Chromatin is a dynamic structure, and chromatin remodeling enzymes regulate chromatin structure to control gene expression and proper lineage specification. Tip60-p400 is a multi-subunit chromatin remodeling complex containing two biochemical activities: the Tip60 subunit is a lysine acetyltransferase (KAT) that targets histones and non-histone proteins, and p400 catalyzes ATP-dependent incorporation of histone variant H2AZ into chromatin. Both of these chromatin modifications have been widely studied with respect to gene expression, DNA damage repair, and apoptosis. Ablation of these catalytic subunits causes defects in normal embryonic development, ESC self-renewal, and gene expression. My goal has been to understand the multiple independent functions of Tip60-p400 acetyltransferase in ESC maintenance and embryonic development. I showed that Tip60 KAT function is dispensable for gene expression, chromatin accessibility, and ESC self-renewal, which is different from Tip60 knockdown phenotype. Interestingly, KAT deficient mutants exhibited defect in differentiation towards mesoderm and endoderm lineages. Consistent with this defect, I also observed gastrulation defect in mice lacking Tip60 KAT activity. Together, these data demonstrate that Tip60 KAT dependent function is only required during later stages of embryonic development, and it is dispensable for ESC self-renewal and pre-implantation development. Tip60 KAT contains four isoforms generated from alternative splicing, whose individual functions are poorly characterized. In the second part of this thesis, I investigated the developmental role of one of the isoforms of Tip60, called Tip55. Unlike Tip60 knockout mice, which lack all the isoforms and causes pre-implantation lethality, I found that ablation of Tip55 results in post-implantation lethality. I further found that loss of Tip55 causes defects in heart, and neural tube development, demonstrating the essential function of Tip55 isoform for organogenesis during embryonic development. Together, these studies have provided new insight into the functions of Tip60-p400 and the mechanisms by which this complex regulates gene expression, ESC pluripotency, and embryonic development. Furthermore, these studies set the stage for future work to identify how the catalytic and non-catalytic functions are directed to perform distinct regulatory functions, as well as how each Tip60 isoform individually contributes to formation of the mammalian body plan.

A subset of eukaryotic heterochromatin is located around the nucleoli, and this localization is correlated with gene silencing. Although there is some evidence for trans-acting factors organizing genomic loci around the nucleolus, the characterization of proteins and /or RNAs involved in perinculeolar heterochromatin localization and maintenance is incomplete. Notably, the mammalian female inactive X chromosome, a well-studied model of facultative heterochromatin, frequently resides in the perinucleolar regions during mid to late S phase. The disruption of the Xi–nucleolus association results in the erosion of heterochromatin compartment and silencing, which renders it a good model to investigate the mechanism and biological relevance of heterochromatin organization around the nucleolus. This dissertation will present evidence showing that Ki-67 regulates inactive X (Xi) chromosome association with nucleoli, maintains Xi heterochromatic structures, and regulates cell cycle progression, in cell-type-specific manner dependent on checkpoint proficiency. Ki-67 protein plays roles in heterochromatin organization during interphase. Upon Ki-67 depletion, a subset of Xi in human female hTERT-RPE1 moved away from nucleolus and displayed several features of compromised heterochromatin maintenance. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in virally transformed 293T cells upon Ki-67 depletion. Furthermore, I demonstrated that the different Xi heterochromatin alteration responses result from cell-type-specific reduced proportion of cells in S phase upon Ki-67 depletion. In human hTERT-RPE1, WI-38, IMR90, hTERT-BJ cell lines, depletion of Ki-67 slowed entry into S phase and coordinately downregulated genes related to DNA replication. These cell lines are able to induce p21 expression upon Ki-67 depletion. On the contrary, alteration of transcription and cell cycle progression were not observed in tumor-derived HeLa, U2OS and 293T cell lines. These cell lines do not induce p21 expression either. I additionally examined the Ki-67 function in mouse cell cultures. Depletion of Ki-67 neither redistributes inactive X chromosome nor regulates S phase progression in primary female mouse embryonic cells.

