Modificações nas histonas são conhecidas por regular a estrutura conformacional da cromatina e a expressão gênica em células adultas e células-tronco pluripotentes. Tem sido postulado que a acetilação e deacetilação das histonas podem influenciar a expressão de genes envolvidos na iniciação, progressão e metástase tumoral, além de contribuir para o desenvolvimento de resistência à quimioterapia. Assim, buscou-se avaliar a influência das modificações nas histonas sobre a biologia do carcinoma epidermoide de cabeça e pescoço (CECP) e sua respectiva subpopulação de células semelhantes às células-tronco (CSC). Inicialmente, foi checado os níveis de acetilação da histona H3 (membro das histonas nucleares associado à compactação da cromatina) em um painel representativo de linhagens celulares de CECP. Posteriormente, para estudar a influência do estroma tumoral no padrão de acetilação da histona H3, o microambiente do tumor foi mimetizado através da utilização de meio condicionado derivado do cultivo de fibroblastos e cultura primária de células endoteliais humanas. Além disso, validamos esses resultados in vitro por meio de amostras humanas de CECP. Finalmente, a acetilação e deacetilação da cromatina foi induzida, respectivamente, pela administração dos inibidores das enzimas histona deacetilase tricostatina A (TSA) e histona acetiltransferase curcumina, em linhagens celulares de CECP. Foi feita a análise da formação de esferas (ensaio funcional de células-tronco), juntamente com a verificação dos níveis de ALDH, marcador de células-tronco (citometria de fluxo - FACS), além da determinação do índice de proliferação tumoral (Ki-67) e realização dos ensaios de invasão e migração celular. Linhagens celulares de CECP apresentaram níveis baixos de acetilação da histona H3 e demonstraram capacidade de retenção de uma subpopulação de CSC. Apenas o meio condicionado de células endoteliais humanas foi capaz de alterar a conformação da cromatina, uma vez que induziu o aumento da acetilação da histona H3. Interessantemente, foi também notado um concomitante aumento da agressividade de linhagens celulares de CECP (aumento dos níveis de BMI-1 e vimentina). Esses resultados foram confirmados em amostras humanas de CECP que mostraram, apenas no fronte de invasão, células com cromatina acetilada. Curiosamente, essas mesmas células também expressaram vimentina. Os tratamentos com TSA e curcumina resultaram na diminuição significativa da subpopulação de CSC, interrompendo a formação de esferas e reduzindo os níveis de ALDH. Além disso, o tratamento com curcumina mostrou resultados muito interessantes, uma vez que gerou uma redução evidente da invasão celular e impactou por completo o potencial de migração tumoral, sendo nesse sentido mais eficiente que a cisplatina, droga antineoplásica bem estabelecida. Por outro lado, o tratamento com TSA induziu a transição epitélio-mesenquimal nas linhagens celulares de CECP, detectada pelo aumento da expressão de vimentina e indução de um fenótipo fusiforme, juntamente com o aumento da invasão tumoral e os níveis de BMI-1. Portanto, a organização da cromatina está envolvida na modulação da presença de CSC e os altos níveis de acetilação das histonas intensificam o comportamento agressivo de células de CECP. / Histone modifications are known to regulate chromatin conformation structure and gene expression in adult cells and pluripotent stem cells. It has been postulated that histone acetylation and deacetylation could influence the expression of genes involved in cancer initiation, progression, metastasis, and development of resistance to chemotherapies. Here, we sought to evaluate the influence of histone modifications over the biology of head and neck squamous cell carcinoma (HNSCC) and its stem cell-like subpopulation (CSC). Initially, we checked the status of histone H3 acetylation (a member of the core histones associated to chromatin compaction) in a representative set of HNSCC cell lines. Subsequently, to analyze the influence of tumor stroma over the histone H3 acetylation, we mimicked the tumor microenvironment by using conditioned medium from fibroblasts and primary human endothelial cells. Further we validated these in vitro findings through human samples of HNSCC. Finally, we induced chromatin acetylation and deacetylation by the administration of the histone deacetylase inhibitor trichostatin A (TSA) and histone acetyltransferase inhibitor curcumin, respectively, in HNSCC cell lines. The analysis of spheres formation (stem cell functional assay), along with