Dissertations / Theses on the topic 'Chromatin'

Les arguments en faveur d’un rôle important de l'architecture des chromosomes en interphase pour la régulation des gènes et la maintenance du génome s’accumulent rapidement. Au cours de l'interphase, les chromosomes sont positionnés de façon non aléatoire l’un par rapport à l'autre et fournissent ainsi des points de repère nucléaires. Deux types d'interactions contribuent probablement à ce positionnement non aléatoire: (i) des domaines subchromosomiques interagissent avec des structures nucléaires telles l'enveloppe nucléaire (EN) et (ii) des interactions intrachromosomiques s’établissent entre des loci situés de façon linéairement distante en cis sur un même chromosome. Contribuant à l’expansion de ce domaine de recherche, nous avons poursuivi le développement d’une technique préalablement établie au laboratoire pour détecter des interactions protéine-protéine. Le développement de cette technique nouvelle a constitué une part de ce travail de thèse accompli sur des cellules humaines. Elle se base sur le marquage par la biotine de composants de la chromatine qui en interphase se trouvent à proximité immédiate de l’EN. Les cellules ont été traitées pour exprimer (i) la biotine ligase BirA fusionnée à l’émerine, une protéine de l’EN, conjointement avec (ii) une variante d’histone, l’histone macroH2A, en fusion avec un peptide accepteur de biotine. L'étiquette biotine déposée sur l’histone macroH2A pendant l'interphase est ensuite détectée par microscopie à fluorescence sur des cellules en mitose étalées sur lames. Les chromosomes mitotiques marqués peuvent en outre être caractérisés par des techniques plus classiques de caryotypage. Nous avons nommé cette technique «topokaryotypage» car elle peut fournir des informations d’ordre à la fois topologique et caryotypique. Son développement pas à pas a nécessité la production d'une lignée cellulaire ad hoc et une optimisation fine du protocole. Ce travail de thèse peut déboucher sur des questions biologiques explorées sur cellules uniques. A titre d’application, une analyse comparative a été réalisée par topokaryotypage sur des cellules cultivées in vitro dans diverses conditions de stress expérimentales. L’utilisation du topocaryotypage pourrait fournir des informations précieuses sur les mécanismes à la base de l’organisation et de la dynamtique des noyaux cellulaires
Evidence is rapidly accumulating that the architecture of interphase chromosomes is important for both gene regulation and genome maintenance. During interphase, chromosomes are nonrandomly positioned with respect to each other and thus they provide nuclear landmarks. Two kinds of interactions are likely to contribute to this nonrandom positioning: (i) subchromosomal domains interact with nuclear structures such as the nuclear envelope (NE) and ii) intrachromosomal interactions take place between linearly distant loci positioned in cis on the same chromosome. As a contribution to this expanding research domain, we have built upon an existing approach previously established in the laboratory to detect protein-protein interactions. The new technique was developed in human cells as part of the present PhD research. It is based on biotin labelling of chromatin components which are in close proximity with the nuclear envelope (NE) in interphase cells. Cells were made to express (i) the biotin ligase BirA fused to the NE protein emerin together with (ii) a fusion between a biotin acceptor peptide and macroH2A, a variant core histone. The biotin label deposited on the macroH2A histone during interphase is then detected by fluorescence microscopy on mitotic cells spread on slides. The biotin-labelled mitotic chromosomes can be further characterized using more classical karyotyping techniques. We refer to this new technique as “Topokaryotyping” since it can provide both topological and karyotypic information. Its step-by-step development has required the establishment of an ad hoc cell line and a fine protocol optimization. This PhD work could pave the way for biological questions explored at a single cell level. As an illustration, a comparative topokaryotyping analysis was performed on cells cultivated in vitro in various experimental stress conditions. It is envisioned that using this technique can provide valuable mechanistic insights relevant to the organization and dynamics of cell nuclei

Development and application of genomic approaches based on 3C methods combined with increasingly powerful imaging approaches have enabled high-resolution genome-wide analysis of the spatial organization of chromosomes in genome function. In this thesis, I first describe an updated protocol for Hi-C (Hi-C 2.0), integrating recent improvements that significantly contribute to the efficient and high-resolution capture of chromatin interactions. Secondly, I present an assessment of the epigenetic landscape and chromosome conformation around the MYC gene in acute myeloid leukemia (AML) cells before and after small molecule, AI-10-49, treatment. MYC is up-regulated upon inhibition of the RUNX1 repressor by the fusion oncoprotein CBFβ-SMMHC. Treatment of AML cells with AI-10-49 blocks the RUNX1-CBFβ-SMMHC interaction, restoring RUNX1 at MYC regulatory elements. We demonstrate that the established loop is maintained and exchange between activating and repressive chromatin complexes at the regulatory elements, rather than altered chromatin topology, mediates disruption of target gene expression. Finally, Hi-C interaction maps represent the population-averaged steady-states. To understand the forces that promote and maintain the association of loci with specific sub-nuclear structures genome-wide, we developed liquid chromatin Hi-C. Detection of intrinsic locus-locus interaction stabilities and chromatin mobility are enabled by fragmenting chromosomes prior to fixation and Hi-C, thus removing strong polymeric constraints. Nuclear compartmentalization was found to be stable for average fragment lengths are 10-25 kb while fragmentation below 6kb led to a gradual loss of spatial genome organization. Dissolution kinetics of chromatin interactions vary widely for different domains and are analyzed in detail in the final chapter of this thesis., with lamin-associated domains being most stable, and speckle-associated loci most dynamic.