Dans les cellules souches embryonnaires de souris (mESCs), le groupe de protéines Polycomb (PcG) répriment les gènes de développement en participant ainsi à la maintenance de l’état de pluripotence. Ce complexe dépose la H3K27me3au niveau des éléments régulateurs induisant une compaction de la chromatine. Cette marque forme en plus des marquesactives H3K4me3 présentes des domaines bivalents. Etrangement, des boucles d’ADN dites entre le promoteur et enhancer, généralement associé à l’activation du gènes, sont observées au niveau des gènes bivalents avant leur activation.On suppose que la fonction du PcG pourrait être de neutraliser l'enhancer conférant une future activation rapide des gènes.Au cours de ma thèse, j’ai identifié les boucles d’ADN formé par les réprimés par PcG dans les mESCs. Pour cela,j’ai effectué un profilage épigénomique de 4 marques d'histones et identifié près de 2500 promoteurs bivalents et 13000enhancers. En utilisant des données publiées de Hi-C à haute résolution, j’ai identifié toutes les boucles formées par les domaines bivalents. Etonnement, j’ai pu identifier que de nombreux gènes réprimés par PcG interagissent avec des enhancers actifs. Cette observation a été suivie d'une validation par le 4C-seq. De plus, j’ai effectué une caractérisation fonctionnelle des boucles en utilisant deux approches. Tout d'abord, j'ai mis en place, en collaboration avec D. Bourc'his(Institut Curie), un système de culture de mESCs (2i + VitC) où le taux de H3K27me3 est réduit. J'ai effectué un profilage épigénomique similaire révélant que les promoteurs réprimés par PcG ont perdu la marque H3K27me3. En RNA-seq, j’ai démontré que l’expression des gènes ne change pas après le PcG soit détacher des promoteurs.. Ensuite, par la réalisation de plusieurs validations en 4C-seq j’ai démontré que les interactions avec les enhancers ne sont pas affecté alors que la moitié des enhancers interagissant perdent leurs marques activatrices. Dans le système 2i+VitC, ces gènes semblent être réprimés par un autre mécanisme suite à la perte du PcG. De plus, j’utilise une approche ciblée pour enlever localement laH3K27me3 de deux gènes bivalents en utilisant le système Cette technique est en cours d’optimisation.Notre étude est la plus systématique au niveau génomique des boucles d'ADN dans le cadre de la régulation des gènes PcG. Notre étude révèle une nouvelle fonction du PcG qui est la répression de boucle d’ADN déjà établies entre promoteurs et enhancers. / In the mouse embryonic stem cells (mESCs), Polycomb Group Proteins (PcG) repress developmental genes and thereby participating in the maintenance of the pluripotency. PcG repress genes by depositing the H3K27me3 histone marks on their regulatory elements, followed by chromatin compaction. In addition to the H3K27me3 marks, those genes carry H3K4me3 active marks and were characterized as bivalent. Intriguingly, at many PcG repressed genes, DNA loops can be observed with enhancer elements, which are normally thought to have an activating function. The aim of my project is to both describe and mechanistically dissect the function of Polycomb repressed promoter – enhancer loops.During my PhD, I aimed firstly to identify all promoter–enhancer loops involved by PcG repressed genes in mESCs. I have performed ChIP-seq profiling of 4 histone marks and identified around 2500 PcG repressed promoters and 13000 enhancers. Using a recently published high-resolution Hi-C data in mESCs, I have identified all DNA loops that are formed by PcG repressed promoters. Surprisingly, a high percentage of bivalent promoters were found to contact active enhancers. The presence of those loops were validated by ultra-high 4C-seq on selected genes and imply a small significant increase of the gene expression without leading to a complete activation of the gene. I have established a more physiological ESC model (2i+VitC) where H3K27me3 is reduced at all promoters. I have performed ChIP-seq, where bivalent promoters were all classified as H3K27me3 negative. RNA-seq experiments have showed that those genes do not become activated. 4C-seq experiments have revealed that those loops do not disappear after PcG removal, whereas the half of interacted enhancer loose their H3K27ac active marks. Those genes seem to remain repressed by an unknown mechanism. These results argue for a possible role of PcG in preparing the gene for their activation by blocking the productivity of such DNA loops. Secondly, I aimed to functionally characterize those DNA loops by using a CRISPR/dCas9 approach to completely remove H3K27me3 from two PcG repressed genes that contact active enhancers Pax6 and Nkx1-1 genes. This system is still under optimization steps.My project revealed the most systematic characterization of DNA loops under the regulation of PcG, providing important insight how PcG function to inactivate such loops. I have highlighted an additional function of PcG which the involvement in the repression of already establish loops between active enhancers and promoters and thereby blocking the productivity of such activating loops. This function is an addition to the already described repressive function of PcG on both promoters and poised enhancers.

