Dentre as tecnologias de reprodução assistida (TRAs), a produção in vitro de embriões (PIVE) bovinos é a mais amplamente estudada, além de possuir um grande interesse econômico. Entretanto, existem fatores que influenciam negativamente a taxa de produção embrionária e o sucesso da gestação. Sabe-se que embriões provenientes das TRAs apresentam frequentemente alterações epigenéticas, relacionadas principalmente ao microambiente no qual os gametas e embriões se desenvolvem. A suplementação com ácido fólico parece prevenir tais erros epigenéticos, visto que além de ser um importante composto vitamínico, ele pode atuar como doador de grupamentos metil e possui ação antioxidante. A adição de ácido fólico durante a maturação oocitária parece influenciar positivamente a PIVE, além de existir evidências de que ele possa alterar os níveis de metilação do DNA e a expressão de genes importantes ao desenvolvimento embrionário. Sendo assim, este trabalho teve como objetivo, avaliar a influência de diferentes concentrações de ácido fólico durante a maturação in vitro (MIV) de oócitos de diferentes qualidades (graus I e III) na produção embrionária e na expressão dos genes IGF2 e LIT1 em blastocistos bovinos. Para tanto, três diferentes concentrações de ácido fólico (10, 30 e 100µM) foram avaliadas durante a MIV, em que 2159 oócitos (1087 oócitos grau I e 1072 oócitos grau III) foram maturados, fertilizados e cultivados por sete dias. Os blastocistos foram avaliados quanto à (1) taxa de produção nos diferentes tratamentos e origem dos oócitos e (2) a expressão dos genes IGF2 e LIT1 foi investigada utilizando a transcrição reversa associada a PCR quantitativa. A análise estatística foi realizada através do teste t de Student. A avaliação da produção embrionária mostrou que a adição de 10µM de ácido fólico influencia positivamente a produção de embriões provenientes de oócitos grau III (p=0,017), enquanto em embriões provenientes de oócitos grau III não foi observada influência. O segundo tratamento mostrou que a adição de 30µM de ácido fólico parece não influenciar tanto embriões provenientes de oócitos grau I quanto grau III. Por outro lado, a adição de 100µM de ácido fólico prejudica o desenvolvimento de embriões provenientes de oócitos grau I (p=0,043), mas parece não influenciar os embriões provenientes de oócitos grau III. Quando avaliada a expressão gênica, o gene IGF2 foi expresso em 25% das amostras, sendo que, com exceção de uma amostra, todas eram provenientes de oócitos grau I. Apesar do gene LIT1 ser expresso em todas as amostras analisadas, não foram observadas diferenças entre os embriões cujos oócitos foram maturados na ausência ou na presença de ácido fólico, independente da concentração adicionada ou origem oocitária (graus I ou III). A adição de ácido fólico durante a maturação de oócitos bovinos parece ser interessante para a PIVE. No entanto, a concentração e a qualidade oocitária parecem ser fatores importantes para a produção embrionária, visto que menores concentrações favorecem oócitos de baixa qualidade enquanto que maiores concentrações prejudicam oócitos de maior qualidade. Por fim, as alterações na produção embrionária devido à adição de ácido fólico parecem não estar relacionada com os níveis de expressão dos genes IGF2 e LIT1. / Among assisted reproductive Technologies (ARTs), in vitro production (IVP) of bovine embryos is the most studied, besides exist a great economic interest. However, there are many factors that negatively influences production rates and pregnancy success. It\'s know that embryos produced by ARTs frequently present epigenetics alterations, related, specially, to the microenvironment where occurs the development of gametes and embryos. Supplementation with folic acid seems to prevent these epigenetics damages, since it is an important component and may act like a methyl donor and has antioxidant action. Folic acid addition during oocitary maturation seems to positively influences embryos IVP, in addition there are evidences that it may alter DNA methylation levels and expression of important genes during embryo development. Thus, this paper had as objective; evaluate different concentrations of folic acid during in vitro maturation (IVM) of different bovine oocytes (Grade I and III) on the embryo production and expression of IGF2 and LIT1 gene in the bovine blastocyst. For this purpose, three different folic acid concentrations (10, 30, and 100µM) were evaluated during IVM, when 