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1
Meng, Wei. "DNA Mutation/Methylation Screening Method for Colon Cancer Screening." Cleveland State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=csu1290364705.
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Trimarchi, Michael Paul Trimarchi. "Identification of endometrial cancer methylation features using a combined methylation analysis method." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461302615.
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ZERILLI, FRANCESCO. "Development of an isothermal method for the detection of DNA hypermethylation." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/7481.
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This thesis relates to the development of an isothermal method for the detection of DNA hypermethylation. The function of DNA methylation is to silence specific areas of the human genome and its maintenance is necessary to regulate key functions of the cell and of the organism. Aberrant hypermethylation of the promoter regions of several tumor suppressor genes can lead to formation of tumors and its detection, even at very early stages, can be an useful and powerful instrument for the diagnosis of the cancer, the determination of the prognosis and also to gauge the therapy. In this work we developed a molecular assay (MS-LAMP), based on the Loop-mediated Isothermal Amplification, for the rapid and specific amplification and detection of the methylated DNA in three human gene promoters after a sodium bisulfite treatment. The assay, showed PCR-level performance in terms of specificity, sensitivity and selectivity both on synthetic genes and fully methylated and unmethylated genomic DNA. The method was also validated on DNA extracted from clinical samples in comparison with a reference technique. Finally the notable specificity of LAMP was also exploited for the development of a triplex reaction, tested successfully on control DNA and clinical samples, with two possible signal detection approaches.
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Hu, Ke. "METHODS AND ANALYSES IN THE STUDY OF HUMAN DNA METHYLATION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1522760441838452.
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Van, Heerden Chrisna. "Establishing a method for measuring the DNA methylation status of specific human genes / Chrisna van Heerden." Thesis, North-West University, 2006. http://hdl.handle.net/10394/1443.
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Antunes, Joana AP. "The Study of Tissue-Specific DNA Methylation as a Method for the Epigenetic Discrimination of Forensic Samples." FIU Digital Commons, 2017. https://digitalcommons.fiu.edu/etd/3676.
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In forensic sciences, the serological methods used to determine which body fluid was collected from the crime scene are merely presumptive or labor intensive since they rely on protein detection or on microscopic identification of cells. Given that certain forensic cases may need the precise identification of a body fluid to determine criminal contact, such is the example of a suspected sexual assault of a minor; certainty in the body fluid of origin may depict a precise picture of the events. The identification of loci that show differences in methylation according to the tissue of origin can aid forensic analysts in determining the origin of a DNA sample. The process of DNA methylation occurs naturally in the genome of living organisms and consists in the presence of a methyl group on the carbon 5 of a cytosine, which is typically followed by a guanine (CpG). Analyzing patterns of DNA methylation in body fluids collected from a crime scene is preferential to the analysis of proteins or mRNA since the same extracted DNA used for STR typing can be used for DNA methylation analysis. We have validated and identified loci able to discriminate blood, saliva, semen and vaginal epithelia. In the current study, we have also established the minimum amount of DNA able to provide reliable results using methodologies such as pyrosequencing and high-resolution melt (HRM) analysis for the different markers identified. Lastly, we performed an alternative bioinformatic analysis of data collected using an array that studied methylation in over 450,000 individual cytosines on the human genome. We were able to sort the locations that showed potentially higher methylation differences between body fluids and investigated over 100 of them using HRM analysis. The results of that study, allowed the identification of three new loci able to distinguish blood and two new loci able to distinguish saliva and vaginal epithelia, respectively. The use of DNA methylation patterns to aid forensic investigations started with a publication in 2010, therefore each small contribution such as this work may, similarly to what occured in the biochemistry field, result in the discovery of a method able to put the technology in the hands of forensic analysts.
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Capparuccini, Maria. "Inferential Methods for High-Throughput Methylation Data." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/156.
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The role of abnormal DNA methylation in the progression of disease is a growing area of research that relies upon the establishment of sound statistical methods. The common method for declaring there is differential methylation between two groups at a given CpG site, as summarized by the difference between proportions methylated db=b1-b2, has been through use of a Filtered Two Sample t-test, using the recommended filter of 0.17 (Bibikova et al., 2006b). In this dissertation, we performed a re-analysis of the data used in recommending the threshold by fitting a mixed-effects ANOVA model. It was determined that the 0.17 filter is not accurate and conjectured that application of a Filtered Two Sample t-test likely leads to loss of power. Further, the Two Sample t-test assumes that data arise from an underlying distribution encompassing the entire real number line, whereas b1 and b2 are constrained on the interval . Additionally, the imposition of a filter at a level signifying the minimum level of detectable difference to a Two Sample t-test likely reduces power for smaller but truly differentially methylated CpG sites. Therefore, we compared the Two Sample t-test and the Filtered Two Sample t-test, which are widely used but largely untested with respect to their performance, to three proposed methods. These three proposed methods are a Beta distribution test, a Likelihood ratio test, and a Bootstrap test, where each was designed to address distributional concerns present in the current testing methods. It was ultimately shown through simulations comparing Type I and Type II error rates that the (unfiltered) Two Sample t-test and the Beta distribution test performed comparatively well.
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Kretzmer, Helene. "Methods for DNA Methylation Sequencing Analysis and their Application on Cancer Data." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-203416.
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The fundamental subject of this thesis is the development of tools for the analysis of DNA methylation data as well as their application on bisulfite sequencing data comprising a large number of samples. DNA methylation is one of the major epigenetic modifications. It affects the cytosines of the DNA and is essential for the normal development of cells and tissues. Unusual alterations are associated with a variety of diseases and, specially, in cancergeneous tissues global changes in the DNA methylation level have been detected. To sequence DNA methylation on single nucleotide resolution, the sequences are treated with sodium bisulfite before sequencing, whereby unmethylated cytosines are represented as thymines. Thus, specialized techniques are required to process and analyze these kind of data.
Here, the bisulfite analysis toolkit BAT is introduced, that is designed to facilitate an quick analysis of bisulfite treated DNA methylation sequencing data. It covers all steps of processing raw sequencing data up to calling of differential DNA methylation. At the begin of analysis, sodium bisulfite treated sequence data are aligned and DNA methylation rates for each covered cytosine in the reference genome are called. Subsequently, BAT integrates annotation data and performs basic analysis, i. e., methylation rate distribution plots and hierarchical clustering of the samples. In addition, calling of differentially methylated regions is performed and statistics of called regions are automatically created. Finally, DNA methylation and gene expression data integration is covered by the calculation of correlating regions.
Secondly, a novel algorithm, metilene, for the calculation of differentially methylated regions (DMRs) between two groups of samples is introduced. Existing methods are limited in terms of detection sensitivity as well as time and memory consumption. Our approach is based on a circular binary segmentation, using a scoring function to detect sub-regions that show a stronger difference between the mean methylation levels of two groups than the surrounding background. These sub-regions are tested using a two-dimensional Kolmogorov Smirnov test (2D-KS test) [Fasano 1987] for significant differences taking all samples of each group into account. The use of the non-parametric 2D-KS test allows to avoid assumptions about a background distribution. Furthermore, the two dimensions of the problem, i. e., (i) the detection of a region, such that (ii) the methylation rates of the samples in the groups are significantly different, are taken into account in a single test. The algorithm calls DMRs in sufficiently short time on single sample comparisons as well as on about 50 samples per group. Furthermore, it works on whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) data and is able so estimate missing data points from the methylation rates of other samples in the group. Benchmarks on simulated and real data sets show that metilene outperforms other existing methods and is especially suitable for noisy datasets often found for example in cancer analysis.
In the framework of this thesis, the previously introduced methods and algorithms are used to analyze a WGBS dataset of two different subtypes of germinal-center derived B-cell lymphomas and healthy controls. In both lymphoma subgroups genome-wide hypomethylation was found, with an exception for a specific type of promoter regions, i. e., poised promoters, that were frequently found to be hypermethylated. Using the previously presented algorithm, DMRs were called between the three entities. A strong enrichment of DMRs immediately downstream of the transcription start site was observed, indicating the regulatory relevance of this regions. The integration of gene expression data of the same samples, revealed that a considerable amount of the DMRs showed significant correlation between gene expression and DNA methylation. Finally, transcription factor binding sites and mutation data were combined with the methylation and expression data analysis. This identified strongly altered signaling pathways and cancer subtype specific genes. Furthermore, the data integration indicates that mutations and DNA methylation changes may act complementary to another.
Finally, findings from the lymphoma study regarding the hypermethylation of poised promoters in cancer were extended to a huge data set comprising a variety of cancers. We could show that the relation of DNA methylation at a small set of frequently poised regions with respect to the background methylation level is sufficient to classify almost all samples based on DNA methylation data from 450k BeadChips into cancer or non-cancer probes. In addition, we found that the increase in methylation co-occurs with upregulated gene expression of several poised promoter regulated genes in almost all fresh cancer samples, implying a de-poising of poised regions. This upregulated gene expression is in contrast to the silencing of those genes in cancer cell lines, indicating that the upregulated gene expression might be a temporary status and possibly contributes to cancerogenesis.
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Han, Chenggong. "Statistical models and computational methods for studying DNA differential methylation and 3D genome structure." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595417277891892.
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Jackel, Jamie Nicole. "GEMINIVIRUSES AS MODELS TO STUDY THE ESTABLISHMENT AND MAINTENANCE OF DNA METHYLATION." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1367494030.
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Lutsik, Pavlo [Verfasser], and Jörn [Akademischer Betreuer] Walter. "Bioinformatic tools and computational methods for mapping DNA methylation variability / Pavlo Lutsik ; Betreuer: Jörn Walter." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2017. http://d-nb.info/1128148501/34.
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Lutsik, Pavlo Verfasser], and Jörn [Akademischer Betreuer] [Walter. "Bioinformatic tools and computational methods for mapping DNA methylation variability / Pavlo Lutsik ; Betreuer: Jörn Walter." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:291-scidok-67884.
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Yip, Wai-Ki. "Statistical Methods for Analyzing DNA Methylation Data and Subpopulation Analysis of Continuous, Binary and Count Data for Clinical Trials." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226106.
