Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-16T14:51:26Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-16T14:51:39Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-03-16T14:51:39Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2017-11-25 / Introduction: The colloid goiter (CG) is a heterogeneous disorder and represents the main cause of benign thyroid nodule. Prevalent in geographical areas with iodine deficiency, in most cases carried out expectant conduct or surgically. Classified according to the morphological and functional characteristics, it may present as diffuse or nodular, toxic or nontoxic (with or without functional autonomy). Can reach large volume and compression of neck structures or even turn into malignant disease. Dependent on environmental and genetic factors, with iodine deficiency being the main determinant for the impact on incidence, other environmental and hereditary factors are listed, but little is known about the molecular pathogenesis. Objectives: To map the Epigenetic alterations of the five different surgical parts of CG from patients submitted to total thyroidectomy in Manaus, Amazonas, to analyze, compare and describe the specific pattern for this population. Methods: Five specimens of CG with histopathological confirmation were obtained from the “Normal Thyroid Tissue Bank and with Goiter” from the UFAM. Were used to identify the global methylation patterns of DNA and histone 3 in trimethylated lysine 4, 9 and 27 (H3K4me3, H3K9me3 and H3K27me3). Was used an ELISA type specific immunoassay method, with the results compared between the patients, between the different region of the gland in the same individual and between the individuals. Results: there was no evidence there were significant differences (5%) in the mean global DNA methylation patterns between the patients (p = 0.114) or between the thyroid regions (p = 0.843) considered similar. The mean pan-methylation patterns of histones H3K4me3, H3K9me3 and H3K27me3 are significantly different among patients (p <1%), although there were no significant differences between parts in the same individual (p> 5%). Conclusion: The results of the analyzes of this study revealed that CG parts obeyed a pattern of distribution of significant DNA hypermethylation between patients and parts of CG, differing only among patients the pattern of histone methylation. As these findings are more common in cancer, we may suggest that at the epigenetic level CG may have some common etiologic factor that leads to this tissue response or that responds by phenotypic expressions and malignant transformation. There are now no efficient markers that accurately detect the risk of CG malignancy, much less the possibility of defining its tissue progression, it is very likely that multiple genes will participate in this process. Therefore, the findings found in these patients may make way for the future application of the CG epigenetic study, which may imply the viability of new therapeutic or preventive forms of progression, since the methylation status can be reversed, but other studies in the population are needed to understand better those finding. / Introdução: O bócio coloide (BC) constitui numa desordem heterogênea e representa a principal causa de nódulo benigno da tireoide. Prevalente em áreas geográficas com deficiência de iodo, sendo, na maioria dos casos, conduzido de forma expectante ou cirúrgica. Classificado de acordo com as características morfológicas e funcionais, pode se apresentar difuso ou nodular, tóxico ou atóxico (com ou sem autonomia funcional), podendo alcançar grandes volumes e ocasionar compressão de estruturas do pescoço, ou, não muito raramente, sofrer transformação maligna. Dependente de fatores ambientais e genéticos, com a deficiência de iodo sendo o principal fator determinante para o impacto na incidência, outros fatores ambientais e hereditários são arrolados, porém pouco se conhece da patogênese molecular. Objetivos: mapear as alterações epigenéticas de diferentes regiões de 05 espécies cirúrgicas de BC de pacientes submetidos a tireoidectomia total em Manaus, Amazonas, com o objetivo de analisar, comparar e descrever o padrão específico para esta população. Metodologia: Foram utilizados 05 espécimes de BC com confirmação histopatológica, provenientes do “Banco de Tecidos de Tireoide Normal e com Bócio” da UFAM, e para identificar os padrões de metilação global do DNA e da histona 3 na lisina trimetiladas 4, 9 e 27 (H3K4me3, H3K9me3 e H3K27me3) foram utilizados método de imunoensaio específico do tipo ELISA, com os resultados comparados entre as pacientes, entre as diferentes regiões da glândula num mesmo indivíduo e entre os indivíduos. Resultados: não houveram evidências de que existam diferenças significativas (ao nível de 5%) dos padrões médios de metilação global do DNA entre os pacientes (p = 0,114), ou entre as regiões da tireoide (p = 0,843), sendo, portanto, consideradas similares. Os padrões médios de pan-metilação das histonas H3K4me3, H3K9me3 e H3K27me3 são significativamente diferentes entre os pacientes (p < 1%), apesar de não apresentar diferenças significativas entre as regiões num mesmo indivíduo, (p > 5%). Conclusão: Os resultados das análises deste estudo, revelou que os espécimes de BC obedeceram a um padrão de distribuição de hipermetilação do DNA significativa entre os pacientes e as regiões do BC, diferindo somente entre os pacientes o padrão de metilação das histonas. Como estes achados são mais comuns no câncer, podemos sugerir que em nível epigenético, o BC possa ter algum fator etiológico comum que leve a esta resposta tecidual ou que responda pelas expressões fenotípicas e transformação maligna. Como, até o momento, não existem marcadores eficientes que detecte com precisão a existência de risco de malignização do BC, muito menos a possibilidade de definir sua progressão tecidual, é muito provável que participem múltiplos genes neste processo. Portanto, os achados encontrados nestas pacientes podem abrir caminho na aplicação futura do estudo epigenético do BC, podendo implicar na viabilização de novas formas terapêuticas ou preventivas da progressão, uma vez que, o status de metilação pode ser revertido, porém outros estudos ampliados na população em questão são necessários para entender melhor estes achados.