2159 oocytes (1087 grade I and 1072 grade III) were matured, fertilized and cultured during seven days. Blastocysts were evaluated for (1) production rates in different treatments and oocyte type and (2) expression of IGF2 and LIT1 was investigated using quantitative reverse transcription PCR. Statistical analysis was realized using student t test. Embryo production analysis revealed that addition of 10µM of folic acid positively influences production of embryos from oocytes grade III (p=0,017), whereas in embryos from oocytes grade I this influence wasn\'t observed. The second treatment showed that 30µM of folic acid didn\'t influence both embryos from oocytes grade I and embryos from oocytes grade III. On the other hand, addition of 100µM of folic acid damages the development of embryos provide from oocytes grade I (p=0,043), but this wasn\'t observed in embryos provide from oocytes grade III. When evaluated gene expression, IGF2 gene were expressed in 25% of all samples, being that, with exception of one sample, all of them were embryos from oocytes grade I. Despite LIT1 expression were detected in all samples, wasn\'t observed statistical difference between embryos whose oocytes were matured in the absence or presence of folic acid, independent of concentration added or oocytes types (grades I ou III). Folic acid addition during bovine oocytes maturation seems to be interesting to embryo IVP. However, folic acid concentration and oocyte quality seems to be important factors to embryo production, since lower concentration its beneficial for lower quality oocytes, while higher folic acid concentration damage better oocytes. Finally, embryos production rates alterations due folic acid addition appears to be unrelated with expression levels of IGF2 and LIT1.

O nucléolo é uma organela subnuclear formada pela atividade transcricional dos genes ribossomais 18S-5.8S-26S (rDNA 45S) e consequente biogênese dos ribossomos. A atividade destes genes resulta na região organizadora do nucléolo (NOR), na forma de uma constrição secundária em cromossomos metafásicos. As constrições secundárias se condensam progressivamente durante a mitose e se descondensam ao final da telófase quando a reestruturação do nucléolo se inicia. Genomas que apresentam mais de um locus de rDNA 45S deve apresentar, obrigatoriamente, pelo menos um par de NORs, enquanto os demais loci poderão ou não serem expressos. O controle da expressão dos genes ribossomais e a formação da cromatina nucleolar são modulados por eventos epigenéticos. Embora alguns pontos sobre o funcionamento dos genes ribossomais e a formação do nucléolo estejam bem estabelecidos, questões como o padrão de condensação da cromatina nucleolar durante a mitose, o padrão de funcionamento de sítios adicionais de genes ribossomais, o papel das modificações epigenéticas na dinâmica da cromatina nucleolar e na expressão do rDNA 45S e o mecanismo de herança dos genes ativos, permanecem abertas. A espécie Crotalaria juncea (Leguminosae-Papilionoideae), com 2n=2x=16 cromossomos, que possui um locus de rDNA 45S no braço curto do cromossomo 1, que sempre forma constrição secundária, e um sítio adicional com atividade facultativa no braço curto do cromossomo 4, é um excelente modelo para o estudo destas questões. No contexto apresentado, foram estudadas a dinâmica de condensação das NORs durante o ciclo celular e sua correlação com a atividade dos genes ribossomais, incluindo o locus adicional, e ainda o papel da metilação da citosina do DNA durante estes processos. Os resultados demonstram que a cromatina da região organizadora do nucléolo segrega em um estado descondensado durante a mitose, na forma de constrição secundária, ou seja, tal estrutura não se condensa durante a metáfase e não volta a se distender no início da telófase. Aparentemente, o que causa correlações equivocadas entre a atividade nucleolar e a observação morfológica da constrição secundária na metáfase é a contração forçada da cromatina da NOR causada por agentes antimitogênicos. Este modelo de segregação em um estado aberto pode ser explicado pela descrição de diversas proteínas que permanecem diretamente ligadas ou indiretamente associadas à região da NOR durante a mitose, funcionando como uma barreira física para a compactação. Ambos os sítios, principais e adicionais, do rDNA 45S presentes em Crotalaria juncea apresentam atividade transcricional, embora o locus do cromossomo 4 mostre atividade facultativa. Ao contrário