Chromatin is the combination of DNA and proteins in the nucleus that is used to aid in the compaction of DNA. Histones are a group of proteins used to condense DNA by forming a complex (nucleosome) around which DNA wraps around; there are two of each type of histone in a nucleosome: H2A, H2B, H3 and H4. Once the DNA is wrapped around the histones, the genome is further compacted. A shortened, "clipped" version of histone H3 has been found in some organisms including yeasts, flies, mammalian stem cells, and the ciliated protozoan, Tetrahymena thermophila. In each organism, clipping occurs at a different site on the N-terminus, usually before an alanine residue. Clipping is important as it may affect other epigenetic modifications and gene regulation in cell differentiation, but the regulation of this histone proteolysis has remained largely unstudied. In Tetrahymena thermophila, approximately half of the histone H3 molecules are clipped between residues 6 and 7 on histone H3, solely in the transcriptionally silent micronucleus. The histones in the micronuclei are deacetylated, while histones in the macronuclei can be acetylated or deacetylated. It is hypothesized that the post-translational acetylation modification to the histone tails may inhibit histone H3 clipping. Immunoblot analyses were carried out with acetylated and deacetylated micronuclei, demonstrating an increase of clipping when acetylated. Additionally, mutations were created at lysine 9 upstream of the clip site on the histone H3 tails to mimic acetylation and deacetylation to study whether the modification of that site has a regulatory effect.

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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.

A principal característica do diabetes mellitus (DM) é a perda da homeostasia glicêmica. O músculo esquelético desempenha papel chave e a adequada expressão do transportador de glicose GLUT4 (gene Slc2a4) é fundamental. Regulações epigenéticas do Slc2a4, como acetilação/trimetilação de histona H3, nunca foram investigadas no DM; e o resveratrol, sugerido como sensibilizador da insulina, poderia modular essas regulações, pois ativa a desacetilase sirtuína 1 (SIRT1). O objetivo foi avaliar em modelos de DM o efeito do tratamento com resveratrol sobre a homeostasia glicêmica, a expressão de Slc2a4/GLUT4 em músculo esquelético, a regulação epigenética do Slc2a4, e a possível participação da SIRT1. Os dados revelam a ocorrência de regulações epigenéticas no gene Slc2a4 em músculo de animais diabéticos e mostra que o tratamento com insulina ou resveratrol modula algumas dessas alterações, melhorando o controle glicêmico. Esses resultados apoiam o resveratrol como um sensibilizador da insulina, e constroem bases para o desenvolvimento de terapias epigenéticas para o DM. / The main characteristic of diabetes mellitus (DM) is the loss of glycemic homeostasis. The skeletal muscle plays a key role and the maintenance expression of the GLUT4 glucose transporter (encoded by the Slc2a4 gene) is fundamental. Epigenetic regulations of Slc2a4, such as histone H3 acetylation/trimethylation, have never been investigated in DM; and resveratrol, suggested as an insulin sensitizer, could modulate these regulations, as it is an activator of the deacetylase sirtuin 1 (SIRT1). The aim was to evaluate in skeletal muscle of diabetic animals the effect of resveratrol treatment on glycemic homeostasis, Slc2a4/GLUT4 expression in skeletal muscle, the epigenetic regulation of Slc2a4, and the possible participation of SIRT1. The data reveals the occurrence of epigenetic regulation in the Slc2a4 gene in muscle of diabetic animals and the insulin or resveratrol treatment modulates some of these changes, improving glycemic control. These results support resveratrol as an insulin sensitizer, and build bases for the development of epigenetic therapies for DM.

Les interactions protéine–protéine jouent un rôle essentiel dans le fonctionnement cellulaire et sont impliquées dans diverses pathologies. L'étude de ces interactions est donc primordiale. Nous avons entrepris de développer une méthode de « footprinting » basée sur la différence d'accessibilité à l'eau des acides aminés d'une protéine selon qu'elle est seule ou en interaction. Le principe de cette méthode de caractérisation des zones d'interactions protéine–ligand, est basé sur une étape de génération de radicaux carbo-centrés sur les chaînes latérales des acides aminés de la protéine, et sur une étape de réparation de ces radicaux par un atome de tritium.<br /> <br />La première étape a été de déterminer la réactivité des 20 acides aminés communs vis-à-vis de notre méthode : <br />Lys>Leu>Arg>Ile>Trp>Phe>Val>Cys>Met>His>Tyr>Glu>Thr>Asp><br />Gln>Pro>Ala>Asn> Ser>Gly. Notre méthode ensuite appliquée à l'étude du complexe entre la protéine hAsf11-156 et un fragment de l'histone H3 a permis de caractériser sans ambiguïté les trois résidus principaux de H3 (L126, R129 et I130) impliqués dans cette interaction. De plus, nous avons mis en évidence que notre méthode de caractérisation des interactions protéique est à la fois sensible aux phénomènes d'interaction et de repliement.

