

    Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

  16. Reelin (RELN) DNA methylation in the peripheral blood of schizophrenia.

    PubMed

    Nabil Fikri, Rahim Mohd; Norlelawati, A Talib; Nour El-Huda, Abdul Rahim; Hanisah, Mohd Noor; Kartini, Abdullah; Norsidah, Kuzaifah; Nor Zamzila, Abdullah

    2017-05-01

    The epigenetic changes of RELN that are involved in the development of dopaminergic neurons may fit the developmental theory of schizophrenia. However, evidence regarding the association of RELN DNA methylation with schizophrenia is far from sufficient, as studies have only been conducted on a few limited brain samples. As DNA methylation in the peripheral blood may mirror the changes taking place in the brain, the use of peripheral blood for a DNA methylation study in schizophrenia is feasible due to the scarcity of brain samples. Therefore, the aim of our study was to examine the relationship of DNA methylation levels of RELN promoters with schizophrenia using genomic DNA derived from the peripheral blood of patients with the disorder. The case control studies consisted of 110 schizophrenia participants and 122 healthy controls who had been recruited from the same district. After bisufhite conversion, the methylation levels of the DNA samples were calculated based on their differences of the Cq values assayed using the highly sensitive real-time MethyLight TaqMan(®) procedure. A significantly higher level of methylation of the RELN promoter was found in patients with schizophrenia compared to controls (p = 0.005) and also in males compared with females (p = 0.004). Subsequently, the RELN expression of the methylated group was 25 fold less than that of the non-methylated group. Based upon the assumption of parallel methylation changes in the brain and peripheral blood, we concluded that RELN DNA methylation might contribute to the pathogenesis of schizophrenia. However, the definite effects of methylation on RELN function during development and also in adult life still require further elaboration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. 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

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

    PubMed

    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

  19. Initiation of DNA replication at CpG islands in mammalian chromosomes.

    PubMed Central

    Delgado, S; Gómez, M; Bird, A; Antequera, F

    1998-01-01

    CpG islands are G+C-rich regions approximately 1 kb long that are free of methylation and contain the promoters of many mammalian genes. Analysis of in vivo replication intermediates at three hamster genes and one human gene showed that the CpG island regions, but not their flanks, were present in very short nascent strands, suggesting that they are replication origins (ORIs). CpG island-like fragments were enriched in a population of short nascent strands from human erythroleukaemic cells, suggesting that islands constitute a significant fraction of endogenous ORIs. Correspondingly, bulk CpG islands were found to replicate coordinately early in S phase. Our results imply that CpG islands are initiation sites for both transcription and DNA replication, and may represent genomic footprints of replication initiation. PMID:9545253

  20. Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved

    PubMed Central

    Long, Hannah K.; King, Hamish W.; Patient, Roger K.; Odom, Duncan T.; Klose, Robert J.

    2016-01-01

    DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. PMID:27084945

  1. The effect of paternal methyl-group donor intake on offspring DNA methylation and birth weight.

    PubMed

    Pauwels, S; Truijen, I; Ghosh, M; Duca, R C; Langie, S A S; Bekaert, B; Freson, K; Huybrechts, I; Koppen, G; Devlieger, R; Godderis, L

    2017-03-06

    Most nutritional studies on the development of children focus on mother-infant interactions. Maternal nutrition is critically involved in the growth and development of the fetus, but what about the father? The aim is to investigate the effects of paternal methyl-group donor intake (methionine, folate, betaine, choline) on paternal and offspring global DNA (hydroxy)methylation, offspring IGF2 DMR DNA methylation, and birth weight. Questionnaires, 7-day estimated dietary records, whole blood samples, and anthropometric measurements from 74 fathers were obtained. A total of 51 cord blood samples were collected and birth weight was obtained. DNA methylation status was measured using liquid chromatography-tandem mass spectrometry (global DNA (hydroxy)methylation) and pyrosequencing (IGF2 DMR methylation). Paternal betaine intake was positively associated with paternal global DNA hydroxymethylation (0.028% per 100 mg betaine increase, 95% CI: 0.003, 0.053, P=0.03) and cord blood global DNA methylation (0.679% per 100 mg betaine increase, 95% CI: 0.057, 1.302, P=0.03). Paternal methionine intake was positively associated with CpG1 (0.336% per 100 mg methionine increase, 95% CI: 0.103, 0.569, P=0.006), and mean CpG (0.201% per 100 mg methionine increase, 95% CI: 0.001, 0.402, P=0.049) methylation of the IGF2 DMR in cord blood. Further, a negative association between birth weight/birth weight-for-gestational age z-score and paternal betaine/methionine intake was found. In addition, a positive association between choline and birth weight/birth weight-for-gestational age z-score was also observed. Our data indicate a potential impact of paternal methyl-group donor intake on paternal global DNA hydroxymethylation, offspring global and IGF2 DMR DNA methylation, and prenatal growth.

  2. 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

  3. Methylation of inorganic arsenic in different mammalian species and population groups.

    PubMed

    Vahter, M

    1999-01-01

    Thousands of people in different parts of the world are exposed to arsenic via drinking water or contaminated soil or food. The high general toxic of arsenic has been known for centuries, and research during the last decades has shown that arsenic is a potent human carcinogen. However, most experimental cancer studies have failed to demonstrate carcinogenicity in experimental animals, indicating marked variation in sensitivity towards arsenic toxicity between species. It has also been suggested that there is a variation in susceptibility among human individuals. One reason for such variability in toxic response may be variation in metabolism. Inorganic arsenic is methylated in humans as well as animals and micro-organisms, but there are considerable differences between species and individuals. In many, but not all, mammalian species, inorganic arsenic is methylated to methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which are more rapidly excreted in urine than is the inorganic arsenic, especially the trivalent form (AsIII, arsenite) which is highly reactive with tissue components. Absorbed arsenate (AsV) is reduced to trivalent arsenic (AsIII) before the methyl groups are attached. It has been estimated that as much as 50-70% of absorbed AsV is rapidly reduced to AsIII, a reaction which seems to be common for most species. In most experimental animal species, DMA is the main metabolite excreted in urine. Compared to human subjects, very little MMA is produced. However, the rate of methylation varies considerably between species, and several species, e.g. the marmoset monkey and the chimpanzee have been shown not to methylate inorganic arsenic at all. In addition, the marmoset monkey accumulates arsenic in the liver. The rat, on the other hand, has an efficient methylation of arsenic but the formed DMA is to a large extent accumulated in the red blood cells. As a result, the rat shows a low rate of excretion of arsenic. In both human subjects and rodents

  4. DNA Methylation Dynamics in Blood after Hematopoietic Cell Transplant

    PubMed Central

    Rodriguez, Ramon M.; Suarez-Alvarez, Beatriz; Salvanés, Rubén; Muro, Manuel; Martínez-Camblor, Pablo; Colado, Enrique; Sánchez, Miguel Alcoceba; Díaz, Marcos González; Fernandez, Agustin F.; Fraga, Mario F.; Lopez-Larrea, Carlos

    2013-01-01

    Epigenetic deregulation is considered a common hallmark of cancer. Nevertheless, recent publications have demonstrated its association with a large array of human diseases. Here, we explore the DNA methylation dynamics in blood samples during hematopoietic cell transplant and how they are affected by pathophysiological events during transplant evolution. We analyzed global DNA methylation in a cohort of 47 patients with allogenic transplant up to 12 months post-transplant. Recipients stably maintained the donor’s global methylation levels after transplant. Nonetheless, global methylation is affected by chimerism status. Methylation analysis of promoters revealed that methylation in more than 200 genes is altered 1 month post-transplant when compared with non-pathological methylation levels in the donor. This number decreased by 6 months post-transplant. Finally, we analyzed methylation in IFN-γ, FASL, IL-10, and PRF1 and found association with the severity of the acute graft-versus-host disease. Our results provide strong evidence that methylation changes in blood are linked to underlying physiological events and demonstrate that DNA methylation analysis is a viable strategy for the study of transplantation and for development of biomarkers. PMID:23451113

  5. Genetic variants of methionine metabolism and DNA methylation.

    PubMed

    Bleich, Stefan; Semmler, Alexander; Frieling, Helge; Thumfart, L; Muschler, Marc; Hillemacher, Thomas; Kornhuber, Johannes; Kallweit, Ulf; Simon, Matthias; Linnebank, Michael

    2014-01-01

    Altered DNA methylation is associated with important and common pathologies such as cancer. The origin of altered DNA methylation is unknown. The methyl groups for DNA methylation are provided by methionine metabolism. This metabolism is characterized by a high interindividual variability, which is in part explained by genetic variants. In a cohort of 313 individuals derived from a family-based study with index cases of cerebrovascular disease, we analyzed whether global methylation of leukocyte DNA was associated with age, gender, homocysteine plasma levels or functionally relevant genetic variants. We observed an association of the G-allele of the methionine synthase variant c.2756A>G (D919G) with global methylation (% methylation ± 1 SD, AA: 41.3 ± 14.9; AG: 36.4 ± 18.2; GG: 30.8 ± 16.9; F = 4.799; p = 0.009). The methionine synthase variant c.2756A>G is associated with various types of cancer. Our data suggest that an impact on DNA methylation may contribute to the clinical relevance of the methionine synthase variant.

  6. 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

  7. Genome-Wide Mapping of DNA Methylation in Chicken

    PubMed Central

    Hu, Xiaoxiang; Li, Jinxiu; Du, Zhuo; Chen, Li; Yin, Guangliang; Duan, Jinjie; Zhang, Haichao; Zhao, Yaofeng; Wang, Jun; Li, Ning

    2011-01-01

    Cytosine DNA methylation is an important epigenetic modification termed as the fifth base that functions in diverse processes. Till now, the genome-wide DNA methylation maps of many organisms has been reported, such as human, Arabidopsis, rice and silkworm, but the methylation pattern of bird remains rarely studied. Here we show the genome-wide DNA methylation map of bird, using the chicken as a model organism and an immunocapturing approach followed by high-throughput sequencing. In both of the red jungle fowl and the avian broiler, DNA methylation was described separately for the liver and muscle tissue. Generally, chicken displays analogous methylation pattern with that of animals and plants. DNA methylation is enriched in the gene body regions and the repetitive sequences, and depleted in the transcription start site (TSS) and the transcription termination site (TTS). Most of the CpG islands in the chicken genome are kept in unmethylated state. Promoter methylation is negatively correlated with the gene expression level, indicating its suppressive role in regulating gene transcription. This work contributes to our understanding of epigenetics in birds. PMID:21573164

  8. Role of DNA Methylation in Modulating Transcription Factor Occupancy.

    PubMed

    Maurano, Matthew T; Wang, Hao; John, Sam; Shafer, Anthony; Canfield, Theresa; Lee, Kristen; Stamatoyannopoulos, John A

    2015-08-18

    Although DNA methylation is commonly invoked as a mechanism for transcriptional repression, the extent to which it actively silences transcription factor (TF) occupancy sites in vivo is unknown. To study the role of DNA methylation in the active modulation of TF binding, we quantified the effect of DNA methylation depletion on the genomic occupancy patterns of CTCF, an abundant TF with known methylation sensitivity that is capable of autonomous binding to its target sites in chromatin. Here, we show that the vast majority (>98.5%) of the tens of thousands of unoccupied, methylated CTCF recognition sequences remain unbound upon abrogation of DNA methylation. The small fraction of sites that show methylation-dependent binding in vivo are in turn characterized by highly variable CTCF occupancy across cell types. Our results suggest that DNA methylation is not a primary groundskeeper of genomic TF landscapes, but rather a specialized mechanism for stabilizing intrinsically labile sites. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. 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

  10. High resolution DNA content measurements of mammalian sperm

    SciTech Connect

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.

    1982-01-01

    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  11. Obesity-related DNA methylation at imprinted genes in human sperm: Results from the TIEGER study.