Une caractéristique majeure de l’infection par Toxoplasma gondii est le contrôle rapide de la population parasitaire par une réponse immunitaire engageant des cellules résidentes et recrutées ainsi que des cytokines pro- et anti-inflammatoire. Dans ce contexte, l’IFNγ active une multitude d’activité anti- T. gondii des cellules immunes et non-immunes, mais peut aussi contribuer à l’immunopathologie. T. gondii a élaboré des mécanismes pour contrer les défenses de l’hôte en interférant avec la transcription des gènes stimulés par l’IFNγ. Nous avons identifié TgIST (T. gondii inhibitor of STAT1 transcriptional activity) comme un interrupteur moléculaire exporté par les parasites intracellulaires et qui est localisé dans le noyau des cellules hôtes, où il inhibe l’expression des gènes pro-inflammatoires dépendants de STAT1. Nous avons montré que TgIST séquestre STAT1 à des sites spécifiques, et promeut la formation de chromatine non permissive grâce à sa capacité à recruter le remodeleur chromatinien NuRD. Nous avons montré que durant l’infection aiguë en souris, les parasites déficients pour TgIST sont rapidement éliminés par les monocytes pro-inflammatoires GR1+, ce qui montre le rôle protecteur de TgIST contre les défenses médiées par l’IFNγ. En révélant les fonctions de TgIST, cette étude montre de nouvelles évidences sur la façon dont T.gondii a élaboré une arme moléculaire de choix pour prendre le contrôle sur la réponse immune, de façon à promouvoir le parasitisme à long terme / An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii–targeting activities in immune and nonimmune cells but can also con- tribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ–stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ–mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.

No description available.

Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.

No description available.

No description available.

<p>Regulatory interactions evolve to incorporate new genomic material and contribute to bacterial diversity. These regulatory interactions are flexible and likely provide bacteria with a means of rapid environmental adaptation. In this thesis, the RpoS regulon is used as a model system to investigate the hypothesis that regulon composition and expression are modified according to environmental pressures. Several novel findings are presented, namely the distribution of RpoS homologs in bacteria, the flexibility of the RpoS regulon, and the effect of diverse environmental pressures on RpoS regulon expression. Based on phylogenetic and reciprocal best hits analyses, RpoS was determined to be conserved in gamma-, beta-, and delta-proteobacteria, likely because it confers a selective advantage in many bacterial niches. Regulon composition, however, was highly flexible. Even between species of the same class, <em>Escherichia coli</em> and <em>Pseudomonas aeruginosa</em>, only 12 of 50 orthologs were regulated in common by RpoS. RpoS regulon flexibility may thus be the result of adaptation to different bacterial habitats. Indeed, mutations in <em>rpoS</em> and differential regulon expression could be identified among environmental <em>E. coli</em> isolates collected from diverse sources. Among environmental <em>E. coli</em> isolates, RpoS mutant frequency was found to be 0.3%, and activity of KatE, a prototypical RpoS regulon member, was undetectable in some isolates despite the presence of functional RpoS. Modulated RpoS regulon expression among environmental <em>E. coli</em> isolates is consistent with environment as a key factor shaping regulatory interactions. Regulon flexibility was similarly apparent in oxidative stress regulons, OxyR and SoxRS, of <em>E. coli</em>. SoxRS regulon function is weakly conserved, possibly due to low selective pressure for a superoxide stress response regulon in some bacterial species. Environment, therefore, is a crucial element that defines the dynamics of regulatory networks.</p> / Doctor of Philosophy (PhD)

