Vertebrate genomes are punctuated by CpG islands regions, which have an elevated frequency of CpG dinucleotides. CpG islands are associated with over 70% of mammalian promoters suggesting they may contribute to the regulation of transcription. However, despite being discovered over 30 years ago, the function of CpG islands is still not understood. Unlike the majority of the genome, CpG islands are resistant to DNA methylation. This provides a binding site for CFP1 which binds specifically to non-methylated DNA via its zinc-finger CXXC (zf-CXXC) domain. CFP1 is a subunit of the SET1 methyltransferase complex, and is thought to direct the activating histone modification H3K4me3 to CpG islands. Interestingly, CFP1 also contains a PHD domain which is proposed to bind the H3K4me3 mark, potentially producing a feedback loop between H3K4me3 and the SET1 complex. Although the structural basis for discrimination of non-methylated CpGs is known, it is not clear how zf-CXXC proteins distinguish CpG islands amongst the irregular nucleosomal landscape which exists within the nucleus. This thesis is focused on the role of CFP1 in the relationship between CpG islands, SET1 and H3K4me3. To address these questions, it was important to mechanistically dissect the contribution of the PHD and zf-CXXC domains. The proposal that the PHD domain of CFP1 binds selectively to H3K4me3 was confirmed by in vitro experiments, however this study demonstrates that the PHD domain is insufficient for stable interactions with chromatin. Using complementary genome-wide and live cell imaging approaches, the zf-CXXC domain shown to be required for PHD-dependent interactions. Genome-wide snapshots of binding interactions, together with spatial and temporal details, expose a surprising contribution of the SET1 complex to the nuclear mobility of CFP1, providing a new perspective on the role of CFP1 in H3K4 methylation.

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Toxoplasma gondii is an intracellular parasite that causes a persistent infection in 10- 80% of the world's population, depending on geographic location. Infection with this parasite causes changes in behavior and physiological functions of an infected host, including warm- blooded animals including humans. Recent studies also show influence of infection with T.gondii on male reproductive fitness in mice and rat and to investigate this issue further became a task of my diploma thesis. In this study, detailed histometric screening of testicular function along with levels of a pituitary luteinizing hormone (LH) and screening of epigenetic modification of testicular DNA were analysed in infected and control mice. Testicular function and sperm production was significantly decreased in T. gondii positive group after a 30-day infection. The level of LH in urine of T. gondii infected mice was decreased compares to control and the number of leptotene primary spermatocytes and spermatids was lowered, but also number of Sertoli cells and tubule diameter were elevated. In epigenetic study the testicular epigenom was tested to measure global methylation and methylation of specific genes Hspa1, Crem and Creb1 which are essential for successfully ongoing spermatogenesis. The global level of methylation of...

Au sein des tumeurs, y compris pour le carcinome hépatocellulaire (CHC), des sous-populations de cellules néoplasiques ont révélé une grande capacité à initier de nouvelles tumeurs et à induire des métastases. Les premières études sur ces cellules ont rapidement montré que la présence de ces cellules était déterminante dans le développement tumoral et elles ont donc été renommées « cellules souches cancéreuses » (CSCs). Malheureusement les mécanismes impliqués dans la maintenance de ces CSCs ne sont que partiellement compris. Par ailleurs dans le CHC un lien a été établi entre les signaux du facteur de croissance de transformation (Transforming Growth Factor, TGF-ß) provenant du microenvironnement tumoral et certaines populations de cellules cancéreuses dont la présence est corrélée à un faible pronostic. La façon dont TGF-ß peut ainsi établir et modifier un phénotype cellulaire dans le CHC reste néanmoins obscure. La méthylation de l’ADN étant un acteur majeur dans la mise en place des programmes cellulaires, notre but a été de caractériser le méthylome de CSCs hépatiques et son lien avec la capacité de TGF-ß à induire des CSCs. Nous nous sommes appuyés sur l’expression du marqueur CD133 pour définir la population de CSCs hépatiques. Afin comprendre l’importance des marques de méthylation de l’ADN dans les CSCs hépatiques, nous avons dans un premier temps déterminé quelle était la signature des cellules CD133+ au niveau de la méthylation de l’ADN en utilisant des puces de méthylation à grande échelle. Les sites CpG différentiellement méthylés ont montré un enrichissement pour d’une part des voies de signalisation déjà identifiées dans les CSCs et, d’autre part, pour des voies de signalisation associées au processus inflammatoire dont la voie TGF-ß/SMAD. Par la suite, nous avons montré que TGF-ß pouvait induire de façon permanente les cellules CD133+ contrairement à une autre cytokine influente dans le cancer du foie, l’interleukine 6. Cette augmentation de cellules CD133+ induite par TGF-ß est associée à des changements de méthylation de l’ADN sur l’ensemble du génome et qui sont, de plus, maintenus au cours des divisions cellulaires. La comparaison entre les deux méthylomes (liés