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1
Müller, Sara [Verfasser]. "Biologische Funktionsanalyse und Identifizierung neuer Substrate der Methyltransferase Dnmt2 / Sara Müller." Kassel : Universitätsbibliothek Kassel, 2012. http://d-nb.info/101926845X/34.
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Kiani, Jafar. "Hérédité épigénétique et méthylation des ARNs : rôle de la méthyltransférase Dnmt2." Nice, 2011. http://www.theses.fr/2011NICE4093.
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Epigenetics deals with heritable alterations in gene expression that is not based on changes n DNA sequence. It was thought that DNA methylation and chromatin-encoded epigenetic information were the only epigenetic marks transmitted to the following generations. Recently, our group described the role of RNA in hereditary epigenetic variation, paramutation in mice. The role of RNA was demonstrated by the establishment of a heritable phenotype following microinjection into one-cell embryos of Kit heterozygous sperm RNA. It was further confirmed by induction of hereditary phenotypes after microinjection of an oligoribonucleotide with a Kit RNA sequence or small noncoding RNAs (miR-222 and miR-124). We now report that, Dnmt2, a RNA methyltransferase, is required for induction by small non-coding RNAs of hereditaty epigenetic variation of expression of the Kit and Sox9 genes, inactivation of the Dnmt2 gene precluded their occurrence. Kit* paramutants, which maintain a mutant phenotype with a Kit+/+ genotype, were not observed in the progeny of crosses between Dnmt2-/- Kittm1al∫+ heterozygotes, no were they generated by microinjection in fertilized eggs of RNAs of the Dnmt2-negative Kit heterozygotes. The Sox9 “giant” phenotype was similary not generated by miR-124 RNA in Dnmt2-/- embryos. Interaction of the Dnmt2 protein with Kit RNA was evidenced by co-immunoprecipitation assays. Bisulfite sequencing assays detected Dnmt2-dependent cytosine methylation in Kit RNA, exclusively in embryos undergoing the modification. RNA methylation effected by Dnmt2 appears as a key feature of the induction of epigenetic variations by non-coding RNAs. In the other hand, growing evidence indicates thet ncRNAs play a key role in regulation of basal transcription machinery. Several studies have recently revealed that noncoding RNAs such as B2RNA, 7SK and U1 snRNA are involved in the regulation of CTD phosphorylation of RNA polymerase II by modulating kinase activity of Cyclin H/T1. Here we reported cardiac hypertrophy in Dnmt2 mutant mice which is accompanied by enhanced activity of RNA polymerase II. Our results showed significant changes in the expression profiles of B2 RNA abd ASK in wild type compare to Dnmt2 mutant mice. In this study, we are attempting to determine that methyl transferase activity of Dnmt2 has a potential role in epigenetic inheritance and pathology.
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Schuster, Isabelle [Verfasser]. "Strukturelle und funktionelle Charakterisierung des Dnmt2-Homologs DnmA von Dictyostelium discoideum / Isabelle Schuster." Kassel : Universitätsbibliothek Kassel, 2016. http://d-nb.info/1101616091/34.
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Hartmann, Mark [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Centromeric tRNA and Dnmt2-mediated Methylation in Mitotic Chromosome Segregation / Mark Hartmann ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177148862/34.
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Durdevic, Zeljko [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Characterization of the Biological Function of Dnmt2 in Drosophila melanogaster / Zeljko Durdevic ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://d-nb.info/1177249774/34.
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Kaiser, Steffen [Verfasser]. "Investigations on DNA methylation by Dnmt2 and impact of tRNA modifications on TLR7 stimulation / Steffen Kaiser." Mainz : Universitätsbibliothek Mainz, 2015. http://d-nb.info/1080401431/34.
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Shanmugam, Raghuvaran [Verfasser], and Albert [Akademischer Betreuer] Jeltsch. "Biochemical characterisation of tRNA-Asp methyltransferase Dnmt2 and its physiological significance / Raghuvaran Shanmugam. Betreuer: Albert Jeltsch." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2014. http://d-nb.info/1049931661/34.
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Vieira, Gilberto Cavalheiro. "Modelagem molecular e imunodetecção de DNA Metiltransferases 2 de Drosofilídeos : uma abordagem evolutiva da enigmática DNMT2." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2015. http://hdl.handle.net/10183/117889.
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A metilação do DNA genômico é um dos principais mecanismos de regulação epigenética nos organismos. Dentre as diferentes classes de DNA MTase, as m5C-MTase são as que se distribuem amplamente de procariotos a eucariotos. Em vertebrados existem três diferentes famílias: DNMT1, DNMT2 e DNMT3a e 3b. A DNMT1 possui atividade junto ao DNA hemimetilado. As DNMT3a e 3b são responsáveis pela metilação de novo. Já a subfamília DNMT2 possui seus sítios catalíticos altamente conservados, desde procariotos até eucariotos, possuindo propriedades que permitem executar funções tanto de metilação de novo, assim como de manutenção de metilação. Além disso, as enzimas da família DNMT2 podem atuar metilando citosinas genômicas ou de tRNAs. Em mamíferos, invertebrados e plantas a DNMT2 é classificada, prioritariamente in vivo como uma tRNA MTase. Entretanto, já foi descrita atividade de DNA MTase por parte dessas enzimas, mesmo que em baixos níveis. O que se discute são as atividades preferenciais da DNMT2 e os mecanismos que modulam sua atividade, pois se reconhece que em organismos que não possuem as MTases canônicas (DNMT1 e DNMT3), mas apresentam metilação em seu genoma, é a DNMT2 que atua como MTase em ambos os substratos. Espécies de Drosophila são conhecidas como de Dnmt2-only, justamente por possuírem apenas a DNMT2 na função de metilação de citosinas. A importância de seu papel no âmbito ecológico e evolutivo nesse grupo de espécies se reflete na presença de fenômenos peculiares, como a metilação sexo-específica presente em espécies do subgrupo willistoni de Drosophila, descrito por nosso grupo de pesquisa. No presente trabalho realizou-se a modelagem das enzimas DNMT2 de D. melanogaster, D. willistoni e Mus musculus com diferentes metodologias. A partir desses modelos realizaram-se análises comparativas com as estruturas cristalográficas de DNMT2 depositadas no banco de dados PDB, com o objetivo de estabelecer as relações evolutivas e funcionais entre as diferentes enzimas. Adicionalmente, de posse de modelos de DNMT2 - de validada qualidade - de duas espécies pertencentes a diferentes grupos evolutivos de drosofilídeos, realizaram-se estudos de caracterização evolutiva e estrutural das 22 espécies que tiveram seus genomas sequenciados e depositados no bando de dados Flybase, somando-se a essas a sequência de DNMT2 de Drosophila tropicalis (subgrupo willistoni), sequenciado por nosso grupo de pesquisa. Os resultados das análises evolutivas e estruturais sugerem propriedades diferenciais entre as DNMT2 de espécies do subgrupo willistoni em relação às demais. Estes resultados indicam que mesmo em espécies que possuem relações evolutivas próximas possam ocorrer mecanismos adaptativos que estabeleçam gradações na afinidade das DNMT2 por diferentes substratos, sem que para isso ocorram drásticas mudanças na arquitetura da enzima.The methylation of genomic DNA is a major mechanism of epigenetic regulation in organisms. Among the different classes of DNA MTase the M5C-MTase are as widely distributed in prokaryotes to eukaryotes. In vertebrates there are three different families: DNMT1, DNMT2 and DNMT3a and 3b. The DNMT1 has activity with the hemimethylated DNA. The DNMT3a and 3b are responsible for de novo methylation. While the DNMT2 subfamily has its catalytic sites highly conserved from prokaryotes to eukaryotes, having properties that allow performing functions of both de novo and maintenance methylation. Furthermore, the DNMT2 family can act methylating cytokines, tRNAs or DNA. In mammals, invertebrates and plants, DNMT2 is classified primarily in vivo as a tRNA MTase. However,DNA-MTase activity by these enzymes has been described, even at low levels. The question is the preferred activities of DNMT2 and mechanisms that modulate its activity, because in organisms that do not have the canonical DNA-MTases (DNMT1 and DNMT3), but have cytokines methylated in its genome, the DNMT2 acts as MTase on both substrates. Drosophila species are known as Dnmt2-only. The importance of DNMT2’s role in ecological and evolutionary context in this species group is reflected by presence of a peculiar phenomenon: the sex-specific methylation described by our research group, presents in willistoni subgroup of Drosophila. In this study, DNMT2 of D. melanogaster, D. willistoni and Mus musculus were modeled by different methodologies. comparative analyzes with the crystallographic DNMT2 structures deposited in the PDB database were performed from these models, in order to establish the evolutionary and functional relationships between the different enzymes. Additionally, evolutionary and structural characterization studies of the 22 species, with the validated DNMT2 models of two species belonging to different evolutionary drosophilids groups, were conducted that have had their genomes sequenced and deposited in FlyBase data pack, adding the DNMT2 sequence of Drosophila tropicalis (willistoni subgroup) sequenced by our research group. The results of evolutionary and structural analyzes suggest differences between the DNMT2 properties of subgroup willistoni species from the other drosophilids. These results indicate that even species that have close evolutionary relationships may have adaptive mechanisms that establish gradations of DNMT2 affinity for different substrates, without the need of drastic changes in enzyme architecture.
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Liebers, Reinhard Kai [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Dnmt2 in RNA methylation, RNA inheritance, and environmental responses in the mouse / Reinhard Kai Liebers ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180614186/34.
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Liebers, Reinhard [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Dnmt2 in RNA methylation, RNA inheritance, and environmental responses in the mouse / Reinhard Kai Liebers ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2016. http://d-nb.info/1180614186/34.
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FILIP, Kamila Maria. "Effects of 5-Azacitidine on Dnmt2/Trdmt1 expression levels and endoplasmic reticulum stress in cellular models of insulinoma." Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/514949.
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Diabetes mellitus affects people all over the world of all ages or social groups making it a
worldwide healthcare challenge. Moreover, untreated or badly treated diabetes carries a risk
of serious complications including premature death (IDF Diabetes Atlas 9th edition, 2019).
The main symptom of diabetes is insulin secretion and/or action disorder leading to
hyperglycemia what results in impaired carbohydrate, fat, and protein metabolism
(“Diagnosis and Classification of Diabetes Mellitus,” 2013).
Dnmt2/ Trdmt1 in its structure and sequence is similar to DNA methyltransferases, however,
it has been shown that mainly methylates aspartic acid transfer RNA, specifically at the
cytosine-38 residue (Goll et al., 2006; Okano et al., 1998). tRNA methylation ensures
accurate protein synthesis, tRNA structure stabilization, codon-anticodon interaction
strengthening, and prevention from any mistakes in a frameshift (Hori, 2014). Different
tRNA deficiency and various enzymes modifying ribonucleotides in tRNAs can disturb ER
function. In the presented study we wanted to explore the potential role of 5-Azacitidine and
Dnmt2/Trdmt1 in pancreatic β-cells.
To investigate Trdmt1, ER stress, senescence, and aging and potential implications in
diabetes insulin production and but also possible promotion of senescent cells elimination,
insulinoma pancreatic β-cells were treated with two compounds: hydrogen peroxide, a wellknown pro-oxidant inducing aging, and SISP, and 5-Azacitidine, an inhibitor of DNA
methylation and tRNA methylation suppressor at C38 position which is a major Trdmt1
target (Lewinska et al., 2018; Schaefer et al., 2009).