Die Modifikation von Chromatin und Histonen sind vielmehr zentrale Ereignisse in der Regulation der Geneexpression. Es ist gut etabliert, dass die Trimethylierung von Histon H3 Lysin 27 (H3K27me3) durch ‚Polycomb group’ (PcG) Proteine epigenetisches Gedächtnis aufrechterhält. Genomweite Analysen haben jedoch gezeigt, dass ein großer Teil des nicht-exprimierten Genoms die H3K27me3 Modifikation trägt. In dieser Arbeit habe ich RNA-seq und ChIP-seq auf Drosophila Plasmatozyten angewendet und ein hochauflösendes Chromatinprofil generiert. Ich konnte zeigen, dass eine Gruppe von Immungenen, die durch H3K27me3 markiert sind, nach einer Immunstimulierung rasch hochreguliert wird. Weiterhin konnte ich durch die Anwendung eines neu entwickelten Genomanalyse-Algorithmus zeigen, dass diese H3K27me3 positiven Gene in einem Chromatinzustand sind, der sich von kanonischem Polycomb Chromatin unterscheidet. Dieser Chromatinzustand hat zwei wichtige Eigenschaften: Erstens beweise ich, dass H3K27me3 als Antwort auf physiologische Stimuli dynamisch reguliert wird. Zweitens weise ich nach, dass es für den Erhalt eines reprimierten Genezustands instruktiv ist. Daher bezeichne ich diesen Chromatinzustand als dynamisches Polycomb Chromatin. Meine weiteren Analysen haben gezeigt, dass dieses dynamisches Polycomb Chromatin in Drosophila Plasmatozyten mit einer großen Zahl anderer dynamisch regulierter Gene assoziieret ist. Einige dieser Gene wurden ebenfalls nach der Depletion von H3K27me3 hochreguliert. Deshalb schlage ich vor, dass dynamisches Polycomb Chromatin einen neuartigen Chromatinzustand darstellt, der an Genen zu finden ist, deren Transkription durch unvorhersehbare Ereignisse ausgelöst wird. Die Reprimierung durch dynamisches Polycomb Chromatin bildet demnach eine Aktivierungschwelle gegen aberrante Transkription, die aber trotzdem eine rasche Geninduktion in physiologisch relevanten Situationen erlaubt. / Modification of chromatin and histones are central events in regulating gene expression. It is clearly established that histone H3 lysine 27 trimethylation (H3K27me3) by Polycomb group (PcG) proteins maintains the repressed state in epigenetic memory. Genome wide analysis revealed that much of the non-transcribed genome carries the H3K27me3 modification. This raises the question, whether this modification is functionally relevant outside of development to preclude activation of genes through physiological signaling. Here, I applied RNA-seq and ChIP-seq to Drosophila plasmatocytes generating a high resolution epigenetic landscape. Thereby, I demonstrated that a set of H3K27me3 marked immune genes was rapidly transcriptionally induced upon challenge. By applying a newly developed genome clustering algorithm, I demonstrated that these H3K27me3 positive genes are in a chromatin state that is distinct from the canonical Polycomb chromatin. This state has two important properties: First, by demonstrating that in plasmatocytes H3K27me3 is depleted specifically at immune genes after activation, I showed that it is dynamically regulated in response to physiological stimulation. Second, by establishing that immune genes were up-regulated when H3K27me3 was depleted, I confirmed that it is instructive in maintaining a silenced gene state. Therefore, I termed this novel chromatin state dynamic Polycomb chromatin. Further analysis revealed that this dynamic Polycomb chromatin state is also associated with a large number of other dynamically regulated genes. Some of these genes were also up-regulated when H3K27me3 is depleted by genetic manipulation. Hence, I propose that dynamic Polycomb chromatin is a novel chromatin state that targets genes which are triggered by non-predetermined signaling events. Silencing by dynamic Polycomb chromatin thresholds such genes against aberrant gene activation, but permits rapid induction in physiologically relevant situations.