aux cellules CD133+ et à l’action de TGF-ß) a exposé une signature commune significative indiquant que TGF-ß pourrait promouvoir le phénotype de CSC via le processus de méthylation de l’ADN. Mais nous avons également déterminé qu’une grande partie des effets sur la méthylation induits par TGF-ß était totalement indépendante de l’induction de cellules CD133+. Enfin, nous avons observé que les sites de méthylation sensibles au signal de TGF-ß étaient regroupés de façon significative au niveau de régions « enhancer » qui régulent la transcription des gènes. Par ailleurs, ces sites incluaient également des gènes précédemment identifiés comme cibles de TGF-ß mais aussi des gènes codant pour des acteurs épigénétiques de premier ordre comme les méthyltransférases de l’ADN. Ces résultats constituent la première description d’une signature de méthylation de l’ADN induite par TGF-ß permettant une reprogrammation stable vers un profil épigénétique de CSC hépatiques. / Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display a pronounced ability to initiate new tumors and induce metastasis. Investigations on theses cells rapidly described them as essential for tumor growth and based on theses observations they have been named “cancer stem cells” (CSCs). Unfortunately, the mechanisms involved in sustaining their programs are only partially known. In HCC, there is an established link between microenvironmental signals from Transforming Growth Factor beta (TGF-ß) and survival of certain cell subpopulations which is results in a bad prognosis. However, how TGF-ß establishes and modifies cell behavior in HCC is not fully understood. As DNA methylation is involved in establishing cellular programs, our aim was to characterize the methylome of putative liver CSCs, and its link to the ability of TGF-ß to induce liver CSCs. We used CD133 expression as a positive marker for liver CSC. To understand the relevance of DNA methylation programs in liver CSCs, we first defined the methylome signature of CD133+ cells in liver cancer cells using methylation bead arrays. Differentially methylated CpG sites were enriched in known pathways related to CSC survival and to inflammation, including the TGF-ß/SMAD pathway. Next, we showed that TGF-ß persistently induces CD133+ cells in opposition to another cytokine related to HCC, interleukin 6. We observed that this increase is associated with genome-wide changes in the methylome induced by TGF-ß and that are perpetuated through cell division. We observed a significant overlap between the CD133+ methylome and the methylome induced by TGF-β, indicating that TGF-ß may induce CSC phenotype through DNA methylation reprogramming. Additionally, we observed genome-wide effects of TGF-ß that are independent of the induction of CD133. Finally, TGF-ß methyl-sensitive sites were significantly concentrated in enhancer regions of the genome, and include well-known targets of TGF-ß, and epigenetic players, such as de novo DNA methyl-transferases. In conclusion our results are the first indication of the ability of TGF-ß to induce genome-wide changes of DNA methylation, leading to a stable switch to a liver cancer stem cell epigenetic program.

La diversité génétique est à l’origine de l'évolution et de l'adaptation des pop. et des espèces. Dans les agrosystèmes, la div. gén. intra-population est d'une importance majeure : d'une part, elle peut fournir un effet tampon contre les variations climatiquesinterannuelles et les stress biotiques, et d'autre part cette div. peut permettre l’adaptation locale des pop., du fait de leur évolution sous l’effet des pressions sélectives spécifiques aux conditions locales de la région, particulièrement dans le cas d’uneintrod. dans un nouvel environnement. En raison de son importance socio-économique et de son aire de culture étendue, le blé a été choisi comme espèce modèle dans cette étude, en se focalisant sur l’étude de la précocité de de floraison, un caractère adaptatif majeur qui permet au blé de croître sur une large gamme de conditions écologiques et climatiques. Ce projet de thèse a pour objet l’analyse de l'impact de la diversité int.-pop. sur la réponse adaptative à court terme de pop. soumises à des conditions agro-climatiques contrastées, ce par l'étude des variations génétiques, épigénétiques et phénotypiques.L'absence de marqueurs épig. disponible pendant la thèse a conduit à développer 2 études complémentaires. Dans une 1ère partie, sept var. pays. (pop. conservées à la ferme) et une variété moderne ont été distribuées et cultivées pendant trois ans dans sept fermes localisées dans trois pays d'Europe, puis étudiées pour leur réponse aux différentes conditions agro-climatiques, sous l’angle de leurs variations phénotypiques et génotypiques. Dans une 2nde partie, l'effet de la vernalisation sur le profil de méth. de l'ADN du gène VRN-A1 a été étudié, constituant une 1ère étape vers le développement de marqueurs épig. Les résultats de la 1ère partie de l'étude ont révélé que l'histoire de la conservation des var. pays. a fortement influencé leur div. gén. et leur structure génétique fine. Les var. pays. conservées ex situ montrent une faible div. gén., avec une struct. génétique simple. Les var. pays. et les mélanges conservés in situ révèlent une div. gén. plus élevée, avec une struct. génétique complexe. Une différenc. spatio-temporelle génétique et phénotypique a été observée, en relation avec le niveau de diversité initial et avec la complexité