    PubMed

    Soubry, Adelheid; Guo, Lisa; Huang, Zhiqing; Hoyo, Cathrine; Romanus, Stephanie; Price, Thomas; Murphy, Susan K

    2016-01-01

    Epigenetic reprogramming in mammalian gametes resets methylation marks that regulate monoallelic expression of imprinted genes. In males, this involves erasure of the maternal methylation marks and establishment of paternal-specific methylation to appropriately guide normal development. The degree to which exogenous factors influence the fidelity of methylation reprogramming is unknown. We previously found an association between paternal obesity and altered DNA methylation in umbilical cord blood, suggesting that the father's endocrine, nutritional, or lifestyle status could potentiate intergenerational heritable epigenetic abnormalities. In these analyses, we examine the relationship between male overweight/obesity and DNA methylation status of imprinted gene regulatory regions in the gametes. Linear regression models were used to compare sperm DNA methylation percentages, quantified by bisulfite pyrosequencing, at 12 differentially methylated regions (DMRs) from 23 overweight/obese and 44 normal weight men. Our study population included 69 volunteers from The Influence of the Environment on Gametic Epigenetic Reprogramming (TIEGER) study, based in NC, USA. After adjusting for age and fertility patient status, semen from overweight or obese men had significantly lower methylation percentages at the MEG3 (β = -1.99; SE = 0.84; p = 0.02), NDN (β = -1.10; SE = 0.47; p = 0.02), SNRPN (β = -0.65; SE = 0.27; p = 0.02), and SGCE/PEG10 (β = -2.5; SE = 1.01; p = 0.01) DMRs. Our data further suggest a slight increase in DNA methylation at the MEG3-IG DMR (β = +1.22; SE = 0.59; p = 0.04) and H19 DMR (β = +1.37; SE = 0.62; p = 0.03) in sperm of overweight/obese men. Our data support that male overweight/obesity status is traceable in the sperm epigenome. Further research is needed to understand the effect of such changes and the point of origin of DNA methylation differences between lean and

  12. Tissue-specific patterns of allelically-skewed DNA methylation

    PubMed Central

    Marzi, Sarah J.; Meaburn, Emma L.; Dempster, Emma L.; Lunnon, Katie; Paya-Cano, Jose L.; Smith, Rebecca G.; Volta, Manuela; Troakes, Claire; Schalkwyk, Leonard C.; Mill, Jonathan

    2016-01-01

    ABSTRACT While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ∼220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood. PMID:26786711

  13. Tissue-specific patterns of allelically-skewed DNA methylation.

    PubMed

    Marzi, Sarah J; Meaburn, Emma L; Dempster, Emma L; Lunnon, Katie; Paya-Cano, Jose L; Smith, Rebecca G; Volta, Manuela; Troakes, Claire; Schalkwyk, Leonard C; Mill, Jonathan

    2016-01-01

    While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ∼220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.

  14. Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

    PubMed

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

    2017-04-01

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

  15. Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of Chlamydomonas

    PubMed Central

    Sager, Ruth; Grabowy, Constance

    1983-01-01

    In Chlamydomonas, the maternal inheritance of chloroplast genes correlates with the differential methylation of chloroplast DNA (chlDNA) in females (mt+) but not in males (mt-). Our previous studies have supported our methylation-restriction model in which the maternal transmission is accounted for by the differential methylation in gametes which protects female but not male chlDNA from degradation during zygote formation. In the mutant me-1 [Bolen, P. L., Grant, D. M., Swinton, D., Boynton, J. E. & Gillham, N. W. (1982) Cell 28, 335-343], chlDNA of vegetative cells of both mating types is heavily methylated even before gametogenesis; nonetheless, maternal inheritance occurs in mutants as in wild type. To investigate the mechanism of maternal inheritance in the me-1 mutant, we have compared restriction fragment patterns after agarose gel electrophoresis of chlDNAs from mutant vegetative cells and gametes with those from wild type, by using a set of 32 restriction enzymes of which 17 were methylation-sensitive in this system. We find that additional methylation occurs during gametogenesis in the mutant female (mt+) but not in the corresponding male (mt-). Thus, gamete-specific, mating-type-specific methylation occurs in the me-1 mutant as in the wild type, consistent with our methylation-restriction model. In the me-1 mutant, gametic methylation occurs on a background of vegetative cell methylation not present in wild-type cells and irrelevant to the regulation of chloroplast inheritance. Comparison of the me-1 mutation with the mat-1 mutation [Sager, R., Grabowy, C. & Sano, H. (1981) Cell 24, 41-47] provides evidence for the existence of two different chlDNA methylation control systems: mat-1, linked to the mating type locus and regulating the mating-type-specific methylation that correlates with maternal inheritance, and me-1, unlinked to the mating type locus and unrelated to the regulation of maternal inheritance. Images PMID:16593314

  16. DNA methylation signature of long noncoding RNA genes during human pre-implantation embryonic development

    PubMed Central

    Shen, Xiaoli; Han, Shubiao; Ye, Hong; Huang, Guoning

    2017-01-01

    DNA methylation have crucial roles in regulating the expression of developmental genes during mammalian pre-implantation embryonic development (PED). However, the DNA methylation dynamic pattern of long noncoding RNA (lncRNA) genes, one type of epigenetic regulators, in human PED have not yet been demonstrated. Here, we performed a comprehensive analysis of lncRNA genes in human PED based on public reduced representation bisulphite sequencing (RRBS) data. We observed that both lncRNA and protein-coding genes complete the major demethylation wave at the 2-cell stage, whereas the promoters of lncRNA genes show higher methylation level than protein-coding genes during PED. Similar methylation distribution was observed across the transcription start sites (TSS) of lncRNA and protein-coding genes, contrary to previous observations in tissues. Besides, not only the gamete-specific differentially methylated regions (G-DMRs) but also the embryonic developmental-specific DMRs (D-DMRs) showed more paternal bias, especially in promoter regions in lncRNA genes. Moreover, coding-non-coding gene co-expression network analysis of genes containing D-DMRs suggested that lncRNA genes involved in PED are associated with gene expression regulation through several means, such as mRNA splicing, translational regulation and mRNA catabolic. This firstly provides study provides the methylation profiles of lncRNA genes in human PED and improves the understanding of lncRNA genes involvement in human PED. PMID:28915634

  17. 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

  18. DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

    PubMed

    Koike, Tasuku; Wakai, Takuya; Jincho, Yuko; Sakashita, Akihiko; Kobayashi, Hisato; Mizutani, Eiji; Wakayama, Sayaka; Miura, Fumihito; Ito, Takashi; Kono, Tomohiro

    2016-06-01

    The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

  19. Genome-wide analysis of DNA methylation dynamics during early human development.

    PubMed

    Okae, Hiroaki; Chiba, Hatsune; Hiura, Hitoshi; Hamada, Hirotaka; Sato, Akiko; Utsunomiya, Takafumi; Kikuchi, Hiroyuki; Yoshida, Hiroaki; Tanaka, Atsushi; Suyama, Mikita; Arima, Takahiro

    2014-12-01

    DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

  20. 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

  1. Cancer diagnostic classifiers based on quantitative DNA methylation

    PubMed Central

    2014-01-01

    Epigenetic change is part of the carcinogenic process and a deep reservoir for biomarker discovery. Reversible methylation of cytosines is noteworthy because it can be measured accurately and easily by various molecular methods and DNA methylation patterns are linked to important tumourigenic pathways. Clinically relevant methylation changes are known in common human cancers such as cervix, prostate, breast, colon, bladder, stomach and lung. Differential methylation may have a central role in the development and outcome of most if not all human malignancies. The advent of deep sequencing holds great promise for epigenomics, with bioinformatics tools ready to reveal large numbers of new targets for prognosis and therapeutic intervention. This review focuses on two selected cancers, namely cervix and prostate, which illustrate the more general themes of epigenetic diagnostics in cancer. Also discussed is differential methylation of specific human and viral DNA targets and laboratory methods for measuring methylation biomarkers. PMID:24649818

  2. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis.

    PubMed

    Tang, Binhua

    2016-01-01

    Genome-wide deciphering intercellular differential DNA methylation as well as its roles in transcriptional regulation remains elusive in cancer epigenetics. Here we developed a toolkit META2 for DNA methylation annotation and analysis, which aims to perform integrative analysis on differentially methylated loci and regions through deep mining and statistical comparison methods. META2 contains multiple versatile functions for investigating and annotating DNA methylation profiles. Benchmarked with T-47D cell, we interrogated the association within differentially methylated CpG (DMC) and region (DMR) candidate count and region length and identified major transition zones as clues for inferring statistically significant DMRs; together we validated those DMRs with the functional annotation. Thus META2 can provide a comprehensive analysis approach for epigenetic research and clinical study.

  3. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis

    PubMed Central

    2016-01-01

    Genome-wide deciphering intercellular differential DNA methylation as well as its roles in transcriptional regulation remains elusive in cancer epigenetics. Here we developed a toolkit META2 for DNA methylation annotation and analysis, which aims to perform integrative analysis on differentially methylated loci and regions through deep mining and statistical comparison methods. META2 contains multiple versatile functions for investigating and annotating DNA methylation profiles. Benchmarked with T-47D cell, we interrogated the association within differentially methylated CpG (DMC) and region (DMR) candidate count and region length and identified major transition zones as clues for inferring statistically significant DMRs; together we validated those DMRs with the functional annotation. Thus META2 can provide a comprehensive analysis approach for epigenetic research and clinical study. PMID:28116291

  4. 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.

  5. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

    PubMed Central

    Yang, Allen S.; Estécio, Marcos R. H.; Doshi, Ketan; Kondo, Yutaka; Tajara, Eloiza H.; Issa, Jean-Pierre J.

    2004-01-01

    We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation. PMID:14973332

  6. Dynamic DNA Methylation Regulates Levodopa-Induced Dyskinesia

    PubMed Central

    Figge, David A.; Eskow Jaunarajs, Karen L.

    2016-01-01

    Levodopa-induced dyskinesia (LID) is a persistent behavioral sensitization that develops after repeated levodopa (l-DOPA) exposure in Parkinson disease patients. LID is a consequence of sustained changes in the transcriptional behavior of striatal neurons following dopaminergic stimulation. In neurons, transcriptional regulation through dynamic DNA methylation has been shown pivotal to many long-term behavioral modifications; however, its role in LID has not yet been explored. Using a rodent model, we show LID development leads to the aberrant expression of DNA demethylating enzymes and locus-specific changes to DNA methylation at the promoter regions of genes aberrantly transcribed following l-DOPA treatment. Looking for dynamic DNA methylation in LID genome-wide, we used reduced representation bisulfite sequencing and found an extensive reorganization of the dorsal striatal methylome. LID development led to significant demethylation at many important regulatory areas of aberrantly transcribed genes. We used pharmacologic treatments that alter DNA methylation bidirectionally and found them able to modulate dyskinetic behaviors. Together, these findings demonstrate that l-DOPA induces widespread changes to striatal DNA methylation and that these modifications are required for the development and maintenance of LID. SIGNIFICANCE STATEMENT Levodopa-induced dyskinesia (LID) develops after repeated levodopa (l-DOPA) exposure in Parkinson disease patients and remains one of the primary obstacles to effective treatment. LID behaviors are a consequence of striatal neuron sensitization due to sustained changes in transcriptional behavior; however, the mechanisms responsible for the long-term maintenance of this cellular priming remain uncertain. Regulation of dynamic DNA methylation has been shown pivotal to the maintenance of several long-term behavioral modifications, yet its role in LID has not yet been explored. In this work, we report a pivotal role for the

  7. Factors underlying variable DNA methylation in a human community cohort

    PubMed Central

    Lam, Lucia L.; Emberly, Eldon; Fraser, Hunter B.; Neumann, Sarah M.; Chen, Edith; Miller, Gregory E.; Kobor, Michael S.

    2012-01-01

    Epigenetics is emerging as an attractive mechanism to explain the persistent genomic embedding of early-life experiences. Tightly linked to chromatin, which packages DNA into chromosomes, epigenetic marks primarily serve to regulate the activity of genes. DNA methylation is the most accessible and characterized component of the many chromatin marks that constitute the epigenome, making it an ideal target for epigenetic studies in human populations. Here, using peripheral blood mononuclear cells collected from a community-based cohort stratified for early-life socioeconomic status, we measured DNA methylation in the promoter regions of more than 14,000 human genes. Using this approach, we broadly assessed and characterized epigenetic variation, identified some of the factors that sculpt the epigenome, and determined its functional relation to gene expression. We found that the leukocyte composition of peripheral blood covaried with patterns of DNA methylation at many sites, as did demographic factors, such as sex, age, and ethnicity. Furthermore, psychosocial factors, such as perceived stress, and cortisol output were associated with DNA methylation, as was early-life socioeconomic status. Interestingly, we determined that DNA methylation was strongly correlated to the ex vivo inflammatory response of peripheral blood mononuclear cells to stimulation with microbial products that engage Toll-like receptors. In contrast, our work found limited effects of DNA methylation marks on the expression of associated genes across individuals, suggesting a more complex relationship than anticipated. PMID:23045638

  8. Horizontal transfer of DNA methylation patterns into bacterial chromosomes.

    PubMed

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

    2016-05-19

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Self-reported smoking, serum cotinine, and blood DNA methylation.

