Heterochromatin in the budding yeast Saccharomyces cerevisiae is composed of polymers of the SIR (Silent Information Regulator) complex bound to nucleosomal DNA. Assembly of heterochromatin requires all three proteins of the Sir complex: the histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. Heterochromatin establishment requires passage through at least one cell cycle, but is not dependent on replication. Inhibition of chromatin modifying enzymes may be a mechanism for how cells limit assembly. Dot1 dependent methylation of H3K79 is suggested to inhibit de novo assembly. Halving the levels of Sir4 in cells causes a loss of silencing, suggesting that Sir4 protein abundance regulates the assembly of heterochromatin. We examine de novo assembly using a single cell assay. Half the level of Sir4 affects establishment, but not the maintenance, of silencing at HM loci. Additional Sir4 accelerates the rate of assembly. Epistasis analysis suggests that Dot1 dependent chromatin modification may act upstream of Sir4 abundance. We hypothesize that dot1Δ mutants speed assembly by disrupting telomeric heterochromatin, which liberates Sir4 to act at the HM loci. Deletion of YKU70, which specifically disrupts telomeric silencing, also speeds de novo assembly, without altering the methylation of histone H3. Consistent with our model, we have shown that Sir4 abundance falls during pheromone and stationary phase arrests after which several cell cycles are required before silencing can be reestablished.

Afin d’étudier la prise en charge des histones H3 jusqu’à l’ADN et pour comprendre l’influence de leur dynamique dans l’organisation d’ordre supérieur de la chromatine, une analyse des chaperonnes d’histones a été menée. Nous avons identifié et caractérisé les sous-unités du complexe HIR, impliqué dans l’assemblage de la chromatine réplication-indépendante chez Arabidopsis. La perte d’AtHIRA, la sous-unité centrale du complexe, affecte le niveau d’histone soluble, l’occupation nucléosomale des régions euchromatiniennes et héterochromatiniennes ainsi que la mise sous silence transcriptionnel des séquences d’ADN répétées. Alors que le complexe HIR ne participe pas à l’organisation d’ordre supérieur de la chromatine, j’ai montré que CAF-1, impliqué dans l’assemblage de la chromatine au cours de la réplication, joue un rôle central dans la formation des chromocentres. Lors du développement post-germinatif des cotylédons, les séquences d’ADN répétées centromériques et péricentromériques se concentrent dans les chromocentres et s’enrichissent en histone H3.1 de manière CAF-1 dépendante. Cet enrichissement, associé à des modifications post-traductionnelles d’histones associées à un état répressif de la transcription, participe à la formation des chromocentres et met en évidence l’importance de l’assemblage de la chromatine par CAF-1 dans la structure et le maintien du génome. Alors que la perte individuelle de HIR ou de CAF-1 n’affecte pas la viabilité, l’absence des deux complexes altère fortement l’occupation nucléosomale et le développement des plantes. Ceci suggère que la compensation fonctionnelle entre ces complexes de chaperonnes ainsi que la plasticité des voies de dépôt des histones restent limitées. / To understand how histones H3 are handled and how histone dynamics impact higher-order chromatin organization such as chromocenter formation in Arabidopsis, a comprehensive analysis of the different histone chaperone complexes is required. We identified and characterized the different subunits of the Arabidopsis HIR complex. AtHIRA is the central subunit and its loss affects non-nucleosomal histone levels, reduces nucleosomal occupancy not only at euchromatic but also at heterochromatic targets and alleviates transcriptional gene silencing. While the HIR complex-mediated histone deposition is dispensable for higher-order organization of Arabidopsis heterochromatin, I show that CAF-1 plays a central role in chromocenter formation. During postgermination development in cotyledons when centromeric and pericentromeric repeats cluster progressively into chromocenter structures, these repetitive elements but not euchromatic loci become enriched in H3.1 in a CAF-1- dependent manner. This enrichment, together with the appropriate setting of repressive histone post-translational marks, contributes to chromocenter formation, identifying chromatin assembly by CAF-1 as driving force in formation and maintenance of genome structure. Finally, while absence of HIR or CAF-1 complexes sustains viability, only the simultaneous loss of both severely impairs nucleosomal occupancy and plant development, suggesting a limited functional compensation between the different histone chaperone complexes and plasticity in histone variant interaction and deposition in plants.

Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T20:32:58Z (GMT). No. of bitstreams: 1 Mampumbu_AndreRoberto_D.pdf: 659647 bytes, checksum: 40d0a48fa1d02750dffb30ae80b25445 (MD5) Previous issue date: 2006 / Resumo: O gênero Melipona (abelhas sem ferrão) tem sido dividido em dois grupos, com base no seu conteúdo em heterocromatina revelada com a técnica de banda-C em cromossomos mitóticos. Melipona quadrifasciata e Melipona rufiventris apresentam, respectivamente, níveis baixos e altos de heterocromatina. Na suposição de que cromatina condensada possa ser rica em seqüências de DNA metiladas, M. quadrifasciata e M. rufiventris poderiam então apresentar diferenças em conteúdo de seqüências CpG metiladas. Se isso acontecesse, as diferenças poderiam ser reveladas pela comparação de valores Feulgen-DNA obtidos por análise de imagem de células tratadas com as enzimas de restrição Msp I e Hpa II, que distinguem entre seqüências metiladas e não metiladas. Msp I e Hpa II clivam as seqüências ¿CCGG-, porém não há clivagem pela Hpa II se a citosina do dinucleotídeo central CG for metilada. Neste trabalho, túbulos de Malpighi de larva de último estádio de M. quadrifasciata e M. rufiventris submetidos à reação de Feulgen precedida pelo tratamento com Msp I e Hpa II tiveram suas células analisadas por microespectrofotometria de varredura automática. Para esse material houve necessidade do desenvolvimento prévio de um ajuste metodológico que tornasse a reação de Feulgen reveladora apenas de DNA, visto que ocorria reação plasmal; isto foi conseguido com um tratamento por boridreto de sódio a 5% e acetona/clorofórmio (1:1, v/v) antecedendo a reação de Feulgen. Também, embora a definição de altos e baixos conteúdos de heterocromatina em Melipona pela técnica de banda-C não fosse extensível à cromatina de núcleos interfásicos dos túbulos de Malpighi dessas abelhas, demonstrou-se que a depurinação do DNA em M. quadrifasciata era mais rápida do que a de M. rufiventris, confirmando, maiores teores de cromatina condensada em M. rufiventris. Os valores Feulgen-DNA para a heterocromatina de Melipona rufiventris e para a pouca heterocromatina somada a alguns domínios de eucromatina de Melipona quadrifasciata diminuíram após tratamento com Msp I, porém ficaram inalterados após tratamento com Hpa II. Conclui-se que seqüências CpG metiladas podem estar contidas em diferentes compartimentos cromatínicos, conforme a espécie do gênero Melipona considerada, e que os seus efeitos silenciadores possam atuar induzindo uma mesma fisiologia celular / Abstract: The genus Mellipona has been divided into two groups based on its heterochromatin content revealed by C-banding pattern in mitotic chromosomes. Melipona quadrifasciata and Melipona rufiventris show low and high heterochromatin content, respectively. Supposing that condensed chromatin may be rich in DNA methylated sequences, M quadrifasciata and M. rufiventris could, thus, show differences regarding their content of CpG methylated sequences. In this situation, such differences could be revealed by comparing the Feulgen-DNA values acquired after image analysis of cells treated with restriction enzymes Msp I and Hpa II, which distinguish between methylated and nonmethylated sequences. Msp I and Hpa II break the CCGG sequences. Nevertheless, Hpa II is ubable to break the DNA strand if the cytosine from the central nucleotide pair CG is methylated. In this work, Malpighian tubules from larvae from the last stage of M. quadrifasciata and M. rufiventris, subjected to the Feulgen reaction after by treatment with Msp I and Hpa II, were analysed in automatic scanning microspectrophotometry. Since a plasmal reaction was observed in this material, it was previously necessary the development of a methodological adjustement to make the Feulgen reaction specific to DNA. This was achieved by treatment of material with 5% sodium borohydrade followed by acetone-chloroform (1:1, v/v) before the Feulgen reaction. Also, although the definition of high and low heterochromatin content in Melipona after C-banding technique is not applicable to the chromatin of interphasic nuclei in Malpighian tubules of bees, it was demonstrated that DNA depurination in M. quadrifasciata was faster than that of M. rufiventris, thus confirming that this species has a higher condensed chromatin content. The Feulgen-DNA values for the heterochromatin of Melipona rufiventris, and for the heterochromatin besides some euchromatic domains of Melipona quadrifasciata, decreased after treatment with Msp I, remaining, however, unaltered after treatment with Hpa II. In conclusion, methylated CpG sequences may be part of different chromatin compartments, according to the considered species of the genus Melipona, and that their silencing effects may act by inducing the same cell physiology / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

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- ABSTRACT - Among vertebrates, reptiles represent the ideal group for the study of sex determination. Reptiles include lineages with environmental sex determination (ESD) as seen in crocodiles and tuatara, lineages with genotypic sex determination (GSD), like e.g. iguanas, chameleons, skinks, lacertid lizards and birds, and few groups which possess variability in sex determination mechanisms, i.e. geckos, dragon lizards and turtles. This thesis is focused on the evolution of sex chromosomes and sex determination in turtles. The majority of turtle species exhibit ESD, which is considered the ancestral sex determination system of this group, while GSD either as male or female heterogamety evolved independently at least five times. We investigated the presence of sex chromosomes in representative species of turtles by cytogenetic analyses. The analyses included the reconstruction of karyotypes, distribution of constitutive heterochromatin (C-banding, methylation analysis) and repetitive elements (fluorescence in situ hybridization) and comparative genome hybridization (CGH), which often characterize the degenerated Y or W and can be helpful in the identification of "cryptic" sex chromosomes. We described XX/XY sex chromosomes in seven previously unstudied Australasian chelids (Pleurodira) from the genera...