de structure des var. pays. Les variétés traditionnelles se différencient plus nettement que les var. modernes, ce qui plaide en faveur de utilisation dans des systèmes d'agriculture biologique et à bas intrants. De façon intéressante, une différenc. phénotypique significative a été observée pour les var. qui présentaient une div. gén. initiale très faible, ce qui suggère que d’autres facteurs, par exemple épig., pourraient intervenir dans les adaptations misesen évidence. La 2nde partie a permis de mettre en évidence un profil de méth. intéressant de l’ADN de VRN-A1 : sur plantes non-vernalisées, ce gène présente des niveaux élevés de méth. dans la partie centrale du gène, mais pas en début et fin de gène. De plus, une partie du 1er intron montre une augmentation significative du niveau de méth. de l'ADN suite au traitement au froid. Ce changement de méth. est positivement associé au niveau d'expression du gène. Si la compréhension du rôle de cette méth. sur la régulation de VRN-A1 nécessite des analyses complémentaires, cette étude a permis de caractériser les modifications de méth. de VRN-A1 en réponse au froid et constitue une 1ère étape vers l’identification de possibles epiallèles dans nos pop. et fournit une base à la construction de marqueurs permettant de suivre la variabilité épig. dans différentes pop. En conclusion, cette étude apporte des connaissances utiles pour une meilleure compréhension de l’origine et l'évolution de la div. gén. présente dans les var. pays. Elles permettront de dvper des méthodes de conservation et desélection à la ferme, en tenant compte de l'importance de la div. int.-pop., afin de rép. aux contraintes posées par l'agriculture bio. / Biodiversity provides the raw material for evolution and adaptation of populations and species. In agricultural biodiversity, the within-population genetic diversity is of major importance. On one hand, it can provide a buffering effect against the year-to-year variation of climate or biotic pressures and on the other hand diversity serves as a resource for the population to respond to selective pressures due to specific local conditions, thus allowing for local adaptation, particularly in the case where a population is introduced into a new location. Due to its wide geographic distribution indicating a high adaptiveotential and its socio-economic importance, wheat was chosen as model crop in this study. Flowering time is a major adaptive trait which has allows wheat to grow over a wide range of ecological and climatic conditions. This PhD study was designed to gain insights about the influence of within population diversity on the short term response of populations to contrasting agro-climatic conditions by studying the genetic, epigenetic and phenotypic variation. But due to the lack of prior existence of epigenetic markers, this thesis study is divided of two parts: In the first part, European wheat populations coming from a set of seven farmer and one modern varieties, each of which was grown on seven farms (distributed across Europe) for three years, were used to study their short term response to contrasting agro-climatic conditions in Europe by analysing their phenotypic and genotypic variations. For the second part the effect of vernalization on the DNA methylation profile of theVRN-A1 gene in winter wheat was studied as a first step towards the development for the epigenetic marker in this gene.The results from the first part of the study revealed that conservation history of these farmer varieties strongly influenced the genetic diversity and fine genetic structure. Ex situ conserved farmer varieties showed low genetic diversity and simpler structure whereas in situ conserved farmer varieties and mixtures revealed higher level of genetic diversity and complex genetic structure. Genetic and phenotypic spatio-temporal differentiation depending upon the level of diversity and structural complexity of the farmer variety was observed. The traditional varieties tend to become more differentiated than the modern variety arguing in favour of use of these diverse traditional (farmer) varieties in organic and low input agriculture systems. Interestingly, a significant phenotypic differentiation for varieties with very low genetic diversity has also been observed in this study, which gives indication of a possible role of epigenetic variation in the process of evolution.From the second part of the study (effect of vernalization on the DNA methylation profile of the VRN-A1 gene), it was found that in addition to the detection of gene body methylation across the VRN-A1 gene, we identified a region within intron 1 that shows significant increase in DNA methylation in response to vernalization treatment that is positively correlated with the gene expression. Although the role of this shift in gene regulation is still unclear due to time limitations in the thesis and the small number of genotypes analysed, this study will provide a good material towards future identification of new epialleles and the development of epigenetic markers to study the epigenetic variability of these populations.This study at large provides useful knowledge on the understanding of farmers' varieties evolutionary response to be used in the development of different breeding and conservation approaches for organic agriculture, taking into consideration of the importance of within population diversity, to satisfactorily address the problems of organic agriculture.