    PubMed

    Zhang, Yan; Florath, Ines; Saum, Kai-Uwe; Brenner, Hermann

    2016-04-01

    Epigenome-wide profiling of DNA methylation pattern with respect to tobacco smoking has given rise to a new measure of smoking exposure. We investigated the relationships of methylation markers with both cotinine, an established marker of internal smoking exposure, and self-reported smoking. Blood DNA methylation levels across the genome and serum cotinine were measured in 1000 older adults aged 50-75 years. Epigenome-wide scans were performed to identify methylation markers associated with cotinine. The inter-dose-response relationships between the number of cigarettes smoked per day, cotinine concentration, and DNA methylation were modeled by restricted cubic spline regression. Of 61 CpGs that passed the genome-wide significance threshold (p<1.13×10(-7)), 40 CpGs in 25 chromosomal regions were successfully replicated, showing 0.2-3% demethylation per 10ng/ml increases in cotinine. The strongest associations were observed for several loci at AHRR, F2RL3, 2q37.1, 6p21.33, and GFI1 that were previously identified to be related to self-reported smoking. One locus at RAB34 was newly discovered. Both cotinine and methylation markers exhibited non-linear relationships with the number of cigarettes smoked per day, where the highest rates of increase in cotinine and decreases in methylation were observed at low smoking intensity (1-15 cigarettes/day) and plateaued at high smoking intensity (>15-20 cigarettes/day). A clear linear relationship was observed between cotinine concentration and methylation level. Both cotinine and methylation markers showed similar accuracy in distinguishing current from never smoker, but only methylation markers distinguished former from never smoker with high accuracy. Our study corroborates and expands the list of smoking-associated DNA methylation markers. Methylation levels were linearly related to cotinine concentration and provided accurate measures for both current and past smoking exposure. Copyright © 2016 Elsevier Inc. All rights

  10. Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

    PubMed

    Wu, Yiping; Zhang, Bintian; Guo, Liang-Hong

    2013-07-16

    Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes.

  11. Conserved DNA methylation in Gadd45a(-/-) mice.

    PubMed

    Engel, Nora; Tront, Jennifer S; Erinle, Toyin; Nguyen, Nghi; Latham, Keith E; Sapienza, Carmen; Hoffman, Barbara; Liebermann, Dan A

    2009-02-16

    Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha) plays a pivotal role in cellular stress responses and is implicated in DNA repair, cell cycle arrest and apoptosis.(1) Recently, it was proposed that GADD45A is a key regulator of active DNA demethylation by way of its role in DNA repair.(2) Barreto et al. reported that Gadd45a overexpression activated transcription from methylation-silenced reporter plasmids and promoted global DNA demethylation. siRNA-mediated knockdown of Gadd45a levels resulted in increased levels of DNA methylation at specific endogenous loci. Based on these exciting results, Gadd45a(-/-) mice might be predicted to have a hypermethylation phenotype. We report here that neither global nor locus-specific methylation is increased in Gadd45a(-/-) mice.

  12. A genome-wide DNA methylation study in azoospermia.

    PubMed

    Ferfouri, F; Boitrelle, F; Ghout, I; Albert, M; Molina Gomes, D; Wainer, R; Bailly, M; Selva, J; Vialard, F

    2013-11-01

    The objective of this study was to assess genome-wide DNA methylation in testicular tissue from azoospermic patients. A total of 94 azoospermic patients were recruited and classified into three groups: 29 patients presented obstructive azoospermia (OA), 26 displayed non-obstructive azoospermia (NOA) and successful retrieval of spermatozoa by testicular sperm extraction (TESE+) and 39 displayed NOA and failure to retrieve spermatozoa by TESE (TESE-). An Illumina Infinium Human Methylation27 BeadChip DNA methylation array was used to establish a testicular DNA methylation pattern for each type of azoospermic patient. The OA and NOA groups were compared in terms of the relative M-value (the log2 ratio between methylated and non-methylated probe intensities) for each CpG site. We observed significantly different DNA methylation profiles for the NOA and OA groups, with differences at over 9000 of the 27 578 CpG sites; 212 CpG sites had a relative M-value >3. The results highlighted 14 testis-specific genes. Patient clustering with respect to these 212 CpG sites corresponded closely to the clinical classification. The DNA methylation patterns showed that in the NOA group, 78 of the 212 CpG sites were hypomethylated and 134 were hypermethylated (relative to the OA group). On the basis of these DNA methylation profiles, azoospermic patients could be classified as OA or NOA by considering the 212 CpG sites with the greatest methylation differences. Furthermore, we identified genes that may provide insight into the mechanism of idiopathic NOA.

  13. An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells.

    PubMed

    Walter, Marius; Teissandier, Aurélie; Pérez-Palacios, Raquel; Bourc'his, Déborah

    2016-01-27

    DNA methylation is extensively remodeled during mammalian gametogenesis and embryogenesis. Most transposons become hypomethylated, raising the question of their regulation in the absence of DNA methylation. To reproduce a rapid and extensive demethylation, we subjected mouse ES cells to chemically defined hypomethylating culture conditions. Surprisingly, we observed two phases of transposon regulation. After an initial burst of de-repression, various transposon families were efficiently re-silenced. This was accompanied by a reconfiguration of the repressive chromatin landscape: while H3K9me3 was stable, H3K9me2 globally disappeared and H3K27me3 accumulated at transposons. Interestingly, we observed that H3K9me3 and H3K27me3 occupy different transposon families or different territories within the same family, defining three functional categories of adaptive chromatin responses to DNA methylation loss. Our work highlights that H3K9me3 and, most importantly, polycomb-mediated H3K27me3 chromatin pathways can secure the control of a large spectrum of transposons in periods of intense DNA methylation change, ensuring longstanding genome stability.

  14. An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells

    PubMed Central

    Walter, Marius; Teissandier, Aurélie; Pérez-Palacios, Raquel; Bourc'his, Déborah

    2016-01-01

    DNA methylation is extensively remodeled during mammalian gametogenesis and embryogenesis. Most transposons become hypomethylated, raising the question of their regulation in the absence of DNA methylation. To reproduce a rapid and extensive demethylation, we subjected mouse ES cells to chemically defined hypomethylating culture conditions. Surprisingly, we observed two phases of transposon regulation. After an initial burst of de-repression, various transposon families were efficiently re-silenced. This was accompanied by a reconfiguration of the repressive chromatin landscape: while H3K9me3 was stable, H3K9me2 globally disappeared and H3K27me3 accumulated at transposons. Interestingly, we observed that H3K9me3 and H3K27me3 occupy different transposon families or different territories within the same family, defining three functional categories of adaptive chromatin responses to DNA methylation loss. Our work highlights that H3K9me3 and, most importantly, polycomb-mediated H3K27me3 chromatin pathways can secure the control of a large spectrum of transposons in periods of intense DNA methylation change, ensuring longstanding genome stability. DOI: http://dx.doi.org/10.7554/eLife.11418.001 PMID:26814573

  15. Statistical approaches for the analysis of DNA methylation microarray data.

    PubMed

    Siegmund, Kimberly D

    2011-06-01

    Following the rapid development and adoption in DNA methylation microarray assays, we are now experiencing a growth in the number of statistical tools to analyze the resulting large-scale data sets. As is the case for other microarray applications, biases caused by technical issues are of concern. Some of these issues are old (e.g., two-color dye bias and probe- and array-specific effects), while others are new (e.g., fragment length bias and bisulfite conversion efficiency). Here, I highlight characteristics of DNA methylation that suggest standard statistical tools developed for other data types may not be directly suitable. I then describe the microarray technologies most commonly in use, along with the methods used for preprocessing and obtaining a summary measure. I finish with a section describing downstream analyses of the data, focusing on methods that model percentage DNA methylation as the outcome, and methods for integrating DNA methylation with gene expression or genotype data.

  16. DNA methylation testing and marker validation using PCR: diagnostic applications.

    PubMed

    Egger, Gerda; Wielscher, Matthias; Pulverer, Walter; Kriegner, Albert; Weinhäusel, Andreas

    2012-01-01

    DNA methylation provides a fundamental epigenetic mechanism to establish and promote cell-specific gene-expression patterns, which are inherited by subsequent cell generations. Thus, the epigenome determines the differentiation into a cell lineage but can also program cells to become abnormal or malignant. In humans, different germline and somatic diseases have been linked to faulty DNA methylation. In this article, we will discuss the available PCR-based technologies to assess differences in DNA methylation levels mainly affecting 5-methylcytosine in the CpG dinucleotide context in hereditary syndromal and somatic pathological conditions. We will discuss some of the current diagnostic applications and provide an outlook on how DNA methylation-based biomarkers might provide novel tools for diagnosis, prognosis or patient stratification for diseases such as cancer.

  17. DNA methylation mapping by tag-modified bisulfite genomic sequencing.

    PubMed

    Han, Weiguo; Cauchi, Stephane; Herman, James G; Spivack, Simon D

    2006-08-01

    A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p<0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.

  18. CGGBP1 mitigates cytosine methylation at repetitive DNA sequences.

    PubMed

    Agarwal, Prasoon; Collier, Paul; Fritz, Markus Hsi-Yang; Benes, Vladimir; Wiklund, Helena Jernberg; Westermark, Bengt; Singh, Umashankar

    2015-05-16

    CGGBP1 is a repetitive DNA-binding transcription regulator with target sites at CpG-rich sequences such as CGG repeats and Alu-SINEs and L1-LINEs. The role of CGGBP1 as a possible mediator of CpG methylation however remains unknown. At CpG-rich sequences cytosine methylation is a major mechanism of transcriptional repression. Concordantly, gene-rich regions typically carry lower levels of CpG methylation than the repetitive elements. It is well known that at interspersed repeats Alu-SINEs and L1-LINEs high levels of CpG methylation constitute a transcriptional silencing and retrotransposon inactivating mechanism. Here, we have studied genome-wide CpG methylation with or without CGGBP1-depletion. By high throughput sequencing of bisulfite-treated genomic DNA we have identified CGGBP1 to be a negative regulator of CpG methylation at repetitive DNA sequences. In addition, we have studied CpG methylation alterations on Alu and L1 retrotransposons in CGGBP1-depleted cells using a novel bisulfite-treatment and high throughput sequencing approach. The results clearly show that CGGBP1 is a possible bidirectional regulator of CpG methylation at Alus, and acts as a repressor of methylation at L1 retrotransposons.

  19. Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

    PubMed Central

    Boon, Kathy; Tomfohr, John K; Bailey, Nathaniel W; Garantziotis, Stavros; Li, Zhuowei; Brass, David M; Maruoka, Shuichiro; Hollingsworth, John W; Schwartz, David A

    2008-01-01

    Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK), a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05) after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models of complex human diseases in

  20. Mapping DNA methylation with high-throughput nanopore sequencing.

    PubMed

    Rand, Arthur C; Jain, Miten; Eizenga, Jordan M; Musselman-Brown, Audrey; Olsen, Hugh E; Akeson, Mark; Paten, Benedict

    2017-04-01

    DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in Escherichia coli.

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

    PubMed Central

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

    2016-01-01

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

  2. DNA Methylation Indicates Susceptibility to Isoproterenol-Induced Cardiac Pathology and Is Associated With Chromatin States

    PubMed Central

    Chen, Haodong; Orozco, Luz; Wang, Jessica; Rau, Christoph D.; Rubbi, Liudmilla; Ren, Shuxun; Wang, Yibin; Pellegrini, Matteo; Lusis, Aldons J.; Vondriska, Thomas M.

    2016-01-01

    provides the first single base-resolution map of the mammalian cardiac DNA methylome and the first case-control analysis of the changes in DNA methylation with heart failure. The findings demonstrate marked genetic differences in DNA methylation that are associated with disease progression. PMID:26838786

  3. De novo DNA methylation during monkey pre-implantation embryogenesis

    PubMed Central

    Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

    2017-01-01

    Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

  4. The 'golden age' of DNA methylation in neurodegenerative diseases.

    PubMed

    Fuso, Andrea

    2013-03-01

    DNA methylation reactions are regulated, in the first instance, by enzymes and the intermediates that constitute the 'so called' one-carbon metabolism. This is a complex biochemical pathway, also known as the homocysteine cycle, regulated by the presence of B vitamins (folate, B6, B12) and choline, among other metabolites. One of the intermediates of this metabolism is S-adenosylmethionine, which represent the methyl donor in all the DNA methyltransferase reactions in eukaryotes. The one-carbon metabolism therefore produces the substrate necessary for the transferring of a methyl group on the cytosine residues of DNA; S-adenosylmethionine also regulates the activity of the enzymes that catalyze this reaction, namely the DNA methyltransferases (DNMTs). Alterations of this metabolic cycle can therefore be responsible for aberrant DNA methylation processes possibly leading to several human diseases. As a matter of fact, increasing evidences indicate that a number of human diseases with multifactorial origin may have an epigenetic basis. This is also due to the great technical advances in the field of epigenetic research. Among the human diseases associated with epigenetic factors, aging-related and neurodegenerative diseases are probably the object of most intense research. This review will present the main evidences linking several human diseases to DNA methylation, with particular focus on neurodegenerative diseases, together with a short description of the state-of-the-art of methylation assays.