do que foi anteriormente proposto, uma vez ativo, o locus adicional permanece descondensado durante todo o ciclo mitótico, seguindo o mesmo comportamento dos sítios principais. As constrições secundárias e a cromatina nucleolar são hipermetiladas em nível citológico, independentemente de sua atividade. A aparente hipometilação observada no rDNA 45S em cromossomos mitóticos e núcleos interfásicos se deve ao menor grau de compactação da região organizadora do nucléolo e, consequentemente, à baixa densidade de cromatina. / The nucleolus is a subnuclear organelle formed as a result of transcriptional activity of ribosomal RNA genes 18S-5.8S-26S (45S rDNA) and subsequent ribosome biogenesis. This activity forms the nucleolar organizing region (NOR) as a secondary constriction in metaphase chromosomes. The secondary constrictions progressively condense during mitosis and decondense at the end of telophase, when nucleoli start to reassemble. Genomes presenting more than one 45S rDNA locus must have at least one pair of NOR bearing chromosomes, while other loci may be expressed or not. Ribosomal gene expression and nucleolar chromatin assembly are modulated by specific epigenetic events. Although some topics related to rDNA gene activity and nucleolus formation are well understood, questions such as the behavior of nucleolar chromatin condensation during mitosis, standard functions associated with rDNA additional sites, role of epigenetic modifications in nucleolar chromatin and 45S rDNA expression processes, and inheritance mechanism of active genes, remain to be solved. Crotalaria juncea (Leguminosae - Papilionoideae) has 2n=2x=16 chromosomes and carries a 45S rDNA locus at the short arm of chromosome 1, always presenting a secondary constriction, and an additional site with facultative activity at the short arm of chromosome 4, being an excellent model to resolve these questions. Thus, this study aimed to study NOR condensation dynamics during the cell cycle and its correlation with ribosomal gene activity, including the additional locus, while analyzing the role of rDNA cytosine methylation during this process. The results show that NOR chromatin segregate in a decondensed way throughout mitosis, as a secondary constriction. In other words, this structure does not condense during metaphase and the NOR is not reassembled at the beginning of telophase. Misinterpretations relating nucleolar activity with morphological observations of secondary constrictions, appear to be induced by the artificial contraction of NOR chromatin caused by antimitotic drugs. This segregation model in an open state may be supported by strong diversity of proteins that are maintained attached to NORs during mitosis, serving as a physic barrier for condensation. Both principal and additional 45S rDNA sites of C. juncea are transcriptionally active, although the additional locus in chromosome 4 presented facultative activity depending upon ribosomal request. Unlike what was previously proposed, once the additional site is activated, it remains in an open configuration throughout the cell cycle, similarly to principal site behavior. Secondary constrictions and nucleolar chromatin are hypermethylated at cytological level, regardless of their activity. The seeming hipomethylated state of 45S rDNA in interphase nucleus and mitotic chromosomes is due to a lower compaction level of nucleolar organizing regions and subsequent low chromatin density.

Em mamíferos a inativação do cromossomo X (ICX) consiste no silenciamento gênico de um dos dois X presentes nas células somáticas normais das fêmeas, garantindo a compensação de dose transcricional em relação aos machos. Existem duas formas de ICX: aleatória, na qual a escolha do cromossomo X inativado se dá ao acaso (X paterno ou materno); e de maneira completamente desviada, na qual a atividade do cromossomo X dependerá de sua origem parental. Nas fêmeas marsupiais a inativação ocorre de forma completamente desviada, sendo o X paterno preferencialmente inativado em todas as células, já nas células embrionárias de eutérios, o que se observa é a ICX aleatória. Entretanto, naquelas células que darão origem aos tecidos extra-embrionários, de camundongos e bovinos, a ICX se dá de forma equivalente à dos marsupiais, ou seja, o X paterno é preferencialmente inativado. Há mais de 30 anos o padrão de ICX em tecidos extra-embrionários humanos tem sido alvo de intenso debate. A crítica que se faz aqui é que tais