- ABSTRACT - Snakes (Serpentes) are a group of squamate reptiles (Squamata) that represents more than one third of the total reptile species diversity. Snake karyotype is generally conserved with the most common chromosome number of 36 (16 macro- and 20 microchromosomes) in diploid state. It is believed that this karyotype was also present in the common ancestor of all snakes. The majority of snake species belong to the group Caenophidia and share homologous ZW sex chromosomes. Snakes from the groups "Scolecophidia" and "Henophidia" have mostly poorly differentiated, homomorphic sex chromosomes, which made them impossible to distinguish from the autosomes in the past. These snakes were for many years assumed to have ZW sex chromosomes as well. However, recent studies demonstrated not only ZW but also two non- homologous XY sex chromosome systems in non-caenophidian snakes and thus the sex determination systems in snakes are much more variable than previously thought. In this thesis, eight species of henophidian snakes (representatives from the genera Eryx, Cylidrophis, Python and Tropidophis) and one caenophidian species (Ophiophagus hannah) were examined using conventional and molecular cytogenetic methods. However, sex chromosomes were not detected in the henophidian species, only in Ophiophagus hannah,...

L'hétérochromatine est une caractéristique conservée des chromosomes eucaryotes, avec des rôles centraux dans la régulation de l'expression des gènes et le maintien de la stabilité du génome. Comment la réparation de l'ADN est régulée par l'hétérochromatine reste mal compris. Chez Saccharomyces cerevisiae, le complexe SIR (Silent Information Regulator) assemble une fibre de chromatine compacte. La chromatine SIR limite la résection aux cassures double-brin (DSB) protégeant les extrémités chromosomiques endommagées contre la perte d'informations génétiques. Toutefois, lesquels des trois complexes de résection redondants, MRX-Sae2, Exo1 et Sgs1-Dna2 sont inhibés et par quel(s) mécanisme(s) reste à decouvrir. Nous montrons que Sir3, le facteur de fixation des histones de l’hétérochromatine de Saccharomyces cerevisiae, interagit physiquement avec Sae2 et inhibe toutes ses fonctions. Cette interaction limite notamment la résection médiée par Sae2, stabilise MRX à la DSB et augmente le Non-Homologous End Joining (NHEJ). De plus, la chromatine répressive SIR inhibe partiellement les deux voies de résection extensive médiées par Exo1 et Sgs1-Dna2 par des mécanismes distincts. L'inhibition par les SIR de la résection extensive et de Sae2 favorise la NHEJ et limite le Break-Induced Replication (BIR), prévenant ainsi de la perte d'hétérozygotie au niveau des subtélomères. / Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in regulation of gene expression and maintenance of genome stability. How DNA repair occurs in heterochromatin remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles a compact chromatin fibre. SIR-mediated repressive chromatin limits Double Strand Break (DSB) resection protecting damaged chromosome ends against the loss of genetic information. However, which of the three redundant resection complexes, MRX-Sae2, Exo1 and Sgs1-Dna2 are inhibited and by which mechanism remains to be deciphered. We show that Sir3, the histone-binding factor of yeast heterochromatin, physically interacts with Sae2-mediated resection and inhibits all its functions. Notably, this interaction limits Sae2-mediated resection, delays MRX removal from DSB ends and promotes Non-Homologous End Joining (NHEJ). In addition, SIR-mediated repressive chromatin partially inhibits the two long range resection pathways mediated by Exo1 and Sgs1-Dna2 by distinct mechanisms. Altogether SIR mediated inhibition of extensive resection and of Sae2 promotes NHEJ and limits Break-Induced Replication (BIR) preventing loss of heterozygosity at subtelomeres.