  5. 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

  6. Genome-wide analysis of DNA methylation associated with HIV infection based on a pair of monozygotic twins

    PubMed Central

    Zhang, Yinfeng; Li, Sai-Kam; Tsui, Stephen Kwok-Wing

    2015-01-01

    Alteration of DNA methylation in mammalian cells could be elicited by many factors, including viral infections [1]. HIV has shown the ability to interact with host cellular factors to change the methylation status of some genes [2], [3], [4]. However, the change of the DNA methylation associated with HIV infection based on the whole genome has not been well illustrated. In this study, a unique pair of monozygotic twins was recruited: one of the twins was infected with HIV without further anti-retroviral therapy while the other one was healthy, which could be considered as a relatively ideal model for profiling the alterations of DNA methylation associated with HIV infection. Therefore, using methylated DNA immunoprecipitation–microarray method (MeDIP–microarray), we found the increased DNA methylation level in peripheral blood mononuclear cells from HIV infected twin compared to her normal sibling. Moreover, several distinguished differential methylation regions (DMRs) in HIV infected twin worth further study. The raw data has been deposited in Gene Expression Omnibus (GEO) datasets with reference number GSE68028. PMID:26697319

  7. 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

  8. DNA methylation and cognitive functioning in healthy older adults.

    PubMed

    Schiepers, Olga J G; van Boxtel, Martin P J; de Groot, Renate H M; Jolles, Jelle; Kok, Frans J; Verhoef, Petra; Durga, Jane

    2012-03-01

    Long-term supplementation with folic acid may improve cognitive performance in older individuals. The relationship between folate status and cognitive performance might be mediated by changes in methylation capacity, as methylation reactions are important for normal functioning of the brain. Although aberrant DNA methylation has been implicated in neurodevelopmental disorders, the relationship between DNA methylation status and non-pathological cognitive functioning in human subjects has not yet been investigated. The present study investigated the associations between global DNA methylation and key domains of cognitive functioning in healthy older adults. Global DNA methylation, defined as the percentage of methylated cytosine to total cytosine, was measured in leucocytes by liquid chromatography-MS/MS, in 215 men and women, aged 50-70 years, who participated in the Folic Acid and Carotid Intima-Media Thickness (FACIT) study (clinical trial registration number NCT00110604). Cognitive performance was assessed by means of the Visual Verbal Word Learning Task, the Stroop Colour-Word Interference Test, the Concept Shifting Test, the Letter-Digit Substitution Test and the Verbal Fluency Test. Using hierarchical linear regression analyses adjusted for age, sex, level of education, alcohol consumption, smoking status, physical activity, erythrocyte folate concentration and 5,10-methylenetetrahydrofolate reductase 677 C → T genotype, we found that global DNA methylation was not related to cognitive performance on any of the domains measured. The present study results do not support the hypothesis that global DNA methylation, as measured in leucocytes, might be associated with cognitive functioning in healthy older individuals.

  9. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    PubMed

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  10. 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

  11. Aberrant DNA methylation imprints in aborted bovine clones.

    PubMed

    Liu, Jing-He; Yin, Shen; Xiong, Bo; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-04-01

    Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.

  12. Genome-Wide Analysis of DNA Methylation in Human Amnion

    PubMed Central

    Kim, Jinsil; Pitlick, Mitchell M.; Christine, Paul J.; Schaefer, Amanda R.; Saleme, Cesar; Comas, Belén; Cosentino, Viviana; Gadow, Enrique; Murray, Jeffrey C.

    2013-01-01

    The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies. PMID:23533356

  13. Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells.

    PubMed

    Castro-Smirnov, Fidel Antonio; Piétrement, Olivier; Aranda, Pilar; Bertrand, Jean-Rémi; Ayache, Jeanne; Le Cam, Eric; Ruiz-Hitzky, Eduardo; Lopez, Bernard S

    2016-11-03

    Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

  14. Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells

    NASA Astrophysics Data System (ADS)

    Castro-Smirnov, Fidel Antonio; Piétrement, Olivier; Aranda, Pilar; Bertrand, Jean-Rémi; Ayache, Jeanne; Le Cam, Eric; Ruiz-Hitzky, Eduardo; Lopez, Bernard S.

    2016-11-01

    Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

  15. Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells

    PubMed Central

    Castro-Smirnov, Fidel Antonio; Piétrement, Olivier; Aranda, Pilar; Bertrand, Jean-Rémi; Ayache, Jeanne; Le Cam, Eric; Ruiz-Hitzky, Eduardo; Lopez, Bernard S.

    2016-01-01

    Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications. PMID:27808269

  16. Inferring chronological age from DNA methylation patterns of human teeth.

    PubMed

    Giuliani, Cristina; Cilli, Elisabetta; Bacalini, Maria Giulia; Pirazzini, Chiara; Sazzini, Marco; Gruppioni, Giorgio; Franceschi, Claudio; Garagnani, Paolo; Luiselli, Donata

    2016-04-01

    Current methods to determine chronological age from modern and ancient remains rely on both morphological and molecular approaches. However, low accuracy and the lack of standardized protocols make the development of alternative methods for the estimation of individual's age even more urgent for several research fields, such as biological anthropology, biodemography, forensics, evolutionary genetics, and ancient DNA studies. Therefore, the aim of this study is to identify genomic regions whose DNA methylation level correlates with age in modern teeth. We used MALDI-TOF mass spectrometry to analyze DNA methylation levels of specific CpGs located in the ELOVL2, FHL2, and PENK genes. We considered methylation data from cementum, dentin and pulp of 21 modern teeth (from 17 to 77 years old) to construct a mathematical model able to exploit DNA methylation values to predict age of the individuals. The median difference between the real age and that estimated using DNA methylation values is 1.20 years (SD = 1.9) if DNA is recovered from both cementum and pulp of the same modern teeth, 2.25 years (SD = 2.5) if DNA is recovered from dental pulp, 2.45 years (SD = 3.3) if DNA is extracted from cementum and 7.07 years (SD = 7.0) when DNA is recovered from dentin only. We propose for the first time the evaluation of DNA methylation at ELOVL2, FHL2, and PENK genes as a powerful tool to predict age in modern teeth for anthropological applications. Future studies are needed to apply this method also to historical and relatively ancient human teeth. © 2015 Wiley Periodicals, Inc.

  17. 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

  18. 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.

  19. DNMT1-interacting RNAs block gene specific DNA methylation

    PubMed Central

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

    2013-01-01

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

  20. Intra- and extra-cellular DNA damage by harmine and 9-methyl-harmine.

    PubMed

    Vignoni, Mariana; Erra-Balsells, Rosa; Epe, Bernd; Cabrerizo, Franco M

    2014-03-05

    It is known that β-carbolines are able to produce photosensitized damage in cell-free DNA, but there is little information on their effects on cellular DNA. Therefore, we have analyzed the DNA damage produced by harmine and 9-methyl-harmine under UVA irradiation in V79 cells, together with the associated generation of micronuclei and photocytotoxicity. The results indicate that the most frequent photoproducts generated in the cellular DNA are modified purines such as 8-oxo-7,8-dihydroguanine. Only relatively few single-strand breaks were observed. CPDs were absent, although they were generated in cell-free DNA irradiated under the same conditions. The overall extent of DNA damage in the cells was considerably smaller than the one observed in cell free DNA. The generation of cellular DNA damage was associated with a significant generation of micronuclei and decreased cell proliferation. The data indicate that β-carbolines act as photosensitizers in mammalian cells. The spectrum of DNA modification, and therefore the mechanism of DNA damage generation, differs considerably from that observed with cell-free DNA.

  1. Leukocyte DNA as Surrogate for the Evaluation of Imprinted Loci Methylation in Mammary Tissue DNA

    PubMed Central

    Barault, Ludovic; Ellsworth, Rachel E.; Harris, Holly R.; Valente, Allyson L.; Shriver, Craig D.; Michels, Karin B.

    2013-01-01

    There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31–77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearman's correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue. PMID:23409079

  2. Studies of the repair of O6-alkylguanine and O4-alkylthymine in DNA by alkyltransferases from mammalian cells and bacteria.

    PubMed Central

    Pegg, A E; Dolan, M E; Scicchitano, D; Morimoto, K

    1985-01-01

    O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3910413

  3. DNA methylation divergence and tissue specialization in the developing mouse placenta.

    PubMed

    Decato, Benjamin E; Lopez-Tello, Jorge; Sferruzzi-Perri, Amanda; Smith, Andrew D; Dean, Matthew D

    2017-04-04

    The placental epigenome plays a vital role in regulating mammalian growth and development. Aberrations in placental DNA methylation are linked to several disease states, including intrauterine growth restriction and preeclampsia. Studying the evolution and development of the placental epigenome is critical to understanding the origin and progression of such diseases. Although high resolution studies have found substantial variation between placental methylomes of different species, the nature of methylome variation has yet to be characterized within any individual species. We conducted a study of placental DNA methylation at high resolution in multiple strains and closely related species of house mice (Mus musculus musculus, Mus m. domesticus, and M. spretus), across developmental timepoints (embryonic days 15 to 18), and between two distinct layers (labyrinthine transport and junctional endocrine).We observed substantial genome-wide methylation heterogeneity in mouse placenta compared to other differentiated tissues. Species-specific methylation profiles were concentrated in retrotransposon subfamilies, specifically RLTR10 and RLTR20 subfamilies. Regulatory regions such as gene promoters and CpG islands displayed cross-species conservation, but showed strong differences between layers and developmental timepoints. Partially methylated domains exist in the mouse placenta and widen during development. Taken together, our results characterize the mouse placental methylome as a highly heterogeneous and deregulated landscape globally, intermixed with actively regulated promoter and retrotransposon sequences.

  4. 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

  5. Use of DNA methylation for cancer detection and molecular classification.

    PubMed

    Zhu, Jingde; Yao, Xuebiao

    2007-03-31

    Conjugation of the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3' sequence (DNA methylation) is the best studied epigenetic mechanism, which acts together with other epigenetic entities: histone modification, chromatin remodeling and microRNAs to shape the chromatin structure of DNA according to its functional state. The cancer genome is frequently characterized by hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methyl cytosine, the pathological implication of which to the cancerous state has been well established. While the latest genome-wide technologies have been applied to classify and interpret the epigenetic layer of gene regulation in the physiological and disease states, the epigenetic testing has also been seriously explored in clinical practice for early detection, refining tumor staging and predicting disease recurrence. This critique reviews the latest research findings on the use of DNA methylation in cancer diagnosis, prognosis and staging/classification.

  6. Oxidative stress and DNA methylation regulation in the metabolic syndrome.

    PubMed

    Yara, Sabrina; Lavoie, Jean-Claude; Levy, Emile

    2015-01-01

    DNA methylation is implicated in tissue-specific gene expression and genomic imprinting. It is modulated by environmental factors, especially nutrition. Modified DNA methylation patterns may contribute to health problems and susceptibility to complex diseases. Current advances have suggested that the metabolic syndrome (MS) is a programmable disease, which is characterized by epigenetic modifications of vital genes when exposed to oxidative stress. Therefore, the main objective of this paper is to critically review the central context of MS while presenting the most recent knowledge related to epigenetic alterations that are promoted by oxidative stress. Potential pro-oxidant mechanisms that orchestrate changes in methylation profiling and are related to obesity, diabetes and hypertension are discussed. It is anticipated that the identification and understanding of the role of DNA methylation marks could be used to uncover early predictors and define drugs or diet-related treatments able to delay or reverse epigenetic changes, thereby combating MS burden.

  7. DNA methylation levels associated with race and childhood asthma severity.

    PubMed

    Chan, Marcia A; Ciaccio, Christina E; Gigliotti, Nicole M; Rezaiekhaligh, Mo; Siedlik, Jacob A; Kennedy, Kevin; Barnes, Charles S

    2016-12-08

    Asthma is a common chronic childhood disease worldwide. Socioeconomic status, genetic predisposition and environmental factors contribute to its incidence and severity. A disproportionate number of children with asthma are economically disadvantaged and live in substandard housing with potential indoor environmental exposures such as cockroaches, dust mites, rodents and molds. These exposures may manifest through epigenetic mechanisms that can lead to changes in relevant gene expression. We examined the association of global DNA methylation levels with socioeconomic status, asthma severity and race/ethnicity. We measured global DNA methylation in peripheral blood of children with asthma enrolled in the Kansas City Safe and Healthy Homes Program. Inclusion criteria included residing in the same home for a minimum of 4 days per week and total family income of less than 80% of the Kansas City median family income. DNA methylation levels were quantified by an immunoassay that assessed the percentage of 5-methylcytosine. Our results indicate that overall, African American children had higher levels of global DNA methylation than children of other races/ethnicities (p = 0.029). This difference was more pronounced when socioeconomic status and asthma severity were coupled with race/ethnicity (p = 0.042) where low-income, African American children with persistent asthma had significantly elevated methylation levels relative to other races/ethnicities in the same context (p = 0.006, Hedges g = 1.14). Our study demonstrates a significant interaction effect among global DNA methylation levels, asthma severity, race/ethnicity, and socioeconomic status.