O estado de metilação é suscetível a mudanças quando os organismos são expostos a agentes ambientais tais como componentes dos alimentos e medicamentos. Uma dieta rica em metionina (Met) poderia modular a concentração de S-adenosilmetionina (SAM) e S-adenosilhomocisteína (SAH) e alterar o estado de metilação da região promotora de genes supressores de tumores. Tanto a hipometilação global quanto a hipermetilação de genes específicos estão envolvidas na instabilidade genômica e poderiam resultar em dano ao DNA. Este estudo avalia se a dieta suplementada com Met associada a doxorrubicina (DXR), um fármaco antitumoral que induz espécies reativas, resulta em alterações no estado de metilação da região promotora do gene TP53, na razão SAM/SAH, na concentração de glutationa (GSH) e em dano ao DNA. Quarenta ratos machos Wistar foram separados em dois grupos: dieta suplementada com Met (ração comercial acrescida de 2% Met) e dieta controle (ração comercial) por seis semanas. Cada grupo foi subdividido em dois subgrupos que receberam DXR (1mg/Kg) ou solução salina intraperitoneal na terceira e sexta semanas de tratamento. Os rins e fígado foram utilizados para isolamento do DNA, determinação da concentração de SAM, SAH e GSH, e análise da instabilidade genômica. Todos os grupos apresentaram o mesmo estado de metilação da região promotora do gene TP53, determinado pelo método de análise de restrição combinada com bissulfito (COBRA). Este fato poderia ser explicado pelo índice de metilação (razão SAM/SAH) que permaneceu inalterado, possivelmente devido a uma adaptação do ciclo da Met que manteve a concentração de SAM. A depleção de GSH não ocorreu quando DXR foi associada a dieta suplementada com Met. Portanto, a suplementação com Met manteve a concentração de GSH em ratos tratados com DXR. A dieta suplementada com Met não induziu instabilidade genômica e não alterou o dano ao DNA induzido pela DXR. Em conclusão, DXR induz depleção de GSH que é inibida pela suplementação com Met. Entretanto, a mesma suplementação não previne a instabilidade genômica induzida pela DXR. A dieta suplementada com Met aumenta a concentração de SAH renal sem alterar a concentração de SAM e GSH. Tanto a dieta suplementada quanto a DXR não induzem hipermetilação na região promotora do gene TP53. / The DNA methylation status is susceptible to changes when organisms are exposed to environmental agents such as food components and drugs. A methionine-rich (Met) diet may modulate S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations, which could change the DNA methylation status in the promoter region of tumor suppressor genes. Global hipomethylation and gene-specific hipermethylation are involved in genomic instability and it could result in DNA damage. This study intends to evaluate if a Met-rich diet associated with doxorubicin (DXR), an antitumoral drug that induces reactive species, result in changes in the methylation status of the TP53 gene promoter, in the SAM/SAH ratio, in glutathione levels (GSH) and in DNA damage. Forty male Wistar rats were separated into two groups: Met-rich diet (standard chow plus 2% Met), and control diet (standard chow) for six weeks. Each group was subdivided into another two groups that received DXR (1mg/kg) or saline intraperitoneally in the third and sixth weeks of the experiment. The kidneys and the liver were removed for DNA isolation, SAM, SAH and GSH determination, and genomic instability assay. All groups showed the same unmethylated status in the TP53 promoter according to the Combined Bisulfite Restriction Analysis (COBRA). This could be explained by the fact that the methylation index (SAM/SAH ratio) remained unchanged, possibly because of an adaptive Met pathway that maintains SAM levels. GSH depletion did not occur when DXR was associated with the Met-rich diet. As a matter of fact, the Met-rich diet improved GSH concentration in DXR-treated rats. Met-rich diet did not induce genomic instability, and it did not alter DNA damage induced by DXR. In conclusion, DXR induces GSH depletion which is inhibited by Met supplementation. However, Met-rich diet may not prevent genomic instability induced by DXR. A Met-rich diet increases SAH levels; however, it does not change GSH and SAM levels. Neither Met supplementation nor DXR induced DNA hypermethylation in the TP53 gene promoter.