Homocysteine-Methionine (HM) cycle produces a universal methyl group donor S-adenosylmethionine (SAM), a competitive methylation inhibitor S-adenosylhomocysteine (SAH), and an intermediate amino acid product homocysteine (Hcy). Elevated plasma levels of Hcy is termed as hyperhomocycteinemia (HHcy) which is an established risk factor for cardiovascular disease (CVD) and neural degenerative disease. We were the first to describe methylation inhibition as a mediating biochemical mechanism for endothelial injury and inflammatory monocyte differentiation in HHcy-related CVD and diabetes. We proposed metabolism-associated danger signal (MADS) recognition as a novel mechanism for metabolic risk factor-induced inflammatory responses, independent from pattern recognition receptor (PRR)-mediated pathogen-associated molecular pattern (PAMP)/danger-associated molecular pattern (DAMP) recognition. In this study, we examined the relationship of HM cycle gene expression with methylation regulation in human disease. We selected 115 genes in the extended HM cycle, including 31 metabolic enzymes and 84 methyltransferases (MT), examined their mRNA levels in 35 human disease conditions using a set of public databases. We discovered that: 1) HM cycle senses metabolic risk factor and controls SAM/SAH-dependent methylation. 2) Most of metabolic enzymes in HM cycle (8/11) are located in cytosol, while most of the SAM-dependent MTs (61/84) are located in the nucleus, and Hcy metabolism is absent in the nucleus. 3) 11 up-regulated, 3 down-regulated and 24 differentially regulated SAM/SAH-responsive signal pathways are involved in 7 human disease categories. 4) 8 SAM/SAH-responsive H3/H4 hypomethylation sites are identified in 8 disease conditions. We conclude that HM cycle is a key metabolic sensor system which mediates receptor-independent MADS recognition and modulates SAM/SAH-dependent methylation in human disease. We propose that HM metabolism takes place in cytosol and that nuclear methylation equilibration requires nuclear-cytosol transfer of SAM, SAH and Hcy. CD40 is a cell surface molecule which is expressed on antigen presenting cells such as monocyte, macrophage, dendritic cells and neutrophils. The costimulatory pair, CD40 and CD40L, enhances T cell activation and induce chronic inflammatory disease. Also, DNA hypomethylation on CD40 promotor induces inflammatory monocyte differentiation in chronic kidney disease. In order to figure out if CD40 is a prototypic HM cycle regulated master gene, RNA-seq analysis were performed for CD40+ and CD40- monocytes from mouse peripheral blood and 1,093 differentially expressed genes (DEGs) were selected from those two groups. All the DEGs modulate as much as 15 functional gene groups such as cytokines, enzymes and transcriptional factors. Furthermore, CD40+ monocytes activated trained immunity pathways especially in Acetyl-CoA generation and mevalonate pathway. In HM cycle, CD40 is a prototypic HM cycle regulated master gene to induce the most of the Hcy metabolic enzymes as well as MT, which can further modulate the methylation-regulated pathological signaling. / Biomedical Sciences

The N-terminal tails of histone proteins protrude from the nucleosome core and are extensively post-translationally modified. These modifications are proposed to affect many DNA-based processes such as transcription, DNA replication and repair. Post-translational modifications on histone tails do not act independently but are subject to crosstalk. One example of crosstalk is on histone H3 between lysine 14 (H3K14) and trimethylated lysine 4 (H3K4me3), a modification found at the 5’ end of most active or poised genes. In this work, Western blots and chromatin immunoprecipitation (ChIP) experiments show that different amino acid substitutions at histone H3 position 14 cause varying degrees of H3K4me3 loss, indicating that H3K14 is not essential for H3K4me3 but acts as a modulator of H3K4me3 levels. A neighbouring residue, H3P16 is also important for H3K4me3 and may operate in concert with H3K14 to control H3K4me3. These crosstalk pathways have gene-specific effects and the levels of H3K4me3 are influenced to different extents on genes that fall into functionally distinct classes. A model is proposed to explain how H3K14/H3P16 may exert these varying effects on H3K4me3 at individual genes. In addition to its ability to regulate H3K4me3, H3K14 also influences the levels of two modifications on H3K18, acetylation and monomethylation. A ChIP-sequencing experiment has shown that H3K18me1, a previously uncharacterised modification in S. cerevisiae, is widely distributed throughout the genome and correlates strongly with histone H3 levels. The potential for a functional acetyl/methyl switch at H3K18 is explored. Together, these data indicate that, with gene-specific effects, crosstalk between histone modifications may be even more complex than originally thought.