estudos foram realizados com base na expressão de apenas um ou dois genes ligados ao X com amostras de tecidos extra-embrionários em diferentes idades gestacionais e, por vezes, em poucas amostras, o que deve ter levado às contradições entre as conclusões. O diferencial deste trabalho foi a utilização de técnicas de genotipagem de SNPs presentes em regiões codificadoras, para analisar o padrão de atividade alelo-específica de um grande número de genes presentes ao longo de todo o cromossomo X, gerando um panorama mais representativo da ICX em placenta humana. Neste estudo é comprovado o padrão aleatório de ICX em placenta humana a termo e demonstrado que este órgão se apresenta como um 65 mosaico em relação à escolha do X inativo. A análise global da atividade gênica no cromossomo X indicou ainda que a manutenção do estado epigenético do X inativo parece ser heterogêneo. Em conjunto, os dados gerados são capazes de explicar as incongruências entre as conclusões previamente publicadas. Este trabalho também ilustra as diferenças nos mecanismos de ICX entre humanos e camundongos e reforça a importância de se avaliar esse tema em outras espécies de mamíferos eutérios na tentativa de se elucidar os processos evolutivos envolvidos na compensação de dose em mamíferos / Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 23 X-linked genes, including XIST, using 28 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this chromosome-wide analysis indicated heterogeneous maintenance of the epigenetic state along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals

When conspecific individuals are crossed, the ensuing hybridization creates a spectrum of phenotypes in the resulting offspring. Many of hybrid traits will be additive, similar to the parental phenotypes. In some cases however, transgressive phenotypes are formed, outside the range of that of the parental phenotypes. Transgressive phenotypes can either be restricted to the F1 generation or be heritable throughout the hybrid lineage. While the mechanism behind heritable transgressive phenotyped is yet to be determined, transgressive gene expression is thought to be the root cause of their formation. Epigenetics modifications, heritable variation separate to the DNA code, can alter gene expression, persist through generations, and vary between individuals and over time. This makes them ideal candidates to be involved in the formation of transgressive phenotypes. RNA silencing is an epigenetic mechanism of gene regulation relying on 20Q24nt single stranded small RNAs (sRNAs). Small RNAs, due to their ability to set up persistent epigenetic marks at a locus, have the potential to create heritable transgressive gene expression. For example, when genetic variation from one parental genome presents novel targets to the sRNAs of the other parental genome, new epigenetic marks such as DNA methylation or secondary sRNAs can be created at target sites. In order to understand the potential of small RNAs to influence hybrid phenotype, I designed crossing experiments with Chlamydomonas reinhardtii, choosing this unicellular alga due to the genetic tools available and the haploid nature of its vegetative cells. The specific aim of the experiment was to identify transgressively expressed sRNA populations. Crossing two geographically distinct strains of C. reinhardtii, and sequencing both the genomes and sRNAomes of parents and recombinants, I was able catalogue both genetic and epigenetic variation in the parental strains providing unique insight into the inheritance of small RNAs in this alga. In this thesis, I first compare the genomes of the parental strains, identifying polymorphisms and assessing genetic variation in RNA silencing pathway components. I then describe the sRNA profiles of the parental strains, identifying differentially expressed sRNA loci. I then describe my approach to identifying transgressively expressed sRNA loci in the hybrids. While many sRNA loci in the recombinants exhibit additive sRNA expression, I found multiple transgressively expressed sRNA loci. Using the available bioinformatics tools, I identified potential miRNAs and phased secondary sRNAs within the list of transgressively expressed loci. Target analysis of one of the transgressively expressed miRNAs linked it with the transgressive expression of certain phased loci, suggesting a potential for sRNAs to be able to set up heritable epigenetic marks in recombinant C. reinhardtii cells.