INTRODUÇÃO: A expansão e disseminação do consumo de crack e cocaína no Brasil vem se tornando um grave problema de saúde pública nos últimos vinte anos. Diferentes abordagens biológicas têm sido investigadas utilizando o fenótipo de abuso/dependência de crack/cocaína, cujos resultados têm demonstrado a participação importante do substrato genético, assim como a sua interação com os fatores ambientais no desenvolvimento desse transtorno. OBJETIVOS: Investigar o padrão de metilação do DNA do genoma de indivíduos que apresentam abuso/dependência de cocaína e de crack, comparando ao padrão de metilação de indivíduos controles. MÉTODOS: Foram selecionados 24 dependentes de cocaína e crack e 24 controles saudáveis, pareados por sexo e idade. Utilizando amostras de DNA extraídas de sangue periférico de cada um dos participantes, foi realizada a técnica de metilação global com o ensaio Illumina Infinium Human Methylation450 (450K) BeadChip. Os resultados iniciais foram normalizados considerando a heterogeneidade celular e analisados utilizando o pacote ChAMP (Chip Analysis Methylation Pipeline) para identificar genes e/ou regiões gênicas diferencialmente metiladas que possam representar fatores de vulnerabilidade para o comportamento de abuso/dependência do crack e da cocaína. Os processos biológicos e vias celulares com os quais os sítios diferencialmente metilados estão envolvidos foram explorados usando as ferramentas disponibilizadas pelo \"WebGestalt\" e pelo \"UCSC Genome Browser\". RESULTADOS: Foram observados 250 sítios diferencialmente metilados, associados a 246 genes na comparação dos perfis de metilação entre os casos e controles, sendo que 49% destes estavam localizados nas regiões promotoras dos genes, sugerindo que esses sítios podem estar relacionados com a expressão gênica. Alterações estatisticamente significantes no padrão de metilação entre casos x controles foram observadas em 23 sítios CpG (p-valor ajustado < 10-5 e |?beta| = 0,1). Observou-se também que três regiões diferencialmente metiladas foram associadas a genes hipometilados (BMP8A, GPR88 e RNF166) (p-valor ajustado < 0.05), cada uma com pelo menos três sítios. Alterações estatisticamente significantes também foram observadas em seis genes hiper-representados: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, envolvidos em processos biológicos e moleculares. CONCLUSÕES: Observou-se diferenças estatisticamente significativas no padrão de metilação genômico de usuários/dependentes de crack e cocaína quando comparados aos controles saudáveis em amostra de DNA extraídas de sangue periférico. Comparação de estudos de expressão em tecido cerebral correlacionaram-se parcialmente com os achados apresentados. Outros estudos utilizando amostras independentes são necessários para confirmar esses achados. A confirmação desses resultados poderá contribuir na identificação e compreensão dos mecanismos biológicos envolvidos na dependência do crack/cocaína / BACKGROUND: The expansion and dissemination of crack and cocaine in Brazil has become a progressive and serious public health problem during the last twenty years. Different biological approaches have been investigated using the crack/cocaine abuser/dependent phenotype, with results confirming the important role of the genetic component, as well as its interaction with environmental factors. OBJECTIVES: To investigate the DNA methylation pattern levels in the genome of individuals with crack and cocaine abuse/dependence and comparing it with the DNA methylation pattern of the genome of control subjects. METHODS: 24 crack and cocaine abusers/dependents and 24 healthy controls were selected and matched by sex and age. Using DNA samples from peripheral blood of each participant, a global methylation technique was performed using the Illumina Infinium Human Methylation450 (450K) Bead Chip assay. The initial results were normalized for cellular heterogeneity and re-analyzed using ChAMP package (Chip Methylation Analysis Pipeline) to identify differentially methylated genes or/and DNA regions that may represent biological/genetic risk factors for crack and cocaine abuse/dependence behavior. The biological processes and cellular pathways were explored using tools provided by \"WebGestalt\" and \"UCSC Genome Browser\". RESULTS: 250 differentially methylated sites associated with 246 genes in methylation comparison profiles between cases and controls were identified, of which almost half were located in the promoter regions of genes (49%), suggesting that these sites may be related to gene expression. Statistically significant changes in the methylation patterns between cases and controls were observed in 23 CpG sites (adjust p-value < 10-5 and |deltabeta| = 0.1). In addition, three differentially methylated regions were associated with hipomethylated genes (BMP8A, GPR88 e RNF166) (adjust p-value < 0.05), each one with at least three sites. Statistically significant changes were also observe with six hiper-represented genes: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, which are involved in biological and molecular processes. CONCLUSIONS: Crack and cocaine users/dependents presented significant statistical differences in the methylation pattern when compared to healthy controls in DNA samples extracted from peripheral blood. Results from gene expression studies using brain tissue can be correlated with our results. In order to confirm the present findings, future studies should be replicated using independent samples. The confirmation of these results will contribute to the understanding of the biological mechanisms involved in crack/cocaine dependence