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DNA methylation may represent an important contributor to the missing heritability described in complex trait genetics. However, technology to measure DNA methylation has
outpaced statistical methods for analysis. Novel methodologies are required to accommodate this growing volume of DNA methylation data. In this dissertation, I propose two
novel methods to analyze DNA methylation data: (1) a new statistic based on spatial location information of DNA methylation sites to detect differentially methylated regions
in the genome in case and control studies; and (2) a principal component approach for the detection of unknown substructure in DNA methylation data. For each method, I review existing ones and demonstrate the efficacy of my proposed method using simulation and data application.
Medical research is increasingly focused on personalizing the care of patients. A better understanding of the interaction between treatment and patient specific prognostic factors
will enable practitioners to expand the availability of tailored therapies improving patient outcomes. The Subpopulation Treatment Effect Pattern Plot (STEPP) approach
was developed to allow researchers to investigate the heterogeneity of treatment effects on survival outcomes across increasing values of a continuously measured covariate, such
as biomarker measurement. I extend the STEPP approach to continuous, binary and count outcomes which can be easily modeled with generalized linear models (GLM). The statistical significance of any observed heterogeneity of treatment effect is assessed using permutation tests. The method is implemented in the R software package (stepp) and is
available in R version 3.1.1. The efficacy of my STEPP extension is demonstrated by using simulation and data application.
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Kretzmer, Helene [Verfasser], Peter F. [Gutachter] Stadler, and Martin [Gutachter] Vingron. "Methods for DNA Methylation Sequencing Analysis and their Application on Cancer Data / Helene Kretzmer ; Gutachter: Peter F. Stadler, Martin Vingron." Leipzig : Universitätsbibliothek Leipzig, 2016. http://d-nb.info/1240481691/34.
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Rohde, Christian [Verfasser]. "Development of experimental and bioinformatics methods for high resolution DNA methylation analysis of gene promoters on human chromosome 21 / Christian Rohde." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2009. http://d-nb.info/1034996371/34.
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Manser, Paul. "Methods for Integrative Analysis of Genomic Data." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3638.
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In recent years, the development of new genomic technologies has allowed for the investigation of many regulatory epigenetic marks besides expression levels, on a genome-wide scale. As the price for these technologies continues to decrease, study sizes will not only increase, but several different assays are beginning to be used for the same samples. It is therefore desirable to develop statistical methods to integrate multiple data types that can handle the increased computational burden of incorporating large data sets. Furthermore, it is important to develop sound quality control and normalization methods as technical errors can compound when integrating multiple genomic assays. DNA methylation is a commonly studied epigenetic mark, and the Infinium HumanMethylation450 BeadChip has become a popular microarray that provides genome-wide coverage and is affordable enough to scale to larger study sizes. It employs a complex array design that has complicated efforts to develop normalization methods. We propose a novel normalization method that uses a set of stable methylation sites from housekeeping genes as empirical controls to fit a local regression hypersurface to signal intensities. We demonstrate that our method performs favorably compared to other popular methods for the array. We also discuss an approach to estimating cell-type admixtures, which is a frequent biological confound in these studies. For data integration we propose a gene-centric procedure that uses canonical correlation and subsequent permutation testing to examine correlation or other measures of association and co-localization of epigenetic marks on the genome. Specifically, a likelihood ratio test for general association between data modalities is performed after an initial dimension reduction step. Canonical scores are then regressed against covariates of interest using linear mixed effects models. Lastly, permutation testing is performed on weighted correlation matrices to test for co-localization of relationships to physical locations in the genome. We demonstrate these methods on a set of developmental brain samples from the BrainSpan consortium and find substantial relationships between DNA methylation, gene expression, and alternative promoter usage primarily in genes related to axon guidance. We perform a second integrative analysis on another set of brain samples from the Stanley Medical Research Institute.
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Moreland, Blythe S. "Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu153452875946939.
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Joulie, Michaël. "Recherche de nouvelles protéines humaines se liant à l'ADN méthylé." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112157/document.
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L'épigénétique est un composant essentiel du fonctionnement des génomes eucaryotes. Les divers phénomènes épigénétiques modifient l’état chromatinien et participent à la plasticité du génome, mais aussi au maintien de son identité fonctionnelle à travers les générations cellulaires. Parmi ces processus, la méthylation de l’ADN joue un rôle fondamental dans la régulation de l’expression des gènes.Chez les mammifères, la méthylation de l'ADN est associée à la répression transcriptionnelle, et elle remplit au moins trois fonctions essentielles. Premièrement, elle permet de réprimer les séquences répétées afin de préserver l’intégrité du génome. Deuxièmement, la méthylation contrôle l’expression des gènes soumis à l’empreinte parentale, qui sont des régulateurs cruciaux du développement et de la vie adulte. Enfin, la méthylation permet de réprimer certains gènes tissu-spécifiques dans les organes où ils doivent être silencieux. En plus de ces rôles physiologiques, la méthylation est liée au cancer. En effet, des patrons de méthylation anormaux sont fréquemment observés dans les cellules tumorales, et ces anomalies participent à la transformation cellulaire par plusieurs mécanismes.La méthylation exerce ces effets par l'intermédiaire de protéines dédiées, qui reconnaissent spécifiquement l'ADN méthylé et contrôlent la transcription en modulant la chromatine. Trois familles de protéines liant l'ADN méthylé sont connues chez les mammifères, et elles totalisent entre elles neuf membres. De nombreux arguments suggèrent que cette liste est encore incomplète, et que des protéines humaines liant l'ADN méthylé restent à découvrir. Dans cette optique, nous avons opté pour deux types d’approches distinctes, une approche basée sur la littérature et une approche génétique. L’étude des protéines candidates ne nous a pas permis d’identifier de nouvelles protéines liant l’ADN méthylé et l’approche génétique par phage display a révélé deux protéines intéressantes, CHD3 et HMGB1 qui doivent désormais être validées par des approches in vivo et in vitro.Par ailleurs, nous avons entrepris l’étude de la régulation des éléments répétés par la protéine Zbtb4 chez la souris. Les expériences préliminaires indiquent une possible régulation des satellites mineurs par Zbtb4. Le rôle de cette régulation sera, par la suite, approfondiEpigenetic phenomena are key contributors to the function of eukaryotic genomes. These processes act on chromatin, and they are used to render the genome dynamic, but also stable throughout successive rounds of cell division. Among epigenetic processes, DNA methylation is especially well known for its role in the regulation of gene expression.In mammals, DNA methylation is strongly correlated with transcriptional repression, and fulfills at least three essential roles. First, it maintains repeated sequences transcriptionally silenced, thus ensuring the stability of the genome. Second, it is responsible for the proper regulation of parentally imprinted genes, which are crucial regulators of embryonic development and adult life. Finally, DNA methylation ensures that some tissue-specific genes are kept inactive in the organs in which they should be repressed. Besides these roles in the physiology of normal cells, DNA methylation has strong links to cancer. Indeed the pattern of DNA methylation on the genome is frequently altered in cancer cells, and these anomalies contribute to transformation by several mechanisms.DNA methylation does not control transcription directly, but instead acts via a set of dedicated proteins that specifically recognize methylated DNA and repress transcription by acting at the chromatin level. At present, three families of such proteins, totalling 9 members altogether, are known in humans. However, several lines of evidence suggest that the list is not exhaustive, and that other human proteins that bind methylated DNA remain to be found. This was the goal of the current project.To this end, we opted for two distinct types approaches, an approach based on literature and a genetic approach. The study of candidate proteins does not allow us to identify new methylated DNA binding proteins and the genetic approach by phage display revealed two proteins of interest, HMGB1 and CHD3 that must now be validated by in vivo and in vitro approaches.Furthermore, we studied the regulation of DNA repeats by Zbtb4 in mice. Preliminary results show a regulation of minor satellites by Zbtb4. The role of this regulation will be analyse further in the future
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Marchioretto, Lisa. "Development and validation of methods for genome-wide epigenetic analyses of human myogenic cells." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3423853.
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Epigenetics is subjected to a pressing attention from the scientific community, because of its potential to explain the mechanisms of gene activation or repression. In this thesis I present a discovery-driven project aimed to the investigation of the epigenetic role in human myogenesis (and in particular the differentiation of myoblasts in myotubes).
Studying epigenetics still presents significant hurdles, both experimental and computational. Therefore my first task was the establishment of robust protocols for investigating the role of epigenetics players during skeletal muscle differentiation. In particular, I focused on setting up the tools for studying DNA methylation and protein-DNA interactions, through bisulfite sequencing and chromatin immunoprecipitation. In this thesis, together with the description of the protocols that I developed, I report the first results that were obtained on myogenic cells.
DNA methylation was investigated with bisulfite treatment of the DNA coupled with next generation sequencing. A novel method for studying the whole methylome was conceived and applied on one myoblast sample using SOLiD 5500xl platform giving reliable results.
However, since the cost of methylome sequencing is still very high, instead of producing data for the whole methylome, I decided to focus on selected regions that could be relevant in methylation studies. For this reason I used the SureSelect MethylSeq Target Enrichment kit (Agilent) that effectively captures more than 2,700,000 CpG sites in the human genome.
We identified less than 600 differentially methylated sites (DMS) in myoblasts compared to myotubes, however we observed that the activation of muscle specific genes seem to be poorly correlated with DNA methylation changes. Therefore, we argue that DNA methylation does not show a major role in the control of muscle specific genes. Interestingly, further analysis revealed that a high percentage of differentially methylated regions (DMR) localizes near novel non-coding RNA genes. On the one hand, this observation suggests a role for novel regulatory RNAs in the epigenetics of muscle differentiation; on the other hand, DNA methylation might have a role in the regulation of these RNAs.
Together with DNA methylation, chromatin compaction is a major epigenetics player. In order to describe the epigenetic landscape of muscle differentiation, I optimized the ChIP-seq approach to define the localization of specific histone modifications on DNA. After setting-up the protocol for ChIP-seq, H3K4me3, H3K27me3 and H3K9ac histone modifications were mapped on myoblast DNA. As I started integrating gene expression and ChIP-seq data, I verified that a great concordance exists between gene expression and ChIP-seq results. In particular, euchromatin-associated histone modifications are found in transcription start sites of active genes, and heterochromatic signature spans promoters and bodies of inactive genes. Further investigation show that genes for non-coding RNAs have an euchromatic signature. This observation, together with the findings of DMRs in novel non-coding RNA genes, endorses the hypothesis of a role for novel regulatory RNAs in myogenic differentiation.