Les cellules souches embryonnaires (CSE) sont un outil essentiel pour la recherche biomédicale. Elles ont à la fois la particularité de se multiplier de manière indéfinie tout en gardant leurs propriétés souches et l’incroyable capacité de donner naissance à tous les types cellulaires de l’organisme. Ces caractéristiques ouvrent de nouvelles perspectives pour la médecine régénérative mais également pour la mise au point de nouveaux essais thérapeutiques. Une des révolutions majeures reposant sur la reprogrammation cellulaire des cellules somatiques adultes en cellules souches, permet notamment d’entrevoir de nouvelles applications thérapeutiques. Les mécanismes moléculaires, tels que la méthylation de l’ADN, les modifications d’histones, et l’intervention de facteurs épigénétiques dans le remodelage de la chromatine, jouent un rôle essentiel dans la reprogrammation cellulaire et le contrôle de la pluripotence des cellules souches. L’épigénome des CSE doit non seulement maintenir l’expression des gènes associés à la pluripotence, mais également permettre une activation rapide et spécifique des gènes impliqués dans les étapes de différenciation cellulaire. Une des modifications ayant un rôle important dans l’homéostasie des CSE correspond aux méthylations d’histones H3K9 et H3K27 essentiellement associées à une répression transcriptionnelle. Ces modifications sont effectuées par des lysines méthyltransferases (HKM) dont G9a, ou bien EZH2 appartenant au complexe Polycomb PRC2. Elles recrutent également ces complexes protéiques permettant le maintien et la propagation de la modification le long du génome. Ainsi, dans ce contexte, mes travaux de thèse ont eu pour objectif de caractériser deux facteurs épigénétiques potentiels reconnaissant les histones H3K9me et H3K27me et interagissant avec le complexe PRC2. Ces études ont permis de mieux comprendre le rôle de ces protéines dans la régulation des cellules souches embryonnaires murines. Nos premières données ont montré que nos gènes candidats sont fortement exprimés dans les cellules souches embryonnaires murines (mESC) contrairement aux cellules différenciées. Par la suite, l’expression forcée d’un de ces facteurs altère la différenciation des CSE induite par le retrait de la cytokine LIF. Pour mieux comprendre comment le maintien de l’expression de notre facteur empêche la différenciation des cellules souches embryonnaires, nous avons analysé l’expression des facteurs de pluripotence Oct4, Nanog, Sox2 et Klf4. Nous avons noté un maintien de l’expression de ces facteurs ainsi que le maintien de la régulation des signaux intracellulaires intervenant en amont tels que: l’activation de la voie JAK-STAT3 pour le maintien à l’état pluripotent des ESC, et la diminution de la voie MAPK-ERK impliquée dans les processus de différenciation / Embryonic stem cells (ES cells) are an essential tool for biomedical research. They have the particularity to multiply indefinitely while keeping their stemness properties, and the incredible ability to generate all cell types of the body. These characteristics open up new perspectives for regenerative medicine but also for the development of new therapeutic trials. One of the major revolutions based on the cellular reprogramming of adult somatic cells into stem cells makes it possible to glimpse new therapeutic applications. Molecular mechanisms, such as DNA methylation, histone modifications, and the intervention of epigenetic factors in chromatin remodeling, play a critical role in cell reprogramming and pluripotency. The CSE epigenome must maintain not only the expression of genes associated with pluripotency but also allow rapid and specific activation of genes involved in cell differentiation. One of the modifications having an important role in the homeostasis of the CSE corresponds to histone methylations H3K9 and H3K27 essentially associated with transcriptional repression. These modifications are carried out by lysine methyltransferases (HKM) including G9a, or EZH2 belonging to the Polycomb PRC2 complex. They also recruit these protein complexes to maintain and propagate the change along the genome. Thus, in this context, my thesis work aimed to characterize two potential epigenetic factors recognizing histones H3K9me and H3K27me and interacting with the PRC2 complex. These studies have provided a better understanding of the role of these proteins in the regulation of murine embryonic stem cells. Our first data showed that our candidate genes are strongly expressed in murine embryonic stem cells CGR8 (mESC), unlike differentiated cells. Subsequently, the forced expression of one of these factors alters the CSE differentiation (CGR8) induced by LIF cytokine withdrawal. To better understand how maintaining the expression of our factor prevents the differentiation of embryonic stem cells, we analyzed the expression of pluripotency factors Oct4, Nanog, Sox2 and Klf4. We noted maintenance of the expression of these factors as well as the maintenance of the regulation of signals intervening upstream: including the maintenance of the activation of the JAK-STAT3 pathway for the maintenance of the pluripotent state, and the decrease of the MAPK-ERK pathway involved in differentiation processes