O nucléolo é uma organela nuclear responsável pela produção dos ribossomos, através das Regiões Organizadoras do Nucléolo (NORs). Espécies que possuem mais de um par de cromossomos contendo NORs terão, obrigatoriamente, pelo menos um par ativo, sendo as demais NORs funcionais de acordo com a demanda celular. O mecanismo de compensação de dose é visualizado e bem estabelecido em híbridos interespecíficos, conhecido como dominância nucleolar, com a inativação de NORs de um dos parentais por outras homeólogas ativas que as dominam. A arquitetura da cromatina nas NORs e o controle da sua expressão foram estudados com o objetivo de se entender os mecanismos envolvidos no fenômeno da dominância nucleolar em espécies diplóides que possuem múltiplas NORs. A espécie modelo utilizada neste estudo foi Crotalaria juncea (Leguminosae-Papilionoideae), caracterizada por conter 2n=2x=16, e NORs no braço curto do cromossomo 1, sendo este o principal organizador do nucléolo, e no braço longo do cromossomo 4 adjacente à heterocromatina centromérica, sendo este um sítio adicional (sítio menor) e de expressão facultativa, previamente determinada. Nas raízes de C. juncea sincronizadas, observou-se que a nucleologênese tem seu início durante o final da telófase, em que os 4 sítios de genes ribossomais podem ter atividade e formar até 4 nucléolos, os quais tendem a se fundir durante a interfase. A Hibridação in situ fluorescente (FISH) permitiu estudos da arquitetura da cromatina, com a visualização dos territórios cromossômicos, onde a cromatina não está organizada de forma aleatória dentro do núcleo, e consequentemente o rDNA 45S dentro do nucléolo. Observou-se também que todos os sítios de rDNA 45S possuem diferença no tamanho do arranjo repetitivo. Assim sendo, a hierarquia de dominância está de acordo com o tamanho de cada arranjo (sítio), e estes são ativados de acordo com a demanda celular. As análises das modificações nas histonas mostraram que a H3K9Met1 apresentou marcas fracas no nucléolo, enquanto no restante da cromatina nuclear sua marcação foi intensa. Já a H3K9Met2 apresentou marcação fortemente associada à cromatina presente no nucléolo, com alguns pequenos pontos heterocromáticos dispersos no núcleo. Pela observação entende-se que ambas metilações controlam diferentes tipos de heterocromatinas, ou seja, a H3K9Met2 controla principalmente heterocromatinas associadas aos genes ribossomais, e a H3K9Met controla heterocromatinas não associadas ao rDNA. O rDNA é hiperacetilado dentro do nucléolo para a H3K14. Não foi observada marcação nucleolar para H4K8ac, mas pôde ser observadas regiões hiperacetiladas em outras regiões da cromatina. A metilação do DNA esteve diretamente associada à diferentes níveis de organização da cromatina das NORs. As heterocromatinas adjacentes ao nucléolo apareceram fortemente metiladas, enquanto a cromatina distendida dentro do nucléolo apresentou marcação dispersa, com algumas regiões mais fortemente marcadas, onde a cromatina apresentava-se mais condensada e provavelmente não associados com a cromatina ativa. As fibras estendidas permitiram uma análise de alta resolução, onde foi possível observar que regiões não metiladas apareciam intercaladas entre grandes regiões fortemente metiladas, sugerindo que estas regiões hipometiladas estão, possivelmente, associadas com as alças de transcrição dentro do nucléolo. 12 Esses resultados contribuem para o entendimento sobre o controle genético e epigenético na arquitetura da cromatina ribossomal, bem como seu controle na expressão dos genes ribossomais no genoma das plantas. / The nucleolus is a nuclear organelle responsible for the ribosomes production, by Nucleolus Organizer Regions (NORs). Species presenting more than one chromosome pair with NORs should present, one pair expressing the genes, at least; while the other pairs expressing their genes accordingly to cellular demand. Dosage compensation mechanism is visualized and well established of interspecific hybrids as a well-described phenomena named nucleolar dominance, where a NOR from one parental could lead to inactivation of a NOR from the other parental which is dominated. The chromatin architecture and expression of the NORs were studied to address the mechanism involved in the nucleolar dominance of diploid species containing multiple sites of 45S rDNA. The model species used in the present study was the crop Crotalaria juncea (Leguminosae-Papilionoideae) characterized by 2n=2x=16 chromosomes, being the main NOR mapped into chromosome 1 short arm and presenting an additional site (minor site) in the chromosome 4 long arm adjacent to a centromeric heterochromatin and facultatively expressed. Synchronized meristematic root tip cells determined to nucleologenesis starts during the late-telophase, often expressing every ribosomal gene sites, when up to four nucleoli could be observed and these become merged during interphases. FISH allowed nucleolar chromatin architecture be accessed revealing distinct chromosomal domains (territories), suggesting a non-random distribution of the 45S rDNA, even between homologous chromosomes, into the nucleolus. The 45S rDNA sites from both chromosome pairs 1 and 4 of C. juncea showed differences in their array sizes. The differences in the 45S rDNA array sizes and the order of loci expression suggest a hierarchy of dominance, a feature of nucleolar dominance; being the small RONs activated only on demand. Immunodetection of histone modifications showed different patterns to methylation distribution across the chromatin as a whole; where H3K9Met1 was found mainly distributed along the nuclear chromatin without an evident signal into nucleolus, while H3K9Met2 was detected as conspicuous dots in the nuclear chromatin and highly accumulated into the nucleolus. The results indicate different control on heterochromatin establishment and maintenance, being the modifications specific to certain chromosomal regions. Indeed, H3K9Met is a key component in the nucleolus chromatin architecture and expression. The chromatin inside the nucleolus showed a high accumulation of H3K14ac, with a weak fluorescent signal along the nucleus; on the other hand H4K8ac showed a strong signal homogenously distributed across the nuclear chromatin, but without evident signals inside the nucleolus. DNA methylation was directly associated with different levels of chromatin organization of the NORs. The heterochromatic regions associated to RON are highly methylated, while the chromatin inside the nucleolus showed weaker signals, with some bright spots probably in condensed regions and related to chromatin inactivity. Extended DNA fiber allowed a higher resolution mapping that revealed long methylated regions intermingled by nomethylated ones, being the last probably associated to transcriptional loops of rRNA genes into the nucleolus. The results presented herein contributes to a better understand about the nucleolar chromatin architecture and the genetic and epigenetic control of the ribosomal genes expression on plant genomes.