the levels of stem cells marker ALDH (showed by flow cytometry - FACS), tumor proliferation index (Ki-67), invasion and migration cellular potencial were verified. HNSCC cell lines showed lower levels of histone H3 acetylation and ability to retain a subpopulation of CSC. Only conditioned media from human endothelial cells was able to alter the conformation of chromatin, since it induced the increase of histone H3 acetylation. Interestingly, it was also noted a concomitant augment of HNSCC cell lines aggressiveness (enhanced BMI-1 and vimentin levels). These findings were confirmed in human samples of HNSCC that showed, only at the invasive front, cells with acetylated chromatin. Curiously, these same cells also expressed vimentin. TSA and curcumin treatments resulted in significant decrease of the CSC subpopulation by disrupting the spheres and reducing the levels of ALDH. Also, curcumin treatment showed exciting results since it caused an evident reduction of cellular invasion and it impacted the tumoral migration potential, being more efficient than cisplantin, a well-established antineoplastic drug. However, TSA induced epithelial to mesenchymal transition in HNSCC cell lines detected by the upregulation of vimentin and the induction of a fusiform phenotype along with augmented tumor invasion and the levels of BMI-1. Chromatin organization is involved in the modulation of CSC where high levels of histone acetylation intensify the aggressive behavior of HNSCC cells.

Modificações nas histonas são conhecidas por regular a estrutura conformacional da cromatina e a expressão gênica em células adultas e células-tronco pluripotentes. Tem sido postulado que a acetilação e deacetilação das histonas podem influenciar a expressão de genes envolvidos na iniciação, progressão e metástase tumoral, além de contribuir para o desenvolvimento de resistência à quimioterapia. Assim, buscou-se avaliar a influência das modificações nas histonas sobre a biologia do carcinoma epidermoide de cabeça e pescoço (CECP) e sua respectiva subpopulação de células semelhantes às células-tronco (CSC). Inicialmente, foi checado os níveis de acetilação da histona H3 (membro das histonas nucleares associado à compactação da cromatina) em um painel representativo de linhagens celulares de CECP. Posteriormente, para estudar a influência do estroma tumoral no padrão de acetilação da histona H3, o microambiente do tumor foi mimetizado através da utilização de meio condicionado derivado do cultivo de fibroblastos e cultura primária de células endoteliais humanas. Além disso, validamos esses resultados in vitro por meio de amostras humanas de CECP. Finalmente, a acetilação e deacetilação da cromatina foi induzida, respectivamente, pela administração dos inibidores das enzimas histona deacetilase tricostatina A (TSA) e histona acetiltransferase curcumina, em linhagens celulares de CECP. Foi feita a análise da formação de esferas (ensaio funcional de células-tronco), juntamente com a verificação dos níveis de ALDH, marcador de células-tronco (citometria de fluxo - FACS), além da determinação do índice de proliferação tumoral (Ki-67) e realização dos ensaios de invasão e migração celular. Linhagens celulares de CECP apresentaram níveis baixos de acetilação da histona H3 e demonstraram capacidade de retenção de uma subpopulação de CSC. Apenas o meio condicionado de células endoteliais humanas foi capaz de alterar a conformação da cromatina, uma vez que induziu o aumento da acetilação da histona H3. Interessantemente, foi também notado um concomitante aumento da agressividade de linhagens celulares de CECP (aumento dos níveis de BMI-1 e vimentina). Esses resultados foram confirmados em amostras humanas de CECP que mostraram, apenas no fronte de invasão, células com cromatina acetilada. Curiosamente, essas mesmas células também expressaram vimentina. Os tratamentos com TSA e curcumina resultaram na diminuição significativa da subpopulação de CSC, interrompendo a formação de esferas e reduzindo os níveis de ALDH. Além disso, o tratamento com curcumina mostrou resultados muito interessantes, uma vez que gerou uma redução evidente da invasão celular e impactou por completo o potencial de migração tumoral, sendo nesse sentido mais eficiente que a cisplatina, droga antineoplásica bem estabelecida. Por outro lado, o tratamento com TSA induziu a transição epitélio-mesenquimal nas linhagens celulares de CECP, detectada pelo aumento da expressão de vimentina e indução de um fenótipo fusiforme, juntamente com o aumento da invasão tumoral