In the presented study two insulin secreting pancreatic β-cell lines were treated with
hydrogen peroxide and 5-Azacitidine. NIT-1 is an insulinoma cell line established from
NOD/Lt (nonobese diabetic) mice and transgenic for the SV40 T-antigen. BetaTC6 is
derived from insulinoma transgenic mice expressing SV40 T-antigen. Both cell lines secrete
insulin in a response to the presence of glucose in the medium. Moreover, NIT-1
spontaneously develops type 1 diabetes what makes it a good model to understand T1D
pathogenesis and treatment (Pearson et al., 2016; Poitout et al., 1995).
Presented results indicate 5-AzaC mediated endoplasmic reticulum stress in pancreatic βcells thus UPR and UPS activation. 5-AzaC induced tRNA methylation inhibition at the
Trdmt1 target site in T1D representative leads to ER stress mediated autophagy pathway
which prevents apoptosis and maintains proliferation rate. Furthermore, 5-AzaC boosted 5
insulin production in the non-obese diabetic cell line. It has been shown that BetaTC6 cells
are more vulnerable to stress and HP induced excessive NO production, ER stress, and
related activation of apoptotic cell death pathway and decreased proliferation. Moreover, HP
mediated premature senescence, signaling inhibition, and proliferation blockage of senescent
cells indicates its senostatic activity and HP/5-AzaC mediated selective elimination of
senescent cells treatment demonstrates senolytic activity. Additionally, Trdmt1 may help to
cope with the cellular stress induction either by augmented translocation to the cytoplasm or
increased biosynthesis. 5-AzaC modulates insulin secretion by senescent insulinoma cells
and enhanced insulin production in the NOD cell line may be considered as a factor
preventing T1D. The combined therapy of prooxidants and 5-AzaC can be studied as a
promising candidate for insulinoma treatment.
12
Becker, Maria [Verfasser], Ann [Akademischer Betreuer] Ehrenhofer-Murray, and Bernhard [Akademischer Betreuer] Horsthemke. "Characterization of the Dnmt2 homolog Pmt1 in Schizosaccharomyces pombe / Maria Becker. Gutachter: Ann Ehrenhofer-Murray ; Bernhard Horsthemke. Betreuer: Ann Ehrenhofer-Murray." Duisburg, 2013. http://d-nb.info/1041831803/34.
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Windhof-Jaidhauser, Indra Maria [Verfasser]. "Zelluläre und biochemische Charakterisierung der bifunktionalen Methyltransferase Dnmt2 / Indra Maria Windhof-Jaidhauser. Kassel, Universität, FB 10, Mathematik und Naturwissenschaften, Institut für Biologie." Kassel : Universitätsbibliothek Kassel, 2014. http://d-nb.info/1058420232/34.
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Baudouy, Delphine. "Rôles des gènes PPARβ/δ, Wt1, Cyp51 et Dnmt2 dans l'angiogenèse et la fonction cardiaque chez la souris adulte saine et dans un modèle d'infarctus du myocarde." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4148/document.
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La coronaropathie est une cause majeure de mortalité, motivant la recherche de stratégies limitant le remodelage cardiaque ou stimulant la néovascularisation après un infarctus du myocarde (IDM). Ce travail vise à étudier chez la souris adulte le rôle, sur la fonction cardiaque, de gènes régulant l'angiogenèse et le métabolisme cellulaire en modulant leur expression endothéliale en conditions basales ou en post-IDM (après ligature coronaire) : PPARβ/δ, Wt1, Cyp51 et Dnmt2. Les paramètres échocardiographiques ont été mesurés pré et post-IDM, des analyses histochimiques réalisées, et l’expression de gènes cibles comparée selon le génotype. La surexpression de PPARβ/δ stimule l'angiogenèse basale, causant une hypertrophie ventriculaire gauche (VG). En post-IDM, elle induit un remodelage VG pathologique et majore la taille de l'IDM, posant la question des interactions entre endothélium et cardiomyocytes. En post-IDM, l'invalidation de Wt1 limite l'angiogenèse coronaire, majore le remodelage VG et la taille de l'IDM. A l'état basal, l'invalidation de Cyp51 est à l'origine d'une insuffisance cardiaque dilatée, via une perméabilité vasculaire accrue et une activation endothéliale. La modification de la composition membranaire en stérols peut expliquer la dysfonction de l'endothélium, modifiant ses interactions avec les cardiomyocytes. Ainsi, Cyp51 possède un rôle essentiel dans la structure et la fonction cardiaque, ouvrant le champ de son étude en post-IDM. Enfin, l'expression de Dnmt2 est indispensable pour limiter l'hypertrophie cardiaque, via le contrôle de l'activité de l'ARN polymérase II par la méthylation de l’ARN non codant Rn7skCoronary heart disease is a major cause of mortality, explaining the increasing interest in therapeutics targeting cardiac remodeling and neovascularization after myocardial infarction (MI). Using endothelial expression modulation in adult mice in basal or post-MI conditions (after coronary artery ligation), this work studied several genes involved in angiogenesis and cardiac metabolism, PPARβ/δ, Wt1, Cyp51 and Dnmt2, and their role in cardiac function. Echocardiographic structural and functional parameters were measured before and after MI, histochemistry analyses performed, and target genes expression compared between different genotypes. PPARβ/δ basal overexpression resulted in an increased angiogenesis and cardiac hypertrophy. After MI, it caused MI expansion through increased cardiac remodelling. This discrepancy raises the issue of communication between endothelial cells and cardiomyocytes. Endothelial Wt1 expression is essential for cardiac repair after MI : deletion was responsible for neovascularization impairment, poorer cardiac remodeling and MI enlargement. Endothelial Cyp51 expression is necessary for basal cardiac structure and function. After Cyp51 deletion, membrane and cell junction disorganization caused increased vascular permeability and endothelium activation, resulting in dilated cardiomyopathy. The accumulation of toxic oxysterols or lack of cholesterol might account for endothelial dysfunction, through abnormal endothelial cells to cardiomyocytes signalling. Dnmt2 deletion caused cardiac hypertrophy. through methylation of non-coding RNA Rn7sk and control of RNA polymerase II activity
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Rosa, Cristiane de Santis Alves [UNESP]. "Análise funcional do papel da enzima DNA metiltransferase 2 (DNMT2) no desenvolvimento e resposta à estresses e identificação e caracterização de fragmentos derivados de tRNA (tRFs) em Arabidopsis thaliana." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/144083.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A metilação do DNA está relacionada à regulação gênica, memória celular, silenciamento de elementos transponíveis, imprinting genômico e repressão de pseudoelementos provenientes de sequências duplicadas. Os padrões de metilação são estabelecidos, mantidos e traduzidos em estados funcionais apropriados da maquinaria de metilação do DNA, a qual inclui uma família classificada em três grupos de enzimas do tipo metiltransferases: DNMT1, DNMT3 e DNMT2. A DNA metiltransferase 2 (DNMT2) foi identificada na busca de novos candidatos à uma segunda DNA metiltransferase. Esta enzima não possui função biológica definida, porém, é capaz de metilar tanto DNA quanto RNA, em especial RNA transportadores (tRNAs). A DNMT2 está localizada tanto no núcleo quanto no citoplasma em células humanas, sendo capaz de migrar do núcleo para o citoplasma em resposta a estresses celulares. É provável que a enzima metile o tRNA no citoplasma, possivelmente para protegê-lo contra clivagens em situações de estresse. Quando estas clivagens ocorrem de forma específica, pequenos fragmentos de RNA são gerados (denominados tRFs), fato observado em diversas espécies, incluindo Arabidopsis thaliana. Aparentemente, estes fragmentos de RNA fazem parte de uma nova via de interferência por RNA (RNAi). Contudo, seu papel biológico ainda não foi definido. O objetivo deste trabalho foi determinar a função da enzima DNMT2 de plantas durante o desenvolvimento e em resposta a estresses, além de estabelecer seu possível papel na proteção de tRNAs. Até o momento, foi demonstrado que a enzima AtDNMT2 possuí localização, tanto nuclear quanto citoplasmática e também pode ser visualizada em estruturas que aparentam ser citoesqueletos. Foi possível determinar que AtDNMT2 não atua na proteção do tRNA AspGTC durante estresse oxidativo, porém é positivamente regulada durante diferentes tipos de estresse. A planta mutante dnmt2 não possui...
DNA methylation is associated with genetic regulation, cell memory, silencing of transposable elements, genomic imprinting and repression of pseudo-elements coming from duplicate sequences. Methylation patterns are established, kept and translated via an appropriate functional DNA methylation machinery, which includes a family of proteins classified into three methyltransferase enzyme groups: DNMT1, DNMT3 e DNMT2. DNA methyltransferase 2 (DNMT2) was first identified by searching for novel DNA methyltransferase candidates. DNMT2 is highly conserved in different kingdoms and does not have a biological function well defined so far; however, it has been shown that DNMT2 can methylate both DNA and RNA in animal cells, most specifically transfer RNA (tRNA). In human cells, DNMT2 is localized both in the nucleus and in the cytoplasm, being capable to migrate from nucleus to cytoplasm under stress conditions. In the cytoplasm, DNMT2 methylates tRNAs, possibly to protect against cleavage events that occur under stress conditions. When these cleavages occur in a specific pattern, small RNA fragment emerges (tRFs). tRFs are found in several species, including Arabidopsis thaliana. It seems that these tRNA fragments are part of a new RNAi pathway. However, its biological role has not been reveal yet. The aim of this work is to evaluate the possible role(s) of DNMT2 in plant development and stress response and also establish its possible role in tRNA protection. So far we demonstrated that AtDNMT2 has both nuclear and cytoplasmic cellular localization and can also be visualized in what seen to be the cytoskeleton. We determined that AtDNMT2 does not play role in tRNA AspGTC protection under oxidative stress, though AtDNMT2 is up regulated in different stresses. The mutant plant Atdnmt2 does not have obvious phenotype, what makes harder to understand its biological role, leading us to deeper molecular studies. In this context, the present work reveals...
FAPESP: 13/11579-0(modelo:caso nao houver deletar/se tiver 2 n s abrir outro C)
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Rosa, Cristiane de Santis Alves. "Análise funcional do papel da enzima DNA metiltransferase 2 (DNMT2) no desenvolvimento e resposta à estresses e identificação e caracterização de fragmentos derivados de tRNA (tRFs) em Arabidopsis thaliana." Botucatu, 2015. http://hdl.handle.net/11449/144083.
Full textAbstract:
Orientador: Fabio Tebaldi Silveira NogueiraBanca:??
