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Functional consequences of cytosine methylation in mitochondrial DNA catalyzed by DNA methyltransferase 1Shock, Lisa 01 January 2011 (has links)
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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Intrinsic and extrinsic regulation of DNA methylation during malignant transformationWu, Bo-Kuan 01 July 2014 (has links)
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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Análise funcional dos genes Xist e DNMT1 na manutenção do processo de inativação do cromossomo X humano através do silenciamento gênico por RNAi / Functional analysis of XIST and DNMT1 genes in the maintenance of X chromosome inactivation process in human through gene silencing by RNAiStabellini, Raquel 27 June 2008 (has links)
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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Análise funcional dos genes Xist e DNMT1 na manutenção do processo de inativação do cromossomo X humano através do silenciamento gênico por RNAi / Functional analysis of XIST and DNMT1 genes in the maintenance of X chromosome inactivation process in human through gene silencing by RNAiRaquel Stabellini 27 June 2008 (has links)
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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Manipulating Somatic Cells to Remove Barriers in Induced Pluripotent Stem Cell ReprogrammingChung, Julia 07 June 2014 (has links)
Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.
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Functional and genomic characterization of patient-derived xenograft model to study adaptation to mTORC1 inhibitor in clear cell renal cell carcinoma / 淡明細胞型腎細胞癌におけるmTORC1阻害剤耐性機序解明のための患者由来ゼノグラフトモデルの機能およびゲノム的解析Sakamoto, Hiromasa 23 March 2021 (has links)
京都大学 / 新制・論文博士 / 博士(医学) / 乙第13400号 / 論医博第2224号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 松田 文彦, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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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 / Functional interaction between Poly(ADP-ribose) polymerase-1 (PARPl) and heterochromatin proteins : impact on heterochromatin function and DNA repairDe Vos, Mike 14 March 2014 (has links)
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 homologue. / We 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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Alteration of p53 and NF-kB pathways by E7 protein from cutaneous Human Papillomavirus type 38 / Dérégulation des voies de signalisation p53 et NF-kB par la protéine E7 du Papillomavirus Humain de type 38Saidj, Djamel 21 November 2013 (has links)
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 HPV38 / Viral 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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Regulation of DNA methylation during developmentAguirre-Arteta, Ana Maria 28 June 2000 (has links)
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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Mécanismes d'interaction de l'intégrateur épigénétique UHRF1 avec l'acétyltransférase TIP60 / Interaction mechanisms of epigenetic integrator UHRF1 with TIP60 acetyltransferaseAshraf, Waseem 18 June 2018 (has links)
UHRF1 est une protéine nucléaire responsable du maintien et de la régulation de l'épigénome des cellules. Elle favorise la prolifération cellulaire et est surexprimée dans la plupart des cancers. TIP60, l'un des partenaires le plus important d’UHRF1, est impliqué dans le remodelage de la chromatine et la régulation transcriptionnelle grâce à son activité acétyltransférase. Ensemble, les deux protéines régulent la stabilité et l'activité d'autres protéines telles que la DNMT1 et la p53. Le but de cette étude était d'explorer le mécanisme d'interaction entre UHRF1 et TIP60 en visualisant cette interaction dans les cellules. La microscopie par imagerie à temps de vie de fluorescence et d'autres techniques de biologie moléculaire ont été utilisées. Les résultats ont montré que UHRF1 interagit directement avec le domaine MYST de TIP60 et cette interaction se produit dans la phase S du cycle cellulaire. Les deux protéines ont également montré une réponse similaire aux dommages à l'ADN, ce qui prédit une cohérence dans leur fonction dans le mécanisme de réparation de l'ADN. La surexpression de TIP60 a également induit la baisse du niveau d’UHRF1 et de DNMT1 ainsi qu’une induction d'apoptose dans les cellules ce qui suggère un rôle de TIP60 dans la régulation des fonctions oncogéniques d’UHRF1. / UHRF1 is a nuclear protein maintaining and regulating the epigenome of cells. Its promotes proliferation and is found upregulated in most of cancers. TIP60 is one of the important interacting partner of UHRF1 and is involved in chromatin remodeling and transcriptional regulation through its acetyltransferase activity. Together they regulate the stability and activity of other proteins such as DNMT1 and p53. The aim of this thesis was to explore the mechanism of interaction between UHRF1 and TIP60 by visualizing this interaction in cells. Fluorescent lifetime imaging microscopy and other molecular biology techniques were employed for this purpose. Results of this study showed that UHRF1 interacts directly to the MYST domain of TIP60 and this interaction prevails in the S-phase of cell cycle. Both proteins also showed a similar response to DNA damage predicting a coherence in their function in DNA repair mechanism. Overexpression of TIP60 also downregulated UHRF1 and DNMT1 and induced apoptosis in cells suggesting a role of TIP60 in regulation of oncogenic functions of UHRF1.
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