e os níveis de BMI-1. Portanto, a organização da cromatina está envolvida na modulação da presença de CSC e os altos níveis de acetilação das histonas intensificam o comportamento agressivo de células de CECP. / Histone modifications are known to regulate chromatin conformation structure and gene expression in adult cells and pluripotent stem cells. It has been postulated that histone acetylation and deacetylation could influence the expression of genes involved in cancer initiation, progression, metastasis, and development of resistance to chemotherapies. Here, we sought to evaluate the influence of histone modifications over the biology of head and neck squamous cell carcinoma (HNSCC) and its stem cell-like subpopulation (CSC). Initially, we checked the status of histone H3 acetylation (a member of the core histones associated to chromatin compaction) in a representative set of HNSCC cell lines. Subsequently, to analyze the influence of tumor stroma over the histone H3 acetylation, we mimicked the tumor microenvironment by using conditioned medium from fibroblasts and primary human endothelial cells. Further we validated these in vitro findings through human samples of HNSCC. Finally, we induced chromatin acetylation and deacetylation by the administration of the histone deacetylase inhibitor trichostatin A (TSA) and histone acetyltransferase inhibitor curcumin, respectively, in HNSCC cell lines. The analysis of spheres formation (stem cell functional assay), along with the levels of stem cells marker ALDH (showed by flow cytometry - FACS), tumor proliferation index (Ki-67), invasion and migration cellular potencial were verified. HNSCC cell lines showed lower levels of histone H3 acetylation and ability to retain a subpopulation of CSC. Only conditioned media from human endothelial cells was able to alter the conformation of chromatin, since it induced the increase of histone H3 acetylation. Interestingly, it was also noted a concomitant augment of HNSCC cell lines aggressiveness (enhanced BMI-1 and vimentin levels). These findings were confirmed in human samples of HNSCC that showed, only at the invasive front, cells with acetylated chromatin. Curiously, these same cells also expressed vimentin. TSA and curcumin treatments resulted in significant decrease of the CSC subpopulation by disrupting the spheres and reducing the levels of ALDH. Also, curcumin treatment showed exciting results since it caused an evident reduction of cellular invasion and it impacted the tumoral migration potential, being more efficient than cisplantin, a well-established antineoplastic drug. However, TSA induced epithelial to mesenchymal transition in HNSCC cell lines detected by the upregulation of vimentin and the induction of a fusiform phenotype along with augmented tumor invasion and the levels of BMI-1. Chromatin organization is involved in the modulation of CSC where high levels of histone acetylation intensify the aggressive behavior of HNSCC cells.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Human epidermis relies on a population of adult stem cells to maintain its homeostasis. Stem cells transit from a dormant to an active state and undergo a tightly regulated process of differentiation that replenishes the tissue according to its needs. This process either replaces cells that get shed away, or contributes to tissue healing upon injuries, such as wounding. Distinct molecular mechanisms are required to keep human epidermal stem cells localized in their niche and for their active proliferation and mobilization, while others regulate their differentiation status. However, little is known about the proper global chromatin modifications that ensure the correct transition between these stem cell states. This work shows that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the active stem cell state. Interestingly, Cbx4 also represses genes required for neuronal fate repression, suggesting that it might have a role in ectoderm patterning during development. Cbx proteins are differently expressed during epidermal differentiation and the activity of Cbx4 towards promoting human epidermal stem cell quiescence is unique among the Cbx proteins. This suggests that different Polycomb complexes are assembled, based on the availability of its core member, and balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. / La homeostasis de la epidermis humana depende de una población de células troncales adultas (CTAs). Las CTAs alternan ciclos de quiescencia y actividad, seguidos por una regulación estricta