Resumo: A metilação do DNA está relacionada à regulação gênica, memória celular, silenciamento de elementos transponíveis, imprinting genômico e repressão de pseudoelementos provenientes de sequências duplicadas. Os padrões de metilação são estabelecidos, mantidos e traduzidos em estados funcionais apropriados da maquinaria de metilação do DNA, a qual inclui uma família classificada em três grupos de enzimas do tipo metiltransferases: DNMT1, DNMT3 e DNMT2. A DNA metiltransferase 2 (DNMT2) foi identificada na busca de novos candidatos à uma segunda DNA metiltransferase. Esta enzima não possui função biológica definida, porém, é capaz de metilar tanto DNA quanto RNA, em especial RNA transportadores (tRNAs). A DNMT2 está localizada tanto no núcleo quanto no citoplasma em células humanas, sendo capaz de migrar do núcleo para o citoplasma em resposta a estresses celulares. É provável que a enzima metile o tRNA no citoplasma, possivelmente para protegê-lo contra clivagens em situações de estresse. Quando estas clivagens ocorrem de forma específica, pequenos fragmentos de RNA são gerados (denominados tRFs), fato observado em diversas espécies, incluindo Arabidopsis thaliana. Aparentemente, estes fragmentos de RNA fazem parte de uma nova via de interferência por RNA (RNAi). Contudo, seu papel biológico ainda não foi definido. O objetivo deste trabalho foi determinar a função da enzima DNMT2 de plantas durante o desenvolvimento e em resposta a estresses, além de estabelecer seu possível papel na proteção de tRNAs. Até o momento, foi demonstrado que a enzima AtDNMT2 possuí localização, tanto nuclear quanto citoplasmática e também pode ser visualizada em estruturas que aparentam ser citoesqueletos. Foi possível determinar que AtDNMT2 não atua na proteção do tRNA AspGTC durante estresse oxidativo, porém é positivamente regulada durante diferentes tipos de estresse. A planta mutante dnmt2 não possui...
Abstract: DNA methylation is associated with genetic regulation, cell memory, silencing of transposable elements, genomic imprinting and repression of pseudo-elements coming from duplicate sequences. Methylation patterns are established, kept and translated via an appropriate functional DNA methylation machinery, which includes a family of proteins classified into three methyltransferase enzyme groups: DNMT1, DNMT3 e DNMT2. DNA methyltransferase 2 (DNMT2) was first identified by searching for novel DNA methyltransferase candidates. DNMT2 is highly conserved in different kingdoms and does not have a biological function well defined so far; however, it has been shown that DNMT2 can methylate both DNA and RNA in animal cells, most specifically transfer RNA (tRNA). In human cells, DNMT2 is localized both in the nucleus and in the cytoplasm, being capable to migrate from nucleus to cytoplasm under stress conditions. In the cytoplasm, DNMT2 methylates tRNAs, possibly to protect against cleavage events that occur under stress conditions. When these cleavages occur in a specific pattern, small RNA fragment emerges (tRFs). tRFs are found in several species, including Arabidopsis thaliana. It seems that these tRNA fragments are part of a new RNAi pathway. However, its biological role has not been reveal yet. The aim of this work is to evaluate the possible role(s) of DNMT2 in plant development and stress response and also establish its possible role in tRNA protection. So far we demonstrated that AtDNMT2 has both nuclear and cytoplasmic cellular localization and can also be visualized in what seen to be the cytoskeleton. We determined that AtDNMT2 does not play role in tRNA AspGTC protection under oxidative stress, though AtDNMT2 is up regulated in different stresses. The mutant plant Atdnmt2 does not have obvious phenotype, what makes harder to understand its biological role, leading us to deeper molecular studies. In this context, the present work reveals...
Doutor
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BAIAMONTE, Concetta. "Reactivation of SNURF-SNRPN gene by DNA Methyltransferase inhibitors in a Prader-Willi lymphoblastoid cell line." Doctoral thesis, Università degli Studi di Palermo, 2014. http://hdl.handle.net/10447/91240.
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Unterberger, Alexander. "The role of DNMT1 regulation in cellular function." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92154.
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Disruption of the epigenome and its components is a hallmark of all forms of cancer. Typically observed in cancer is an alteration of the DNA methylation pattern, with silencing of tumour suppressor genes, as well as an increase in DNA methyltransferase 1 (activity or expression). However it has yet to be determined exactly how DNMT1 increases in cancer and how this increase might serve as therapeutic target. This thesis focuses on the regulation of DNMT1 in the cell cycle and the consequences of depleting DNMT1 in cancer cells.During the cell cycle DNMT1 levels increase as the cell enters into S-phase. It has previously been shown that this cyclical regulation of DNMT1 occurs by destabilization of DNMT1 mRNA in G0/G1 through the action of a protein, identified to be the mRNA binding protein AUF1. AUF1 binds a regulator element located in the 3'UTR of DNMT1 mRNA and recruits the exosome, the RNA degradation complex, to degrade it.
When AUF1 is depleted in these cells, DNMT1 mRNA is stabilized which leads to increased DNMT1 protein levels, methyltransferase activity and genomic methylation. The changes of DNMT1 mRNA levels in the cell cycle were determined to occur as an inverse function of AUF1 protein levels. AUF1 levels were observed to decrease in S-phase which lead to increased stability in DNMT1 mRNA. This cell cycle regulation of AUF1 was determined to occur as a function of Rb. Rb actively stabilizes AUF1 protein. Indeed, upon elimination of Rb, AUF1 is degraded through the function of Hsp70 and the proteasome. This consequently leads to an elevation in DNMT1 protein levels which in turn increases genomic methylation levels. Elevated DNMT1 levels resulted in greater association with EZH2, which in turn leads to increased methylation of EZH2 targeted promoters, including p16 and CNR1. This promoter hypermethylation occurred as a function of DNMT1 and EZH2.These observations indicate that regulation of DNMT1 is tied into the cell cycle function of Rb and upon disruption of this system, a characteristic of cancer, site-specific methylation occurs at tumour suppressors, another characteristic of cancer.
Furthermore, we examined the effect of depleting DNMT1 in cancer cells. Upon depletion of DNMT1, a signaling pathway known as the replication arrest/DNA damage checkpoint was induced. Activation of this pathway results in arrest of cell growth and cell cycle blockage and occurred independently of the catalytic activity of DNMT1 and instead responded to the absence of DNMT1. This supports a role for DMNT1 as a negative regulator of the replication arrest/DNA damage checkpoint through the action of interaction with an unknown protein. Moreover, suppression of the replication arrest/DNA damage checkpoint has been determined to be a necessary step in the proliferation of cancer cells. Taken together, the data from this thesis determined that common events in cancer, such as inactivation of Rb, lead to deregulation of DNMT1 mRNA, through AUF1, leading to site-specific methylation of tumour suppressors and could potentially serve to block growth arresting checkpoints like the replication arrest/DNA damage checkpoint. The novel functions of DNMT1, such as cell cycle regulation, site-specific methylation and role in the replication arrest/DNA damage checkpoint discovered in this thesis could serve to help better understand how cancer develops. The results of this thesis could serve to develop novel strategies to target these events and better treat cancer.
L'altération de l'épigénome et de ses composants est une marque caractéristique de tous types de cancer. Une altération des profils de méthylation de l'ADN, associée à une inactivation de gènes suppresseurs de tumeurs ainsi qu'une augmentation de l'(activité/expression) de la méthyltransférase de l'ADN (DNMT1) sont largement observés dans les cancers. Cependant, les causes de cette augmentation de DNMT1 (expression/activité) dans le cancer et l'utilisation potentielle de cette augmentation comme cible thérapeutique n'ont pas encore été déterminées.
Au cours du cycle cellulaire, le niveau de DNMT1 augmente dès lors que la cellule entre en phase S. Il a été montré précédemment qu'une régulation cyclique de DNMT1 se met en place grâce à une déstabilisation de son ARN messager en phase G0/G1 sous l'action d'une protéine non identifiée. Cette protéine a été identifié comme AUF1. AUF1 interagit avec un élément régulateur situé dans la partie 3'-UTR de l'ARNm de DNMT1 et entraîne la dégradation de cet ARNm en recrutant l'exosome, un complexe de dégradation de l'ARN. La déplétion d'AUF1 stabilise l'ARNm de DNMT1 ce qui conduit à une augmentation de l'expression de cette protéine, de son activité méthyltransférase ainsi que de la méthylation du génome. Il a été également montré que le niveau d'expression de l'ARNm de DNMT1 au cours du cycle cellulaire est inversement corrélé à celui de la protéine AUF1. Ce niveau d'AUF1 est diminué en phase S ce qui traduit par une stabilité accrue de l'ARNm de DNMT1. Il a été montré que cette régulation d'AUF1 au cours du cycle cellulaire est fonction de la protéine Rb. Rb stabilise activement la protéine AUF1. En effet, AUF1 est dégradée par l'intermédiaire de la protéine Hsp70 et du protéasome. Cette dégradation a pour conséquence une augmentation du niveau d'expression de DNMT1 lequel conduit à une augmentation du niveau de méthylation du génome. De plus, cette augmentation de DNMT1 résulte en une plus grande association avec la protéine EZH2 entraînant une hyperméthylation de promoteurs de gènes ciblés par EZH2 (ex : p16, CNR1 et PCNA). Ces observations démontrent que la régulation de DNMT1 est étroitement liée aux fonctions de Rb dans le cycle cellulaire. Caractéristique dans les cancers, une rupture de cette relation DNMT1-Rb, entraîne ainsi une méthylation site-spécifique de gènes suppresseurs de tumeurs, une autre caractéristique des cancers.
En parallèle, nous avons étudié l'effet d'une déplétion de DNMT1 dans des cellules cancéreuses. Suite à une déplétion de DNMT1, une voie de signalisation connue comme un point de contrôle de l'arrêt de la réplication/lésions de l'ADN est induite. L'activation de cette voie de signalisation entraîne l'arrêt de la croissance cellulaire et le blocage du cycle cellulaire. L'activation de cette voie répond à l'absence de DNMT1 et de façon indépendante de son activité catalytique. Ceci est en faveur d'un rôle pour DNMT1 de régulateur négatif du contrôle de l'arrêt de la réplication/lésions de l'ADN via l'interaction avec une protéine qui reste encore à identifier. De plus, la suppression des points de contrôle de l'arrêt de la réplication/lésion de l'ADN a été montré comme étant une étape nécessaire à la prolifération des cellules cancéreuses. L'ensemble des données de cette thèse démontre que des événements communs aux cancers, telle que l'inactivation de Rb, peuvent conduire à la dérégulation, via AUF1, de l'ARNm de DNMT1, laquelle entraîne la méthylation site-spécifique de gènes suppresseurs de tumeurs. Cette dérégulation de DNMT1 pourrait potentiellement servir à bloquer les points de contrôle d'arrêt du cycle cellulaire/lésions de l'ADN.
Les nouvelles fonctions de DNMT1, telles que la régulation du cycle cellulaire, la méthylation site-spécifique et le contrôle de la réplication/lésions de l'ADN découverts dans cette thèse devraient permettre de mieux comprendre le développement cancéreux et de développer de nouvelles stratégies thérapeutiques.
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Balinang, Joyce. "The Regulation of Mitochondrial DNMT1 During Oxidative Stress." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2826.