In conclusion, the integration of BS-seq, ChIP-seq and RNA-seq data are opening interesting scenarios concerning the involvement of regulatory RNAs, while recent reports are suggesting to extend our investigation even to DNA hydroxymethylation in the epigenetics of muscle differentiation.Negli ultimi anni l’epigenetica ha raccolto un sempre crescente interesse da parte della comunità scientifica, grazie al suo potenziale di spiegare i meccanismi di attivazione e repressione dell’espressione genica. In questa tesi si presentano i risultati di un progetto di analisi del ruolo dell’epigenetica nella miogenesi umana, mediante approcci genomici. Gli studi di epigenetica presentano tuttora ostacoli significativi, sia dal punto di vista sperimentale che di analisi computazionale del dato prodotto. Il mio primo obiettivo è stato quindi la messa a punto di un protocolli robusti per l’analisi del ruolo dei meccanismi epigenetici durante il differenziamento del muscolo scheletrico, in particolare la metilazione del DNA e le interazioni DNA-proteine (mediante il sequenziamento di DNA trattato con bisulfito e immunoprecipitazione della cromatina). In questa tesi, oltre alla descrizione dei protocolli sviluppati, sono riportati i primi risultati ottenuti applicando i suddetti protocolli alle cellule miogeniche. La metilazione è stata analizzata mediante trattamento con bisulfito del DNA, sequenziato successivamente con tecniche di nuova generazione (NGS). A tal riguardo è stato messo a punto un nuovo metodo per lo studio del metiloma intero, che è stato applicato ad un campione di mioblasti e successivamente sequenziato con la piattaforma SOLiD 5500xl. Questo approccio richiede tuttavia una quantità massiva di sequenze, che ad oggi risultano ancora eccessivamente costose. È stato quindi affiancato allo studio del metiloma il sequenziamento delle regioni più comunemente analizzate in studi di metilazione (ovvero regioni promotoriali ed isole CpG) con un kit di arricchimento di regioni target che cattura selettivamente più di 2.700.000 siti CpG nel genoma umano. Con questo approccio sono stati identificati meno di 600 siti differenzialmente metilati (DMS) nei mioblasti confrontati con i miotubi. Questo studio ha permesso di osservare che l’attivazione di geni muscolari sembra poco correlata con cambiamenti nella metilazione del DNA, permettendo di ipotizzare che la metilazione del DNA non abbia un ruolo centrale nel controllo dell’attivazione dei geni muscolo-specifici. L’analisi di questi dati ha inoltre permesso di rilevare che una significativa frazione di regioni differenzialmente metilate (DMR) localizza in prossimità di geni codificanti non-coding RNA. Da un lato quest’osservazione suggerisce che gli RNA regolatori potrebber avere un ruolo nell’epigenetica nel differenziamento muscolare, e inoltre che la metilazione del DNA potrebbe avere un ruolo nella regolazione di questi RNA. Un altro aspetto importante della regolazione epigenetica, oltre alla metilazione del DNA, è lo stato di condensazione della cromatina. Per vagliarne il contributo nell’ambito del differenziamento muscolare, è stato ottimizzato un approccio di immunoprecipitazione della cromatina seguito dal sequenziamento (ChIP-Seq) per definire la localizzazione nel DNA di specifiche modificazioni istoniche: H3K4me3, H3K27me3 and H3K9ac. I risultati di questi esperimenti di ChIP-Seq sono stati confrontati con quelli di analisi del profilo di espressione (RNA-Seq), permettendo di verificare un ampio margine di sovrapposizione fra il livello di espressione ed i risultati di ChIP-Seq. In particolare le modificazioni istoniche associate all’eucromatina localizzano nei pressi del sito di inizio di trascrizione di geni espressi, mentre i marker di eterocromatina fiancheggiano i promotori dei geni non attivi. Questa osservazione, assieme all’osservazione di DMR in nuovi RNA non codificanti, supporta l’ipotesi di un ruolo per nuovi circuiti regolatori di RNA nel differenziamento miogenic.
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Bienvenu, Carine. "Oxydations radicalaires de la 5-méthyl-2'-désoxycytidine." Université Joseph Fourier (Grenoble ; 1971-2015), 1997. http://www.theses.fr/1997GRE10039.
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Les reactions d'oxydation radicalaire des bases de l'adn peuvent etre initiees par differents agents du stress oxydant tels que les rayonnements du proche uv et ionisant. Ces dommages oxydants de l'adn sont susceptibles d'etre a l'origine de processus de cancerogenese et de mutagenese. La 5-methylcytosine est une base rare de l'adn impliquee dans de nombreuses fonctions biologiques cles pour la vie cellulaire. Cette base methylee est presente principalement au niveau des sequences repetitives cpg qui sont le siege de frequentes mutations. Nous nous sommes interesses a l'oxydation photosensibilisee de la 5-methyl-2'-desoxycytidine (m#5dcyd) en solution aqueuse aeree. Le cation radical pyrimidique initialement forme par un mecanisme de type i est implique dans deux voies competitives de transformation, a savoir un processus de deprotonation et une reaction d'hydratation. Les produits finals ont ete isoles par chromatographie liquide a haute performance et caracterises au moyen de diverses methodes spectroscopiques (uv, spectrometrie de masse, rmn du proton et du carbone, dichroisme circulaire). L'action du radical hydroxyle (oh) sur la 5-methyl-2'-desoxycytidine a egalement ete etudiee. Les resultats obtenus montrent que l'oxydation de ce nucleoside par le radical oh conduit, au moins partiellement, a la formation d'intermediaires radicalaires identiques a ceux produits par photosensibilisation. Des experiences d'irradiation uv et gamma de solutions aqueuses de m#5dcyd en presence d'oxygene 18 ont permis de proposer des mecanismes pour la formation des principaux produits d'oxydation de la m#5dcyd. Un autre volet de ce travail a concerne la mesure de produits d'oxydation de la m#5dcyd dans l'adn isole. Pour ce faire, une technique d'analyse sensible et specifique, la chromatographie gazeuse couplee a la spectrometrie de masse a ete mis en uvre. Ces mesures ont egalement necessite la synthese de molecules etalon enrichies avec des isotopes stables (#2h et #1#5n)
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Tasanasuwan, Piyama. "Targeted DNA methylation." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251476.
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Mathot, Pauline. "Mécanismes épigénétiques et réponse des cellules cancéreuses au microenvironnement : implication de la méthylation de l’ADN et de l’un de ses interprètes, MBD2." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1163/document.
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Les cancers du sein ont la particularité de développer un microenvironnement tumoral important où les fibroblastes associés au cancer (CAF) jouent un rôle crucial dans la tumorigénèse via la sécrétion de différents facteurs de croissance, cytokines, protéases et composants de la matrice extracellulaire. Ces différents facteurs secrétés par les CAFs sont impliqués dans de nombreuses voies de signalisation suggérant que la reprogrammation des cellules cancéreuses par les CAFs peut affecter de nombreux gènes.Le séquençage des ARN messagers (RNAseq) de lignées cellulaires de cancer du sein cultivées en présence de milieux conditionnés de CAF, nous a permis d'identifier 372 gènes surexprimés in vitro par les facteurs sécrétés par les CAF et in vivo selon la teneur en cellules stromales des tumeurs mammaires. De façon inattendue, nous avons pu constater que les facteurs sécrétés par les CAF ainsi que le contenu en cellules stromales des tumeurs n'induisent pas de changements significatifs de méthylation de l'ADN mais activent de manière spécifique des gènes caractérisés par une signature méthylation. Différentes approches expérimentales, telles que l'inhibition de la méthylation de l'ADN, l'inhibition de l'expression de la protéine MBD2 (protéines de liaison à l'ADN méthylé) et des expériences d'immuno-précipitation de la chromatine (ChIP) ont permis de montrer l'implication de ces marques de méthylation et des protéines de liaison à l'ADN méthylé dans la réponse des cellules cancéreuses aux facteurs sécrétés par les CAFs. Ces résultats ont permis l'identification d'événements moléculaires impliqués dans la réponse des cellules tumorales aux signaux sécrétés par les cellules stromales dans les tumeurs mammaires mettant en lumière l'importance des marques épigénétiques dans la reprogrammation des cellules cancéreuses induites par les cellules stromalesBreast cancers develop in complex tissue environments where cancer associated fibroblasts (CAF) play a crucial role in tumorigenesis by secreting various growth factors, cytokines, proteases and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation, and epigenetic modulation suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation signature: hypermethylation at transcription start site (TSS) and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of MBD2, and ChIP assays) demonstrated the implication of DNA methylation and methyl DNA binding protein in the response of cancers cells to CAF-secreted factors. These data put in light the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicate that the interpreters of the DNA methylation signal play a major role in the response of the cancer cells to the microenvironment
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MacLeod, A. Robert (Robert Alan) 1966. "DNA methylation and oncogenesis." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39956.
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DNA methylation is a postreplicative covalent modification of the DNA which is catalysed by the DNA methyltransferase enzyme. DNA methylation plays an important role in controlling the gene expression profile of mammalian cells. The hypothesis presented in this thesis is that the expression of the DNA methyltransferase gene is upregulated by cellular oncogenic pathways, and that this induction of MeTase activity results in DNA hypermethylation and plays a causal role in cellular transformation. Novel DNA methyltransferase inhibitors may inhibit the excessive activity of DNA methyltransferase in cancer cells and induce the original cellular genetic program. These inhibitors may also be used to turn on alternative gene expression programs. Therefore specific DNA methyltransferase antagonists might provide us with therapeutics directed at a nodal point in the regulation of genetic information.
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Tavares, de Araujo Felipe. "DNA replication and methylation." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37847.
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One of the main questions of modern biology is how our cells interpret our genetic and epigenetic information. DNA methylation is a covalent modification of the genome that is essential for mammalian development and plays an important role in the control of gene expression, genomic imprinting and X-chromosome inactivation (Bird and Wolffe, 1999; Szyf et al., 2000). Furthermore, changes in DNA methylation and DNA methyltransferase 1 (DNMT1) activity have been widely documented in a number of human cancers (Szyf, 1998a; Szyf et al., 2000).In Escherichia coli, timing and frequency of initiation of DNA replication are controlled by the levels of the bacterial methyltransferase and by the methylation status of its origin of replication (Boye and Lobner-Olesen, 1990; Campbell and Kleckner, 1990). In mammalian cells, however, the importance of methyltransferase activity and of DNA methylation in replication is only now starting to emerge (Araujo et al., 1998; Delgado et al., 1998; DePamphilis, 2000; Knox et al., 2000).