  8. DNA methylation profiling can classify HIV-associated lymphomas.

    PubMed

    Matsunaga, Akihiro; Hishima, Tsunekazu; Tanaka, Noriko; Yamasaki, Maria; Yoshida, Lui; Mochizuki, Makoto; Tanuma, Junko; Oka, Shinichi; Ishizaka, Yukihito; Shimura, Mari; Hagiwara, Shotaro

    2014-02-20

    HIV-positive patients have a 60-fold to 200-fold increased incidence of non-Hodgkin lymphomas, including Burkitt lymphoma, diffuse large B-cell lymphoma, and primary central nervous system lymphoma. HIV-associated lymphomas frequently have features such as extranodal involvement, decreased responses to standard chemotherapy, and high relapse rates, which indicate a poor prognosis. General pathological features do not clearly differentiate HIV-associated lymphomas from non-HIV lymphomas. To investigate the features of HIV-associated lymphomas, we performed genome-wide DNA methylation profiling of HIV and non-HIV lymphomas using Illumina GoldenGate Methylation Cancer Panel I and Illumina Infinium HumanMethylation450 BeadChip microarrays. DNA methylation profiles in HIV-associated and non-HIV lymphomas were characterized using unsupervised hierarchical clustering analyses. The analyses of promoter regions revealed unique DNA methylation profiles in HIV-associated lymphomas, suggesting profile differences compared with non-HIV lymphomas, which implies specific gene regulation in HIV-associated lymphoma involving DNA methylation. Based on HumanMethylation450 BeadChip data, 2541 target sites were selected as differing significantly in comparisons between HIV-associated and non-HIV-associated lymphomas using Wilcoxon's rank-sum test (P <0.05) and Δβ values more than 0.30. Recurrent cases of HIV-associated lymphoma had different profiles compared with nonrecurrent HIV lymphomas. DNA methylation profiling indicated that 2541 target sites differed significantly in HIV-associated lymphoma, which may partly explain the poor prognosis. Our data indicate that the methylation profiles of target genes have potential in elucidating HIV-associated lymphomagenesis and can serve as new prognostic markers.

  9. 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.

  10. 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

  11. Large conserved domains of low DNA methylation maintained by Dnmt3a

    PubMed Central

    Jeong, Mira; Sun, Deqiang; Luo, Min; Huang, Yun; Challen, Grant A.; Rodriguez, Benjamin; Zhang, Xiaotian; Chavez, Lukas; Wang, Hui; Hannah, Rebecca; Kim, Sang-Bae; Yang, Liubin; Ko, Myunggon; Chen, Rui; Göttgens, Berthold; Lee, Ju-Seog; Gunaratne, Preethi; Godley, Lucy A.; Darlington, Gretchen J.; Rao, Anjana; Li, Wei; Goodell, Margaret A.

    2014-01-01

    Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into mechanisms that could promote shifts in DNA methylation and contribute to cell fate changes, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified murine hematopoietic stem cells. We discovered extended regions of low methylation (Canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. The genes in about half of these methylation Canyons are coated with repressive histone marks while the remainder are covered by activating histone marks and are highly expressed in HSCs. Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for Canyon-associated genes. The novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development. PMID:24270360

  12. DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells

    SciTech Connect

    Olive, P.L.

    1988-01-01

    When mammalian cells are lysed in 2% sodium dodecyl sulphate detergent followed by addition of an equal volume of 0.12 M potassium chloride, a precipitate forms that can be collected by low-speed centrifugation. This precipitate contains the cell protein and nucleic acid in close association with protein. In the absence of DNA damage, most of the DNA precipitates, but when DNA strand breaks are created by exposing cells to ionizing radiation or toxic chemicals, DNA is released from the protein and remains in the supernatant after centrifugation. The proportion of DNA remaining in the supernatant is thus a measure of the amount of DNA damage. This assay is characterized in terms of optimum cell number and pH and dose-response curves for DNA damage and cell survival following ionizing radiation, MNNG, BCNU, and VP-16 are shown. Sensitivity, simplicity, speed, and large sample handling capacity should allow wide application of this new assay to a variety of questions concerning DNA damage and repair.

  13. Covering Your Bases: Inheritance of DNA Methylation in Plant Genomes

    PubMed Central

    Schmitz, Robert J.

    2014-01-01

    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. PMID:24270503

  14. Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

    PubMed

    Schübeler, D; Lorincz, M C; Cimbora, D M; Telling, A; Feng, Y Q; Bouhassira, E E; Groudine, M

    2000-12-01

    We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

  15. Genomic Targeting of Methylated DNA: Influence of Methylation on Transcription, Replication, Chromatin Structure, and Histone Acetylation

    PubMed Central

    Schübeler, Dirk; Lorincz, Matthew C.; Cimbora, Daniel M.; Telling, Agnes; Feng, Yong-Quing; Bouhassira, Eric E.; Groudine, Mark

    2000-01-01

    We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure. PMID:11094062

  16. [Methods for detection of methylated cytosine residues in DNA].

    PubMed

    Smirnikhina, S A; Lavrov, A V

    2009-01-01

    The article provides analysis of common methods for DNA methylation detection. Advantages and limitations of methods used for different purposes are compared. Clue step for most common methods is bisulfite treatment of DNA samples and its protocol is described in details. Recommendations are formulated for each method best in solving specific problems.

  17. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    PubMed

    Speit, Günter; Schütz, Petra; Bausinger, Julia

    2016-06-01

    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  18. 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.

  19. Intragenic DNA methylation in transcriptional regulation, normal differentiation and cancer.

    PubMed

    Kulis, Marta; Queirós, Ana C; Beekman, Renée; Martín-Subero, José I

    2013-11-01

    Ever since the discovery of DNA methylation at cytosine residues, the role of this so called fifth base has been extensively studied and debated. Until recently, the majority of DNA methylation studies focused on the analysis of CpG islands associated to promoter regions. However, with the upcoming possibilities to study DNA methylation in a genome-wide context, this epigenetic mark can now be studied in an unbiased manner. As a result, recent studies have shown that not only promoters but also intragenic and intergenic regions are widely modulated during physiological processes and disease. In particular, it is becoming increasingly clear that DNA methylation in the gene body is not just a passive witness of gene transcription but it seems to be actively involved in multiple gene regulation processes. In this review we discuss the potential role of intragenic DNA methylation in alternative promoter usage, regulation of short and long non-coding RNAs, alternative RNA processing, as well as enhancer activity. Furthermore, we summarize how the intragenic DNA methylome is modified both during normal cell differentiation and neoplastic transformation.

  20. Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing.

    PubMed

    Vaca-Paniagua, Felipe; Oliver, Javier; Nogueira da Costa, Andre; Merle, Philippe; McKay, James; Herceg, Zdenko; Holmila, Reetta

    2015-01-01

    To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

  1. Persistence of cytosine methylation of DNA following fertilisation in the mouse.

    PubMed

    Li, Yan; O'Neill, Chris

    2012-01-01

    Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote.

  2. Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse

    PubMed Central

    Li, Yan; O'Neill, Chris

    2012-01-01

    Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote. PMID:22292019

  3. SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

    PubMed Central

    Lopez-Mosqueda, Jaime; Maddi, Karthik; Prgomet, Stefan; Kalayil, Sissy; Marinovic-Terzic, Ivana; Terzic, Janos; Dikic, Ivan

    2016-01-01

    Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)— a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients. DOI: http://dx.doi.org/10.7554/eLife.21491.001 PMID:27852435

  4. 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.

  5. 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

  6. Rapid DNA Methylation Changes after Exposure to Traffic Particles

    PubMed Central

    Baccarelli, Andrea; Wright, Robert O.; Bollati, Valentina; Tarantini, Letizia; Litonjua, Augusto A.; Suh, Helen H.; Zanobetti, Antonella; Sparrow, David; Vokonas, Pantel S.; Schwartz, Joel

    2009-01-01

    Rationale: Exposure to particulate air pollution has been related to increased hospitalization and death, particularly from cardiovascular disease. Lower blood DNA methylation content is found in processes related to cardiovascular outcomes, such as oxidative stress, aging, and atherosclerosis. Objectives: We evaluated whether particulate pollution modifies DNA methylation in heavily methylated sequences with high representation throughout the human genome. Methods: We measured DNA methylation of long interspersed nucleotide element (LINE)-1 and Alu repetitive elements by quantitative polymerase chain reaction–pyrosequencing of 1,097 blood samples from 718 elderly participants in the Boston area Normative Aging Study. We used covariate-adjusted mixed models to account for within-subject correlation in repeated measures. We estimated the effects on DNA methylation of ambient particulate pollutants (black carbon, particulate matter with aerodynamic diameter ≤ 2.5 μm [PM2.5], or sulfate) in multiple time windows (4 h to 7 d) before the examination. We estimated standardized regression coefficients (β) expressing the fraction of a standard deviation change in DNA methylation associated with a standard deviation increase in exposure. Measurements and Main Results: Repetitive element DNA methylation varied in association with time-related variables, such as day of the week and season. LINE-1 methylation decreased after recent exposure to higher black carbon (β = −0.11; 95% confidence interval [CI], −0.18 to −0.04; P = 0.002) and PM2.5 (β = −0.13; 95% CI, −0.19 to −0.06; P < 0.001 for the 7-d moving average). In two-pollutant models, only black carbon, a tracer of traffic particles, was significantly associated with LINE-1 methylation (β = −0.09; 95% CI, −0.17 to −0.01; P = 0.03). No association was found with Alu methylation (P > 0.12). Conclusions: We found decreased repeated-element methylation after exposure to traffic particles. Whether

  7. Methylated DNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or DNA single-strand breaks and results in DNA damage.

    PubMed

    Groth, Petra; Ausländer, Simon; Majumder, Muntasir Mamun; Schultz, Niklas; Johansson, Fredrik; Petermann, Eva; Helleday, Thomas

    2010-09-10

    Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks. Copyright 2010 Elsevier Ltd. All rights reserved.

  8. DNA Methylation and Genome Evolution in Honeybee: Gene Length, Expression, Functional Enrichment Covary with the Evolutionary Signature of DNA Methylation

    PubMed Central

    Zeng, Jia; Yi, Soojin V.

    2010-01-01

    A growing body of evidence suggests that DNA methylation is functionally divergent among different taxa. The recently discovered functional methylation system in the honeybee Apis mellifera presents an attractive invertebrate model system to study evolution and function of DNA methylation. In the honeybee, DNA methylation is mostly targeted toward transcription units (gene bodies) of a subset of genes. Here, we report an intriguing covariation of length and epigenetic status of honeybee genes. Hypermethylated and hypomethylated genes in honeybee are dramatically different in their lengths for both exons and introns. By analyzing orthologs in Drosophila melanogaster, Acyrthosiphon pisum, and Ciona intestinalis, we show genes that were short and long in the past are now preferentially situated in hyper- and hypomethylated classes respectively, in the honeybee. Moreover, we demonstrate that a subset of high-CpG genes are conspicuously longer than expected under the evolutionary relationship alone and that they are enriched in specific functional categories. We suggest that gene length evolution in the honeybee is partially driven by evolutionary forces related to regulation of gene expression, which in turn is associated with DNA methylation. However, lineage-specific patterns of gene length evolution suggest that there may exist additional forces underlying the observed interaction between DNA methylation and gene lengths in the honeybee. PMID:20924039

  9. 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

  10. DNA methylation and hormone receptor status in breast cancer.

    PubMed

    Benevolenskaya, Elizaveta V; Islam, Abul B M M K; Ahsan, Habibul; Kibriya, Muhammad G; Jasmine, Farzana; Wolff, Ben; Al-Alem, Umaima; Wiley, Elizabeth; Kajdacsy-Balla, Andre; Macias, Virgilia; Rauscher, Garth H

    2016-01-01

    We examined whether differences in tumor DNA methylation were associated with more aggressive hormone receptor-negative breast cancer in an ethnically diverse group of patients in the Breast Cancer Care in Chicago (BCCC) study and using data from The Cancer Genome Atlas (TCGA). DNA was extracted from formalin-fixed, paraffin-embedded samples on 75 patients (21 White, 31 African-American, and 23 Hispanic) (training dataset) enrolled in the BCCC. Hormone receptor status was defined as negative if tumors were negative for both estrogen and progesterone (ER/PR) receptors (N = 22/75). DNA methylation was analyzed at 1505 CpG sites within 807 gene promoters using the Illumina GoldenGate assay. Differential DNA methylation as a predictor of hormone receptor status was tested while controlling for false discovery rate and assigned to the gene closest to the respective CpG site. Next, those genes that predicted ER/PR status were validated using TCGA data with respect to DNA methylation (validation dataset), and correlations between CpG methylation and gene expression were examined. In the training dataset, 5.7 % of promoter mean methylation values (46/807) were associated with receptor status at P < 0.05; for 88 % of these (38/46), hypermethylation was associated with receptor-positive disease. Hypermethylation for FZD9, MME, BCAP31, HDAC9, PAX6, SCGB3A1, PDGFRA, IGFBP3, and PTGS2 genes most strongly predicted receptor-positive disease. Twenty-one of 24 predictor genes from the training dataset were confirmed in the validation dataset. The level of DNA methylation at 19 out 22 genes, for which gene expression data were available, was associated with gene activity. Higher levels of promoter methylation strongly correlate with hormone receptor positive status of breast tumors. For most of the genes identified in our training dataset as ER/PR receptor status predictors, DNA methylation correlated with stable gene expression level. The predictors performed well when

  11. Pulmonary endothelial cell DNA methylation signature in pulmonary arterial hypertension.

    PubMed

    Hautefort, Aurélie; Chesné, Julie; Preussner, Jens; Pullamsetti, Soni S; Tost, Jorg; Looso, Mario; Antigny, Fabrice; Girerd, Barbara; Riou, Marianne; Eddahibi, Saadia; Deleuze, Jean-François; Seeger, Werner; Fadel, Elie; Simonneau, Gerald; Montani, David; Humbert, Marc; Perros, Frédéric

    2017-08-08

    Pulmonary arterial hypertension (PAH) is a severe and incurable pulmonary vascular disease. One of the primary origins of PAH is pulmonary endothelial dysfunction leading to vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Our objective was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. DNA was extracted from cultured PEC from idiopathic PAH (n = 11), heritable PAH (n = 10) and controls (n = 18). DNA methylation was assessed using the Illumina HumanMethylation450 Assay. After normalization, samples and probes were clustered according to their methylation profile. Differential clusters were functionally analyzed using bioinformatics tools. Unsupervised hierarchical clustering allowed the identification of two clusters of probes that discriminates controls and PAH patients. Among 147 differential methylated promoters, 46 promoters coding for proteins or miRNAs were related to lipid metabolism. Top 10 up and down-regulated genes were involved in lipid transport including ABCA1, ABCB4, ADIPOQ, miR-26A, BCL2L11. NextBio meta-analysis suggested a contribution of ABCA1 in PAH. We confirmed ABCA1 mRNA and protein downregulation specifically in PAH PEC by qPCR and immunohistochemistry and made the proof-of-concept in an experimental model of the disease that its targeting may offer novel therapeutic options. In conclusion, DNA methylation analysis identifies a set of genes mainly involved in lipid transport pathway which could be relevant to PAH pathophysiology.