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Epigenetics is the study of heritable gene expression due to alterations in the DNA structure other than the underlying DNA sequence. DNA methylation is one of the three types of epigenetic modifications found in the eukaryotic system. It involves the incorporation of a methyl group at the 5-position of cytosine residues in the DNA. DNA methylation is associated with several notorious disorders and diseases including Fragile X Syndrome, neurodegenerative disease (Parkinson’s, Alzhiemer, etc), diabetes and cancer. Cytosine methylation of mitochondrial DNA (mtDNA) was first demonstrated several decades ago but the mechanism of generating cytosine modification and its functional importance remain elusive. Our laboratory recently demonstrated that the enzyme involved in cytosine modification of mtDNA is a novel mitochondrial isoform of DNA Methyltransferase 1, mtDNMT1. This protein is encoded in the nucleus and targeted to the mitochondria via a N-terminal targeting sequence. Bioinformatic analysis of the DNMT1 coding sequence showed a consensus NRF1 binding site that coincidently overlaps a p53 binding site within the promoter region, previously shown by this group to repress DNMT1 expression. Previous studies in the Taylor laboratory showed that mtDNMT protein expression was regulated by the transcription factor NRF1 as well as its coactivator PGC1α. PGC1α and NRF1 stimulate a large body of genes that are involved in mitochondrial biogenesis and cellular respiration in response to environmental stress. Considering the previous findings in our laboratory regarding mtDNMT1 regulation and the importance of PGC1α and NRF1 in oxidative homeostasis, we asked whether there is a mitochondrial epigenetic component in the cell’s response to cellular stress and whether up-regulation of mtDNMT1 might be part of the general response to this stress. To investigate the relationship between mtDNA methylation and oxidative homeostasis we examined the regulation of mtDNMT1 by transcription factors that respond to oxidative stress. Conditions that induced oxidative stress were applied to HCT 116 and SH-SY5Y cell lines and the protein expression of DNMT1 was observed. Ethanol and hypoxia- induced oxidative stress were observed to increase to protein level of mtDNMT1 while total DNMT1 level either remained constant or decreased. The protein level of PGC1α and NRF1 remained low in HCT 116 cells exposed to hypoxic stress, despite elevated mtDNMT1 protein level. ChIP analysis of HCT 116 cells exposed to hypoxic stress demonstrated that NRF1 and PGC1α are not regulating the transcription of DNMT1i in the mitochondria. However, we observed that p53 dissociated from the DNMT1 promoter upon hypoxic stress, indicating that the up-regulation of mtDNMT1 is through the relief of p53 suppression. The findings of this investigation proved that mtDNMT1 is receptive to oxidative stress through the regulation by p53 and suggested that mitochondrial epigenetics may be playing an integral role in the cellular stress response toward hypoxia.
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Dunne, Philip D. "DNA damage response in MLH1-and DNMT1-depleted cells." Thesis, University of Ulster, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.551590.
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Every day each cell in the body is under threat from DNA damaging agents that have the potential to disrupt the passing of intact genetic information from one generation to the next. The presence of a wide variety of threats has led to the evolution of numerous DNA damage response pathways in order to deal with the damage that they may cause. The mismatch repair system is primarily responsible for the removal of incorrect base insertions and deletions occurring during replication and its importance is highlighted by its conservation from bacteria to humans. MLHl is one of the main components of this repair pathway and loss of this protein has been associated with a number of cancers, particularly hereditary colon cancer. Defects in the mismatch repair machinery have also been associated with resistance to a number of chemotherapeutic drugs and instability of the genome at repeat sequences. The present data initially examines the role of MLHl in DNA damage responses following induction of damage by a number of different treatments in telomerase- immortalised human fibroblasts stably depleted of MLHl using an integrated shRNA plasmid. The importance of a number of specific pathways, namely ATM/ATR, caspase, p53 and PARP, involved in the damage response is assessed through the use of inhibitors. A number of studies have shown that loss of the DNA methyltransferase maintenance protein DNMTl initiates p53-dependent cell death in differentiated cells. While the absence of DNMTl is tolerated in undifferentiated embryonic stem cells in terms of viability, these cells exhibit the hallmarks of mismatch repair deficiency, i.e. drug resistance and genomic instability, although transcription levels of the mismatch genes remains unaffected. Depletion of DNMTl in the colon cancer cell line RT29 resulted in induction of rapid cell death which could be ablated through the inhibition of not just p53 but also P ARP. Analysis of the mismatch repair components showed that although transcription levels were not affected, a reduction in DNMTl resulted in the depletion of a number of DNA repair proteins, suggesting that DNMTl is a key protein involved not only in DNA methylation, but also in the stability of the mismatch repair system.
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Horowitz, Evan Richard Kopp. "Dnmt1 Expression is Required for Lens Epithelial Cell Survival." Miami University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=miami1438379221.
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Stabellini, Raquel. "Análise funcional dos genes Xist e DNMT1 na manutenção do processo de inativação do cromossomo X humano através do silenciamento gênico por RNAi." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-26082008-162745/.
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A inativação do cromossomo X (ICX) é o fenômeno através do qual um dos cromossomos X das fêmeas de mamíferos é silenciado para atingir compensação de dose em relação aos machos. Ela envolve a expressão do gene XIST exclusivamente no X inativo, e a associação em cis de seu RNA nesse cromossomo. Isso inicia a imposição de várias marcas epigenéticas no cromossomo X inativo, que garantem a manutenção deste estado de silenciamento transcricional de maneira estável durante todas as mitoses num organismo. Uma dessas modificações epigenéticas é a metilação do DNA, desempenhada principalmente pela enzima DNMT1. Os papéis de XIST e DNMT1 na manutenção da inativação do cromossomo X ainda são controversos em humanos, e nesse sentido foi objetivo desse trabalho analisar a possível função desses genes nesse processo em células humanas não transformadas. Foi otimizado um sistema experimental para o estudo de possíveis perturbações na manutenção da inativação do cromossomo X, onde a re-expressão de genes submetidos a esse processo pode ser monitorada. Nesse sistema foram identificados dois genes, MAOA e GYG2, cujo padrão de expressão no X inativo difere do previamente descrito. Demonstrou-se que baixos níveis de expressão do gene XIST foram suficientes para manter seu RNA associado ao X inativo, conservando o estado silenciado desse cromossomo. Além disso, foram obtidos indicativos de que a inibição de XIST em fibroblastos humanos gera uma diminuição da viabilidade celular. Foi possível demonstrar que DNMT1 é necessária para a manutenção da metilação global do genoma em células humanas não transformadas, e que eXISTe um mecanismo de compensação da inibição desse gene que leva ao aumento da expressão de DNMT3B. Ainda se observou que a repressão de DNMT1 não é suficiente para levar à reativação de genes no cromossomo X inativo. Além disso, a desmetilação encontrada nos promotores de MAOA e XIST não foi suficiente para levar à expressão destes genes nos cromossomo X inativo e ativo, respectivamente. Estes resultados enfatizam a necessidade de se estudar os mecanismos moleculares da ICX em humanos utilizando sistemas experimentais adequados para a análise de herança epigenética.X chromosome inactivation (XCI) is the phenomenon through which one of the X chromosomes in female mammals is silenced to achieve dosage compensation related to males. It involves the expression of XIST gene exclusively from the inactive X, and the association of its RNA in cis in this chromosome. This leads to a series of epigenetic modifications in the chromatin of the inactive X (Xi) that guarantee a stable maintenance of the transcriptional silence through all the mitoses in the organism. One of these epigenetic modifications is DNA methylation, achieved mainly by the maintenance DNA methylase DNMT1. The roles of XIST and DNMT1 in the maintenance phase of XCI are controversial in humans. Therefore, the main goal of this present work was to analyze some of the possible functions of these genes in this process in untransformed human cells. An experimental system was optimized to study possible disturbances in maintenance of XCI, where the re-expression of genes submitted to this process could be monitored. In this system we identified two genes, MAOA and GYG2, whose pattern of expression on the Xi, differed from what had been previously described. It was demonstrated that low levels of XIST expression were sufficient to keep its RNA associated to the Xi, assuring the silenced state of this chromosome. Besides, evidences have been found that XIST inhibition in human fibroblasts reduces cellular viability. It was possible to demonstrate that DNMT1 is necessary to the maintenance of global genome methylation in untransformed human cells, and the eXISTence of a compensation mechanism involving DNMT3B upregulation. It was also observed that repression of DNMT1 was not sufficient to reactivate genes of the Xi chromosome. Additionally, demethylation of MAOA and XIST promoters was not enough to cause expression of these genes on the inactive and active Xs, respectively. All these results emphasize the requirement of studying the molecular mechanisms of XCI in humans using experimental systems appropriate for the analysis of epigenetic inheritance.
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Halby, Ludovic. "Conception et synthèse de nouveaux inhibiteurs de DNMT." Paris 6, 2013. http://www.theses.fr/2013PA066823.
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Ferro, Leonardo Borges. "Imuno-expressão da DNMT1, DNMT3a e DNMT3b nos tumores odontogênicos." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/23/23141/tde-20022014-162146/.
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Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores.Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.
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Pechalrieu, Dany. "Des inhibiteurs de méthyltransférases de l'ADN au développement de sondes chimiques pour l'identification de modulateurs épigénétiques dérégulés dans les cancers." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30185.
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Les méthyltransférases de l'ADN (DNMT) catalysent la méthylation de l'ADN, l'une des marques épigénétiques les plus étudiées. Dans les cancers, on observe une hyperméthylation spécifique de promoteurs de gènes suppresseurs de tumeurs (GST) conduisant à leur extinction génique, ce qui participe au maintien et à la progression de tumeurs. A ce jour, les mécanismes responsables de cette hyperméthylation spécifique des promoteurs de GST dans les cancers sont indéterminés. Ces travaux de thèse sont consacrés à l'inhibition des DNMT dans les cancers afin de restaurer l'expression des GST mais également à l'utilisation d'une approche innovante de chemobiologie pour l'identification de partenaires des DNMT potentiellement responsables de leur adressage vers les régions promotrices des GST. Les partenaires ainsi identifiés peuvent constituer de nouvelles cibles épigénétiques pour le ciblage indirect de la méthylation de l'ADN dans les cancers. Deux séries d'inhibiteurs de DNMT ont été étudiées. La première est la famille des chloronitro-flavanones, précédemment identifiée par criblage, pour laquelle de nouveaux dérivés de type bromonitro-flavanones ont été synthétisés afin d'améliorer la stabilité en conditions physiologiques. J'ai réalisé l'étude des effets pharmacologiques de cette famille de molécules. J'ai également entrepris la synthèse et la caractérisation pharmacologique de nouveaux inhibiteurs de type bi-substrats, analogues de l'adénosine et de la désoxycytidine, conçus par une approche rationnelle. Ces deux études ont permis respectivement d'identifier un dérivé flavanone plus stable et plus actif que le composé de référence et deux dérivés quinazoline-quinoléine très prometteurs, actifs sur les DNMT et dans les lignées cellulaires, à la fois pour la réexpression d'un gène rapporteur mais surtout dans l'induction de la déméthylation du GST CDKN2A et de sa réexpression. Pour identifier les partenaires de DNMT, nous avons employé une approche de chemobiologie (" Activity-Based Protein Profiling - ABPP ") basée sur la conception de sondes chimiques comportant un inhibiteur de DNMT. Ces sondes, utilisées sur des cellules vivantes, permettent, grâce à une étape de fonctionnalisation par chimie bioorthogonale, de purifier les protéines partenaires des DNMT. Vingt sondes ont été synthétisées et leurs activités ont été évaluées sur des modèles enzymatiques et cellulaires. Les sondes sélectionnées ont été utilisées dans des lignées cellulaires cancéreuses pour purifier les protéines partenaires qui ont ensuite été identifiées par analyse protéomique. Suite à leur validation, ces protéines pourront constituer de nouvelles cibles de la méthylation aberrante de l'ADN dans les cancersDNA methyltransferases (DNMTs) catalyse DNA methylation, one of the most studied epigenetic marks. In cancers, a specific hypermethylation of the promoters of the tumour suppressor genes (TSGs) is observed, which leads to their silencing. This abnormal DNA methylation pattern participates to the maintenance and the progression of the tumour. Today, the mechanisms that direct this specific hypermethylation of TSG promoters and their transcriptional repression in cancers are still unknown. The aim of my PhD is to identify DNMT inhibitors that are able to reactivated TSGs in cancer cells but also to identify the DNMT partners that address specifically these enzymes to TSG promoter regions. Such partners can constitute new anticancer "epitargets" to indirectly target DNA methylation specifically in cancer cells. Two families of DNMT inhibitors were studied. The first one starts from the chloronitro-flavanones previously identified by screening. New derivatives including bromonitro-flavanones were synthesised aiming at improving compound stability. I pharmacologically characterised these compounds and show for one of them an increased stability and activities compared to reference compound. In parallel, I synthesised and pharmacologically characterised new bi-substrate analogue inhibitors, mimicking the adenosine and the deoxycytidine. Two very promising quinazoline-quinoline derivatives were identified. They are active against DNMT and in cell lines, both for reexpression of a reporter gene but mostly in CDKN2A TSG demethylation inducing its reexpression. To identify DNMT partners we adopted a chemical biology approach (Activity-Based Protein Profiling (ABPP)) based on the use of chemical probes including in-house non- nucleoside DNMT inhibitors as bait to trap the DNMT partners. We designed and synthesised twenty chemical probes and evaluate them using enzymatic and cellular-based assays. Selected probes were used to carry out ABPP directly in living cells. After functionalization by bioorthogonal chemistry, DNMT protein partners were purified and identified by proteomic analysis. Target validation would enable to determine new targets for the aberran
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Kuch, David. "Synthese und Charakterisierung neuartiger Inhibitoren für die humane DNA Methyltransferase DNMT1 /." München : Dr. Hut Verlag, 2009. http://edoc.ub.uni-muenchen.de/9490/.