The work described in this thesis focuses mainly on understanding the functional relationship between changes in DNA methylation and DNMT1 activity on mammalian DNA replication. In higher eukaryotes, DNA replication initiates from multiple specific sites throughout the genome (Zannis-Hadjopoulos and Price, 1999). The first part of the thesis describes the identification and characterization of novel in vivo initiation sites of DNA replication within the human dnmt1 locus (Araujo et al., 1999). Subsequently, a study of the temporal relationship between DNA replication and the inheritance of the DNA methylation pattern is presented. We also demonstrate that mammalian origins of replication, similarly to promoters, are differentially methylated (Araujo et al., 1998). We then tested the hypothesis that DNMT1 is a necessary component of the replication machinery. The results presented indicate that inhibition of DNMT1 results in inhibition of DNA replication (Knox et al., 2000). Finally, results are presented, demonstrating that the amino terminal region of DNMT1 is responsible for recognizing hemimethylated CGs, DNMT1's enzymatic target. Taken together, the results presented in this thesis demonstrate that DNMT1 is necessary for proper DNA replication and that DNA methylation may modulate origin function.
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Villa, Raffaella. "Role of epigenetic modifications in acute promyelocytic leukemia." Doctoral thesis, Universitat Pompeu Fabra, 2007. http://hdl.handle.net/10803/7144.
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Mi trabajo ha estado enfocado en la implicación de los diferentes mecanismos epigenéticos de PML-RARa en la inducción de la leucemia promielocítica aguda (APL).En particular yo estudié el rol de MBD1, un miembro de la conservada familia de proteinas capaces de unirse al DNA metilado, demostrando que desempeña un papel importante en la progresión de la leucemia. De hecho, mostré que MBD1 es recruida por PML-RARa a sus promotores diana a través de los mecanismos mediados por HDAC3, participando por tanto en la represión transcripcional. Además, investigué hasta donde la metilación de la H3K27 mediada por Polycomb contribuye a la tumorgénesis mediada por PML-RARa. Demostré que PML-RARa dirige al PRC2 hacia el locus del tumor supresor causando la metilación de la H3K27. Fue interesante ser capaz de mostrar que tanto la metilación del DNA como la de las histonas era requerida para mantener el aberrante silencio génico. Esto apuntaba hacia una intercomunicación entre estos diferentes marcadores epigenéticos contribuyendo a la patología molecular de la leucemia. Resumiendo, estos resultados nos proporcionan elementos nuevos para comprender los mecanismos moleculares esenciales en la tumorgénesis y progresión de la APL.
My work was focused on the involvement of different epigenetic mechanisms in PML-RARa-induced acute promyelocytic leukemia (APL). In particular, I studied the role of MBD1, a member of a conserved family of proteins able to bind methylated DNA, demonstrating that has an important function in leukemia progression. Indeed, I showed that MBD1 is recruited by PML-RARa to its target promoters through an HDAC3-mediated mechanism, thus participating in transcriptional repression.. Furthermore, I investigated how far Polycomb-mediated H3K27 methylation contributes to PML-RARa mediated tumorigenesis. I demonstrated that PML-RARa targets the PRC2 to tumor suppressor loci causing H3K27 methylation. Interestingly, I was able to show that both DNA and histone methylation are required to maintain PML-RARa aberrant gene silencing, pointing towards a crosstalk among these different epigenetic layers that contributes to the molecular pathology of leukemia. In summary these results provide new insights into the molecular mechanisms underlying APL tumorigenesis and progression.
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Tsusaka, Takeshi. "Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation." Kyoto University, 2018. http://hdl.handle.net/2433/232305.
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Carrió, Gaspar Elvira. "DNA Methylation Dynamics during Myogenesis." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/296312.
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Myogenesis is the differentiation process which encompasses the formation of skeletal muscle during development, regeneration and tissue homeostasis throughout life. Arising from embryonic or adult stem cells, the myogenic process comprehends the acquisition of a specialized cell identity and the loss of pluri/multipotent and proliferative capacities. Starting with the hypothesis that DNA methylation, together with other epigenetic mechanisms and the transcription factors, orchestrates the transcriptional program, this thesis provides a comprehensive picture of DNA methylation dynamics during murine myogenic progression, addresses their regulatory implications, and identifies relevant differentially methylated regions that define muscle cell identity.
Initially, we performed a genome-scale DNA methylation study comparing embryonic stem cells (ESCs), primary myoblasts (MBs), differentiated myotubes (MTs), and mature myofibers (MFs) using AIMS-seq method. We identified 1,000 differentially methylated regions during muscle-lineage determination and terminal differentiation, mainly located in gene bodies and intergenic regions. As a whole, muscle lineage commitment was characterized by a major gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, hypomethylated sequences were enriched in enhancer-type chromatin regions, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Importantly, we detected a demethylated region overlapping the super-enhancer of the cell-identity factor Myf5. We showed that the activation of My5 super-enhancer took place upon DNA demethylation exclusively in muscle-committed cells resulting in gene expression. ChIP analyses showed that the binding of the Upstream stimulatory factor 1 (Usf1) to Myf5 locus was DNA demethylation-dependent in myogenic committed cells. Moreover, Usf1 binding site contained an embedded CpG conserved in humans and demethylated in human MBs but not in human ESCs, altogether reinforcing the hypothesis that DNA methylation regulates gene expression by modulating transcription factor binding accessibility.
Next, we analyzed by sodium bisulphite sequencing the DNA methylation state of reported regulatory regions (with and without CpG island) of key genes implicated in myogenesis. After analyzing myogenic and non-myogenic cells we concluded that the muscle cell identity comprehends DNA demethylation of lineage-specific CpG-poor regulatory regions leading to a transcriptionally poised or activated state, while myogenic CpG island promoters are totally unmethylated during myogenesis and are regulated by histone modifications. A collaborative work with Charles Keller’s Lab (Oregon Health & Science University, USA) allowed us to conclude that Rhabdomyosarcoma cell lines present a spurious methylation pattern at usually unmethylated CpG islands, consequently with the aberrant methylation associated to tumorigenesis. Furthermore, the study of pluripotency gene promoters during myogenesis pointed that CpG-poor promoters are repressed during differentiation by DNA methylation and by Polycomb complex at CpG island promoters.
Interested in deepen in the DNA demethylation dynamics, we started a collaboration with Rita Perlingeiro’s Lab (University of Minnesota, USA) to study the DNA methylation changes in the myogenic inducible Pax7 ESC-derived model. We showed that the ESC-derived myoblast precursors recreated the DNA methylation signature of in vivo isolated muscle stem cells, supporting this model as a bona fide strategy to generate myogenic precursors in vitro with therapeutic purposes.
Finally, we addressed the involvement of an active demethylating mechanism during myogenesis. Apobec2 down-regulation in inducible ESC-derived myoblast precursors with shRNA strategies affected dramatically the myogenic differentiation by impairing DNA demethylation of the Myogenin promoter and abolishing the expression of Myogenin and MHC proteins. Based on these results, we proposed that Apobec2 might be involved in the active muscle specific DNA demethylation along myogenesis.Partint de la hipòtesi que la metilació de l’ADN, junt amb altres mecanismes epigenètics i els factors de transcripció, orquestra el programa transcripcional, aquesta tesi ofereix un estudi exhaustiu de les dinàmiques de l'ADN durant la progressió miogènica, aborda les seves possibles implicacions reguladores i identifica regions diferencialment metilades que defineixen la identitat de la cèl·lula muscular. L’anàlisi a escala genòmica els perfils de metilació en diferents estadis de la miogènesi va permetre identificar 1000 regions diferencialment metilades, localitzades principalment en regions intergèniques i intragèniques. La majoria de canvis observats eren guanys de metilació i ocorrien durant la determinació de llinatge. D’altra banda, certes regions amb perfils de cromatina associats a enhancers esdevenien desmetilades, suggerint que la metilació de l’ADN pot estar implicada en la regulació de enhancers específics de teixit. L’estudi de gens específics de múscul va mostrar que la identitat de la cèl·lula muscular requereix la desmetilació de l'ADN de regions reguladores pobres en CpGs, alhora que els gens miogènics amb illes CpG a la regió promotora es troben sempre desmetilats i són regulats per modificacions d’histones. Un exemple de la desmetilació específica de múscul és la regió super-enhancer de Myf5. Els assajos d'immunoprecipitació de la cromatina van demostrar que la unió del factor de transcripció Usf1 al locus Myf5 només es donava quan l’ADN estava desmetilat, reforçant la hipòtesi que la metilació de l'ADN regula l'expressió gènica mitjançant la modulació de l'accessibilitat dels factors de transcripció al seu lloc d’unió. Mitjançant l’estudi el perfil de metilació de l'ADN de gens miogènics en un model miogènic derivat de cèl·lules embrionàries, es va observar el mateix perfil observat en mioblasts primaris, indicant que aquest model és una bona estratègia per obtenir mioblasts in vitro amb finalitats terapèutiques. Finalment, el bloqueig de la deaminasa Apobec2 va afectar severament la diferenciació miogènica i la desmetilació de l'ADN del promotor de la Miogenina, indicant que la deaminasa Apobec2 podria estar implicada en la desmetilació activa de l'ADN.
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Wong, Nicholas Chau-Lun. "DNA methylation at the neocentromere /." Connect to thesis, 2006. http://eprints.unimelb.edu.au/archive/00001883.
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Akman, Kemal. "Bioinformatics of DNA Methylation analysis." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-182873.
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陳桂儀 and Kwai-yi Jacqueline Chan. "DNA methylation and pediatric cancer." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31970370.
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Ó, Riain Ciarán Liam. "DNA methylation in follicular lymphoma." Thesis, Queen Mary, University of London, 2010. http://qmro.qmul.ac.uk/xmlui/handle/123456789/1318.