The mammalian epigenome is globally reprogrammed at two stages of development; this involves the erasure and re-establishment of DNA methylation by both passive and active mechanisms, including DNA repair pathways, and occurs concurrently with an increase in developmental potency. In addition to Uhrf1 and the Tet enzymes, the interplay between activation induced cytidine deaminase (AID) and the DNA repair machinery has been implicated in epigenetic reprogramming of various in vivo and in vitro systems including mouse primordial germ cells, zygotes and induced pluripotent stem cells. AID deaminates cytosine to uracil and can also deaminate methylcytosine, whereas the primary role of UNG is to maintain the integrity of the genome through erasure of uracil. In this thesis, I have aimed to investigate the role of DNA repair in demethylation. To do this I have focused on the specific role of AID and UNG in the demethylation of a static system – primed serum ESCs and a dynamic system – serum to 2i (naïve) to epiblast-like ES cells. As the role of both AID and UNG involves genomic uracil, the central theme of my thesis is the impact of accumulation of uracil on DNA methylation levels in the genome. Therefore, my first aim was to develop a quantitative method to detect low levels of genomic uracil in DNA firstly, by mass spectrometry and secondly, by whole genome sequencing. In Chapter Three, I show that the impact of deamination during DNA preparation can be minimised, such that the level of genomic ESC uracil can be accurately determined as around 12,000 uracil per genome and that, as anticipated, Ung null ESCs have almost twice the genomic uracil content of wildtype ESCs. Secondly, I address the main question which is the impact of uracil accumulation on methylation levels. In order to do this, I generate two cell lines: Ung knockout and Aid over expressing, both of which should result in an increase in genomic uracil. I demonstrate that while over expression of Aid stimulates demethylation in static system and in a dynamic demethylating system, the impact of Ung knockout is less clear. In (static) serum ESCs, loss of Ung results in hypomethylation however, in order to transition to 2i (naïve) ESCs, a process which involves demethylation of the genome, it appears the Ung is required as loss of this gene inhibits proper demethylation. As such, I conclude that UNG-mediated DNA repair functions alongside passive demethylation, by reduction of UHRF1 levels, to demethylate 2i ESCs. To probe the mechanism by which accumulation of uracil in the genome alters methylation levels, I investigate the impact of Ung KO and Aid OE on global levels of DNA damage. I show that both cell lines have a greater incidence of double strand breaks compared to a wild type cell line, and accordingly, upregulate their DNA damage response pathway and the expression of certain repair genes. I suggest that increasing genomic levels of uracil causes genomic instability and that DNA demethylation occurs as a consequence of the repair of extensive DNA damage. More broadly, I suggest that ESCs are uniquely poised, due to their heightened DNA damage response, to use uracil as an intermediate of DNA demethylation. Interestingly, I also note that the biological impact on serum ESCs of loss of Ung appears to be an increase in pluripotency.

Senescent cells interact with the surrounding microenvironment achieving both pro- oncogenic and tumour-suppressive outcomes. In addition to autocrine and paracrine signalling mediated by factors of the senescence-associated secretory phenotype (SASP), we have recently identified that NOTCH1 can drive a unique form of senescence in adjacent cells via juxtacrine signalling. Here, we show that NOTCH1 signalling confers a dramatic impact on chromatin structure during senescence. RAS-induced senescent (RIS) fibroblasts often develop chromatin structures called senescence-associated heterochromatic foci (SAHF). We find that NOTCH1 inhibits SAHF formation at least partially through transcriptional repression of a critical structural component, high-mobility group A (HMGA). Using ATAC-sequencing (assay for transposase accessible chromatin) we demonstrate that nucleosome positioning is substantially altered in RIS and that this re-distribution is also antagonised by NOTCH1, resulting in a distinct chromatin landscape. Importantly, normal or cancer cells that express the NOTCH ligand jagged-1 can drive similar chromatin structural changes in adjacent cells in a cell-cell contact dependent manner. In addition, using a highly optimised chromatin immunoprecipitation (ChIP-seq) protocol and the proximity ligation assay ‘Hi-C’, we demonstrate that HMGA proteins are directly involved in the formation of long-range interactions in RIS cells that may underpin SAHF formation. These ChIP-seq data have also allowed us to identify a unique HMGA1 binding profile, potentially suggesting a novel role for HMGA1 in gene regulation. Together, our data indicate that NOTCH signalling, both cell-autonomously and non-cell-autonomously, can repress HMGA1, a multi-faceted protein that regulates nucleosome positioning (1D structure), SAHF formation (3D structure) and potentially mRNA abundance.