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Kuch, David. "Synthese und Charakterisierung neuartiger Inhibitoren für die humane DNA Methyltransferase DNMT1." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-94905.
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Zampieri, Michele. "Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation." Doctoral thesis, La Sapienza, 2007. http://hdl.handle.net/11573/916862.
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Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents.
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Lau, Ho-Tak. "Transcriptional and post-transcriptional control of DNMT1 in oocytes and somatic cells." Thesis, University of Ulster, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.554332.
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In mammals, the DNA methylation patterns are established by the de novo methyltransferases of the DNMT3 family and subsequently maintained DNMTl. The DNA methylation patterns of imprinted genes are established during gametogenesis. Many imprinted genes are found methylated on the maternal allele, and this modification is established during oocyte growth. My primary aim was to develop a simple in vitro system for culturing oocytes during the period of de novo methylation, which would allow us to access this critical developmental period and to manipulate the microenvironment by supplementing growth factors in order to examine the signalling required for initiating de novo methylation. In Chapter 2, I show the development of the primary follicles from primordial follicles in an in vitro environment. By addition of SCF, I confirmed that it acts as a major growth stimulant in early oocyte growth. However, follicular development was arrested at the primary follicle stage, probably due to the absence of a theca layer surrounding the follicle. Secondly, I wanted to investigate if DNA methylation could be established at imprinted genes and repeat sequences in cultured oocytes with a view to investigating factors responsible for these events. Chapter 3 revealed limited establishment of DNA methylation in the cultured oocytes. Chromatin staining illustrated improper chromatin remodelling of the oocytes which accompanied follicle developmental arrest, and may have blocked de novo methylation. Lastly, I aimed to determine whether Dnmtl expression is regulated posttranscriptionally by the cytoplasmic polyadenylation element present in its 3 'UTR. Results in Chapter 4 show the CPE enhances DNMTl expression and also suggest the importance of a 26nt long conserved region in regulating the gene.
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Rutledge, Charlotte Emily. "Investigating the transcriptional control of Dnmt3L and post-transcriptional control of Dnmt1." Thesis, University of Ulster, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589755.
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In this thesis I present an investigation into mechanisms which regulate the expression of the DNA methyltransferase family members DNMT3L (a co-factor for de novo methylation) and DNMTl (the maintenance methyltransferase). I present work demonstrating the involvement of DNA methylation in the regulation of expression of DNMT3L from one of its three promoters, and also show that DNMT3L itself is required for this methylation to occur in murine oocytes. Furthermore, the data demonstrates that DNMT3L is required not only for DNA methylation of imprinted sequences in murine oocytes, as was previously hypothesised, but also for methylation of several non-imprinted single-copy and repeat sequences which were investigated, and that loss of this methylation in the oocyte results in altered gene expression. These altered patterns of expression have the potential to be maintained throughout early development. In separate work, experiments were conducted to investigate the role of RNA-binding proteins in the translational regulation of DNMTl. Highly conserved consensus binding motifs for several RNA-binding proteins are located in the 3'UTR of DNMT1. Work conducted in human cell lines and murine embryonic stem cells indicates a role for ePEB, MSIl and PUM2 (proteins which are reported to function predominantly via regulation of mRNA polyadenylation) in the positive translational regulation of DNMTl. A thorough investigation of cell cycle-dependent polyadenylation changes for the DNMTl mRNA failed to reveal such regulation, despite evidence of polyadenylation at control mRNAs. These experiments further our understanding of the mechanisms which are involved in the correct regulation of the expression of these important epigenetic regulators, dysregulation of which may result in human diseases which incorporate epigenetic alterations, such as imprinting syndromes and cancers.
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Jurkowska, Renata Zofia [Verfasser]. "Biochemical characterization of the mammalian Dnmt3 family of DNA methyltransferases / Renata Zofia Jurkowska." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2009. http://d-nb.info/1034984969/34.
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Wu, Bo-Kuan. "Intrinsic and extrinsic regulation of DNA methylation during malignant transformation." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/1419.
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Cytosine methylation of CpG dinucleotides is an epigenetic modification that cells use to regulate gene expression, largely to promote transcriptional silencing. Focal hypermethylation of tumor suppressor genes (TSGs) accompanied by genomic hypomethylation are epigenetic hallmarks of malignancy. DNA methyltransferase 1 (DNMT1) is the principle vertebrate enzyme responsible for maintenance of DNA methylation and its dysregulation has been found to lead to aberrant methylation in cancer. In addition, recent findings demonstrated that the ten-eleven translocation 1 (TET1) protein functions as a 5-methylcytosine dioxygenase that converts 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC) to mediate active DNA demethylation. Emerging evidence suggests that TET1 might function as a TSG. To understand the dynamic regulation of DNA methylation during cellular transformation, my work focused on intrinsic regulation of DNMT1 and how TET1 regulates DNA demethylation in generating a cancer methylome.
The replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, other data suggested that RFTS may be a positively acting domain. To test biochemical and structural predictions that the RFTS domain of DNMT1 is inhibitory, we established cellular systems to evaluate the function of DNMT1 alleles. The data indicate that deletion of RFTS is necessary and sufficient to promote cellular transformation, focal hypermethylation of specific TSGs, and global hypomethylation. These data and human mutation data suggest that RFTS domain is a target of tumor-specific dysregulation.
RAS mutations are frequently in multiple malignancies. Methylation-associated silencing of TSGs is a hallmark of RAS-driven-tumorigenesis. I discovered that suppression of TET1 by the ERK signaling cascade is responsible for promoter hypermethylation and the malignant phenotype in KRAS-transformed cells. Restoration of TET1 expression reactivates silenced TSGs and reduces colony formation. Moreover, TET1 knockdown in a cell depleted for KRAS is sufficient to rescue the inhibition of colony formation by KRAS knockdown. My findings suggest that dysregulated TET1-mediated DNA demethylation is a target responsible for epigenetic changes in cancers with KRAS activation.
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Zepper, Matthias Lutz [Verfasser], and Frank [Akademischer Betreuer] Rosenbauer. "Epigenetic characterization of murine Dnmt1-deficient MLL-AF9 leukemia : DNA-methylation in large regions and at cis-regulatory elements dissected in the Dnmt1 -/chip mouse model / Matthias Lutz Zepper ; Betreuer: Frank Rosenbauer." Münster : Universitäts- und Landesbibliothek Münster, 2020. http://d-nb.info/1211670813/34.
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Rottach, Andrea. "Analysis of the cell cycle dependent dynamics of Dnmt1 and Np95 in living cells." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-110219.
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Höche, Alexander [Verfasser]. "Expressionsanalyse der DNA-Methyltransferasen DNMT1 und DNMT3b bei akuten Leukämien im Kindesalter / Alexander Höche." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2012. http://d-nb.info/103038097X/34.
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Ulian, Benitez Suzana. "Function of Kek-6 and DNT2 in structural synaptic plasticity in Drosophila." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8440/.
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The Drosophila nervous system undergoes structural synaptic plasticity, however, the mechanisms that govern such event are little understood. Structural synaptic plasticity in mammals is regulated by the neurotrophin brain derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase full length (TrkB-FL). TrkB-FL has a tyrosine kinase domain (TyrK) intracellularly, that is required for its function in structural synaptic plasticity. Trk receptors have long been sought in Drosophila to verify mechanisms of structural synaptic plasticity, but they have not been found. Later, the Kek receptor family was identified as the kinaseless-Trk homologues in flies (Mandai et al., 2009, Bishop, 2013). Here, I validated that Kek-6 is a neurotrophin receptor for DNT2. DNT2 is a novel retrograde factor at the neuromuscular junction (NMJ), and both DNT2 and Kek-6 regulate structural synaptic plasticity. Kek-6 functions in concert with Toll-6. DNT2 and Kek-6 function upstream of CaMKII and Vap33A at the NMJ synapse. Finally, I show that Kek-6 can regulate intracellular levels of calcium in larval motorneurons. In conclusion, I identified a novel mechanism of structural synaptic plasticity in flies that is independent of a TyrK domain. If there are conserved mechanisms, this may also shed light on how truncated Trks function in the adult mammalian brain.
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Viziteu, Elena. "RECQ1 Helicase Involvement in the Resistance to Replication Stress and Chemotherapy in Multiple Myeloma Myélome Multiple." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT008.