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Follicular Lymphoma (FL) is a common B cell Non-Hodgkin Lymphoma with a median survival of 8-10 years. Patients frequently undergo transformation to a more aggressive lymphoma and this is associated with drastically reduced survival. The hallmark of FL is the t(14;18) translocation yet this alone is insufficient for lymphomagenesis. While a number of secondary genetic changes have been described, epigenetic studies have lagged behind. Epigenetics refers to mechanisms that alter gene expression without a change in the primary DNA sequence. DNA methylation was quantitatively profiled at 1505 CpG loci in 164 untreated FL as well as 10 pairs of pre- and post-transformation samples and 24 benign haematopoietic controls. Tumour-specific methylation occurred in >100 genes, preferentially occurring within CpG rich areas known as CpG islands and in genes marked by a repressive histone modification in embryonic stem (ES) cells, trimethylated Lysine 27 of Histone H3 (H3K27Me3). Significant inverse correlation with gene expression was identified for a small number of genes. Significant changes in methylation were not seen in FL samples upon transformation. These findings suggested that widespread DNA methylation occurred as an early 'pre-programmed' event in lymphomagenesis rather than being due to silencing of individual tumour suppressor genes. Methylation profiling of a subset of these samples at >27,000 CpG loci revealed aberrant methylation in FL in >700 CpG islands. These hypermethylated genes were enriched for high-density CpG promoters and for a key set of genes with developmental function which were marked by both repressive (H3K27Me3) and activating (H3K4Me3) marks in ES cells. Examination of H3K27me3 expression by immunohistochemistry in pre- and post-transformation biopsy samples showed wide variation in expression. Furthermore, mutations were identified in the histone methylase EZH2 that catalyses H3K27Me3, in 7 of 20 patients. We can conclude therefore that deregulation of DNA and histone methylation are critical inter-related events for FL pathogenesis.
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Gonçalves, Athanásio Camila. "DNA methylation in Daphnia magna." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/7140/.
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Daphnia magna is gaining interest as a model for epigenetic research. It is easy to maintain under laboratory conditions and has low genetic diversity due to parthenogenetic reproduction. The D. magna genome is responsive to a wide range of stimuli and genomics resources are being developed for this species. Despite these great advantages, information regarding the epigenome of D. magna and its regulation is still lacking. Thus, the main aim of this work was to describe the methylome of D.magna and investigate its regulation and responsiveness to environmentally relevant exposure conditions. Despite the low levels of global DNA methylation, a defined profile could be identified. DNA methylation in D. magna is sporadic and mainly found at coding regions. These data suggest that D. magna encodes a complete set of genes for DNA methylation reactions. Evidence of direct effects on the DNA methylation profile were found in animals exposed to the DNA methylation inhibitor 5-azacytidine and these changes were persistent after the removal of the stressor. Acute and chronic exposures to environmentally relevant concentrations of stressors (arsenic and hypoxia) also induced changes in gene transcription levels and concentrations of onecarbon pathway metabolites. These findings indicate that the epigenome of D. magna is responsive to changes in the environment, supporting its use as an environmentally relevant model organism for epigenetics research. Furthermore, the maintenance of some of the epigenetic changes in the absence of the initial stressor supports the concept of ‘epigenetic memory’ and its potential use in chemical risk assessment.
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McArthur, Michael. "Chromatin structure and DNA methylation." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627534.
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Hunter, Jennifer Margaret. "Reprogramming a DNA methylation mutant." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25874.
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Chemical modification of the cytosine base via the addition of a methyl group to form 5-‐methylcytosine (5-‐mC) is a well-‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-‐mC is not the only DNA modification found within the vertebrate genome. 5-‐hydroxymethylcytosine (5-‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-‐mC base from which it is derived, 5-‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-‐/-‐) as well as and methylation hypomorphic (p53-‐/-‐, Dnmt1 -‐/-‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-‐hmC for both p53-‐/-‐ and (p53-‐/-‐, Dnmt1 -‐/-‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-‐/-‐, Dnmt1 -‐/-‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.
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Chan, Kwai-yi Jacqueline. "DNA methylation and pediatric cancer." Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2515526x.
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Melquist, Stacey Michelle. "DNA methylation signaling in Arabidopsis." Available to US Hopkins community, 2002. http://wwwlib.umi.com/dissertations/dlnow/3068188.
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Poli, Elena. "DNA METHYLATION ANALYSIS IN RHABDOMYOSARCOMA." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424380.
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Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft-tissue sarcoma. It is mainly classified into two major subtypes characterized by alveolar (ARMS) and embryonal (ERMS) histologies. ARMS are characterized by a more aggressive behavior with a higher tendency to present metastasis at diagnosis and to relapse after treatment. Approximately 80% of ARMS harbour the reciprocal chromosomal translocation t(2;13)(q35;q14) and, less commonly, the variant translocation t(1;13)(p36;q14), in which PAX3 and FOXO1, or PAX7 and FOXO1 genes, respectively, are juxtaposed. Unfortunately, no such specific genetic aberrations are known in ERMS, and myogenic factors as myogenin and MyoD1 are the only diagnostic indicators that can be used. Despite aggressive multimodal therapies, the prognosis of high-risk RMS patients has not been improved, with a 5-year overall survival rate less than 20-30%, which prompts a need for new therapeutic strategies. In the last decade many scientific studies have demonstrated that gene expression signature distinguishes PAX3-FOXO1 positive RMS from PAX3-FOXO1 negative, but the reasons of the different expression are still unknown. Aberrant DNA methylation patterning is a hallmark of cancer and could be responsible for the different gene expression of RMS tumor subtypes.
We performed genome-wide methylation profile by microarray experiments followed by Reduced-Representation Bisulfite Sequencing (RRBS).
Microarray analysis demonstrated a different methylation profile between PAX3-FOXO1 positive and negative RMS, besides among metastatic and non-metastatic RMS. We confirmed HOXC11 as one of the gene differentially methylated between PAX3-FOXO1 positive and negative RMS cell lines using in vitro demethylating agents and bisulfite sequencing. Unfortunately, we did not validate the result in the cohort of RMS biopsies. Moreover, we performed another analysis on microarray data comparing metastatic vs non-metastatic RMS. We found an elevated numbers of differentially methylated regions (DMRs) and many of them map to promoter regions of genes implicated in tumors development. In particular we found DMRs linked to clustered protocadherins, known as tumor suppressor genes. We confirmed a different expression pattern of PCDHA4, as well as a different methylation level of its promotorial region, comparing metastatic and non-metastatic RMS samples. Nevertheless, the methylation status and the expression level of PCDHA4 did not have significant correlation with clinical features and are not a predictor of poor prognosis in RMS.
Then, we performed an RRBS sequencing, in order to validate data obtained with microarray platforms. We observed a very low concordance between the two approaches, probably caused by a low quality DNA used in microarray experiments. The RRBS sequencing had demonstrated again that PAX3-FOXO1 positive and negative RMS have a different methylation pattern. Moreover, we demonstrated that GADD45G and NELL1, already described as tumor suppressors in other cancers and often downregulated by methylation processes, had also an involvement in RMS biology. Our experiments confirmed an epigenetic regulation by DNA methylation for GADD45G and NELL1 and that their expression were correlated to RMS histology, presence of fusion status and IRS group staging. Furthermore, GADD45G and NELL1 expression levels affect the progression free survival of RMS patients suggesting their association with a poor prognosis.
In conclusion, we demonstrated that GADD45G and NELL1 could be novel potential biomarkers in RMS and we evidenced that the DNA methylation pattern in RMS could be interesting for new therapeutic strategies.
We hope that our efforts could contribute to a better molecular classification of RMS tumors and to the identification of new targets for improving standard therapy.Il rabdomiosarcoma (RMS) è una sarcoma pediatrico dei tessuti molli altamente aggressivo. Viene classificato principalmente in due sottotipi, caratterizzati da istologia alveolare (RMSA) o embrionale (RMSE). Nei RMSA si osserva un comportamento più aggressivo e una maggiore tendenza a presentare metastasi alla diagnosi e alla ricaduta dopo trattamento. Circa l'80% dei RMSA presentano la traslocazione cromosomica reciproca t(2; 13) (q35; q14) e, meno comunemente, la variante t(1; 13) (p36; q14), in cui i geni PAX3 e FOXO1, o PAX7 e FOXO1, rispettivamente, sono giustapposti. Purtroppo, non si conoscono aberrazioni genetiche specifiche nei RMSE e i fattori miogenici, come miogenina e MyoD1, sono gli unici indicatori diagnostici che possono essere utilizzati. Nonostante l’applicazione di terapie aggressive multimodali, la prognosi dei pazienti affetti da RMS, della categoria alto rischio, non è migliorata, con un tasso di sopravvivenza a 5 anni inferiore al 20-30%. Questo dato indica la necessità di sviluppare nuove strategie terapeutiche. Nell’ultimo decennio molti studi scientifici hanno dimostrato che in base al profilo di espressione genica è possibile distinguere RMS PAX3-FOXO1-positivi e PAX3-FOXO1-negativi, ma le ragioni di questa diversa espressione sono ancora sconosciute. L’anomala metilazione del DNA è un indicatore di neoplasia e potrebbe essere la causa responsabile della diversa espressione genica dei due sottotipi di tumore. In questo studio, per mezzo di esperimenti di microarray, abbiamo realizzato un’analisi dello stato di metilazione del DNA su tutto il genoma, proseguendo poi con esperimenti di sequenziamento sfruttando la tecnica Reduced-Representation Bisulfite Sequencing (RRBS). L’analisi dei risultati ottenuti con gli esperimenti di microarray ha dimostrato, non solo un profilo di metilazione diverso tra i RMS PAX3-FOXO1-positivi e negativi, ma anche tra i RMS metastatici e non metastatici. Abbiamo confermato che il gene HOXC11 risulta essere differenzialmente metilato tra linee cellulari di RMS PAX3-FOXO1-positive e negative, sfruttando trattamenti con agenti demetilanti in vitro e sequenziamento del DNA dopo conversione con bisolfito; purtroppo, non abbiamo confermato il risultato nella coorte di biopsie di RMS. Inoltre, abbiamo effettuato un'ulteriore analisi sui dati di microarray confrontando i RMS metastatici con i non metastatici. Abbiamo trovato un elevato numero di regioni differenzialmente metilate (DMR) e molte di queste sono risultate coincidere con le regioni promotoriali di geni implicati nello sviluppo di tumori; in particolare, abbiamo trovato DMR connesse alla famiglia delle clustered protocaderine, note come geni soppressori di tumore. Abbiamo poi confermato un diverso profilo di espressione del gene PCDHA4, così come un diverso stato di metilazione a livello della sua regione promotoriale, confrontando campioni di RMS metastatici e non metastatici. Tuttavia, lo stato di metilazione e il livello di espressione di PCDHA4 non hanno dimostrato una correlazione significativa con le caratteristiche cliniche del RMS. Il gene PCDHA4 non risulta quindi essere un predittore prognostico nel RMS. Successivamente, abbiamo effettuato un sequenziamento RRBS, al fine di validare i dati ottenuti con le piattaforme dei microarray. Ne è risultata una bassa concordanza tra i due approcci, probabilmente a causa della bassa qualità del DNA utilizzato negli esperimenti di microarray. Il sequenziamento RRBS ha dimostrato ancora una volta che i RMS PAX3-FOXO1-positivi hanno un profilo di metilazione diverso dai RMS PAX3-FOXO1-negativi. Inoltre, abbiamo dimostrato che GADD45G e NELL1, già descritti come soppressori tumorali in altri tipi di tumore e spesso regolati in maniera negativa da processi di metilazione, sono anche coinvolti nella biologia del RMS. Con i nostri esperimenti abbiamo confermato una regolazione epigenetica, mediata dalla metilazione del DNA ,per i geni GADD45G e NELL1, e come la loro espressione sia correlata alla istologia del RMS, alla presenza dei geni di fusione e alla stadiazione in gruppi IRS. Inoltre, abbiamo dimostrato che i livelli di espressione di GADD45G e NELL1 influenzano la sopravvivenza libera da progressione di malattia nei pazienti affetti da RMS, suggerendo la loro associazione con una prognosi sfavorevole. In conclusione, il nostro lavoro ha dimostrato che GADD45G e NELL1 potrebbero essere nuovi potenziali biomarcatori nel RMS, evidenziando come il profilo di metilazione del DNA nel RMS potrebbe favorire lo sviluppo di nuove strategie terapeutiche. Ci auguriamo che i nostri sforzi possano contribuire ad una migliore classificazione molecolare dei tumori nei pazienti affetti da RMS e alla identificazione di nuovi bersagli farmacologici per una terapia più mirata.