  12. 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

    2015-05-18

    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.

  13. 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. © 2014 WILEY PERIODICALS, INC.

  14. 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

  15. Associations between serum perfluoroalkyl acids and LINE-1 DNA methylation.

    PubMed

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

    2014-02-01

    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±11years). 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. © 2013.

  16. Accounting for Population Stratification in DNA Methylation Studies

    PubMed Central

    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.

    2014-01-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex disease 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 (GWAS), 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 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 principal-components 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 SNP-based principal components, followed by methylation-based principal components, which out-perform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based principal components, we find that principal components based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjustment for population stratification in DNA methylation studies when genome-wide SNP data are unavailable. PMID:24478250

  17. Human Body Epigenome Maps Reveal Noncanonical DNA Methylation Variation

    PubMed Central

    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.

    2015-01-01

    Summary 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 play tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns1,2. 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 genome3, 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 multiple genomic contexts varies substantially among human tissues. PMID:26030523

  18. 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

    2015-07-09

    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.

  19. Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles.

    PubMed

    Waterland, Robert A; Kellermayer, Richard; Laritsky, Eleonora; Rayco-Solon, Pura; Harris, R Alan; Travisano, Michael; Zhang, Wenjuan; Torskaya, Maria S; Zhang, Jiexin; Shen, Lanlan; Manary, Mark J; Prentice, Andrew M

    2010-12-23

    Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

  20. Epigenetic Basis of Regeneration: Analysis of Genomic DNA Methylation Profiles in the MRL/MpJ Mouse

    PubMed Central

    Górnikiewicz, Bartosz; Ronowicz, Anna; Podolak, Justyna; Madanecki, Piotr; Stanisławska-Sachadyn, Anna; Sachadyn, PaweŁ

    2013-01-01

    Epigenetic regulation plays essential role in cell differentiation and dedifferentiation, which are the intrinsic processes involved in regeneration. To investigate the epigenetic basis of regeneration capacity, we choose DNA methylation as one of the most important epigenetic mechanisms and the MRL/MpJ mouse as a model of mammalian regeneration known to exhibit enhanced regeneration response in different organs. We report the comparative analysis of genomic DNA methylation profiles of the MRL/MpJ and the control C57BL/6J mouse. Methylated DNA immunoprecipitation followed by microarray analysis using the Nimblegen ‘3 × 720 K CpG Island Plus RefSeq Promoter’ platform was applied in order to carry out genome-wide DNA methylation profiling covering 20 404 promoter regions. We identified hundreds of hypo- and hypermethylated genes and CpG islands in the heart, liver, and spleen, and 37 of them in the three tissues. Decreased inter-tissue diversification and the shift of DNA methylation balance upstream the genes distinguish the genomic methylation patterns of the MRL/MpJ mouse from the C57BL/6J. Homeobox genes and a number of other genes involved in embryonic morphogenesis are significantly overrepresented among the genes hypomethylated in the MRL/MpJ mouse. These findings indicate that epigenetic patterning might be a likely molecular basis of regeneration capability in the MRL/MpJ mouse. PMID:23929942

  1. A Novel Approach to Assay DNA Methylation in Prostate Cancer

    DTIC Science & Technology

    2016-10-01

    the epigenetic envi- ronment at FOXA1-occupied enhancers , we first tested whether TET1 is able to co-occupy FOXA1-bound ge- nomic regions. As human...diagnosis and prognosis, some of which are being developed into clinical tests . However, several seminal studies have recently reported that DNA...expression and interacts with TET1 protein to mediate lineage-specific enhancer activation. 15. SUBJECT TERMS DNA methylation 5mC, DNA hydroxymethylation 5hmC

  2. Salt stress alters DNA methylation levels in alfalfa (Medicago spp).

    PubMed

    Al-Lawati, A; Al-Bahry, S; Victor, R; Al-Lawati, A H; Yaish, M W

    2016-02-26

    Modification of DNA methylation status is one of the mechanisms used by plants to adjust gene expression at both the transcriptional and posttranscriptional levels when plants are exposed to suboptimal conditions. Under abiotic stress, different cultivars often show heritable phenotypic variation accompanied by epigenetic polymorphisms at the DNA methylation level. This variation may provide the raw materials for plant breeding programs that aim to enhance abiotic stress tolerance, including salt tolerance. In this study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to assess cytosine methylation levels in alfalfa (Medicago spp) roots exposed to increasing NaCl concentrations (0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in addition to a salt-tolerant cultivar that was used as a control. There was a slight increase in DNA methylation upon exposure to high levels of soil salinity. Phylogenetic analysis using MSAP showed epigenetic variation within and between the alfalfa landraces when exposed to saline conditions. Based on MSAP and enzyme-linked immunosorbent assay results, we found that salinity increased global DNA methylation status, particularly in plants exposed to the highest level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain reaction indicated that this might be mediated by the overexpression of methyltransferase homolog genes after exposure to saline conditions. DNA demethylation using 5-azacytidine reduced seedling lengths and dry and fresh weights, indicating a possible decrease in salinity tolerance. These results suggest that salinity affects DNA methylation flexibility.

  3. Discordance of DNA Methylation Variance Between two Accessible Human Tissues

    PubMed Central

    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. PMID:25660083

  4. 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

  5. Dynamic DNA methylation controls glutamate receptor trafficking and synaptic scaling.

    PubMed

    Sweatt, J David

    2016-05-01

    Hebbian plasticity, including long-term potentiation and long-term depression, has long been regarded as important for local circuit refinement in the context of memory formation and stabilization. However, circuit development and stabilization additionally relies on non-Hebbian, homeostatic, forms of plasticity such as synaptic scaling. Synaptic scaling is induced by chronic increases or decreases in neuronal activity. Synaptic scaling is associated with cell-wide adjustments in postsynaptic receptor density, and can occur in a multiplicative manner resulting in preservation of relative synaptic strengths across the entire neuron's population of synapses. Both active DNA methylation and demethylation have been validated as crucial regulators of gene transcription during learning, and synaptic scaling is known to be transcriptionally dependent. However, it has been unclear whether homeostatic forms of plasticity such as synaptic scaling are regulated via epigenetic mechanisms. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously largely considered separately: DNA methylation, homeostatic plasticity, and glutamate receptor trafficking. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously considered separately: glutamate receptor trafficking, DNA methylation, and homeostatic plasticity.

  6. The impact of DNA methylation technologies on drug toxicology.

    PubMed

    Dueñas-Gonzalez, Alfonso; Alatorre, Brenda; Gonzalez-Fierro, Aurora

    2014-05-01

    Drug toxicology is central to drug development. Despite improvements in our understanding of molecular and cell biology, high attrition rates in drug development continue, speaking to the difficulties of developing unequivocal methods to predict the efficacy and safety of drugs. In this review, the authors provide a short overview of the 'omics' technologies that have been applied to drug toxicology, with an emphasis on a whole-genome DNA methylation analysis. Preliminary results from DNA methylation analysis technologies that may help in predicting response and efficacy of a drug are discussed. Currently, we cannot fully contextualize the application of epigenetics to the field of drug toxicology, as there are still many challenges to overcome before DNA methylation-based biomarkers can be effectively used in drug development. Comprehensive whole-genome DNA methylation methods for a unbiased analysis based on either microarray or next-generation sequencing need to be evaluated in drug toxicology in an intensive and systematic manner. Additionally, robust analysis systems need to be developed to decode the large amounts of data generated by whole-genome DNA methylation analyses as well as protocol standardization for reproducibility to develop meaningful databases that can be applied to drug toxicology.

  7. Techniques of DNA methylation analysis with nutritional applications.

    PubMed

    Mansego, Maria L; Milagro, Fermín I; Campión, Javier; Martínez, J Alfredo

    2013-01-01

    Epigenetic mechanisms are likely to play an important role in the regulation of metabolism and body weight through gene-nutrient interactions. This review focuses on methods for analyzing one of the most important epigenetic mechanisms, DNA methylation, from single nucleotide to global measurement depending on the study goal and scope. In addition, this study highlights the major principles and methods for DNA methylation analysis with emphasis on nutritional applications. Recent developments concerning epigenetic technologies are showing promising results of DNA methylation levels at a single-base resolution and provide the ability to differentiate between 5-methylcytosine and other nucleotide modifications such as 5-hydroxymethylcytosine. A large number of methods can be used for the analysis of DNA methylation such as pyrosequencing™, primer extension or real-time PCR methods, and genome-wide DNA methylation profile from microarray or sequencing-based methods. Researchers should conduct a preliminary analysis focused on the type of validation and information provided by each technique in order to select the best method fitting for their nutritional research interests.

  8. The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development

    PubMed Central

    Ruzov, Alexey; Savitskaya, Ekaterina; Hackett, Jamie A.; Reddington, James P.; Prokhortchouk, Anna; Madej, Monika J.; Chekanov, Nikolai; Li, Minghui; Dunican, Donncha S.; Prokhortchouk, Egor; Pennings, Sari; Meehan, Richard R.

    2009-01-01

    Summary Mammalian forms of the transcription repressor, Kaiso, can reportedly bind methylated DNA and non-methylated CTGCNA motifs. Here we compare the DNA-binding properties of Kaiso from frog, fish and chicken and demonstrate that only the methyl-CpG-binding function of Kaiso is evolutionarily conserved. We present several independent experimental lines of evidence that the phenotypic abnormalities associated with xKaiso-depleted Xenopus laevis embryos are independent of the putative CTGCNA-dependent DNA-binding function of xKaiso. Our analysis suggests that xKaiso does not play a role in the regulation of either xWnt11 or Siamois, key signalling molecules in the Wnt pathway during X. laevis gastrulation. The major phenotypic defects associated with xKaiso depletion are premature transcription activation before the mid-blastula transition and concomitant activation of a p53-dependent cell-death pathway. PMID:19158185

  9. Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

    PubMed

    Long, Hannah K; King, Hamish W; Patient, Roger K; Odom, Duncan T; Klose, Robert J

    2016-08-19

    DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Predicting DNA methylation level across human tissues

    PubMed Central

    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

    2014-01-01

    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 [R2 increases from 0.38 (original R2 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

  11. Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis

    PubMed Central

    Wang, Keh-Yang; Chen, Chun-Chang; Tsai, Shih-Feng; Shen, Che-Kun James

    2016-01-01

    DNA methylation at C of CpG dyads (mCpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic mCpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-mCpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability. PMID:27886214

  12. 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

  13. Insects as innovative models for functional studies of DNA methylation.

    PubMed

    Lyko, Frank; Maleszka, Ryszard

    2011-04-01

    The emerging field of epigenomics has the potential to bridge the gap between static genomic sequences and complex phenotypes that arise from multigenic, nonlinear and often context-dependent interactions. However, this goal can only be achieved if easily manageable experimental systems are available in which changes in epigenomic settings can be evaluated in the context of the phenotype under investigation. Recent progress in the characterization of insect DNA methylation patterns enables evaluation of the extent to which epigenetic mechanisms contribute to complex phenotypes in easily accessible organisms whose relatively small genomes are not only sparingly methylated, but the methylated sites are also found almost exclusively in gene bodies. The implementation of insect models in the study of DNA methylation will accelerate progress in understanding the functional significance of this important epigenetic mechanism in controlling gene splicing, in environmentally driven reprogramming of gene expression and in adult brain plasticity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Site-specific methylated reporter constructs for functional analysis of DNA methylation.