L'arthrose (OA) est une maladie articulaire dégénérative, classée comme la forme la plus fréquente au monde. Elle est caractérisée par la dégénérescence du cartilage articulaire, l’inflammation de la membrane synoviale, et le remodelage de l’os sous-chondral. Ces changements structurels et fonctionnels sont dues à de nombreux facteurs. Les cytokines, les prostaglandines (PG), et les espèces réactives de l'oxygène sont les principaux médiateurs impliqués dans la pathophysiologie de l'OA. L'interleukine-1β (IL-1β) est une cytokine pro-inflammatoire majeure qui joue un rôle crucial dans l'OA. L'IL-1β induit l'expression de la cyclooxygénase-2 (COX-2), la microsomale prostaglandine E synthase-1 (mPGES-1), la synthase inductible de l'oxyde nitrique (iNOS), ainsi que leurs produits la prostaglandine E2 (PGE2) et l'oxyde nitrique (NO). Ce sont des médiateurs essentiels de la réponse inflammatoire au cours de l'OA qui contribuent aux mécanismes des douleurs, de gonflement, et de destruction des tissus articulaires. Les modifications épigénétiques jouent un rôle très important dans la régulation de l’expression de ces gènes pro-inflammatoires. Parmi ces modifications, la méthylation/ déméthylation des histones joue un rôle critique dans la régulation des gènes. La méthylation/ déméthylation des histones est médiée par deux types d'enzymes: les histones méthyltransférases (HMT) et les histones déméthylases (HDM) qui favorisent l’activation et/ou la répression de la transcription. Il est donc nécessaire de comprendre les mécanismes moléculaires qui contrôlent l’expression des gènes de la COX-2, la mPGES-1, et l’iNOS. L'objectif de cette étude est de déterminer si la méthylation/déméthylation des histones contribute à la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS dans des chondrocytes OA humains induits par l'IL-1β. Nous avons montré que la méthylation de la lysine K4 de l'histone H3 (H3K4) par SET-1A contribue à l’activation des gènes COX-2 et iNOS dans les chondrocytes humains OA induite par l'IL-1β. Nous avons également montré que la lysine K9 de l’histone H3 (H3K9) est déméthylée par LSD1, et que cette déméthylation contribue à l’expression de la mPGES-1 induite par IL-1β dans les chondrocytes humains OA. Nous avons aussi trouvé que les niveaux d'expression des enzymes SET-1A et LSD1 sont élevés au niveau du cartilage OA. Nos résultats montrent, pour la première fois, l'implication de la méthylation/ déméthylation des histones dans la régulation de l’expression des gènes COX-2, mPGES-1, et iNOS. Ces données suggèrent que ces mécanismes pourraient être une cible potentielle pour une intervention pharmacologique dans le traitement de la physiopathologie de l'OA. / Osteoarthritis (OA) is a disabling disease classified as the most common form of arthritis worldwide. It is characterized by cartilage degeneration, synovium inflammation, and subchondral bone remodeling resulting in a loss of joint function. These structural and functional changes are due to numerous factors. Cytokines, prostaglandins (PG), and reactive oxygen species are the major mediators implicated in the pathophysiology of OA. Interleukin-1 (IL-1) is a major pro-inflammatory cytokine that plays a crucial role in OA. IL-1 induces the expression of Cyclo-oxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), inducible nitric oxide synthase (iNOS), as well as their products prostaglandin E2 (PGE2) and nitric oxide (NO). These are critical mediators of the inflammatory response during OA causing pain, swelling, and joint tissue destruction. The activation of these pro-inflammatory genes results from different changes at the level of chromatin known as epigenetic modifications. Epigenetic modifications such as DNA methylation and histone modifications play a crucial role in gene expression. Among these modifications, histone methylation/demethylation is the most critical one. Histone methylation/demethylation is mediated by two types of enzymes: histone methyltransferases (HMT) and histone demethylases (HDM) which can either activate or repress transcription. It is therefore necessary to understand the molecular mechanisms