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Gould, Poppy Aeron. "The role of DNA repair in DNA methylation dynamics." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274360.
Full textAbstract:
The mammalian epigenome is globally reprogrammed at two stages of development; this involves the erasure and re-establishment of DNA methylation by both passive and active mechanisms, including DNA repair pathways, and occurs concurrently with an increase in developmental potency. In addition to Uhrf1 and the Tet enzymes, the interplay between activation induced cytidine deaminase (AID) and the DNA repair machinery has been implicated in epigenetic reprogramming of various in vivo and in vitro systems including mouse primordial germ cells, zygotes and induced pluripotent stem cells. AID deaminates cytosine to uracil and can also deaminate methylcytosine, whereas the primary role of UNG is to maintain the integrity of the genome through erasure of uracil. In this thesis, I have aimed to investigate the role of DNA repair in demethylation. To do this I have focused on the specific role of AID and UNG in the demethylation of a static system – primed serum ESCs and a dynamic system – serum to 2i (naïve) to epiblast-like ES cells. As the role of both AID and UNG involves genomic uracil, the central theme of my thesis is the impact of accumulation of uracil on DNA methylation levels in the genome. Therefore, my first aim was to develop a quantitative method to detect low levels of genomic uracil in DNA firstly, by mass spectrometry and secondly, by whole genome sequencing. In Chapter Three, I show that the impact of deamination during DNA preparation can be minimised, such that the level of genomic ESC uracil can be accurately determined as around 12,000 uracil per genome and that, as anticipated, Ung null ESCs have almost twice the genomic uracil content of wildtype ESCs. Secondly, I address the main question which is the impact of uracil accumulation on methylation levels. In order to do this, I generate two cell lines: Ung knockout and Aid over expressing, both of which should result in an increase in genomic uracil. I demonstrate that while over expression of Aid stimulates demethylation in static system and in a dynamic demethylating system, the impact of Ung knockout is less clear. In (static) serum ESCs, loss of Ung results in hypomethylation however, in order to transition to 2i (naïve) ESCs, a process which involves demethylation of the genome, it appears the Ung is required as loss of this gene inhibits proper demethylation. As such, I conclude that UNG-mediated DNA repair functions alongside passive demethylation, by reduction of UHRF1 levels, to demethylate 2i ESCs. To probe the mechanism by which accumulation of uracil in the genome alters methylation levels, I investigate the impact of Ung KO and Aid OE on global levels of DNA damage. I show that both cell lines have a greater incidence of double strand breaks compared to a wild type cell line, and accordingly, upregulate their DNA damage response pathway and the expression of certain repair genes. I suggest that increasing genomic levels of uracil causes genomic instability and that DNA demethylation occurs as a consequence of the repair of extensive DNA damage. More broadly, I suggest that ESCs are uniquely poised, due to their heightened DNA damage response, to use uracil as an intermediate of DNA demethylation. Interestingly, I also note that the biological impact on serum ESCs of loss of Ung appears to be an increase in pluripotency.
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Tan, Choon Ping. "Control of mammalian DNA methylation system components by protein arginine methylation." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445922/.
Full textAbstract:
DNA methylation is essential for the survival and development of vertebrates. Methylated cytosine in the context of CpG-dinucleotides within the genome is recognized by proteins from the methyl-CpG DNA binding domain (MBD) family. When bound to methyl-CpG DNA, most MBD proteins can recruit histone deacetylase (HDAC) silencing complexes to the site of chromatin to remodel its structure, and this causes transcription repression. Loss of CpG-DNA methylation results in embryonic lethality in mice, but loss of methyl-CpG DNA recognizing MBD proteins produce a viable phenotype. Our interest in MBD proteins arises from our discovery that a subset of them interact with RNA. Two MBD proteins (MBD2 and MeCP2) bind their RNA partners using a domain that contains arginine and glycine (RG) rich motifs. As proteins with RG rich motifs are often substrates of post-translational modifications catalyzed by Protein Arginine Methyltransferase (PRMT), I asked whether the two MBD proteins are methylated at their arginines, and the consequences of such modification. By in vitro and in vivo labeling assays, I ascertained that MBD2 and MeCP2 are substrates of PRMT, and that PRMT1 and PRMT5 are responsible for catalyzing different forms of methylation on the MBD2 protein. The relationship between the two PRMTs with regard to MBD2 methylation was characterized. Subsequently, I identified the significance of MBD2 arginine methylation. Biochemically, methylated species of MBD2 protein have less affinity for the HDAC silencing complex and methyl-CpG DNA. In cells, the loss in repression activity of arginine methylated MBD2 was demonstrated. As MBD2 mediated repression activity can be relieved by arginine methylation, I propose that this mechanism might possibly explains the discrepancy between the phenotypes of CpG-DNA methylation null and MBD null mice. This study provides the first evidence that PRMT participate in the DNA methylation system of chromatin control.
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Hernando, Herráez Irene 1985. "Evolutionary insights into human DNA methylation." Doctoral thesis, Universitat Pompeu Fabra, 2015. http://hdl.handle.net/10803/392140.
Full textAbstract:
DNA methylation is a crucial epigenetic modification involved in numerous biological processes. However, despite its functional importance, the evolutionary history of this modification and the mechanisms diving such changes are poorly understood. The aim of this thesis is to provide a better understanding of DNA methylation in the context of human recent evolution. We identified and described hundreds of regions presenting a human-specific DNA methylation pattern compared to great apes. We also analyzed for the first time the relationship between DNA methylation changes and sequence evolution at both nucleotide and protein level. In summary, this research reveals new insights into the evolutionary properties of DNA methylation and the interpretation of inter-species non-coding variationLa metilación del ADN es una modificación epigenética implicada en numerosos procesos biológicos. Sin embargo, a pesar de su relevancia funcional, se sabe muy poco sobre su historia evolutiva y los mecanismos que generan estos cambios. El objetivo de esta tesis es proporcionar una mejor compresión de la metilación del ADN en el contexto de la evolución humana reciente. Hemos identificado y descrito cientos de regiones que presentan un patrón de metilación especifico de humanos. Así mismo, hemos analizado por primera vez la relación entre los cambios en metilación y la evolución de la secuencia tanto a nivel nucleotídico como proteico. En resumen, esta investigación revela nuevos conocimientos sobre las propiedades evolutivas de la metilación del ADN y la interpretación de la variación no codificante entre especies.
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Mirbahai, Leda. "DNA methylation profiling of fish tumours." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3633/.
Full textAbstract:
Assessment of disease status in fish is used as an indicator of the biological effects of contaminants in the marine environment. At some UK offshore sites the prevalence of liver tumours in Limanda limanda (dab) exceeds 20%. However, the molecular mechanisms of tumour formation and the causative agents are not known. The contribution of epigenetic mechanisms, although well-established in human tumourigenesis, is under-studied in tumours of aquatic species. In this thesis, alteration in the DNA methylation patterns in tumours of two fish species, the model species zebrafish (Danio rerio) and the un-sequenced marine flatfish dab, were investigated. The data presented provided a comprehensive characterisation of DNA methylation pattern in zebrafish liver and the first evidence of alterations in DNA methylation profiles of key genes in tumourigenesis pathways in any aquatic species. A statistically significant lower level of global DNA methylation was demonstrated in hepatocellular adenoma (HCA) and non-cancerous surrounding liver tissue (ST) compared to liver of non-cancer bearing dab. The evidence presented in this thesis suggests that chronic exposure to a mixture of pollutants contribute to global DNA hypomethylation followed by further epigenetic and genomic changes, leading to the development of tumours in dab. These findings suggest a link between the environment, epigenome and cancer in fish tumours in the wild.
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Chan, Michelle M. (Michelle Mei Wah). "DNA methylation in early mammalian development." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81580.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references.
All the cells in the body contain the same genome yet showcase drastically different phenotypes. This is the result of different transcriptional programs, which are partly controlled by epigenetic modifications, including DNA methylation. In this thesis, I analyze genome-scale DNA methylation profiles across pre-implantation development to identify the targets and characterize the dynamics of global demethylation that lead to totipotency and the subsequent changes to embryonic specification. In Chapter 1, I validate and refine the decades old model for DNA methylation in mouse embryogenesis, identify many retrotransposons with active DNA methylation signatures at fertilization, and discover many, novel differentially methylated regions between the gametes that exist transiently during early development. Notably, the majority of epigenetic events unique to mammalian pre-implantation development are characterized in mouse. In Chapter 2, 1 describe the DNA methylation dynamics in human preimplantation development and show that the regulatory principles that operate in mouse are conserved, though some of their targets are species-specific and define regions of local divergence. Finally, in Chapter 3, I compare DNA methylation dynamics of fertilization to an artificial reprogramming process, somatic cell nuclear transfer, in mouse, and find that most dynamics are conserved but occur at a smaller magnitude after artificial reprogramming. I conclude this thesis with a summary of the chapters and a brief discussion of ongoing and future work.
by Michelle M. Chan.