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Le myélome multiple (MM) est une néoplasie B caractérisée par l’accumulation d’un clone plasmocytaire dans la moelle osseuse. Des études ont démontré que les modifications épigénétiques comme la méthylation de l’ADN jouent un rôle dans la régulation d’expression de différents gènes associés au cancer. Dans une étude récente, nous avons pu décrire un score génique de méthylation de l’ADN permettant de prédire la sensibilité des cellules de MM aux inhibiteurs de DNMT (DNA methyltranfexrase) (Moreaux, et al 2012). Parmi les gènes dont l’expression est inhibée par les inhibiteurs de DNMT et associés avec un pronostic péjoratif chez les patients atteints de MM, nous avons identifié RECQ1. RECQ1 est une hélicase de la famille RECQ qui s’associe aux origines de réplication durant la phase S du cycle cellulaire et joue un rôle important dans l’élongation des fourches de réplication. RECQ1 est fortement exprimé dans différents types de tumeurs solides et l’inhibition de RECQ1 conduit à la catastrophe mitotique et inhibe la croissance de tumeurs solides. Le but de notre projet a été de caractériser la fonction de RECQ1 dans la physiopathologie du MM et les mécanismes de résistance aux traitements. Afin d’étudier le rôle biologique de RECQ1 dans les plasmocytes tumoraux, nous avons utilisé des vecteurs lentiviraux pour induire de façon inductible la surexpression ou l'inhibition de RECQ1. La déplétion de RECQ1 dans les cellules de MM entraîne une inhibition de la croissance, une induction significative d’apoptose et la formation de foyers 53BP1 indiquant la présence de cassures d’ADN double brin. Une forte expression de RECQ1 étant associée à un mauvais pronostic et la déplétion de RECQ1 conduisant à une induction de cassures d’ADN double brin, nous nous sommes demandé si l’inhibition de l’expression de RECQ1 pourrait sensibiliser les cellules de MM aux agents génotoxiques utilisés dans le traitement du MM. La déplétion de RECQ1 sensibilise, de façon significative, les cellules de MM au melphalan suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et du melphalan pourrait avoir un effet synergique chez les patients RECQ1++. La surexpression de RECQ1 protège les lignées cellulaires de myélome contra l'apoptose induite par melphalan et bortézomib. De plus, l'épuisement RECQ1 sensibilise les cellules de myélome de traitement est démontré que RECQ1 interagit avec des protéines impliquées dans différentes voies de réparation des dommages de l’ADN : PARP1 (NHEJ/BER), RAD51 (HR), MSH2 et MSH6 (Mismatch repair). RECQ1 interagit avec PARP1 dans la fraction chromatinienne des cellules de MM mais pas avec RAD51 ni MSH2. Cette interaction est significativement induite en présence de melphalan. Des inhibiteurs de PARP sont actuellement en développement préclinique ou en essai clinique. De façon intéressante, la déplétion de RECQ1 sensibilise significativement les cellules de MM à un inhibiteur de PARP in vitro suggérant que l’association d’un inhibiteur de DNMT pour cibler RECQ1 et d’un inhibiteur de PARP pourrait avoir un intérêt thérapeutique dans le MM. Nous avons également confirmé que des doses sous-létales d’inhibiteur de DNMT sensibilisent les cellules de MM au melphalan in vitroMultiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMCs), primarily in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1high patients associated with a poor prognosis
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Menon, Yoann. "Etude des effets pharmacologiques d'inhibiteurs non nucléosidiques de la méthylation de l'ADN." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30004/document.
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Les modifications épigénétiques participent au contrôle de l'expression génique. Il a été montré que la méthylation des désoxycytidines (dC) de l'ADN joue un rôle clé dans la régulation épigénétique chez les mammifères. Cette modification correspond à la marque épigénétique la plus stable. Elle a lieu sur des résidus CpG regroupés en ilôts, essentiellement situés au niveau des séquences promotrices, des séquences répétées et des séquences encadrants les ilôtsCpG. L'hyperméthylation des promoteurs induit une inhibition de l'expression des gènes, tandis qu'une hypométhylation est associée à une expression. Les enzymes responsables de la méthylation de l'ADN sont les méthyltransférases d'ADN (DNMTs). Deux familles de DNMTscatalytiquement actives ont été identifiées: on distingue la DNMT1, principalement responsable de la maintenance de la méthylation de l'ADN lors de la réplication, et les DNMT3A et 3B, qui sont responsables d'une méthylation de l'ADN dite de novo. L'altération des profils de méthylation de l'ADN conduit à diverses maladies telles que le cancer. Les cellules cancéreuses présentent souvent un profil de méthylation de l'ADN différent des cellules saines, on observe en particulier une hyperméthylation spécifique des gènes dits suppresseurs de tumeur. Une restauration de leur expression par l'inhibition de la méthylation de l'ADN représente ainsi une stratégie thérapeutique attrayante. Plusieurs inhibiteurs de DNMTs ont été décrits et deux analogues de nucléosides sont approuvés par la FDA pour traiter certaines leucémies: la 5-azacytidine (VidazaTM) et la 5-azadeoxycytidine (Dacogene(r)). Notre laboratoire développe depuis plusieurs années de nouveaux inhibiteurs non nucléosidiques de DNMTs qui ciblent leur site catalytique. J'ai étudié ici les effets pharmacologiques de ces inhibiteurs catalytiques des DNMTs, en utilisant plusieurs lignées cellulaires cancéreuses (issues d'une leucémie, d'un lymphome et d'un cancer du côlon). J'ai utilisé pour cela différentes technologies permettant d'analyser la méthylation de l'ADN, l'accessibilité de la chromatine, les modifications des histones et l'expression des gènes. Ces nouvelles thérapies épigénétiques visent à la reprogrammation des cellules cancéreuses, c'est pourquoi j'ai exploré les modifications à long terme induites par ces nouveaux composés. Nous avons montré que ces composés sont des inhibiteurs puissants de DNMT3A et qu'ils sont capables d'induire l'expression d'un gène raporteur (la luciférase) sous le contrôle du promoteur CMV, par une déméthylation de ce promoteur et une ouverture de la chromatine. Enfin, ces nouveaux inhibiteurs de DNMTs déméthylent la région promotrice de gènes suppresseurs de tumeurs et induisent leur ré-expressionEpigenetic modifications participate to the control of gene expression. Methylation of deoxycytidines (dC) in the DNA was shown to play a key role in epigenetic regulation in mammals. It is the most stable epigenetic mark and occurs at CpG sites, which are grouped in islands and essentially located in promoters, repeated sequences and CpG island shores. Hypermethylation of promoters induces gene silencing while hypomethylation is associated to gene expression. Enzymes responsible for DNA methylation are the DNA methyltransferases (DNMTs). Two families of catalyticallyactive DNMTs have been identified: DNMT1, mainly responsible for DNA methylation maintenance during replication; and DNMT3A and 3B that perform de novo DNA methylation and support maintenance. Alteration of DNA methylation patterns lead to various diseases such as cancer. Cancerous cells often present aberrant DNA methylation, in particular a specific hypermethylation of tumor suppressor genes is observed. Restoring their expression by inhibition of DNA methylation represents an attractive therapeutic strategy. Several DNMTs inhibitors have been described. Two nucleoside analogs are FDA approved to treat leukemia: 5-azacytidine (VidazaTM) and 5-azadeoxycytidine (Dacogene(r)). Our laboratory develops since several years new inhibitors of DNMT, non-nucleoside analogs, targeting the catalytic site. Here, I studied the pharmacological effects of these DNMTs catalytic inhibitors using several cancer cell lines (leukemia, lymphoma and colon cancer) and different technologies to follow DNA methylation, chromatin accessibility, histone modifications and gene expression. Since epigenetic therapies aim at the reprogramming of cancer cells, I explored the long-term modifications induced by the compounds. We show that these novel compounds are potent inhibitors of DNMT3A and able to induce the expression of a reporter gene (luciferase) under the control of a methylated CMV promoter by demethylation of the promoter and opening of the chromatin. Finally, these new DNMTs inhibitors demethylate the promoter region of tumor suppressor genes and induce their re-expression
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De, Vos Mike. "Interaction fonctionnelle de la Poly(ADP-Ribose) polymérase-1 (PARP1) avec des protéines de l'hétérochromatine : impact sur la fonction de l'hétérochromatine et la réparation de l'ADN." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ001.
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Nous avons identifié une association poly(ADP-ribose) (PAR)-dépendante entre PARP1 et UHRF1. UHRF1 est PARylé par PARP1 et lie le PAR de façon non covalente. L’absence de PARP1 (i) perturbe l’association de UHRF1 et DNMT1, (ii) induit une ubiquitination excessive de DNMT1 par UHRF1 favorisant sa dégradation au cours du cycle, (iii) favorise la transcription des régions de l’hétérochromatine péricentrique (pHC) (iv) et perturbe la localisation de la marque répressive H4K20me3 au niveau des foyers de l’pHC. Dans un deuxième temps, nous avons étudié le rôle de l’association KAP1-HP1 dans la réponse cellulaire aux dommages. L’interaction entre ces deux partenaires est essentielle pour le recrutement de KAP1 sur les sites de cassures. Après induction de cassures, l’absence d’interaction induit un délai dans la réparation des cassures double-brins et une diminution de la survie cellulaire. Une analyse détaillée suggère une déficience du mécanisme de réparation par recombinaison homologueWe identified a poly(ADP-ribose) (PAR)-dependent interaction between PARP1 and UHRF1. UHRF1 is PARylated by PARP1 and binds PAR in a non-covalent way. The absence of PARP1 (i) impairs the UHRF1/DNMT1 interaction, (ii) induces excessive UHRF1-mediated ubiquitination of DNMT1 promoting its degradation during the cell cycle, (iii) increases the transcription of pericentric heterochromatin (pHC) regions (iv) and impairs the localization of the repressive histone mark H4K20me3 on pHC. In a second project we studied the role of the KAP1/HP1 interaction in response to DNA damage. The interaction between the two partners is essential for KAP1 recruitment to DNA damage sites. The absence of the interaction, after damage, induces a delay of the double strand break repair kinetics and decreases the cell survival rate. A more detailed analysis suggests a deficiency of the homologous recombination repair pathway
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Shock, Lisa. "Functional consequences of cytosine methylation in mitochondrial DNA catalyzed by DNA methyltransferase 1." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/271.
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Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.
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Maciel, Izaque de Sousa. "Envolvimento do óxido nítrico na metilação do DNA induzida por estresse." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17133/tde-25072018-094208/.