which underlie the regulation of COX-2, mPGES-1, and iNOS expression. The objective of this study is to investigate whether histone methylation/demethylation can modulate COX-2, mPGES-1, and iNOS expression in IL-1 induced OA human chondrocytes. We demonstrated that histone H3 lysine K4 (H3K4) methylation by SET-1A contributes to IL-1-induced COX-2 and iNOS expression in human OA Chondrocytes. We showed also that LSD1-mediated demethylation of histone H3 lysine 9 (H3K9) contributes to IL-1β-induced mPGES-1 expression in human OA chondrocytes. We found that levels of SET-1A and LSD1 expression are elevated in OA cartilage as compared with normal cartilage. Our data demonstrates, for the first time, the implication of histone methylation/demethylation in COX-2, mPGES-1, and iNOS regulation suggesting that these mechanisms could be a potential target for pharmacological intervention in the treatment of the pathophysiology of OA.

Le cancer, principale cause de mortalité dans le monde, est un problème majeur de santé publique. Malgré les nombreux traitements disponibles, il est nécessaire de développer de nouvelles thérapies plus efficaces et moins envahissantes. Aujourd’hui la connaissance du génome humain a dirigé la recherche vers de nouvelles approches: il est possible de moduler la réponse biologique en contrôlant l'accès aux informations génétiques via la régulation épigénétique.L’épigénétique est l’ensemble des modifications de l’expression des gènes n’entraînant pas de modifications dans la séquence d’ADN, qui mènent à un phénotype héritable et stable. Chez les eucaryotes, la régulation épigénétique implique des modifications covalentes de l'ADN (méthylation) et des histones (acétylation, méthylation…). Ces phénomènes modifient la structure de la chromatine, aboutissant à une configuration "ouverte" ou "fermée" permettant la transcription ou la répression de gènes. Dans une situation cancéreuse, le profil épigénétique est modifié ; la méthylation anormale de l’ADN ou des histones mène à la répression de certains gènes comme des gènes suppresseurs de tumeur, ou à l’expression des oncogènes. Contrairement aux changements génétiques irréversibles, les aberrations épigénétiques sont des modifications chimiques réversibles. Ainsi, des molécules capables de rétablir l'équilibre épigénétique représentent des outils thérapeutiques potentiels contre le cancer.La méthylation et l’acétylation sont les modifications épigénétiques les plus étudiées. La méthylation de l’ADN est catalysée par les ADN méthyltransférases (DNMTs), et la méthylation des histones par les histones méthyltransférases (HMTs).Le sujet de ce projet doctoral est porté sur les HMTs et en particulier sur DOT1L (DOT1 like, disruptor of telomericsilencing), responsable des méthylations du résidu Lys79 de l’histone 3 (H3K79), conduisant à la transcription des oncogènes. En effet, des études ont montré que DOT1L est liée à la leucémie et se révèle être une cible intéressante à inhiber. DOT1L comme les DNMT ont un même cofacteur : le SAM (S-adénosyl-L-méthionine). Certains de leurs inhibiteurs présentent un mécanisme d'inhibition commun : ils entrent en compétition avec SAM.Nous présentons la conception basée sur des études de modélisation moléculaire, et la synthèse multi-étapes des séries des molécules formées par 3 motifs principaux : a) un motif aminopyrimidine, b) un motif de type benzimidazole ou phénylurée, liés par c) un groupement phényle ou hétérocyclique. L’activité des composés synthétisés sur DOT1L a été évaluée et des relations structure-activité (RSA) ont été établies. L’activité sur DNMT et d’autres HMTs a été déterminée également afin d’étudier la spécificité de nos composés.Différents structures ont été identifiées comme point de départ pour aboutir à des inhibiteurs sélectives de DOT1L ou à des inhibiteurs mixtes DOT1L/DNMT. Ces molécules sont considérées comme des outils thérapeutiques intéressants dans le traitement du cancer. / Cancer is a serious issue of public health as it is one of the main causes of mortality worldwide. Despite the multiple available treatments, it is necessary to develop more efficient