Dentre as substâncias químicas secretadas pelas plantas, contra os insetos herbívoros, os inibidores de peptidases são de grande interesse. A atenção dada a esses inibidores deve-se ao fato, de eles serem uma boa alternativa no controle de insetos praga, uma vez que não causam danos ao meio ambiente. Contudo, muitas espécies de insetos são capazes de escapar dos efeitos negativos dos inibidores de peptidases das plantas, via diferentes mecanismos adaptativos. Devido a esse fato, é importante compreender os mecanismos desenvolvidos pelos insetos para burlar os efeitos dos inibidores de peptidases das plantas. Diante desse panorama, este trabalho teve como objetivo estudar o mecanismo adaptativo das serina endopeptidases de Spodoptera frugiperda aos inibidores de endopeptidases de soja. Foram realizadas análises do transcriptoma dos intestinos das lagartas mantidas em exposição crônica ao inibidor. Para averiguar os efeitos causados devido à exposição crônica ao inibidor, foram realizadas comparações da expressão relativa, dos genes de tripsinas e quimotripsinas, de intestinos de lagartas de sexto instar. Contudo, para entender o efeito da exposição aguda ao inibidor, lagartas de S. frugiperda foram criadas em dieta artificial controle até o primeiro dia do sexto instar, após esse período elas foram transferidas para dieta artificial acrescida com 0,5 % dos inibidores de endopeptidases de soja, durante 48 horas. Para verificar a ocorrência de um possível controle epigenético na expressão dos genes, as lagartas foram conduzidas até a fase adulta e os adultos, de cada tratamento, foram acasalados entre si, constituindo uma segunda geração. Dados de expressão relativa foram obtidos, de indivíduos da primeira e segunda geração, e foram então comparados. Foram identificados 14 possíveis genes de quimotripsinas e nove possíveis genes de tripsinas. Os genes de tripsina foram divididos em dois grupos distintos em relação a sua sensibilidade aos inibidores de endopepetidases de soja e expressão relativa. Houve uma resposta diferenciada na ativação dos genes de serina endopeptidases de S. frugiperda, a qual dividiu os genes em dois grupos, os responsivos e os não responsivos ao inibidor. A exposição aguda ao inibidor ativou um pequeno grupo de genes, enquanto que a exposição crônica promoveu uma maior amplitude de expressão gênica, sugerindo mecanismos temporalmente regulados. Por último, evidências indicam, pela primeira vez, a possível ocorrência de um mecanismo epigenético, na resposta das enzimas digestivas aos inibidores de serina endopeptidases de soja. / Among the chemicals secreted by plants against insect herbivores, peptidase inhibitors (PIs) are of great interest. The attention given to PIs is due to the fact that they are a good alternative to control insect pests since they do not cause damage to human health and the environment. However, many species of insects are able to escape the negative effects of PIs plants via different adaptive mechanisms. Because of this, it is important to understand the mechanisms developed by insects to circumvent the effects of PIs plants. Against this background, this research aimed to study the adaptive mechanism of serine endopeptidases Spodoptera frugiperda to soybean endopeptidase inhibitors. For this purpose, larvae of S. frugiperda were reared on artificial diet and control artificial diet plus 0.5% of endopeptidase inhibitors of soybean. We conducted a transcriptome midgut of worms maintained in chronic ingestion of the inhibitor. The relative expression of the genes, trypsin and chymotrypsin, was also compared the midgut of sixth instar larvae kept in these diets. In another experiment, the larvae were conducted to moth, then the treatments were mated forming a second generation. Relative expression data were obtained for individuals of the first and second generation, and then compared. Identified 14 genes with potential of chymotrypsin and 9 trypsin like genes. The trypsin gene were divided into two groups for both their sensitivity to PI soybean endopeptidase, and for their relative expression pattern. There was a differentiated response on the genes activation of S. frugiperda serina endopeptidases. The genes were clustered in 2 groups, the responsive ones and the non responsive to the inhibitor. The acute exposition to the inhibitor activated a small group of genes and the chronic exposition affected several genes, indicating the existence of temporal regulated mechanism. Besides, there is a possible occurance of an epigenetic mechanism, which is related to the digestive serina endopeptidases inhibitors of soybean.