Ph.D.
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Patel, Yogen. "DNA methylation analysis of Alzheimer's disease." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/dna-methylation-analysis-of-alzheimers-disease(f66ad885-3fdd-4c12-a73a-921cc31ccac2).html.
Full textAbstract:
There is evidence for a role for epigenetic mechanisms in Alzheimer's disease (AD), the most common age-dependent neurodegenerative disorder. The most studied epigenetic mark DNA methylation -the addition of a methyl group to cytosines located in CpG dinucleotides (5mC) - is known to change with aging and may reflect subtle changes in gene expression. Recently a second type of modified cytosine - a hydroxylated and methylated form (5hmC) - has been detected in the brain and maybe linked to the regulation of gene expression. Case-control differences in post-mortem brain DNA methylation were sought by examining both global DNA methylation and DNA methylation of two candidate genes relating to AD risk factors. Simultaneous assessment of 5mC and 5hmC methylation at a global level indicate hypomethylation of 5mC and hypermethylation of 5hmC in AD brain relative to controls, consistent with the notion that 5hmC serves as an intermediary form for demethylation of 5mC. Age was separately associated with a decrease in LINEl methylation and an increase in 5hmC methylation. The comorbidity of depression in AD was explored by assessing the methylation status of the serotonin transporter (SERT) gene promoter across several brain areas and showed tentative associations of disease with SERT CpG methylation. These measurable differences are very small and unlikely to represent any biological plausibility. In a subset of AD patients with additional clinical and behavioural measures there was no effect of SERT 5HTTLPR genotype on DNA methylation. The hypothesis that amyloid- deposition in brain is a consequence of amyloid precursor protein (APP) gene over-expression was examined by measuring DNA methylation across the APP gene region. AD status associates with methylation levels of several CpG sites within the 5' region CGI shore and exon 5 of the APP gene. However there are no co-occurring separate associations of total APP protein levels at these CpG sites. This study demonstrates the utility of the Fluidigm microfluidics platform to generate highly parallel bisulphite sequencing/base pair resolution CpG data.
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Currie, Graeme M. "DNA methylation at cytosine position 5." Thesis, Aston University, 1992. http://publications.aston.ac.uk/12603/.
Full textAbstract:
DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.
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Warnecke, Peter. "DNA methylation in early mammalian development." Thesis, The University of Sydney, 1998. https://hdl.handle.net/2123/27569.
Full textAbstract:
In mammalian genomes, the base cytosine is frequently modified to
S-methylcytosine by the action of DNA methyltransferases. DNA methylation may
affect gene expression within the cell by acting to repress transcription, and has been
suggested to be involved in numerous cellular processes including tissue-specific gene
control, repression of transposon activity, X-chromosome inactivation, genomic
imprinting and tumourigenesis. While the pattern of mammalian DNA methylation is
typically stable in the adult, widespread changes occur to genomic methylation levels
during embryonic development, with a generalised demethylation of the genome at
implantation followed by extensive de novo methylation in the postimplantation
embryo. Embryonic changes to genomic methylation levels are important in
determining the adult methylation state, however limitations in the methods used to
detect DNA methylation have prevented a detailed study of DNA methylation in the
embryo.
Bisulfite sequencing is a technique for the detection of 5—methylcytosine that
offers considerable advantages over previously developed methods, and may be used to
characterise DNA methylation in detail. However, bisulfite sequencing has not
previously been used for the detection of DNA methylation in embryonic samples. In
this project, the bisulfite sequencing technique is adapted for the detection of DNA
methylation from mouse embryos, and the methylation state of single—copy genes
determined throughout embryonic development. The various parameters affecting DNA
methylation analysis in mouse embryos are examined.
The CpG island within the promoter of the Rb gene was found to be completely
unmethylated through all stages of development, supporting a model in which the CpG
island is protected from de novo methylation.
The methylation state of an allele-specific methylation imprint located 5‘ to the
imprinted H19 gene was determined, and found to be methylated only at the paternal
allele for all developmental stages examined. However, unlike the Rb CpG island, the
control of allele—specific methylation was not absolute, with a low level of
demethylation at the paternal allele and a low level of methylation at the maternal allele
observed within the area bounded by the methylation imprint. The methylation of a
boundary region for the H19 methylation imprint was also characterised.
The behaviour of the H19 methylation imprint in mutant ES cells deficient in the
DNA methyltransferase Dnmt-I was examined, compared to wild—type ES cells and
"rescued" ES cells in which Dnmt—I function had been restored. The H19 methylation
imprint in mutant ES cells was found to be completely erased, and not reinstated in the
rescued ES cells. The area containing the H19 methylation imprint was found to be
relatively resistant to de novo methylation.
Methylation at the promoter of the skeletal Ot-actin gene, which exhibits a tissuespecific
pattern of expression, was analysed in both embryos and differentiated tissues.
The embryonic methylation profile was found to be broadly similar to previous models
of tissue-specific genes. In the adult, no consistent correlation was found between
promoter demethylation and tissue—specific gene expression.
The implications of these analyses of embryonic DNA methylation are
discussed, in terms of both the technical aspects of DNA methylation deteCtion
techniques and for the regulation of gene expression in the cell.
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Mischke, Mona. "DNA methylation of the POMC gene." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16456.
Full textAbstract:
Adipositas ist eine polymorphe chronische Erkrankung mit epidemischer Prävalenz. Im katabolen Leptin-Melanocortin-Signalweg ist das Proopiomelanocortin Gen (POMC) ein zentrales Element, das bei Dysfunktion massive Adipositas bewirken kann. Auch eine kürzlich identifizierte intragenische Methylierungsvariante des POMC wurde mit Adipositas assoziiert und deutet somit auf eine mögliche epigenetische Modulation des Gewichtsphänotyps hin. Zur Aufklärung der Relevanz, Stabilität und Entwicklung dieser epigenetischen Modifikation wurden die Funktionalität, Ontogenese und Phylogenese der POMC DNA-Methylierung untersucht. In vitro Analysen zeigten DNA-Methylierungsabhängige Promoteraktivität beider CpG-Inseln (CGIs) des POMC. Diese hier erstmals beschriebene Transkriptionsaktivität der intragenischen CGI weist auf einen alternativen Promoter des POMC hin. Hinsichtlich der Ontogenese konnten in Mensch und Maus postnatal stabile DNA-Methylierungsmuster mit interindividueller Konservierung für beide CGIs des POMC identifiziert werden. Zusätzlich erwiesen sich Gewebeunabhängigkeit der DNA-Methylierungsmuster und ihre pränatale Ausbildung zwischen dem Blastocystenstadium und der frühen Organogenese in der Maus. Die POMC DNA-Methylierungsmuster upstream des Exon3 unterscheiden sich in Mensch und Maus. Der mögliche Einfluss von primatenspezifischen Alu-Elementen im Intron2 des POMC hierauf wurde in verschiedenen Primatenfamilien analysiert. Die Ergebnisse zeigen eine bedingte Assoziation der Alu-Elemente mit der DNA-Methylierung in der entsprechenden Region, lassen jedoch auch weitere Einflussfaktoren vermuten. Insgesamt zeigt diese Arbeit, dass die POMC DNA-Methylierung artspezifisch konserviert ist und in der frühen Embryogenese, vermutlich Alu-abhängig, ausgebildet wird. Dabei könnten stochastische Variationen der DNA-Methylierung die POMC-Aktivität beeinflussen und somit das Risiko für Adipositas erhöhen.Obesity is a polymorphic chronic disease with epidemic prevalence. Within the catabolic leptin-melanocortin signaling pathway pre-proopiomelanocortin (POMC) is a pivotal element. Dysfunction of POMC, e.g. due to mutations, can cause severe obesity. Moreover, a recently identified intragenic methylation variant of POMC was found to be associated with obesity. Therefore, this indicates potential epigenetic modulation of the weight phenotype. To gain further insight into the relevance, stability, and origin of this epigenetic modification, the functionality, ontogenesis, and phylogenesis of the POMC DNA methylation patterns were analyzed. In vitro analyses revealed DNA methylation-dependent promoter activity of both CpG islands (CGIs) of POMC. Thereby, the intragenic CGI was identified as a potential alternative promoter of POMC, which has not been described before. Regarding the ontogenesis, postnatally stable POMC DNA methylation patterns with interindividual conservation were detected for both CGIs in humans and mice. In addition, it was observed that the POMC DNA methylation patterns are non-tissue-specific, stable upon long time administration of a high fat diet, and develop prenatally between the blastocystal stage and the early organogenesis. The POMC DNA methylation pattern upstream of exon3 differs in humans and mice. A possible influence of primate-specific Alu elements within the intron2 region of POMC was analyzed in various primate families. Results evince a partial association of the Alu elements with the DNA methylation pattern in this particular region, but also suggest an influence of additional factors. Overall, this work demonstrates that DNA methylation of the POMC locus is species-specific highly conserved, and that it is established during early embryogenesis, possibly Alu-triggered. In the course of this, stochastic variances of the POMC DNA methylation might influence the POMC activity and consequently alter the risk to develop obesity.
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Aguirre-Arteta, Ana Maria. "Regulation of DNA methylation during development." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2000. http://dx.doi.org/10.18452/14509.