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A exposição ao estresse induz um aumento dos níveis de óxido nítrico (NO) e glutamato em estruturas do cérebro de ratos, as quais estão relacionadas com o transtorno de depressão maior (DM) em humanos. Ademais, o estresse está diretamente relacionado com o aumento da metilação do DNA, uma alteração epigenética repressiva, no hipocampo de animais. Estudos anteriores demonstraram o efeito tipo antidepressivo dos inibidores da enzima óxido nítrico sintase (NOS) em animais submetidos ao estresse. Porém não se sabe se há uma relação entre o aumento do NO e glutamato induzido pelo estresse e alteração na metilação do DNA em genes relacionado com a patofisiologia da DM. Assim, o objetivo deste estudo foi investigar os efeitos dos inibidores da NOS nas alterações comportamentais e nos mecanismos intracelulares relacionado com a metilação do DNA no cérebro de ratos submetidos ao teste do desamparo aprendido (learned helplessness - LH) e em cultura celular do hipocampo desafiadas com NMDA e dexametasona. Métodos: Estudo 1: Cultura primária de células do hipocampo ou cultura imortalizada HiB5 foram desafiadas/estressadas com NMDA (30µM,1h), L-arginina (500µM,1h) e/ou dexametasona (1µM, 1h ou 24h) e pré-tratadas com inibidor seletivo da nNOS (NPA, 100nM, 30min antes do desafio) ou com inibidor da DNMT (5-Aza, 10 µM, 30 min antes do desafio). A expressão dos genes para as enzimas DNMTs, BDNF, NT4, TrkB e nNOS foram avaliadas por RT-qPCR, a expressão proteica das enzimas DNMT3b e nNOS foram avaliadas por western blotting. Estudo 2: Ratos foram submetidos à choques inescapáveis (0,4 mA; 40 choques) na sessão de pré-teste do LH, após sete dias os animais foram submetidos a sessão de teste (choques escapáveis de 0,4 mA). Os animais foram tratados com inibidores da NOS 7-nitroindazole (7-NI;60mg/kg,i.p), aminoguanidina (AMG; 30mg/kg,i.p) ou veículo por 7 dias e submetidos a sessão de teste 1h, após a última injeção. A metilação global foi analisada por imunoensaio (ELISA) e a expressão dos genes DNMT3b, BDNF, nNOS e iNOS foram avaliadas por RT-qPCR, nas estruturas: cortex, hipocampo ventral e hipocampo dorsal. Resultados: Estudo 1: O pré- tratamento com NPA, atenuou o aumento da expressão do mRNA para a enzima DNMT3b, em cultura primária do hipocampo desafiada com NMDA, dexametasona e Larginina, e também em cultura HiB5 desafiada com dexametasona. Porém, o NPA não inibiu a diminuição da expressão do BDNF (exon 1, exon 4 e exon 9), em cultura primária de células do hipocampo desafiadas com NMDA. O pré tratamento com 5-Aza, não inibiu as alterações induzidas pelo NMDA em cultura primária de hipocampo. Estudo 2: Ratos submetidos ao estresse dos choques inescapáveis na sessão de pré-teste apresentaram aumento no número de falhas em escapar dos choques na sessão de teste (desamparo aprendido), um efeito que foi atenuado pelo tratamento com AMG ou 7-NI. Interessantemente, o efeito comportamental do estresse foi acompanhado por aumento nos níveis da metilação global do DNA e DNMT3b no hipocampo ventral (vHPC), que foi atenuado pelos pré-tratamentos com AMG e 7-NI, porém não houve diferença estatisticamente significante no córtex e no hipocampo dorsal dos ratos. Conclusão: Os dados apresentados demonstraram que tanto o estresse (in vivo) quanto o desafio com glicocorticóides, NMDA e L-arginina (in vitro) são capazes de modular a expressão daenzima DNMT3b e a metilação de DNA no hipocampo. O tratamento com inibidores da NOS reduzem os efeitos do estresse in vivo (comportamental e molecular) e in vitro. Em conjunto, os dados sugerem que a liberação de glutamato e NO durante o estresse pode modular a expressão da enzima DNMT3b, levando ao aumento da metilação do DNA em genes relacionados com a resposta de adaptação ao estresse. Essa é a primeira evidência de que o NO pode modular metilação do DNA induzida por estresse.Stress exposure increases glutamate and nitric oxide (NO) levels, as well as DNA methylation in the hippocampus. However, it is not yet known if there is a causal relationship between these events. Moreover, both nitric oxide synthase (NOS) inhibitors and DNA methylation inhibitors counteract the behavioral effects of stress. Therefore, our aim was to investigate the effects of NOS inhibitors on stress-induced changes on behaviour, DNA methylation and genes expression in the hippocampus of rats submitted to learned helplessness - LH. Moreover, the effects of direct administration of dexamethasone (glucocorticoid), NMDA and L-arginine was investigated in hippocampal cell cultures. Methods: Study 1: Primary hippocampal cell culture was challenged with NMDA (30µM,1h), L-arginine (500µM,1h) or dexamethasone (1µM,24h) and pretreated with nNOS inhibitor (NPA, 100nM, 30min before the challenge) or with DNMT inhibitor (5-Aza, 10 µM, 30 min before the challenge). DNMTs, BDNF, NT4, TrkB and nNOS gene expression was assessed by RT-qPCR. DNMT3b and nNOS levels were assessed by western blotting. Study 2: Rats were submitted to inescapable footshocks and treated with the NOS inhibitors 7-nitroindazole (7-NI; 60 mg/kg, i.p) or aminoguanidine (AMG; 30 mg/kg, i.p], or vehicle for 7 days and tested 1h after the last injection with escapable footshocks. The number of escape failures during the test, global DNA methylation (ELISA) and DNMT3b, BDNF, nNOS and iNOS mRNA expression (RT-qPCR) was evaluated. Results: NPA pretreatment attenuated DNMT3b mRNA expression in hippocampus primary cell culture challenged with NMDA, dexamethasone or L-arginine. Similarly effects were observed in HiB5 cell challenged with dexamethasone. However, NPA pretreatment did not inhibit the decrease of BDNF (exon 1, exon 4 and exon 9) induced by NMDA. Moreover, pretreatment with 5-Aza did not inhibit the decreased of BDNF induced by NMDA in primary cell culture. Study 2: Stress exposure increased the number of escape failures in the test, which was attenuated by treatment with AMG or 7-NI, an antidepressant-like effect. Interestingly, the increased DNA methylation DNMT3b mRNA expression in the ventral hippocampus (vHPC) of stressed rats were also attenuated by treatment with both AMG and 7-NI. Conclusions: NOS inhibitors attenuated stress-induced depressive-like behavior, DNA methylation and DNMT3b mRNA expression in the vHPC. In vitro, selective nNOS inhibition also blocks corticosterone-, NMDA- and L-arginine-induced DNMT3b mRNA expression in hippocampal cell culture. Altogether, our results suggest that glutamate release, leading to NO production during stress may mediate intracellular mechanisms that regulate DNMT3b expression and DNA methylation. This is the first evidence indicating that NO modulates DNA methylation induced by stress.
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Dudley, Kevin. "The role of DNMT1 inhibition in the identification of epigenetically silenced genes in pituitary tumours." Thesis, Keele University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493649.
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Epigenetic unmasking, either by pharmacological inhibition or direct gene targeting, has been utilised to identify novel tumour suppressor genes that are silenced in association with inappropriate CpG island (CGI) methylation in various types of cancer. In this study, an epigenetic unmasking strategy was developed to identify novel genes that are silenced in association with inappropriate, CGI methylation in pituitary tumours.
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Moreno, Daniel Antunes. "Estudo da Expressão dos Genes DNMT1, DNMT3A, DNMT3B, MGMT e Efeitos da Zebularina em Glioblastoma." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-22042013-102215/.
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Os gliomas são tumores que surgem a partir de células da glia e são considerados os mais comuns do sistema nervoso central. São subdivididos em quatro grupos: astrocitoma pilocítico (grau I), astrocitoma difuso (grau II), astrocitoma anaplásico (grau III) e glioblastoma (grau IV ou GBM). Entre esses, o GBM é o tumor mais agressivo e mais freqüente. Apesar de ser encontrado em qualquer faixa etária, esse tumor é raro em crianças. Atualmente a cirurgia seguida de radioterapia e quimioterapia com temozolomida (TMZ) tem sido utilizado como protocolo de tratamento padrão para a maioria dos pacientes e mesmo assim a sobrevida se mantem extremamente baixa. Além disso, grande parte dos pacientes não respondem ao tratamento com TMZ indicando a necessidade de agentes quimioterápicos alternativos. A zebularina (ZB) é um agente inibidor de DNA metiltransferases (iDNMTs) estável, pouco tóxico, que promove radiosensibilização e tem mostrado efeitos promissores em diversos tipos de neoplasias, entretanto pouco se sabe a respeito dos efeitos da ZB em glioblastoma. Os objetivos deste trabalho foram analisar a expressão dos genes DNMT1, DNMT3A, DNMT3B, MGMT em 5 amostras de substâncias brancas (SB), 6 linhagens de GBM e 33 amostras de gliomas (13 grau I, 2 grau II e 18 grau IV), correlacionar a expressão desses genes com os diferentes graus de gliomas e analisar os efeitos da ZB combinada ou não com TMZ em linhagens de GBM irradiadas e não irradiadas. Para análise da expressão gênica foi realizada a técnica de PCR em tempo Real. Os ensaios de proliferação celular, clonogênico, radiação e apoptose foram realizados em 3 linhagens de GBM (U251, SF188 e T98G) e uma de fibroblastos (MRC5). Também foi realizado o ensaio de proliferação celular em 5 culturas primárias de GBM tratadas com zebularina. Os genes DNMT3A e MGMT mostraram expressão maior nas amostras de SB comparando-se com gliomas e linhagens de GBM. O gene DNMT3B foi mais expresso nas linhagens de GBM comparando-se com as SB. O gene DNMT1 não mostrou diferenças significativas entre as amostras analisadas. Os ensaios de proliferação celular mostraram diminuição na proliferação com doses a partir de 50-100µM de ZB e de 250-500µM de TMZ nas linhagens e a partir de 50µM de ZB para as culturas primárias de GBM. As combinações de ZB com TMZ não mostraram sinergia na grande maioria das doses testadas. A ZB aumenta a apoptose nas 3 linhagens com doses a partir de 100µM. A ZB e TMZ mostraram diminuição na formação de colônias com as doses de 100µM e 10µM nas linhagens U251 e SF188 não irradiadas e irradiadas com 2, 4 e 6 Gy. A linhagem T98G expressa o gene MGMT, mostrou resistência a 10µM de TMZ e respondeu ao tratamento com 100µM de ZB. Também foi observado que 10µM de TMZ é mais citotóxico do que 100µM de ZB em fibroblastos não irradiados e irradiados (2Gy). Os resultados obtidos neste estudo mostram que a ZB pode representar um alvo terapêutico interessante para o estudo em glioblastoma.Gliomas arise from glial cells and are the most common central nervous system tumors. They are divided in four groups: pilocytic astrocytoma (grade I), difuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma (grade IV or GBM). GBM is the most frequent and aggressive glioma. This type of tumor can occur in any age but its rare in children. Actually, surgery, radiotherapy and temozolomide (TMZ) adjuvant/concomitant chemotherapy has been the standard treatment protocol but the survival is extremely poor. In addition most patients do not respond to TMZ indicating the need for alternative chemotherapeutic agents. Zebularine (ZB) is a DNA metiltransferase inhibitor (DNMTi) stable, slight toxic that has been showed promise effects in cancer including radiosensitivity but little is known about ZB in glioblastoma. The objectives of this study were analyze DNMT1, DNMT3A, DNMT3B, MGMT gene expression profile in 5 samples of normal brain, 6 GBM cell lines and 33 glioma samples (13 grade I, 2 grade II e 18 grade IV), correlate with different gliomas grades and analyze the effects of ZB isolate and in combination with TMZ in irradiated and non irradiated GBM cell lines. Gene expression assays was made using Real Time PCR. Proliferation, clonogenic, radiation and apoptosis assays were realized in three GBM cell lines (U251, SF88, T98G) and one fibroblast cell line (MRC5). We also made proliferation assays in 5 primary cultures of samples of GMB. MGMT and DNMT3A genes showed higher expression in normal brain compared to gliomas and GBM cell lines. DNMT3B gene showed higher expression in GBM cell lines compared with normal brain and DNMT1 showed no significant differences among samples analyzed. We observed decrease of cell proliferation from 50-100µM of ZB and 250-500µM of TMZ on GBM cell lines and from 50µM of ZB for primary GBM samples. It was not observed synergy in the most combinations doses of ZB and TMZ (Calcusyn software). It was observed that 100µM of ZB and 10µM of TMZ decrease colony formation on U251 and SF188 cell lines non irradiated and irradiated with 2, 4, and 6Gy. T98G that express MGMT, did not respond to TMZ but showed response to ZB. It was also observed that 10µM of TMZ is more cytotoxic than 100µM of ZB in fibroblast cell line non irradiated and irradiated with 2Gy. ZB increase apoptosis from 100µM on the three GBM cell lines. Results obtained in this study can indicate that ZB may be an interestig therapeutic target for future studies in glioblastoma.
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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.