The transcriptional repressor Polycomb Repressive Complex 1 (PRC1) is critical for stem cell maintenance and proper differentiation and as such is involved in the development and progression of cancer. Canonical PRC1, composed of PCGF, PHC, RING and CBX, binds histone H3 lysine 27 trimethylation (H3K27me3) allowing for ubiquitination, chromatin compaction and subsequently transcriptional silencing. In mammals, each subunit has multiple paralogs creating functional and compositional diversity. The greatest diversity is contributed by the CBX targeting subunit with five mutually exclusive paralogs (CBX2/4/6/7/8). The CBX paralogs contain an N-terminal chromodomain for methyllysine binding. There has been interest in the CBX paralogs due to their misregulation in various cancers and the “druggability” of the chromodomain histone interaction. However, the unique biochemical and transcriptional functions of the paralogs are unclear. Expression changes during lineage specification and the context-dependent misregulation of CBX paralogs in cancers suggest the paralogs have paralog-specific functions. However, little has been done to define differences in paralog-mediated chromatin binding and regulation. This work utilizes a variety of approaches to tease apart the biological and biochemical functions of the CBX paralogs in chromatin binding and oncogenesis. In this dissertation, we identify a combinatorial therapeutic strategy using a CBX chromodomain inhibitor to enhance chemotherapeutic response. Further, this work demonstrates a role for CBX8 and its chromodomain in glioblastoma oncogenesis suggesting it may serve as a therapeutic target. Finally, we identify a binding mechanism for the CBX8 chromodomain in which DNA and H3K27me3 binding contribute to full chromatin association.

Mouse embryonic stem cells (mESCs) are an excellent model to study epigenetics and chromatin structure, owing to their self-renewal capabilities and tolerance of dynamic changes to DNA and histone modifications. Culturing conditions impact on the ability of mESCs to effectively recapitulate in vivo developmental states, and this is exemplified by refined culture conditions (termed 2i) that promote a pluripotent ground state. 2i-cultured mESC populations are homogeneous, naïve, and distinct from conventional (serum/LIF-cultured) cells, which exist as a metastable population. Remarkably, 2i-cultured mESCs also display global DNA hypomethylation, with methylation patterns more comparable to the cells of the E3.5 pre-implantation blastocyst. This is distinct from conventional serum-cultured cells, which display DNA methylation profiles that resemble later-stage E6.5 post-implantation epiblasts. The ability to transition between 2i- and serum-culture states is an attractive model for studying the dynamic role of DNA methylation in a variety of processes. DNA hypomethylation has been linked with depletion of the Polycomb-mediated repressive histone mark H3K27me3 from its normal target loci. Polycomb repressive complexes (PRC1 and PRC2) are important developmental regulators that maintain the repression of lineage-specific genes through generating compact higher-order chromatin structures. Polycomb target sites are primarily unmethylated CpG islands (CGIs). However, under conditions of DNA hypomethylation, new (previously methylated) binding sites are unveiled, and Polycomb is redistributed from its normal CGI target regions to intragenic regions. Thus, shifting mESCs to ground state conditions results in both DNA methylation and Polycomb patterns that are quite distinct from their serum-cultured counterparts. In my PhD, I sought to investigate the effect of DNA hypomethylation and Polycomb redistribution on higher-order chromatin structure in the ground state. I used a targeted, single-locus approach (FISH) as well as a genome-wide approach (Hi-C) to analyse differences in chromatin structure between conventionally cultured and ground state mESCs. My work suggests that chromatin structure is globally altered in hypomethylated 2icultured mESCs, with a similar state present in E3.5 mouse blastocysts. Using mESC lines in which DNA methylation levels can be directly manipulated, I was able to dissect the molecular mechanism driving higher-order structure changes in 2i medium, and showed the importance of DNA methylation in directing Polycomb-mediated chromatin compaction. My results may be important in considering the impact of DNA-methylation mediated reprogramming in multiple developmental, disease and regenerative medicine contexts.