La sclérose en plaques est une maladie auto-immune dirigée contre les protéines de la myéline du cerveau. Plusieurs mécanismes physiopathologiques sont impliqués dans la SEP tels que l'inflammation, la démyélinisation et l’atteinte axonale. La SEP est associée à une expression accrue de cytokines et à une activation des rétrovirus endogènes humains (HERVs). Dans les conditions physiologiques, ces unités transcriptionnelles sont maintenues dans un état réprimé par un même mécanisme répresseur dépendant de la chromatine : la tri-méthylation de la lysine 9 de l’histone H3 (H3K9me3), qui crée un site de liaison aux protéines de la famille HP1. Nous avons trouvé qu’à la fois, les gènes de l’immunité et les HERVs nécessitent les protéines hétérochromatiniennes HP1α pour leur répression transcriptionnelle. Nous avons montré que la peptidylarginine déiminase 4, une enzyme qui joue un rôle dans la SEP, affaiblit la liaison de HP1α à la lysine 9 tri-méthylée de l'histone H3 en citrullinant l’arginine 8. Nous avons apporté la preuve que de multiples événements de la réactivation de la transcription chez les patients atteints de la SEP peuvent être expliqués par un défaut du mécanisme unique de répression génique. Nous avons également montré qu'il est possible de renforcer la répression de HP1 à l'aide de petites molécules. Par exemple, l’EGCG, un composé de thé vert, est en mesure de réduire à la fois l’expression des HERVs et des cytokines en augmentant l'activité de l’histone méthyltransférase SUV39H1. Cela conduit à une accumulation de la marque répressive H3K9me3, qui va favoriser la liaison de HP1.Ensemble, ces résultats suggèrent que HP1 est une composante importante de la SEP au niveau de la régulation des cytokines et des HERVs. / Multiple Sclerosis is an autoimmune disease resulting in damage to myelin structures of the brain. Several physiopathological mechanisms are involved in MS including inflammation, demyelination, and axonal damage. MS is associated with increased cytokine expression and activation of human endogenous retroviruses (HERVs). These two types of transcriptional units are kept in check by chromatin-dependent silencing associated with lysine 9 trimethylation of histone H3, and subsequent of HP1 proteins. We find that both the cytokine genes and the HERVs require the heterochromatin protein HP1 for their transcriptional repression. Furthermore, we have shown that the peptidylarginine deiminase 4, an enzyme with a suspected role in MS, weakens the binding of HP1 to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. We thereby evidence that multiple events of transcriptional reactivation in MS patients can be explained by deficiency of a single mechanism of gene silencing. We have also shown that it is possible to reinforce HP1 repression by using small molecules. For example, EGCG, a green tea compound, is able to reduce both HERVs and cytokines expression by increasing histone methyltransferase activity SUV39H1. This leads to increased accumulation of H3K9me3 repressive marks and favors binding of HP1. All together, these results suggest that HP1 is an important component of the regulation of cytokine genes and HERVs in MS patients.

Neutrophils are major cell type of innate immunity, that can eliminate pathogens by different mechanisms. One of these mechanisms is called NETosis, which leads to release of decondensed chromatin and citrullinated histone proteins. Citrullination is post-translational modification catalysed by peptidylarginine deiminase (PAD) and causing transformation of possitively charged arginin to neutral citrullin and can change expression of cytokine genes. Concetrations of pro-inflammatory cytokines (IL-8, TNF, IL-1) were measured after activation of PAD4 and induction of citrullination. Calcium ionophore was used to induce citrullinaton, Cl-amidine and TDFA were used as inhibitors. Production of cytokines was assessed by ELISA on protein level and by qPCR on mRNA level. It was found that induction of citrullination led to increased concentrations of IL-8 and IL-1. Elevated gene expression of IL-8 was confirmed on mRNA level. Both inhibitors were able to decrease level of histone H3 citrullination and IL-8 and IL-1 concentrations. Expression of TNF was not detected on protein and mRNA level.

Protein Prp45, the yeast ortholog of the human transcription coregulator SNW1/SKIP, has been previously associated only with the regulation of pre-mRNA splicing. However, our laboratory found that protein Prp45 genetically interacts not only with the proteins involved in pre-mRNA splicing, but also with factors important for transcription elongation and with chromatin modifying enzymes. Our data and the information about the human ortholog SNW1/SKIP suggest that Prp45 could serve as a regulator coupling splicing, transcription and chromatin state in S. cerevisiae. The main aim of this diploma thesis was to find out whether the protein Prp45, which is essential for cotranscriptional assembly of the spliceosome, affects posttranslational modifications of chromatin on transcribed genes. Using chromatin immunoprecipitation, the influence of prp45(1-169) mutation on trimethylation of histone H3 at lysine 4 and acetylation of histone H3 at lysines 9, 14 and 18 on transcriptionally active genes was not confirmed. The other aim was to analyse the behavior of cells synchronized by α-factor by using flow cytometry. According to our results, prp45(1-169) mutation leads to the prolongation of the cell cycle. For the purpose of monitoring the dynamics of nucleosomes in S. cerevisiae strains, the system of...

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.