de su diferenciación, según las necesidades celulares del tejido. Este proceso es esencial para repoblar el tejido de células envejecidas o dañadas. Cada estadío por el que transita una CTA está regulado por procesos moleculares específicos. Sin embargo, aún sabemos poco sobre los procesos que regulan la reorganización de la cromatina necesarios para mediar dichas transiciones en la población de las CTAs. Estos resultados demuestran que la proteina Cbx4, pertenciente al complejo Polycomb Repressive Complex-1 (PRC1), es necesaria para mantener a las CTAs de la epidermis humana quiescentes, indiferenciadas, y protegidas de la senescencia. A nivel molecular, la actividad polycomb de Cbx4 es únicamente necesaria para su función antisenescente, pero es dispensable para la regulación de la proliferación y diferenciación de las CTAs. La inhibición de la proliferación y diferenciación celular sin embargo depende de la activdad E3 SUMO ligasa de Cbx4. Analisis del transcriptoma global y de unión a la cromatina (ChIP), demuestran que Cbx4 regula la expresión de moduladores esenciales de la homeostasis de la epidermis, y reprime la expresión de factores necesarios para la activación de las CTAs, tales como Ezh2, Dnmt1 y Bmi1. Cabe destacar que Cbx4 también reprime la expresión de genes que determinam el linage neuronal, lo que sugiere que Cbx4 pueda ser importante para separar el neuroectodermo entre ectodermo y neuronas, durante el desarrollo embrionario. Cbx4 es la única proteina Cbx capaz de inducir entrada en quiescencia de las CTAs, y el resto de proteinas Cbx se expresa de forma diferente durante la diferenciación en la epidermis. Por lo tanto, nuestros estudios sugieren que la actividad de distintos complejos Polycomb actúa en los sucesivos estadíos de quiescencia, proliferación y diferenciación de las CTAs, a la vez que impiden su senescencia de forma constante.

Durant l’hématopoïèse, les cellules souches hématopoïétiques peuvent soit s’autorenouveler soit se différencier dans les divers types de cellules matures constituant le système hématopoïétique. Un des modèles prédominants sur le développement du système hématopoïétique postule une différenciation par étape des différentes lignées le constituant. Ce modèle débute avec les cellules souches hématopoïétiques qui donnent naissance à des précurseurs multipotents qui peuvent à leur tour se différencier en précurseurs dédiées à la lignée lymphoïde ou myéloïde. Bien que la dernière décennie ait apporté de nombreuses connaissances sur les principales signalétiques transcriptionnelles impliquées dans le développement hématopoïétique, le détail des mécanismes moléculaires en jeu expliquant comment les cellules souches hématopoïétiques sont initialement amorcées puis complètement engagées vers une voie de différenciation reste toujours à élucider. Le travail de notre laboratoire indique que l’assemblage combinatoire du complexe de remodelage de la chromatine SWI/SNF est un élément clé parmi les mécanismes épigénétiques qui gouvernent le destin cellulaire et notamment la famille de sous-unités Smarcd qui comporte 3 membre alternatifs Smarcd1/2/3. Des analyses du transcriptome par séquençage haut débit ont montré que l’expression de la sous-unité Smarcd1 du complexe est élevée dans le compartiment des cellules souches, les précurseurs multipotents et les progénitures lymphoïdes tandis que la sous-unité Smarcd2 est principalement exprimée dans les précurseurs myéloïdes. En utilisant des modèles de délétion conditionnelle dans des modèles murins, nous avons démontré que Smarcd1 et Smarcd2 jouent des rôles critiques et lignés spécifiques durant l’hématopoïèse. Dans un premier temps, nous avons pu montrer que Smarcd1 collabore avec le facteur de transcription de la famille bHLH E2A pour spécifier le destin lymphoïde des précurseurs multipotents et qu’elle est donc absolument essentielle pour la lymphopoïèse. Notre travail sur les mécanismes moléculaires en jeu a pu montrer que Smarcd1 interagit directement avec E2A et est nécessaire pour l’accessibilité de la chromatine sur un ensemble de régions enrichies avec les modifications d’histones H3K27ac/H3K3me1 qui marquent des régions activatrices (« enhancer ») impliquées dans l’activation d’une signature lymphoïde dans les précurseurs multipotents. Le blocage de l’interaction entre Smarcd1 et E2A inhibe l’amorce de cette signature lymphoïde et bloque l’émergence de précurseurs destinés à la voie lymphocytaire. Concernant la fonction de Smarcd2, nous avons pu montrer que cette sous-unité est