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La sénescence est un mécanisme de défense antiprolifératif dont la cellule est munie afin de prévenir l’accumulation de mutations pouvant mener à sa transformation et l’éventuel développement d’une tumeur. Ce programme consiste en un arrêt permanent du cycle cellulaire. Il peut être activé par de nombreux stimuli tels que le raccourcissement des télomères, le stress oxydatif, ou l’expression d’un oncogène constitutivement actif. Sa régulation requiert l’activation de protéines appelées des suppresseurs de tumeur dont les plus importants sont p53 et RB. De manière plus spécifique, les sénescences induites par l’expression des oncogène RASV12 ou STAT5A(1*6) sont respectivement caractérisées par l’augmentation de l’expression des protéines PML et CHES1/FOXN3. Le but de cette thèse est, dans un premier temps, de mettre en évidence le mécanisme de régulation de la sénescence par PML. PML est un suppresseur de tumeur dont l’expression dans des cellules diploïdes normales est suffisante pour induire la sénescence. Cette protéine forme des corps nucléaires sphériques au sein desquels est recruté, parmi d’autres molécules, la protéine du rétinoblastome RB. RB est un régulateur négatif du cycle cellulaire capable de lier et inhiber les facteurs de transcription E2F dont les gènes cibles sont nécessaires à la prolifération. Nos travaux démontrent que le mécanisme d’induction de la sénescence par PML implique le recrutement du complexe RB/E2F aux corps de PML afin de renforcer l’inhibition de l’activité des E2F par RB. Également, les E2F sont recrutés aux corps de PML en compagnie de leurs promoteurs ce qui favorise la formation d’hétérochromatine au niveau de ces gènes, aidant à leur répression et donc à l’établissement de la sénescence. D’autre part, cette thèse a pour but de caractériser le rôle de CHES1/FOXN3 dans la régulation du cycle cellulaire. CHES1 est un facteur de transcription de la famille des Forkheads. Son expression dans des cellules cancéreuses provoque un ralentissement de leur prolifération. Afin de comprendre son mécanisme de fonctionnement, une analyse sur micropuce d’ADN de l’expression des gènes de cellules cancéreuses exprimant CHES1 a été réalisée. Cette analyse a montré que, dans la cellule, CHES1 joue un rôle de répresseur transcriptionnel. Plus précisément, CHES1 réprime, entre autres, l’expression de gènes nécessaires à la synthèse des protéines tels que PIM2 et DYRK3. De manière intéressante, la réexpression de PIM2 dans des cellules cancéreuses exprimant CHES1 permet de rétablir partiellement la prolifération cellulaire. Également, l’analyse sur micropuce a révélé que CHES1 régule l’expression de nombreux gènes impliqués dans la formation des cilia dont l’une des fonctions semble être de moduler la synthèse protéique. Pris ensemble, ces résultats suggèrent que le mécanisme antitumoral de CHES1 consiste en une inhibition de la synthèse de protéines. / Senescence is an antiproliferative defense mechanism that protects the cell against the accumulation of mutations and, eventually, her transformation and the development of a tumor. This program consists of a permanent cell cycle arrest. It can be activated by many stimuli, such as telomere shortening, oxidative stress, or the expression of a constitutively active oncogene. Senescence regulation requires activation of proteins called tumor suppressors which the most important are p53 and RB. More specifically, senescences induced by the expression of oncogenic RASV12 or STAT5A (1 * 6) are respectively characterized by increased expression of the proteins PML and CHES1/FOXN3. The purpose of this thesis is, firstly, to identify the mechanism of regulation of senescence by PML. PML is a tumor suppressor whose expression in normal diploid cells is sufficient to induce senescence. This protein forms spherical nuclear bodies in which is recruited, among other molecules, the retinoblastoma protein RB. RB is a negative regulator of the cell cycle due to its capacity to bind and inhibit the E2F transcription factors whose target genes are necessary for proliferation. Our work demonstrates that the mechanism of induction of senescence by PML involves the recruitment of the complex RB/E2F to the PML body to enhance the inhibition of E2F activity by RB. Also, the E2Fs are recruited to PML bodies together with their promoters which promotes the formation of heterochromatin in these genes, helping their repression and thus the establishment of senescence. On the other hand, this thesis aims to characterize the role of CHES1/FOXN3 in regulating the cell cycle. CHES1 is a transcription factor of the Forkheads family which expression in cancer cells causes a growth reduction. To understand the antitumoral mechanism of CHES1, a microarray analysis of cancer cells expressing CHES1 was performed. This analysis shows that CHES1 is a transcriptional repressor. Specifically, CHES1 represses, among others, the expression of genes required for the synthesis of proteins such as PIM 2 and DYRK3. Interestingly, re-expression of PIM 2 in cancer cells expressing CHES1 partially restores cell proliferation. Also, microarray analysis revealed that CHES1 regulates the expression of many genes involved in the formation of cilia which one of the functions seems to be to modulate protein synthesis. Taken together, these results suggest that the antitumor mechanism of CHES1 involves inhibition of protein synthesis.