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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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SONCINI, MATIAS CRISTOBAL. "Epigenetic therapies for acute myeloid leukemias : pre-clinical validation and study of molecular mechanisms." Doctoral thesis, Università degli Studi di Milano, 2008. http://hdl.handle.net/2434/56628.
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Epigenetic therapies of cancer have been intensely studied, because of the high frequency of epigenetic alterations found in tumor cells. Epigenetic modulators, such as histone deacetylases (HDAC) and DNA methyltransferase (DNMT), can be targeted by specific drugs with the intent to revert the epigenetic alterations induced by tumor progression. Although a few DNMT inhibitors have been approved for the therapy of some specific hematopoietic diseases, most of the results of clinical trials on other types of cancer have given limited results. The need for a better understanding of the molecular mechanism(s) underlying sensitivity to epigenetic therapies is therefore very strong. For obvious reasons, the use of preclinical models is mandatory to achieve this deeper understanding, to be translated to better clinical trials. We therefore used one form of acute myeloid leukemias (APL) as a preclinical model for epigenetic therapies. The choice of APL is due to the fact that the APL-associated fusion protein PML/RARα is known to induce epigenetic alterations in APL blasts by recruiting HDACs and DNMTs, making APL blasts obvious candidates for this kind of drugs.
Monotherapies of either HDACi (VPA) or DNMTi (DAC) induced increased survival in APL mice and their combination were able to further prolong survival of APL mice, demonstrating the importance of hitting multiple targets of the epigenetic “Silencing Loop”. Both the studied epigenetic drugs also demonstrated an enhancement of the therapeutic effect of differentiating therapy with All Trans Retinoic Acid (ATRA).
We also tried to identify the molecular mechanisms involved in anti-leukemic activity. Both VPA and DAC demonstrated an induction of the apoptotic response in leukemic cells through the upregulation of members of the Death Receptor pathways. When treating with VPA or DAC, we also observed some modifications in chromatin structure and DNA methylation pattern in a specific CpG rich region within the TRAIL promoter. These results suggest a partial direct epigenetic mechanism underlying TRAIL induction by DAC and VPA.
We were able to confirm the results obtained in the APL model in an unrelated form of AML derived from the expression of AML1/ETO fusion protein, suggesting that a substantial fraction of AML patients could benefit from epigenetic treatments, and further reinforcing our view that the APL model represents a paradigm for the study of epigenetic drugs.
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Saidj, Djamel. "Alteration of p53 and NF-kB pathways by E7 protein from cutaneous Human Papillomavirus type 38." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10237/document.
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Les infections virales sont responsables de 15 à 20 % des cancers humains. L étude des mécanismes moléculaires avec lesquels les virus oncogènes induisent la transformation cellulaire est essentielle pour la compréhension des cancers qui en résultent. Cela permettra également la découverte de nouveaux mécanismes pouvant être impliqués dans le développement de cancers, qui peuvent être ciblés par des approches thérapeutiques. Les virus du papillome humain (HPV) sont des petit virus à ADN qui futs isolés de la peau de patients souffrants de Epidermodysplasia Verruciformis (EV) qui cause un risque élevé d'infection par les HPV et le développement de cancer de la peau non mélanique (NMSC). Certains HPV cutanés, tels que HPV5, 8 et 38, sont suspectés de jouer un rôle dans de développement du cancer de la peau. Cependant, le lien direct entre les HPV cutanés et l'étiologie du cancer n'est pas encore clairement établi. Des études de notre laboratoire ont montré que les oncoprotéines HPV38 E6 et E7 sont capables d'immortaliser des kératinocytes primaires humains in vitro et in vivo. Pour immortaliser des cellules, d'importantes voies de signalisations, telles que les voies de p53 et celle de NF-KB, doivent être affectées. Dans cette étude, nous avons cherché à mettre en évidence les mécanismes moléculaires menant à la dérégulation de p53 et de NF-KB par E6 et E7 de HPV38, dans des kératinocytes humains. Nous avons montré que HPV38 E6 et E7 induisent la formation d'un complexe protéique incluant IKKβ, ΔNp73α, EZH2 et DNMT1. La formation de ce groupement protéique corrèle avec l'inhibition de la transcription de certains gènes cibles de p53, tel que PIG3. Nous avons également mis en évidence l'activation de la voie NF-KB par les oncoprotéins E6 et E7 de HPV38. Cette activation est importante par le rôle joué par NF-KB dans la protection des cellules de l apoptose induite par TNF-α et par l'exposition aux rayonnements UVB. De plus nous avons observé que E7 est la principale oncoprotéine de HPV38 responsable de la dérégulation des voies p53 et NF-KB. Nos études mettent en évidence de nouveaux mécanismes moléculaires qui peuvent être essentiels dans le processus de transformation cellulaire par HPV38Viral infections contribute to 15–20% of all human cancers. Studying the mechanisms employed by the oncogenic viruses to induce cellular transformation is essential for a better understanding of the resulting cancers and the discovery of new mechanisms involved in cancer development which can be targeted in therapeutic approaches. Human papillomaviruses (HPVs) are small dsDNA viruses which have been clearly associated with certain cancers. They were first isolated from the skin of patients suffering from Epidermodysplasia Verruciformis (EV) having an increased susceptibility to infection by specific HPV types and to the development of non-melanoma skin cancer (NMSC). Certain cutaneous HPV types, such as 5, 8, and 38, are suspected to play a role in skin cancer development. However the direct role of cutaneous HPV in the etiology of cancer is still under debate. Previous studies from our laboratory have reported that HPV38 E6 and E7 proteins are able to immortalize human primary keratinocytes in vitro and in vivo. Cellular immortalization can be achieved through the deregulation of important signaling pathways including p53 and NF-KB. In the present work, we have investigated the molecular mechanisms of p53 and NF-KB pathways deregulation by E6 and E7 oncoproteins from HPV38 in human keratinocytes. We show here that HPV38 E6E7 induce the formation of a transcription repressor complex including IKKβ, ΔNp73α, and polycomb group members EZH2 and DNMT1. The formation of this protein complex correlates with the inhibition of several p53-target genes, such as PIG3. We also report in these studies that HPV38 E6E7 activate NF KB pathway, which plays an important role in the survival of HPV38 E6E7-immortalized human keratinocytes upon TNF-α– and UVB-mediated apoptosis. In addition our data highlight E7 being the main HPV38 protein mediating p53 and NF-KB deregulation. Our studies shed light on novel molecular mechanisms that could be important for HPV38-mediated cellular transformation
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Sakamoto, Hiromasa. "Functional and genomic characterization of patient-derived xenograft model to study adaptation to mTORC1 inhibitor in clear cell renal cell carcinoma." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263350.
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BARRA, Viviana. "DNMT1 SILENCING ELICITS DIFFERENT CELL CYCLE RESPONSES IN PRIMARY VERSUS TUMOR CELLS AND IS ASSOCIATED WITH ANEUPLOIDY GENERATION." Doctoral thesis, Università degli studi di Palermo, 2011. http://hdl.handle.net/10447/95092.
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Dahlet, Thomas. "Méthylation de l'ADN : fonctions et ciblage au cours du développement chez la souris." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ075.
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La méthylation des cytosines est une modification épigénétique catalysée par la famille des ADN méthyltransférases (DNMTs). C’est une marque répressive lorsqu’elle est adressée sur les îlots CpG des promoteurs de gènes. Le développement embryonnaire murin est caractérisé par une reprogrammation de la méthylation de l’ADN qui est critique pour le développement de l’embryon. Cependant, la contribution des différentes DNMTs dans la méthylation du génome ainsi que les mécanismes qui ciblent la méthylation de l'ADN vers certains gènes durant le développement embryonnaires sont mal connus. En combinant des approches de cartographie génomique avec des lignées génétiquement modifiées de souris, mes travaux de Thèse ont permis de clarifier la contribution des différentes DNMTs dans la méthylation du génome dans l’embryon : DNMT3A et DNMT3B sont strictement impliqués dans la méthylation de novo, et DNMT1 est strictement impliqué dans son maintien au cours des divisions cellulaires. De plus, l’analyse d’embryons globalement déméthylés a révélé de nombreuses fonctions de la méthylation de l’ADN dans le maintien de l'intégrité transcriptomique de l'embryon en réprimant des gènes gamétiques, des gènes du développement, des promoteurs cryptiques ainsi qu’un large panel de transposons. Dans un deuxième temps, j’ai étudié le rôle du facteur de transcription E2F6 dans le ciblage de la méthylation de l'ADN in vivo chez la souris. Mes résultats démontrent que E2F6 facilite l’acquisition de la méthylation de l’ADN au niveau du promoteurs des gènes gamétiques et est nécessaire pour initier leur répression à long terme au cours de l'embryogenèse. Dans leur ensemble, ces travaux contribuent à mieux comprendre les fonctions et mécanismes de ciblage de la méthylation de l'ADN au cours de l'embryogenèse des mammifèresCytosine methylation is an epigenetic modification catalyzed by the family of DNA methyltransferases (DNMTs). This modification is involved in gene repression when it is addressed to CpG islands in gene promoters. Global DNA methylation reprogramming occurs in mice during the early phases of embryogenesis, which is critical for proper embryo development. However, the contribution of different DNMTs in genome methylation and the mechanisms that target DNA methylation to specific genes during embryonic development are poorly understood. By combining genomic mapping with genetically modified mouse lines, my Thesis work clarified the contribution of the different DNMTs in genome methylation in the embryo: DNMT3A and DNMT3B are strictly involved in de novo methylation, and DNMT1 is strictly involved in the maintenance of DNA methylation during cellular divisions. In addition, the analysis of globally demethylated embryos revealed numerous functions of DNA methylation in maintaining the transcriptomic intergrity of the embryo by repressing germline genes, developmental genes, cryptic promoters as well as a large panel of transposons. In the second part of my Thesis, I studied the role of the E2F6 transcription factor in the targeting of DNA methylation in vivo in mice. My results demonstrate that E2F6 facilitates the acquisition of DNA methylation in the promoters of germline genes and is required to initiate their long-term epigenetic silencing during embryogenesis. Collectively, this work contributes to a better understanding of the functions and targeting mechanisms of DNA methylation during mammalian embryogenesis
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Champion, Christine. "Inhibiteurs de méthyltransférases d'ADN (DNMT) : caractérisation et application à la recherche de partenaires protéiques." Paris 6, 2011. http://www.theses.fr/2011PA066251.
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Dans les cancers, le paysage épigénétique est altéré avec notamment une hyperméthylation ciblée des promoteurs de certains gènes suppresseurs de tumeurs, ce qui participe à leur inactivation. Afin de comprendre ce phénomène, nous avons cherché à identifier de nouveaux partenaires protéiques des enzymes responsables de la méthylation de l’ADN, les méthyltransférases d’ADN (DNMT). L’originalité du travail consiste à développer deux techniques qui utilisent des inhibiteurs de DNMT comme outil moléculaire afin d’identifier les partenaires de la méthylation. La première partie du manuscrit est consacrée à la caractérisation d’inhibiteurs de DNMT (Champion et al. , 2010 ; Cecccaldi et al. , 2011). La seconde partie décrit le développement de deux techniques fondées soit sur le principe de chromatographie par affinité, soit sur l’utilisation d’une sonde chimique photoactivable qui peut ponter l’enzyme ou ses partenaires et être couplée à une étiquette par une réaction de «click chemistry»