absolument nécessaire pour la granulopoïèse. Les souris ayant subi une délétion génétique de Smarcd2 deviennent très rapidement neutropéniques. Ce phénotype découle d'un blocage au stade de différenciation myélocyte/métamyélocyte, tandis que les autres lignées hématopoïétiques restent non affectées par la délétion. Nous avons pu identifier le facteur de transcription C/ebpƐ comme un partenaire essentiel de Smarcd2 qui interagit avec le complexe SWI/SNF sur les promoteurs de gènes de granules secondaires afin d’en activer la transcription. Les analyses du transcriptome que nous avons effectué lorsque l’interaction de Smarcd2 et C/ebpƐ est interrompue dans des précurseurs de granulocytes ont montré une diminution de l’expression des gènes de granules secondaires liée à une maturation incomplète des granulocytes menant au développement d’un syndrome de myélodysplasie au court du temps. / During hematopoiesis, hematopoietic stem cells (HSCs) either selfrenew or differentiate into all mature blood cell types through successive rounds of binary cell fate decisions. The prevailing model of hematopoiesis predicts a step-by-step model of lineage differentiation in which HSCs first give rise to multipotent progenitors that subsequently differentiate into myeloid and lymphoid restricted progenitors. Although key transcriptional pathways controlling hematopoietic development are beginning to be deciphered, detailed molecular mechanisms explaining how HSCs and progenitors are initially primed and then commit to the different hematopoietic cell lineages are lacking. Work from our laboratory indicates that combinatorial assembly of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex is a key epigenetic mechanism that governs cell fate decisions. Transcriptomics analyses revealed that expression of the Smarcd1 subunit is enriched in hematopoietic stem/progenitors and early lymphoid cells, while Smarcd2 is mainly expressed in myeloid progenitors. Using conditional knock-out mouse models, we demonstrated that Smarcd1 and Smarcd2 subunits perform critical and lineage-specific roles during hematopoiesis. First, we found that Smarcd1 collaborates with the bHLH transcription factor E2A to specify lymphoid cell fate during hematopoiesis and, therefore, is absolutely required for lymphopoiesis. Mechanistically, we showed that Smarcd1 physically interacts with E2A and is required for chromatin accessibility of a set of H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hematopoietic stem cells. Impairing the interaction between Smarcd1 and E2A inhibits lymphoid lineage determination and the emergence of lymphoid-primed multipotent progenitors. Conversely, we showed that Smarcd2 is absolutely required for granulopoiesis. Smarcd2-deficient mice quickly become neutropenic due to a XIII block at the myelocyte/metamyelocyte stage of granulocyte maturation while other lineages remain unaffected. We discovered that Smarcd2 interacts with the transcription factor C/ebpε to recruit the mSWI/SNF complex on the promoter of secondary granule genes, thus inducing their transcriptional activation. As shown by transcriptomic analysis, impairing this interaction results in decreased expression of secondary granule genes, improper granulopoietic maturation, and development of a myelodysplastic-like syndrome over time. Altogether, this work identifies the Smarcd1 and Smarcd2 subunits of SWI/SNF complexes as master chromatin remodelers allowing the recruitment of lineage-specific transcription factors at key regulatory loci controlling lymphoid lineage priming and granulocyte development, respectively. More globally, these studies highlight that combinatorial assembly of alternative subunits of mSWI/SNF complexes is a key epigenetic mechanism controlling cell fate decisions during hematopoiesis.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Several common diseases such as atherosclerosis, post-angioplasty restenosis, and graft vasculopathies, are associated with the changes in the structure and function of smooth muscle cells. During the pathogenesis of these diseases, smooth muscle cells have a marked alteration in the expression of many smooth muscle-specific genes and smooth muscle cells undergo a phenotypic switch from the contractile/differentiated status to the proliferative/dedifferentiated one. Serum response factor (SRF) is the major transcription factor that plays an essential role in coordinating a variety of transcriptional events during this phenotypic change. The first goal of my thesis studies is to determine