and less invasive therapies against cancer. The knowledge of the human genome and epigenome has directed research to new cancer treatment approaches: it is possible to modulate the biological outcome by controlling the access to the genetic information by means of the epigenetic regulation.Epigenetics are the changes happening on the genome without modifying its DNA sequence, leading to a heritable andstable phenotype. In the eukaryotic chromatin, epigenetic regulation implies covalent modifications of DNA and histones. These chemical modifications remodel the chromatin structure leading to an “opened” or “closed” configuration, which is related to the expression or repression of genes. The epigenetic landscape is altered in cancers; for example, abnormal methylation leads to the silencing of certain genes (such as tumor suppressor genes), or to the over-expression of oncogenes. Unlike genetic alterations that are irreversible, epigenetic aberrations are reversible. Thus, molecules that can reestablish the epigenetic balance represent potent therapeutic tools for cancer treatment.Methylation and acetylation are the most studied epigenetic modifications. DNA methylation is carried out by the DNAmethyltransferases (DNMTs) and histone methylation by the histone methyltransferases (HMTs).This PhD project was focused on the histone methyltransferase DOT1L (DOT1 like, disruptor of telomeric silencing), responsible of methylation of residue Lys79 of histone 3 (H3K79), which leads to the transcription of some oncogenes. Recent studies have shown that DOT1L is implicated in MLL-rearranged leukemia (MLL-r, Myeloid-Lymphoid Leukemia) thus it is a potent target in cancer. As DOT1L and DNMTs share the same cofactor, S-adenosyl-L-methionine (SAM), DNMT and DOT1L inhibitors can present a common inhibition mechanism by competing with SAM.We present herein the in silico – based design, and the multi-step synthesis of some series of molecules containing 3 main moieties: a) an aminopyrimidine motif and b) a benzimidazole or phenylurea motif, linked by c) a phenyl or heterocycle motif. DOT1L activity was determined for the different compounds synthesized and structure-activity relationships (SAR) were established. The activity on DNMT and other HMTs was determined as well, in other to study the DOT1L specificity of our compounds.Different scaffolds were identified to obtain DOT1L-selective or DOT1L/DNMT dual inhibitors. These molecules are interesting therapeutic tools for cancer treatment.

Apoptose und Seneszenz sind stress-responsive, genetisch verankerte „Failsafe“- Mechanismen, welche die Zelle vor maligner Transformation schützen. Onkogenes Ras induziert zelluläre Seneszenz über den p16/Retinoblastoma (Rb)-Signalweg und führt dabei zu einem permanenten Zellzyklusarrest - das tumorsuppressive Potential von Seneszenz in vivo bleibt jedoch bis heute fraglich. In seneszenten Zellen ist die Expression von S-Phase relevanten Gene durch die lokale Ausbildung von Heterochromatin, bzw. der Methylierung von Histon H3 an Lysin 9 (H3K9me) blockiert. Dies lässt vermuten, dass Seneszenz ein epigenetische kontrollierter Prozess ist und von Proteinen wie der Rb-assozierte Histonmethyltransferase Suv39h1 reguliert wird. In der vorliegenden Arbeit konnte gezeigt werden, dass Eµ-N-Ras transgene Mäuse mit heterozygoten Läsionen im Suv39h1 oder p53 Lokus aggressive T-Zell Lymphome entwickeln, die gegen Suv39h1, bzw. p53-Expression selektieren. Im Gegensatz dazu entwickeln N-Ras-transgene Wildtyp-Tiere („Kontrollen“) vorrangig nicht-lymphoide Tumoren und sterben signifikant später. In primären Lymphozyten induziert onkogenes Ras einen Suv39h1-abhängigen, H3K9me-assoziierten Proliferationsarrest und kann dadurch Lymphomgenese verhindern. Suv39h1-defiziente Lymphomzellen wachsen exponentiell und sind, entgegen p53 defizienten Zellen, sensitiv gegenüber Adriamycin-induzierten Zelltod (Apoptose). Jedoch arretieren nur Kontroll-Lymphome unter Therapie in vitro wenn Apoptose blockiert ist, nicht aber Suv39h1 oder p53-defiziente Lymphomzellen. Diese Resultate identifizieren Ras-induzierte Seneszenz als einen neuen, H3K9me-abhängigen