    PubMed

    Han, Weiguo; Shi, Miao; Spivack, Simon D

    2013-11-01

    Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

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

    PubMed

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

    2016-10-14

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

  16. Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells.

    PubMed Central

    Okano, M; Xie, S; Li, E

    1998-01-01

    We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase. In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast. Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase. However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells. We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells. Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells. PMID:9592134

  17. Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

    PubMed Central

    Estève, Pierre-Olivier; Zhang, Guoqiang; Ponnaluri, V.K. Chaithanya; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Sagum, Cari; Black, Karynne; Corrêa, Ivan R.; Bedford, Mark T.; Cheng, Xiaodong; Pradhan, Sriharsa

    2016-01-01

    Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion. PMID:26553800

  18. DNA methylation in repetitive elements and Alzheimer disease.

    PubMed

    Bollati, V; Galimberti, D; Pergoli, L; Dalla Valle, E; Barretta, F; Cortini, F; Scarpini, E; Bertazzi, P A; Baccarelli, A

    2011-08-01

    Epigenetics is believed to play a role in Alzheimer's disease (AD). DNA methylation, the most investigated epigenetic hallmark, is a reversible mechanism that modifies genome function and chromosomal stability through the addition of methyl groups to cytosine located in CpG dinucleotides to form 5 methylcytosine (5mC). Methylation status of repetitive elements (i.e. Alu, LINE-1 and SAT-α) is a major contributor of global DNA methylation patterns and has been investigated in relation to a variety of human diseases. However, the role of methylation of repetitive elements in blood of AD patients has never been investigated so far. In the present study, a quantitative bisulfite-PCR pyrosequencing method was used to evaluate methylation of Alu, LINE-1 and SAT-α sequences in 43 AD patients and 38 healthy donors. In multivariate analysis adjusting for age and gender, LINE-1 was increased in AD patients compared with healthy volunteers (ADs: 83.6%5mC, volunteers: 83.1%5mC, p-value: 0.05). The group with best performances in mini mental state examination (MMSE) showed higher levels of LINE-1 methylation compared to the group with worst performances (MMSE>22: 83.9%5mC; MMSE≤22: 83.2%5mC; p=0.05). Our data suggest that LINE-1 methylation may lead to a better understanding of AD pathogenesis and course, and may contribute to identify novel markers useful to assess risk stratification. Further prospective investigations are warranted to evaluate the dynamics of DNA methylation from early-stage AD to advanced phases of the disease.

  19. Effects of temperature and relative humidity on DNA methylation.

    PubMed

    Bind, Marie-Abele; Zanobetti, Antonella; Gasparrini, Antonio; Peters, Annette; Coull, Brent; Baccarelli, Andrea; Tarantini, Letizia; Koutrakis, Petros; Vokonas, Pantel; Schwartz, Joel

    2014-07-01

    Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation. We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the Normative Aging Study (1999-2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models. Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°C increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (-8% to -1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days. DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects.

  20. Effects of Temperature and Relative Humidity on DNA Methylation

    PubMed Central

    Bind, Marie-Abele; Zanobetti, Antonella; Gasparrini, Antonio; Peters, Annette; Coull, Brent; Baccarelli, Andrea; Tarantini, Letizia; Koutrakis, Petros; Vokonas, Pantel; Schwartz, Joel

    2014-01-01

    Background Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation. Methods We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the normative aging Study (1999–2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models. Results Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°c increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (−8% to −1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days. Conclusions DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects. PMID:24809956

  1. Sensitive digital quantification of DNA methylation in clinical samples

    PubMed Central

    Li, Meng; Chen, Wei-dong; Papadopoulos, Nickolas; Goodman, Steven; Bjerregaard, Niels Christian; Laurberg, Søren; Levin, Bernard; Juhl, Hartmut; Arber, Nadir; Moinova, Helen; Durkee, Kris; Schmidt, Kerstin; He, Yiping; Diehl, Frank; Velculescu, Victor E; Zhou, Shibin; Diaz, Luis A; Kinzler, Kenneth W; Markowitz, Sanford D; Vogelstein, Bert

    2010-01-01

    Abnormally methylated genes are increasingly being used as cancer biomarkers 1, 2. For clinical applications, it is important to precisely determine the number of methylated molecules in the analyzed sample. We here describe a digital approach that can enumerate one methylated molecule out of ~5000 unmethylated molecules. Individual DNA fragments can be amplified and analyzed either by flow cytometry or next generation sequencing instruments. Using methylated vimentin as a biomarker, we tested 191 plasma samples and detected cancer cases with 59% sensitivity (95% CI, 48%–70%) and 93% specificity (95% CI, 86%–97%). Using the same assay, we analyzed 80 stool samples and demonstrated 45% sensitivity for detecting colorectal adenomas (23%–68%), 41% sensitivity for detecting cancer (21%–64%), and 95% specificity (82%–99%). This digital quantification of rare methylation events should be applicable to diagnostic evaluations of clinical samples, to preclinical assessments of new epigenetic biomarkers, and to quantitative analyses of epigenetic biology. PMID:19684580

  2. The methylating agent streptozotocin induces persistent telomere dysfunction in mammalian cells.

    PubMed

    Paviolo, Natalia S; Santiñaque, Federico F; Castrogiovanni, Daniel C; Folle, Gustavo A; Bolzán, Alejandro D

    2015-12-01

    We analyzed chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the methylating agent and antineoplastic/diabetogenic drug streptozotocin (STZ), to test whether it induces long-term telomere instability (by chromosome end loss and/or telomere dysfunction). Rat cells (ADIPO-P2 cell line, derived from Sprague-Dawley rat adipose cells) were treated with a single concentration of STZ (2mM). Chromosomal aberrations were analyzed 18h, 10 days, and 15 days after treatment, using PNA-FISH with a pan-telomeric probe [Cy3-(CCCTAA)3] to detect (TTAGGG)n repeats. Cytogenetic analysis revealed a higher frequency of chromosomal aberrations in STZ-exposed cultures vs. untreated cultures at each time point analyzed. The yield of induced aberrations was very similar at each time point. Induction of aberrations not involving telomere dysfunction was only observed 18h and 15 days after treatment, whereas induction of telomere dysfunction-related aberrations by STZ (mainly in the form of telomere FISH signal loss and duplications, most of them chromatid-type aberrations) was observed at each time point. Our results show that STZ induces persistent telomere instability in mammalian cells, cytogenetically manifested as telomere dysfunction-related chromosomal aberrations. Neither telomere length nor telomerase activity is related to the telomere dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Increased DNA methylation of Dnmt3b targets impairs leukemogenesis.

    PubMed

    Schulze, Isabell; Rohde, Christian; Scheller-Wendorff, Marina; Bäumer, Nicole; Krause, Annika; Herbst, Friederike; Riemke, Pia; Hebestreit, Katja; Tschanter, Petra; Lin, Qiong; Linhart, Heinz; Godley, Lucy A; Glimm, Hanno; Dugas, Martin; Wagner, Wolfgang; Berdel, Wolfgang E; Rosenbauer, Frank; Müller-Tidow, Carsten

    2016-03-24

    The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.

  4. DNA methylation and hydroxymethylation in hematologic differentiation and transformation

    PubMed Central

    Ko, Myunggon; An, Jungeun; Rao, Anjana

    2015-01-01

    Maintenance of the balance of DNA methylation and demethylation is fundamental for normal cellular development and function. Members of the Ten-Eleven-Translocation (TET) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that catalyze sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent oxidized derivatives in DNA. In addition to their roles as intermediates in DNA demethylation, these oxidized methylcytosines are novel epigenetic modifications of DNA. DNA methylation and hydroxymethylation profiles are markedly disrupted in a wide range of cancers but how these changes are related to the pathogenesis of cancers is still ambiguous. In this review, we discuss the current understanding of TET protein functions in normal and malignant hematopoietic development and the ongoing questions to be resolved. PMID:26595486

  5. DNA methylation remodeling in vitro and in vivo

    PubMed Central

    Clark, Amander T

    2015-01-01

    In mammals, global DNA demethylation in vivo occurs in the pre-implantation embryo and in primordial germ cells (PGCs) where it is hypothesized to create a blank slate or “tabula rasa” upon which new DNA methylation patterns are written. However, global DNA demethylation in vivo is far from complete with a small number of loci protected from demethylation. Failure to demethylate, or overt demethylation results in compromised differentiation. Recent work has shown that reversion of primed human pluripotent stem cells to the naïve state leads to unbridled DNA demethylation which has unknown consequences on the quality differentiated cells created in vitro. Taken together understanding DNA methylation remodeling is critical for understanding the epigenetic foundations of life, and the quality of stem cells for regenerative medicine. PMID:26451496

  6. DNA methylation dynamics in plants and mammals: overview of regulation and dysregulation.

    PubMed

    Elhamamsy, Amr Rafat

    2016-07-01

    DNA methylation is a major epigenetic marking mechanism regulating various biological functions in mammals and plant. The crucial role of DNA methylation has been observed in cellular differentiation, embryogenesis, genomic imprinting and X-chromosome inactivation. Furthermore, DNA methylation takes part in disease susceptibility, responses to environmental stimuli and the biodiversity of natural populations. In plant, different types of environmental stress have demonstrated the ability to alter the archetype of DNA methylation through the genome, change gene expression and confer a mechanism of adaptation. DNA methylation dynamics are regulated by three processes de novo DNA methylation, methylation maintenance and DNA demethylation. These processes have their similarities and differences between mammals and plants. Furthermore, the dysregulation of DNA methylation dynamics represents one of the primary molecular mechanisms of developing diseases in mammals. This review discusses the regulation and dysregulation of DNA methylation in plants and mammals. Copyright © 2016 John Wiley & Sons, Ltd.

  7. DNA Methylation and BMI: Investigating Identified Methylation Sites at HIF3A in a Causal Framework

    PubMed Central

    Richmond, Rebecca C.; Ward, Mary E.; Fraser, Abigail; Lyttleton, Oliver; McArdle, Wendy L.; Ring, Susan M.; Gaunt, Tom R.; Lawlor, Debbie A.; Davey Smith, George; Relton, Caroline L.

    2016-01-01

    Multiple differentially methylated sites and regions associated with adiposity have now been identified in large-scale cross-sectional studies. We tested for replication of associations between previously identified CpG sites at HIF3A and adiposity in ∼1,000 mother-offspring pairs from the Avon Longitudinal Study of Parents and Children (ALSPAC). Availability of methylation and adiposity measures at multiple time points, as well as genetic data, allowed us to assess the temporal associations between adiposity and methylation and to make inferences regarding causality and directionality. Overall, our results were discordant with those expected if HIF3A methylation has a causal effect on BMI and provided more evidence for causality in the reverse direction (i.e., an effect of BMI on HIF3A methylation). These results are based on robust evidence from longitudinal analyses and were also partially supported by Mendelian randomization analysis, although this latter analysis was underpowered to detect a causal effect of BMI on HIF3A methylation. Our results also highlight an apparent long-lasting intergenerational influence of maternal BMI on offspring methylation at this locus, which may confound associations between own adiposity and HIF3A methylation. Further work is required to replicate and uncover the mechanisms underlying the direct and intergenerational effect of adiposity on DNA methylation. PMID:26861784

  8. 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.

  9. Cord Blood DNA Methylation Biomarkers for Predicting Neurodevelopmental Outcomes

    PubMed Central

    Hodyl, Nicolette A.; Roberts, Claire T.; Bianco-Miotto, Tina

    2016-01-01

    Adverse environmental exposures in pregnancy can significantly alter the development of the fetus resulting in impaired child neurodevelopment. Such exposures can lead to epigenetic alterations like DNA methylation, which may be a marker of poor cognitive, motor and behavioral outcomes in the infant. Here we review studies that have assessed DNA methylation in cord blood following maternal exposures that may impact neurodevelopment of the child. We also highlight some key studies to illustrate the potential for DNA methylation to successfully identify infants at risk for poor outcomes. While the current evidence is limited, in that observations to date are largely correlational, in time and with larger cohorts analyzed and longer term follow-up completed, we may be able to develop epigenetic biomarkers that not only indicate adverse early life exposures but can also be used to identify individuals likely to be at an increased risk of impaired neurodevelopment even in the absence of detailed information regarding prenatal environment. PMID:27918480

  10. Associative DNA methylation changes in children with prenatal alcohol exposure.

    PubMed

    Laufer, Benjamin I; Kapalanga, Joachim; Castellani, Christina A; Diehl, Eric J; Yan, Liying; Singh, Shiva M

    2015-01-01

    Prenatal alcohol exposure (PAE) can cause fetal alcohol spectrum disorders (FASD). Previously, we assessed PAE in brain tissue from mouse models, however whether these changes are present in humans remains unknown. In this report, we show some identical changes in DNA methylation in the buccal swabs of six children with FASD using the 450K array. The changes occur in genes related to protocadherins, glutamatergic synapses, and hippo signaling. The results were found to be similar in another heterogeneous replication group of six FASD children. The replicated results suggest that children born with FASD have unique DNA methylation defects that can be influenced by sex and medication exposure. Ultimately, with future clinical development, assessment of DNA methylation from buccal swabs can provide a novel strategy for the diagnosis of FASD.