how SRF regulates the expression of smooth muscle myosin light chain kinase (smMLCK) to mediate changes in contractility. Using a combination of transgenic reporter mouse and knockout mouse models I demonstrated that a CArG element in intron 15 of the mylk1 gene is necessary for maximal transcription of smMLCK. SRF binding to this CArG element modulates the expression of smMLCK to control smooth muscle contractility. A second goal of my thesis work is to determine how SRF coordinates the activity of chromatin remodeling enzymes to control expression of microRNAs that regulate the phenotypes of smooth muscle cells. Using both mouse knockout models and in vitro studies in cultured smooth muscle cells I showed how SRF acts together with Brg1-containing chromatin remodeling complexes to regulate expression of microRNAs-143, 145, 133a and 133b. Moreover, I found that SRF transcription cofactor myocardin acts together with SRF to regulate expression of microRNAs-143 and 145 but not microRNAs-133a and 133b. SRF can, thus, further modulate gene expression through post-transcriptional mechanisms via changes in microRNA levels. Overall my research demonstrates that through direct interaction with a CArG box in the mylk1 gene, SRF is important for regulating expression of smMLCK to control smooth muscle contractility. Additionally, SRF is able to harness epigenetic mechanisms to modulate expression of smooth muscle contractile protein genes directly and indirectly via changes in microRNA expression. Together these mechanisms permit SRF to coordinate the complex phenotypic changes that occur in smooth muscle cells.

Le génome de la levure de boulanger Saccharomyces cerevisiae a évolué à partir d'un ancêtre chez lequel une profonde décompaction du génome s'est produite à la suite de la perte de la méthylation de la lysine 9 de l'histone H3, il y a environ 300 millions d'années. Il a été proposé que cette décompaction du génome a entraîné une capacité accrue des levures à évoluer par des mécanismes impliquant des taux de recombinaison méiotique et de mutation exceptionnellement élevés. La capacité à évoluer accrue qui en résulte pourrait avoir permis des adaptations uniques, qui en ont fait un eucaryote modèle idéal et un outil biotechnologique. Dans cette thèse, je présenterai deux exemples de la façon dont les adaptations locales et globales du génome se reflètent dans les changements des propriétés mécaniques de la chromatine qui, à leur tour, indiquent un phénomène de séparation de phase causée par les modifications post-traductionnelles des histones et des changements dans les taux d'échange des histones. Dans un premier manuscrit, je présente des preuves d'un mécanisme par lequel la relocalisation du locus INO1, gène actif répondant à la déplétion en inositol, du nucléoplasme vers l'enveloppe nucléaire, augmente la vitesse d'adaptation et la robustesse métabolique aux ressources fluctuantes, en augmentant le transport des ARNm vers le cytosol et leur traduction. La répartition d'INO1 vers l'enveloppe nucléaire est déterminée par une augmentation locale des taux d'échange d'histones, ce qui entraîne sa séparation de phase du nucléoplasme en une phase de faible densité plus proche de la périphérie nucléaire. J'ai quantifié les propriétés mécaniques de la chromatine du locus du gène dans les états réprimé et actif en analysant le déplacement de 128 sites LacO fusionnés au gène liant LacI-GFP en calculant diffèrent paramètres tel que la constante de ressort effective et le rayons de confinement du locus. De plus, j'ai mesuré l'amplitude et le taux d'expansion en fonction du temps du réseau LacO et j'ai observé une diminution significative du locus à l'état actif, ce qui est cohérent avec le comportement de ressort entropique de la chromatine décompactée. J'ai montré que les séquences d'éléments en cis dans le promoteur du locus, essentielles à la séparation de phase, sont des sites de liaison pour les complexes de remodelage de la chromatine effectuant l'acétylation des histones. Ces modifications de la chromatine entraînent une augmentation des taux d'échanges des sous-unités des complexes d'histones, et une séparation de phase locale de la chromatine. Enfin, je présente l’analyse de simulations in silico qui montrent que la séparation de phase locale de la chromatine peut être prédite à partir d'un modèle de formation/disruption des interactions multivalentes protéine-protéine et protéine-ADN qui entraîne une diminution de la dynamique de l'ADN. Ces résultats suggèrent un mécanisme général