Tumorsuppressor-Mechanismus, wobei dessen Inaktivierung die Entwicklung von aggressiven, aber dennoch Apoptose-kompetenten Lymphomen herbeiführt. / Cellular “failsafe” programs like apoptosis or senescence are genetically encoded, stress-responsive mechanisms that ultimately counteract malignant transformation. Acute induction of oncogenic Ras provokes cellular senescence that involves the p16/Retinoblastoma (Rb) pathway to induce a permanent arrest, but the tumor suppressive mechanism in vivo still remains questionable. Senescent cells display heterochromatic features on S-phase relevant genes involving methylation of histone H3 on lysine 9 (H3K9me), which may depend on the Rb-associated histone methyltransferase Suv39h1. In the present thesis it was shown that Eµ-N-Ras transgenic mice harboring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wildtype (“control”) animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.

No description available.

Alors que les méthylations sur différents résidus lysine des histones (par exemple H3K4, H3K27 et H3K36) sont bien connues pour exercer diverses fonctions biologiques, leurs interactions et/ou leur mode d’actions demeurent encore peu caractérisés. Par la génétique et des outils de biologie moléculaire, nous visons à étudier les rôles et interconnections des méthylations des H3K4, H3K27 et H3K36 dans la transcription, la croissance de la plante et la régulation du développement chez Arabidopsis thaliana.La première partie de ma thèse est centrée sur les rôles et interconnections des méthylations de H3K4 and K36.ATX1 et ATX2 sont des méthyltransférases de H3K4 alors que SDG8 est une méthyltransférase de H3K36.L’analyse de doubles mutants a révélé que sdg8 est dominant sur atx1 et atx2 pour le temps de floraison et larégulation de la prolifération cellulaire. La triméthylation de H3K36 (H3K36me3) est partiellement dépendante de H3K4me3 mais non-réciproquement. SDG25 a une double activité H3K4me3 et H3K36me3 et les déméthylases de H3K4, LDL1 et LDL2, sont des antagonistes de l’activité de SDG25. Les triples mutants sdg25ldl1ldl2 fleurissent plus tôt que la lignée sauvage, mais plus tard que sdg25 et montrent une augmentation de taille de cellule similaire à celle des mutants ldl1ldl2.La deuxième partie de ma thèse se concentre sur les rôles et interconnections entre les méthylations H3K4/K36 et H3K27. CLF catalyse les H3K27me3 au sein du complexe PRC2. Les doubles mutants sdg8clf etsdg25clf fleurissent plus tôt que les mutants simples et montrent un nombre réduit de cellules par feuille. Unniveau plus élevé de H3K4me3 et dans une moindre mesure de H3K36me3 a été observé dans le cas de déposition de H3K27me3 réduite, et de la même façon, une déposition de H3K4me3/H3K36me3 réduite augmente aussi le niveau de H3K27me3. Distinct du rôle antagoniste rapporté auparavant entre CLF et ATX1,CLF n’a pas montré d’antagonisme avec SDG25 ou SDG8.La dernière partie de ma thèse est centrée sur le mécanisme de SDG26 dans la régulation du temps de floraison. Mes résultats ont montré que SDG26 est une méthyltransférase H3K4 et/ou H3K36 spécifique de la chromatine de SOC1, un intégrateur de la floraison actif. De manière similaire à SDG25 et SDG8, SDG26 ne travaillait pas de façon antagoniste avec CLF. L’analyse de doubles mutants a révélé que sdg26 domine atx2mais sdg25, atx1 and clf est dominant sur sdg26 pours le temps de floraison et la régulation de la prolifération cellulaire. Les triples mutants sdg26ldl1ldl2 fleurissaient encore plus tard que les mutants sdg26 et ldl1ldl2 et a révélé que sdg26 est dominant sur ldl1ldl2 lors de la régulation de la prolifération cellulaire. Les analysesd’interaction avec les autres composants de PRC2, VEL1 et VRN5, ont révélé que sdg26vel1 et sdg26vrn5 fleurissaient encore plus tard que les mutants simples dans des conditions de jours courts et de vernalisation. Ensemble, mes résultats révèlent des couches additionnelles de complexité de redondance et de diversification de fonctions entre et au sein des méthyltransférases et déméthylases, pour la transcription, le temps de floraison et la régulation de la