Full textAbstract:
Die DNA Methyltransferasen sind verantwortlich für die spezifische Methylierung von DNA-Basen. Mehrere DNA Methyltransferasen sind bekannt, wobei die Dnmt1 das hauptsächlich vorkommende Enzym ist. Bei Säugetieren korreliert die DNA-Methylierung mit der Genaktivität und ist essentiell für die Embryonalentwicklung. Eine beeinträchtigte Funktion oder Verfügbarkeit des Enzyms kann zu pathologisch veränderten Zuständen führen. Die Regulation der Dnmt1 und die damit verbundene Bedeutung bei der Entstehung von Krankheiten ist bisher nur unvollständig untersucht. In der Frühphase der Embryonalentwicklung von Säugetieren ändert sich das Methylierungsmuster des Genoms dramatisch. In zeitlich aufeinander folgenden Phasen wird die DNA demethyliert (Verlust der Methylgruppen) und neu methyliert (De-Novo Methylierung). Die Hypothese dieser Arbeit ist, dass verschiedene Isoformen der Dnmt1 in spezifischen Entwicklungsstadien exprimiert werden und zu Veränderungen des Methylierungsmusters der DNA beitragen. Um diese Regulation zu untersuchen, wurde die Struktur der Maus Dnmt1-Gens bestimmt. Außerdem wurde in verschiedenen Gewebetypen die Transkriptionsgröße und die Transkriptionsintensität der mRNA mit Hilfe von Northern-Blots quantifiziert. Mit diesen Experimenten konnte im Hoden- und Skelettmuskelgewebe ein längeres Dnmt1-Transkript als in anderen Geweben identifiziert werden. Dieses neue Dnmt1-Transkript wurde mit Hilfe von RT-PCR und RACE-Techniken kloniert und ist in beiden Geweben identisch. Es unterscheidet sich auf DNA-Ebene in der Sequenz des 5'-Endes von der bisher bekannten Form der Dnmt1 und besitzt einen anderen Startpunkt für die Transkription. Darüber hinaus besitzt das neue Dnmt1-Transkript ein 800 Basenpaar großes erstes Exon, welches sich von dem des bekannten Dnmt1-Transkripts unterscheidet. Die spezifische zelluläre Lokalisation des neuen Transkripts wurde mit Hilfe der In-Situ-Hybridisierung analysiert. Mit dieser Technik wurde das alternative Transkript in stärker spezialisierten, haploiden spermatogenen Zellen (Spermatiden) und zu einem geringen Maß im Skelettmuskel nachgewiesen. Während der Differenzierung von Muskelzellen wurde eine verminderte Expression des bereits bekannten mRNA-Transkripts und eine verstärkte Expression des neu identifizierten mRNA-Transkripts festgestellt. Obwohl die mRNA der alternativen Isoform verschiedene, kurze offene Leserahmen enthält, welche die Translation eines spezifischen Dnmt1 Proteins verhindern könnten, wurde durch Immunofluoreszenz- und Western-Blot Analysen ein Translationsprodukt nachgewiesen. Nach den hier aufgezeigten Ergebnissen werden alternative Dnmt1 Isoformen in vivo exprimiert, welche eine aktive Rolle bei der Regulation der DNA-Methylierung spielen könnten.DNA methyltransferases (DNA MTases) are enzymes responsible for DNA methylation (transfer of methyl groups to a base in the DNA) and are vital for the development of mammals. Several MTases have been identified in eukaryotes but the most abundant is Dnmt1. Furthermore, many pathological conditions are often attributed to an altered availability or function of this enzyme, however the understanding of the regulation of Dnmt1 and the concomitant relationship to diseases is far from being complete. In mammals the methylation of DNA correlates with gene activity, and methylation patterns change dramatically during early development when the genome of the mammalian embryo undergoes consecutive waves of demethylation (loss of methylation) and de novo methylation (methylation of DNA sites that have not been previously methylated). The hypothesis of this study was that alternative Dnmt1 isoforms are expressed at specific developmental stages and thus contribute to changes in the DNA methylation pattern. To study this regulation the structure of the Dnmt1 gene was determined. In this work, the tissue distribution and abundance of Dnmt1 mRNA was analyzed by Northern blot and a new, longer transcript was identified that is present in testis and skeletal muscle tissue. The novel isoform was cloned by a combination of RT-PCR and RACE techniques and found to be identical in both tissues. This new isoform differs from the ubiquitous cDNA in the 5' end, utilizing a new transcriptional start site and an 800 bp long alternative first exon. The cellular localization of this new transcript was determined by in situ hybridization and found to be present in the more specialized haploid spermatogenic cells, spermatids and at lower level in skeletal muscle. During muscle differentiation, the ubiquitous isoform is downregulated while the alternative isoform is upregulated. Although this mRNA codes for several short upstream ORFs which could prevent translation of the Dnmt1-specific ORF, it was found by immunofluorescence and Western blot analyses that this transcript can be translated in vivo producing a shorter Dnmt1 protein. The results shown here indicate that alternative Dnmt1 isoforms are expressed in vivo and might play an active role in the regulation of DNA methylation.
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Ibrahim, Abdulkhaleg. "Regulation of DNA methylation by DNA glycosylases MBD4 and TDG." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ019/document.
Full textAbstract:
Chez les mammifères, la méthylation est une marque épigénétique ciblant la cytosine principalement dans un contexte CpG pour produire une 5mC. 5mC est très sensible à une déamination spontanée ou enzymatique, conduisant à la formation d'un mésappariement G/T. La 5mCpeut également être oxydée pour former successivement la 5hmC, la 5fC et la 5caC. Ces modifications de la 5mC participent aux processus actifs de déméthylation de l’ADN. Chez les mammifères, la thymine, dans le mésappariement G/T, est clivée par TDG et MBD4. TDG est également en mesure d'exciser 5fC et 5caC. Cette thèse avait pour but de clarifier la fonction de TDG et MBD4 dans la dynamique de la 5mC. Nous avons montré que MBD4 est associée aux protéines de réparation des mésappariements. Les tests enzymatiques, in vitro, montrent que le complexe MBD4/MMR a une activité bifonctionnelle (glycosylase/lyase) spécifique pour G/T, qui est régulée par la méthylation. Pour TDG, nous avons ciblé cette enzyme dans les cellules MEF et caractérisé la distribution des cytosines modifiées. Les résultats montrent des profils de méthylation/oxydation d'ADN qui sont régulés par TDG et surviennent principalement au niveau des répétitions de CA et dans les rétroéléments spécifiques de la lignée sourisIn mammals, methylation is an epigenetic mark targeting cytosine mainly in a CpG context, producing 5mC. 5mC is highly sensitive to a spontaneous or enzymatic deamination leading to G/Tmismatch. 5mC can also be oxidized to 5- 5hmC, 5fC and 5caC. These modifications of 5mC participate in the active demethylation processes. In mammals, the thymine in G/T mismatch is cleaved by TDG and MBD4 glycosylases. TDG is able also to excise the 5fC and 5caC.This thesis was to clarify the function of TDG and MBD4 in the dynamics of 5mC. We showed that MBD4 is associated with PMS2, MLH1, MSH2 and MSH6 proteins, four proteins involved in DNA mismatch repair (MMR). The in vitro enzymatic tests show that MBD4/MMR complex has a bifunctional glycosylase/lyase activity specific for G/T and is regulated by methylation.For TDG, we targeted this enzyme in MEF cells and characterized the distribution of modified cytosines. The results show that DNA methylation/oxidation patterns are regulated by TDG and occur mainly at CA repeats and at the mouse-lineage specific retro-elements
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Devailly, Guillaume. "Les protéines MBD2 et ZBTB4 répriment la transcription de nombreux gènes méthylés. MBD2 est redistribuée sur l’ADN méthylé dans des modèles de transformation oncogénique." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10316.
Full textAbstract:
La méthylation de l'ADN est une marque épigénétique répressive impliquée dans de nombreux processus physiologiques et pathologiques. Des hyperméthylations de promoteurs sont ainsi responsables de répressions transcriptionnelles de gènes suppresseurs de tumeurs dans les cancers. La méthylation de l'ADN serait capable d'induire une répression transcriptionnelle par la combinaison de deux mécanismes principaux : l'éloignement de facteurs de transcription activateurs, et le recrutement de protéines répressives liant spécifiquement l'ADN méthylé. MBD2 est une protéine de liaison à l'ADN méthylé capable de recruter les complexes répresseurs NuRD et SIN3A. ZBTB4 est capable de se lier à l'ADN méthylé in vitro et induit une répression de la transcription de plasmides méthylés lorsqu'elle est surexprimée. Son rôle de répresseur transcriptionnel dépendant de la méthylation de l'ADN reste toutefois peu documenté. Nous avons identifiés par RNAseq les modifications du transcriptome induites par une déplétion de MBD2 ou de ZBTB4. Les gènes surexprimés après déplétion de MBD2 ou ZBTB4 sont méthylés sur leur promoteur, et sont aussi surexprimés après traitement avec des agents déméthylants. Des résultats d'immuno-précipitations de chromatine réalisées contre les deux protéines endogènes montrent que la quasi-totalité des sites de fixation de MBD2 et qu'une partie des sites de fixations de ZBTB4 correspondent à des régions méthylés. Ces résultats confirment à l'échelle du génome que MBD2 endogène est bien un interprète majeur de la méthylation de l'ADN, et que ZBTB4 réprime bien la transcription de gènes méthylés. Nous avons aussi observé une redistribution importante de MBD2 sur le génome dans des modèles de progression tumorale. Nos résultats montrent que les gènes réprimés pendant la transformation oncogénique le sont en partie par MBD2. L'expression de certains de ces gènes peut être induite dans les lignées transformées par déplétion de MBD2 par siRNADNA methylation is an epigenetic mark that plays a role in many physiological and pathological processes. Indeed, silencing of tumor suppressor genes in cancer is frequently caused by promoter hypermethylations. Transcriptional repression induced by DNA methylation is likely caused by the combination of two mechanisms: the repulsion of activator transcription factors, and the recruitment of repressor proteins able to specifically recognize methylated DNA. MBD2 is a methyl DNA binding protein that cans recruits NuRD or SIN3A repressor complexes. ZBTB4 is able to bind methylated DNA in vitro, and can repress the transcription of methylated plasmids when overexpressed. Its methylationdependent transcriptional repressor function remains poorly documented. By RNAseq, we have identified transcriptomic modifications induced by the depletion of either MBD2 or ZBTB4. Genes up regulated after MBD2 or ZBTB4 depletion were methylated on their promoter, and were also up regulated after treatment with demethylating agents. Chromatin immunoprecipitations experiments against endogenous proteins showed that almost all MBD2 binding sites, and that a part of ZBTB4 binding sites, correspond to methylated DNA regions. These results confirmed at genome wide scale that endogenous MBD2 is a major reader of DNA methylation and that ZBTB4 does repress the transcription of methylated genes. We observed an important redistribution of MBD2 on the genome in models of tumor progression. Our results showed that MBD2 plays role in gene repressions occurring during oncogenic transformation. Some of those repressed genes can be re-expressed in transformed cell lines after depletion of MBD2 by siRNA
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Pichler, Garwin. "Crosstalk between DNA methylation and histone modifications." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-143799.
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