permettant de contrôler la formation rapide des domaines de la chromatine, bien que les processus spécifiques contribuant à la diminution de la dynamique de l'ADN restent à étudier. Dans un second manuscrit, je décris comment nous avons induit la « retro-évolution » de la levure en réintroduisant la méthylation de la lysine 9 de l'histone H3 par l'expression de deux gènes de la levure Schizosaccaromyces pombe Spswi6 et Spclr4. Le mutant résultant présente une augmentation de la compaction de la chromatine, ce qui entraîne une réduction remarquable des taux de mutation et de recombinaison. Ces résultats suggèrent que la perte de la méthylation de la lysine 9 de l'histone H3 pourrait avoir augmenté la capacité à l'évoluer. La stabilité inhabituelle du génome conférée par ces mutations pourrait être utile pour l'ingénierie métabolique de S. cerevisiae, dans laquelle il est difficile de maintenir des gènes exogènes intégrés pour les applications de nombreux processus biotechnologiques courants tels que la production de vin, de bière, de pain et de biocarburants. Ces résultats soulignent l'influence des propriétés physiques d'un génome sur son architecture et sa fonction globales. / The genome of the budding yeast Saccharomyces cerevisiae evolved from an ancestor in which a profound genome decompaction occurred as the result of the loss of histone H3 lysine 9 methylation, approximately 300 million years ago. This decompaction may have resulted in an increased capacity of yeasts to evolve by mechanisms that include unusually high meiotic recombination and mutation rates. Resultant increased evolvability may have enabled unique adaptations, which have made it an ideal model eukaryote and biotechnological tool. In this thesis I will present two examples of how local and global genome adaptations are reflected in changes in the mechanical properties of chromatin. In a first manuscript, I present evidence for a mechanism by which partitioning of the active inositol depletion-responsive gene locus INO1 from nucleoplasm to the nuclear envelope increases the speed of adaptation and metabolic robustness to fluctuating resources, by increasing mRNA transport to the cytosol and their translation. Partitioning of INO1 to the nuclear envelope is driven by a local increase in histone exchange rates, resulting in its phase separation from the nucleoplasm into a low-density phase closer to the nuclear periphery. I quantified the mechanical properties of the gene locus chromatin in repressed and active states by monitoring mean-squared displacement of an array of 128 LacO sites fused to the gene binding LacI-GFP and calculating effective spring constants and radii of confinement of the array. Furthermore, I measured amplitude and rate of time-dependent expansion of the LacO array, and observed a significant decrease for the active-state locus which is consistent with entropic spring behavior of decompacted chromatin. I showed that cis element sequences in the promoter and upstream of the locus that are essential to phase separation are binding sites for chromatin remodeling complexes that perform histone acetylation among other modifications that result in increased histone complex exchange rates, and consequent local chromatin phase separation. Finally, I present analytical simulations that show that local phase separation of chromatin can be predicted from a model of formation/disruption of multivalent protein-protein and protein-DNA interactions that results in decreased DNA dynamics. These results suggest a general mechanism to control rapid formation of chromatin domains, although the specific processes contributing to the decreased DNA dynamics remain to be investigated. In a second manuscript, I describe how we retro-evolutionarily engineered yeast by reintroducing histone H3 lysine 9 methylation through the expression of two genes from the yeast Schizosaccaromyces pombe Spswi6 and Spclr4. This mutant shows an increase in compaction, resulting in remarkable reduced mutation and recombination rates. These results suggest that loss of histone H3 lysine 9 methylation may have increased evolvability. The unusual genome stability imparted by these mutations could be of value to metabolically engineering S. cerevisiae, in which it is difficult to maintain integrated exogenous genes for applications for many common biotechnological processes such as wine, beer, bread, and biofuels production. These results highlight the influence of the physical properties of a genome on its overall architecture and function.