prolifération cellulaire chez Arabidopsis. / While methylations at different lysine residues of histones (e.g. H3K4, H3K27 and H3K36) are well known to exert diverse biological functions, their interactions and/or ensemble-actions remain poorly characterized so far.Using genetic and molecular biology tools, we aim to investigate roles and ‘crosstalks’ of H3K4, H3K27 andH3K36 methylations in transcription and plant growth and development regulation in Arabidopsis thaliana.The first part of my thesis focuses on the roles and crosstalks between H3K4 and K36 methylations.ATX1 and ATX2 are H3K4 methyltransferases while SDG8 is a H3K36 methyltransferase. Double mutant analysis revealed that sdg8 dominates over atx1 and atx2 in flowering time and cell proliferation regulation.H3K36 trimethylation (H3K36me3) is partially dependent on H3K4me3 but not vice versa. SDG25 has a dualH3K4me3 and H3K36me3 activity and the H3K4-demethylases LDL1 and LDL2 antagonize SDG25 activity.The sdg25ldl1ldl2 triple mutants flowered earlier than wild type but later than sdg25 and showed an increased cell size similarly to ldl1ldl2 mutantsThe second part of my thesis focuses on the roles and crosstalks between H3K4/K36 and H3K27methylations. CLF within PRC2 complex catalyzes H3K27me3. The sdg8clf and sdg25clf double mutants flowered earlier than the single mutants and showed a reduced number of cells per leaf. An increased level ofH3K4me3 and to a less extent H3K36me3 was observed upon impaired H3K27me3 deposition, and similarly impaired H3K4me3/H3K36me3 deposition also enhanced H3K27me3 level. Distinct from previously reported antagonistic role between CLF and ATX1, CLF did not show antagonism with SDG25 or SDG8.The last part of my thesis focuses on mechanism of SDG26 in flowering time regulation. My result showed that SDG26 is a H3K4 and/or H3K36 methyltransferase specific at chromatin of SOC1, an activeflowering integrator. Similarly to SDG25 and SDG8, SDG26 did not work antagonistically with CLF. Double mutant analysis revealed that sdg26 dominates over atx2 while sdg25, atx1 and clf dominate over sdg26 inflowering time and cell proliferation regulation. The sdg26ldl1ldl2 triple mutants flowered even later than thesdg26 and ldl1ldl2 mutants and showed that sdg26 dominates over ldl1ldl2 in cell proliferation regulation.Interaction analysis with the other PRC2 components VEL1 and VRN5 revealed that sdg26vel1 and sdg26vrn5flowered even later than the single mutants under short day and vernalization conditions.Together, my study revealed additional layers of complexity of overlap and non-overlap functions between and within methyltransferases and demethylases in transcription, flowering time and cell proliferation regulation in Arabidopsis.

ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...

Aberrant DNA methylation in stem cells is a hallmark of aging and tumor development. Recently, we have suggested that promoter DNA hyper-methylation originates in DNA repair and that even successful DNA repair might confer this kind of epigenetic long-term change. Here, we ask for interrelations between promoter DNA methylation and histone modification changes observed in the intestine weeks after irradiation and/or following Msh2 loss. We focus on H3K4me3 recruitment to the promoter of H3K27me3 target genes. By RNA- and histone ChIP-sequencing, we demonstrate that this recruitment occurs without changes of the average gene transcription and does not involve H3K9me3. Applying a mathematical model of epigenetic regulation of transcription, we show that the recruitment can be explained by stronger DNA binding of H3K4me3 and H3K27me3 histone methyl-transferases as a consequence of lower DNA methylation. This scenario implicates stable transcription despite of H3K4me3 recruitment, in agreement with our RNA-seq data. Following several kinds of stress, including moderate irradiation, stress-sensitive intestinal stem cell (ISCs) are known to become replaced by more resistant populations. Our simulation results suggest that the stress-resistant ISCs are largely protected against promoter hyper-methylation of H3K27me3 target genes.