O produto comercial CI Disperse Blue 291 (CI DB291) é amplamente utilizado pela industria têxtil. Estudos mostram que corantes dinitrofenilazo são genotóxicos no ensaio de Ames/Salmonella, mas há poucos estudos sobre seus efeitos em células eucariontes. Este trabalho teve como objetivo investigar a genotoxicidade e citotoxicidade do corante DB291 em células humanas em cultura, bem como vias pelas quais o corante atua levando aos efeitos tóxicos. O corante comercial foi purificado por HPLC-DAD e utilizado para incubação com células HepG2 (5-100 &#181;M por 3-72 h). A citotoxicidade foi avaliada pelos ensaios de XTT, corante cristal violeta, lactato desidrogenase extracelular e consumo de glicose. A geração de ROS intracelular foi verificada pela emissão de fluorescência da 2\',7\'-diclorofluoresceína. Análises de ciclo celular, fragmentação do DNA, potencial de membrana mitocondrial (&#936;) e cálcio intracelular foram realizadas por citometria de fluxo. A produção de ATP foi mensurada por quimiluminescência. Níveis de 8-oxodG e 5-mdC foram avaliados por HPLC-ESI-MS/MS e HPLC-UV, respectivamente. A expressão da enzima DNMT1 foi avaliada por western blot. Um produto de biotransformação foi identificado e caracterizado estruturalmente por MS/MS e 1H-RMN. A exposição ao corante DB291 diminuiu significativamente a sobrevivência das células em um modo tempo- e dose-dependente (IC50 = 74 &#181;M), com a concomitante formação de um produto de biotransformação reduzido. A morte celular ocorreu sem lise da membrana plasmática. Nas células expostas, foi observado aumento da atividade enzimática mitocondrial, acompanhado por um aumento da taxa de consumo de glicose. Índices elevados de ATP, &#936; e cálcio foram verificados após a incubação das células com concentrações crescentes do corante. Alterações mitocondriais e o processo de biotransformação impeliram a aumento na produção de ROS intracelular, 8-oxodG e fragmentação do DNA. O dano ao DNA induziu a parada no ciclo celular e super-expressão de DNMT1, seguido por hipometilação global do DNA no ciclo celular subsequente. Os resultados obtidos apontam pela primeira vez para a toxicidade do corante DB291 via biotransformação, com alterações mitocondriais e indução de estresse oxidativo. / The commercial CI Disperse Blue 291 (CI DB291) is widely used by textile industry. It is mutagenic in the Ames/S. typhimurium assay, but there are few studies showing its effects in eukaryotic cells. We evaluated here the toxicity of CI DB291 in the human HepG2 cell line. The commercial dye was purified by HPLC-DAD and used for incubation with HepG2 cells (5-100 &#181;M for 3-72 h). Cytotoxicity was assessed by the XTT, crystal violet dye, extracellular lactate dehydrogenase and glucose consumption assays. ROS formation was assessed by 2\',7\'-dichlorofluorescein fluorescence. Analyses of cell cycle, DNA fragmentation, mitochondrial membrane potential (&#936;) and intracellular calcium were analyzed by flow cytometry. ATP level was measured by chemiluminescence. Levels of 8-oxodG and 5-mdC were evaluated by HPLCESI-MS/MS and HPLC-UV, respectively. DNMT1 expression was assessed by western blot. A biotransformation product was identified and structurally characterized by MS/MS and 1H-NMR. DB291 significantly decreased cell survival in a time- and dose-dependent manner (IC50 = 74 &#181;M), with concomitant formation of a reduced biotransformation product. Plasma membrane lysis did not occur. Increased mitochondrial enzymatic activity, accompanied by increase in glucose consumption rate, was observed in cells incubated with DB291. Elevated ATP, &#936;, and intracellular calcium was verified after cell incubation with increasing dye concentrations. Mitochondrial changes and the biotransformation process accounted for the observed raise in intracellular ROS, 8-oxodG, and DNA fragmentation. DNA damage induced cell cycle arrest and DNMT1 overexpression, followed by DNA hypomethylation in the subsequent cell cycle. Results point for the first time to toxicity of the dye through biotransformation, mitochondrial changes, and oxidative stress.