  11. DNA Methylation in Whole Blood: Uses and Challenges.

    PubMed

    Houseman, E Andres; Kim, Stephanie; Kelsey, Karl T; Wiencke, John K

    2015-06-01

    Due to its convenience, the blood is commonly used in epigenomic studies, but its heterogeneous nature leads to interpretation difficulties, given the now widely recognized potential for confounding by cell composition effects. Many recent publications have reported significant associations between DNA methylation and a variety of health conditions or exposures. In this review, we summarize many of these recent publications, highlighting the findings in the context of potential cell composition effects, particularly findings that are indicative of immune response or inflammation. While there is substantial evidence for confounding by cell composition, there is nevertheless also evidence for differential DNA methylation suggestive of processes that are not cell mediated. We conclude that important biological insights still may be gained from studying DNA methylation in whole blood, either by investigating the cell composition effects themselves or processes that demonstrate associations even after adjusting for cell composition effects.

  12. 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. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. DNA methylation signatures identify biologically distinct thyroid cancer subtypes.

    PubMed

    Rodríguez-Rodero, Sandra; Fernández, Agustín F; Fernández-Morera, Juan Luís; Castro-Santos, Patricia; Bayon, Gustavo F; Ferrero, Cecilia; Urdinguio, Rocio G; Gonzalez-Marquez, Rocío; Suarez, Carlos; Fernández-Vega, Iván; Fresno Forcelledo, Manuel Florentino; Martínez-Camblor, Pablo; Mancikova, Veronika; Castelblanco, Esmeralda; Perez, Marco; Marrón, Pablo Isidro; Mendiola, Marta; Hardisson, David; Santisteban, Pilar; Riesco-Eizaguirre, Garcilaso; Matías-Guiu, Xavier; Carnero, Amancio; Robledo, Mercedes; Delgado-Álvarez, Elías; Menéndez-Torre, Edelmiro; Fraga, Mario F

    2013-07-01

    The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

  14. The DNA methylation profile of activated human natural killer cells.

    PubMed

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-03

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

  15. The DNA methylation profile of activated human natural killer cells

    PubMed Central

    Wiencke, John. K.; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A.; Siegel, Derick; Houseman, E. Andres; Kelsey, Karl T.

    2016-01-01

    ABSTRACT 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

  16. The further development of a mammalian DNA alkaline unwinding bioassay with potential application to hazard identification for contaminants from environmental samples

    SciTech Connect

    Daniel, F.B.; Chang, L.W.; Schenck, K.M.; DeAngelo, A.B.; Skelly, M.F. )

    1989-10-01

    Recently, we have detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells. In this paper we present further development of this assay, including: (1) studies on the relationship between DNA adducts and DNA strand breaks; (2) evaluation of the role of cytotoxicity in DNA strand breaks; and (3) application of the DAUA to cell preparations from the liver of mice dosed with methylating agents. The level of DNA adducts produced in human CCRF-CEM cells by treatment with benzo(a)pyrene diol-epoxide (BPDE), N-acetoxy-2-acetyl aminofluorene (AAAF), and various methylating agents was linear with concentration over several orders of magnitude. Likewise, the level of strand breaks increased with the concentration over the same dose range. The strand breaks/adduct ratio ranged from 0.05 for the methyl adducts to 0.001 for the BPDE adducts. Using these values and the inherent sensitivity of the DAUA (circa 100 to 1000 breaks/cell) the ability of the assay to detect DNA damage induced by various classes of chemical carcinogens can be calculated. The DAUA appears to be useful for assessing the relative potency of various environmental genotoxic effects on mammalian cells. In addition, it can be conducted on cells isolated from target organs of whole animals.

  17. DNA methylation age of human tissues and cell types

    PubMed Central

    2013-01-01

    Background It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure. Results I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance. Conclusions I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research. PMID:24138928

  18. 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.

  19. 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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. 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

  1. DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood.

    PubMed

    Yang, Rongxi; Pfütze, Katrin; Zucknick, Manuela; Sutter, Christian; Wappenschmidt, Barbara; Marme, Frederik; Qu, Bin; Cuk, Katarina; Engel, Christoph; Schott, Sarah; Schneeweiss, Andreas; Brenner, Hermann; Claus, Rainer; Plass, Christoph; Bugert, Peter; Hoth, Markus; Sohn, Christof; Schmutzler, Rita; Bartram, Claus R; Burwinkel, Barbara

    2015-04-15

    Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC. © 2014 UICC.

  2. Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

    PubMed Central

    Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

    2014-01-01

    Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

  3. DNA methylation by N-methyl-N-nitrosourea: methylation pattern changes in single- and double-stranded DNA, and in DNA with mismatched or bulged guanines.

    PubMed Central

    Wurdeman, R L; Douskey, M C; Gold, B

    1993-01-01

    The detection of abnormal DNA base pairing arrangements and conformations is chemically probed in synthetic 32P-end-labeled deoxyribonucleotide oligomers using N-methyl-N-nitrosourea (MNU) and 2,12,-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1 -[17],2,11,13,15 pentaene-Ni (II) (Ni-complex) with KHSO5. The DNA targets studied are single-stranded (s-s) DNA, double-stranded (d-s) DNA, d-s DNA with G-G, G-A and G-T mismatches, d-s DNA with a single bulged G and d-s DNA with two bulged G's. The effect of the non-Watson--Crick structures on the formation of N7-methylguanine (N7-MeG) by MNU and the oxidation of G by Ni-complex is reported along with the Tm's and circular dichroism spectra of the different duplex oligomers. The results for MNU and Ni-complex show that the qualitative and quantitative character of the cleavage patterns at a G3 run change with the nature of the abnormal base pairing motif. Based on the DNA substrates studied, the results indicate that a combination of reagents which report electronic and steric perturbations can be a useful approach to monitor DNA mismatches and bulges. Images PMID:8177747

  4. Twin birth changes DNA methylation of subsequent siblings.

    PubMed

    Li, Shuai; Kim, Eunae; Wong, Ee Ming; Joo, Ji-Hoon Eric; Nguyen, Tuong L; Stone, Jennifer; Song, Yun-Mi; Flander, Louisa B; Saffery, Richard; Giles, Graham G; Southey, Melissa C; Sung, Joohon; Hopper, John L

    2017-08-16

    We asked if twin birth influences the DNA methylation of subsequent siblings. We measured whole blood methylation using the HumanMethylation450 array for siblings from two twin and family studies in Australia and Korea. We compared the means and correlations in methylation between pairs of siblings born before a twin birth (BT siblings), born on either side of a twin birth (B/AT pairs) and born after a twin birth (AT siblings). For the genome-wide average DNA methylation, the correlation for AT pairs (rAT) was larger than the correlation for BT pairs (rBT) in both studies, and from the meta-analysis, rAT = 0.46 (95% CI: 0.26, 0.63) and rBT = -0.003 (95% CI: -0.30, 0.29) (P = 0.02). B/AT pairs were not correlated (from the meta-analysis rBAT = 0.08; 95% CI: -0.31, 0.45). Similar results were found for the average methylation of several genomic regions, e.g., CpG shelf and gene body. BT and AT pairs were differentially correlated in methylation for 15 probes (all P < 10(-7)), and the top 152 differentially correlated probes (at P < 10(-4)) were enriched in cell signalling and breast cancer regulation pathways. Our observations are consistent with a twin birth changing the intrauterine environment such that siblings both born after a twin birth are correlated in DNA methylation.

  5. Maintenance of DNA methylation: Dnmt3b joins the dance.

    PubMed

    Walton, Emma L; Francastel, Claire; Velasco, Guillaume

    2011-11-01

    DNA methylation mostly occurs within the context of CpG dinucleotides and is essential for embryonic development and gene repression. It is generally accepted that DNA methyltransferases carry out specific and non-overlapping functions, Dnmt3a and Dnmt3b being responsible for the establishment of methylation around the time of implantation and Dnmt1 ensuring that methylation is faithfully copied to daughter cells via what has come to be known as "maintenance methylation." This longstanding view has been challenged over the years with the observation that Dnmt1 alone is incapable of perfect maintenance methylation. A new model is emerging that takes into account a contribution of the de novo enzymes Dnmt3a and Dnmt3b in the maintenance of the DNA methylation. We recently showed that certain germ line genes are specific targets of Dnmt3b, and that Dnmt3b remains bound to their promoter regions in somatic cells via interaction with the transcriptional repressor E2F6. It is tempting to consider an ongoing role for Dnmt3b in the methylation of germ line genes in somatic cells. We propose here observations in support of the hypothesis that the maintenance of methylation and subsequent silencing of a handful of germ line genes requires Dnmt3b but not Dnmt1. In addition to suggesting a new role for Dnmt3b in the protection of somatic cells against the promiscuous expression of the germ line program, these observations are of particular interest in the field of carcinogenesis, given that the expression of catalytically inactive Dnmt3b isoforms and aberrant expression of germ line genes are commonly observed in cancer cells.

  6. DNA methylation, ageing and the influence of early life nutrition.

    PubMed

    Lillycrop, Karen A; Hoile, Samuel P; Grenfell, Leonie; Burdge, Graham C

    2014-08-01

    It is well established that genotype plays an important role in the ageing process. However, recent studies have suggested that epigenetic mechanisms may also influence the onset of ageing-associated diseases and longevity. Epigenetics is defined as processes that induce heritable changes in gene expression without a change in the DNA nucleotide sequence. The major epigenetic mechanisms are DNA methylation, histone modification and non-coding RNA. Such processes are involved in the regulation of tissue-specific gene expression, cell differentiation and genomic imprinting. However, epigenetic dysregulation is frequently seen with ageing. Relatively little is known about the factors that initiate such changes. However, there is emerging evidence that the early life environment, in particular nutrition, in early life can induce long-term changes in DNA methylation resulting in an altered susceptibility to a range of ageing-associated diseases. In this review, we will focus on the changes in DNA methylation that occur during ageing; their role in the ageing process and how early life nutrition can modulate DNA methylation and influence longevity. Understanding the mechanisms by which diet in early life can influence the epigenome will be crucial for the development of preventative and intervention strategies to increase well-being in later life.

  7. Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

    PubMed

    Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

    2017-08-17

    DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments.

    PubMed

    Wu, Feinan; Olson, Brennan G; Yao, Jie

    2016-01-27

    The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

  9. Analysis of DNA Methylation in Plasma for Monitoring Hepatocarcinogenesis

    PubMed Central

    Huang, Wenqing; Li, Tong; Yang, Wenli; Chai, Xinjuan; Chen, Kefei; Wei, Ling; Duan, Shuwang

    2015-01-01

    Aim: To explore whether the aberrant DNA methylation status in plasma could be used as a biomarker for hepatocellular carcinoma (HCC) screening among high-risk individuals. Methods: The promoter methylation status of ELF, RASSF1A, p16, and GSTP1 was investigated by methylation-specific polymerase chain reaction (PCR) in 34 paired HCC and nontumor liver tissue from HCC patients and 10 tissues from patients with liver cirrhosis (LC). Plasma samples from 31 HCC patients, 10 LC patients as well as 7 patients with benign hepatic conditions were also collected and characterized using the same method. Results: Among liver specimens, HCC tissues displayed a significantly higher methylation frequency of each gene compared with nontumor tissue (p<0.05). Moreover, the frequency was much higher in tumor tissues than in nontumor tissue, when the data from two or three genes were combined (p=0.001 and p<0.001, respectively). Among plasma samples, either the frequency of at least one methylated gene (p<0.001) or the average number of methylated genes (p<0.05) demonstrated a stepwise increase in patients with benign lesions, LC, and HCC. Furthermore, when positive results, that is, plasma methylation status of at least one gene were combined with the elevated AFP400 level (serum alpha-fetoprotein [AFP] level at a cutoff of 400 ng/mL), the diagnostic sensitivity of HCC could increase to 93.55%. Conclusions: These results suggested that the methylation of tumor suppressor genes may participate in the development and progression of HCC. Additionally, it may be useful to combine the plasma DNA methylation status of a panel of gene markers and the serum AFP for HCC screening. PMID:25923138

  10. Z-DNA-forming sequences generate large-scale deletions in mammalian cells.

    PubMed

    Wang, Guliang; Christensen, Laura A; Vasquez, Karen M

    2006-02-21

    Spontaneous chromosomal breakages frequently occur at genomic hot spots in the absence of DNA damage and can result in translocation-related human disease. Chromosomal breakpoints are often mapped near purine-pyrimidine Z-DNA-forming sequences in human tumors. However, it is not known whether Z-DNA plays a role in the generation of these chromosomal breakages. Here, we show that Z-DNA-forming sequences induce high levels of genetic instability in both bacterial and mammalian cells. In mammalian cells, the Z-DNA-forming sequences induce double-strand breaks nearby, resulting in large-scale deletions in 95% of the mutants. These Z-