Estudos recentes têm revelado que uma fração significativa do transcriptoma de eucariotos é composta por RNAs não codificadores longos (lncRNAs). Este trabalho investigou o padrão de expressão de um conjunto de lncRNAs originados a partir de regiões intrônicas de genes codificadores de proteínas em três linhagens celulares tumorais humanas utilizando microarranjos de DNA customizados. Realizamos uma série de análises in silico com a perspectiva de identificar propriedades globais desses transcritos, tais como a abundância relativa em diferentes tecidos, características evolutivas, estruturais e regulatórias, além de possíveis funções celulares. Avaliamos também a contribuição da metilação do DNA, um mecanismo de silenciamento epigenético da expressão de genes codificadores de proteínas, na regulação da expressão de lncRNAs intrônicos. Observamos que uma fração dos lncRNAs intrônicos detectados nas linhagens estudadas são conservados evolutivamente, tem padrão de expressão tecido específico, e está enriquecida em elementos regulatórios na sua extremidade 5\'. Foram identificados subconjuntos de lncRNAs intrônicos possivelmente atuando sobre genes associados a vias regulatórias importantes para o controle do desenvolvimento de organismos e ciclo celular. Comparativamente a mRNAs, uma menor proporção de lncRNAs intrônicos possui ilhas CpGs (CGIs) na vizinhança de seu início de transcrição. Apesar disso, observamos que um subconjunto desses transcritos teve sua expressão sensível ao tratamento com o agente desmetilante de DNA 5-AZA, demonstrando que lncRNAs intrônicos transcritos podem estar sujeitos a regulação transcricional mediada por metilação do DNA. Dentre os lncRNAs intrônicos regulados por metilação do DNA, destaca-se o lncRNA AS-APP, cuja expressão aumentou em 25 a 80 vezes nas linhagens celulares DU-145 e HEK293, respectivamente, após tratamento com 5-AZA. Este lncRNA possui uma CGI metilada e um promotor ativo a cerca de 4 kb de distância do seu início de transcrição conhecido. O aumento da transcrição do lncRNA AS-APP após desmetilação do DNA correlacionou-se a uma diminuição significativa dos níveis de expressão do mRNA do gene APP. Este resultado sugere uma possível ação regulatória em cis do lncRNA AS-APP no locus APP, um importante gene envolvido na doença de Alzheimer e com expressão associada ao prognóstico de alguns tipos de câncer. Os resultados obtidos neste trabalho reforçam a ideia de que lncRNAs intrônicos constituem unidades transcricionais independentes que se encontram sobre controle regulatório nos diferentes tipos celulares. Foi gerado também um catálogo de lncRNAs intrônicos regulados por metilação que permitirá a seleção de candidatos com maior potencial de relevância funcional para caracterização detalhada. / Recent studies have revealed that a significant fraction of the eukaryotic transcriptome is composed of long noncoding RNAs (lncRNAs). This work investigated the expression pattern in three human tumor cell lines of a set of lncRNAs originated from intronic regions of protein coding RNAs, using custom DNA oligoarrays. In silico analyses were performed to identify global properties of these transcripts such as relative abundance in different human tissues, regulatory, evolutionary and structural aspects, as well as their possible cellular functions. In addition, we evaluated the contribution of DNA methylation, an important epigenetic mechanism that control the expression of protein coding genes, in the regulation of intronic lncRNAs expression. We found that a fraction of the intronic lncRNAs detected in the cell lines are evolutionarily conserved, show a tissue specific expression pattern, and is enriched in regulatory elements at their 5\' end region. Subsets of intronic lncRNAs possibly acting on genes associated to important regulatory pathways controlling organism development and cell cycle were identified. A smaller proportion of intronic lncRNAs relative to mRNAs displayed CpG islands (CGI) in the vicinity of the transcription start site. Notwithstanding, we observed that a subset of these transcripts responded to treatment with the DNA demethylation agent 5-AZA, demonstrating that intronic lncRNAs may be under transcriptional regulation mediated by DNA methylation. Among intronic lncRNAs regulated by DNA demethylation, stands out AS-APP lncRNA, which was up regulated 25 to 80 times in DU-145 and HEK293 cell lines following 5-AZA treatment, respectively,. This lncRNAs has a methylated CGI and an active promoter at 4-kb upstream from its known transcription start site. Increased AS-APP lncRNA transcription following DNA demethylation correlated with a significant decrease of APP gene messenger RNA levels. This finding suggests a possible cis-regulatory action of the lncRNA AS-APP in the APP locus, an important gene involved in Alzheimer disease and whose expression is associated with prognosis of different cancer types. The results obtained in this study reinforce the idea that intronic lncRNAs constitute independent transcriptional units under regulatory control in the different cell types. It was generated a catalog of intronic lncRNAs regulated by DNA methylation that will allow the selection of candidates with higher potential of functional relevance for detailed characterization