L'entrée en sénescence, considérée comme un arrêt irréversible du cycle, se caractérise par une modification de l'organisation de la chromatine formant de véritables foyers d' hétérochromatine spécifiques (SAHF) coordonnée à une modification d'expression génique et à un déclin progressif de la compétence à répliquer le génome. Ainsi, au cours de ma thèse, j'ai voulu comprendre en quoi ces changements d'organisation du génome pouvaient influer sur la distribution et l'activation des origines d e réplication lors de l'entrée en sénescence réplicative ou déclenchée de façon prématurée par l'inhibition d'un modulateur de chromatine, la protéine à activité Histone AcétylTransférase p300. Pour étudier ces régulations, j'ai utilisé le peignage moléculaire d'ADN réplicatif qui permet de suivre les fourches de réplication et d'évaluer la distribution moyenne des origines. De plus, à l'aide de la purification de brins naissants aux origines de réplication couplée à un séquençage haut débit, nous avons cartographié la position de ces origines sur l'ensemble du génome humain et étudier un ensemble de facteurs pouvant intervenir dans ce déterminisme. Grâce à cette étude, nous avons pu suivre finement les modifications d'activité des origines associées à l'entrée en sénescence. De plus, afin de mieux comprendre les mécanismes d'activation des origines de réplication, nous avons étudié en collaboration avec l'équipe du Dr Fisher, le rôle de Cdk1 et de Cdk2 dans l'activation des origines dans le modèle Xénope
Senescence entry, considered as an irreversible cell cycle arrest, is characterized by modifications of chromatin organization forming specific heterochromatin foci (SAHF) coordinated to modification of gene expression and the progressive loss of capacity to replicate the genome. During my PhD, we investigated whether these changes in genome organization might induce modifications in the distribution and the activity of replication origins during replicative senescence entry and in prematurely induced senescence by inhibition of a chromatin modulator, the Histone AcetylTransferase p300. To study these regulations, we used the replicating DNA combing allowing to follow the progression of replication forks and to evaluate the mean distribution of origins. By using the nascent strand purification assay coupled to deep sequencing, we mapped the position of replication origins in the whole human genome and studied some factors which could be involve d with this determinism. Thanks to this study, we followed finely the modifications of activity of replication origins associated to senescence entry. Moreover, in order to better understand the mechanisms of activation of origins, we studied in collaboration with Dr Fisher's team, the role of Cdk1 and Cdk2, in the activity of replication origins in the Xenopus model

Drosophila nucleosome remodelling factor (NURF) is one of the founding members of the ISWI family of ATP-dependent chromatin remodelling enzymes and mediates energy-dependent nucleosome sliding leading to transcription regulation. In previous work (Wysocka et al., 2006), NURF was shown to be recruited to gene targets by binding specific histone modifications. The largest subunit of NURF, NURF301, contains a bromodomain and three PHD finger domains that have the ability to recognize specific histone modifications. Here we determine the histone binding-specificities of these domains, and how NURF histone binding is influenced by histone modification "cross-talk". This has been analyzed by histone peptide library array assays and our study shows that the PHD2 domain specifically recognizes the histone H3K4me3 mark. This binding can be inhibited by phosphorylation of H3 Thr 3, while enhanced by acetylation of H3 Lys 9 and phosphorylation of Ser 10. The binding specificities of bromodomain, PHD and PHD1 domains were also determined. These data were confirmed by peptide pull-down, Biacore and immunofluorescence microscopy assays. Moreover, two different NURF301-A/B and NURF301-C isoforms were CTAP-tagged by recombineering, and we used chromatin immunoprecipitation coupled sequencing (ChIP-Seq) to profile the genome-wide distribution of NURF in vivo. Therefore, our results identify regulatory mechanisms of histone modifications directing recruitment of ATP-dependent chromatin remodelling enzymes.

Les oligodendrocytes (OLs) sont les cellules myélinisantes du système nerveux central, s’enroulant autour des axones et permettant la conduction saltatoire du potentiel d’action. Dans la Sclérose en Plaques, des gaines de myélines sont détruites et l’efficacité de la remyélinisation par les précurseurs d’oligodendrocytes (OPCs) diminue avec la progression de la maladie. Une meilleure compréhension du mécanisme qui contrôle la génération des OPCs et leur différentiation est donc essentielle pour développer des thérapies efficaces de remyélinisation. L’oligodendrogenèse, qui comprend les étapes de génération des OPCs, de différenciation et de maturation des OLs, est un processus contrôlé par des facteurs de transcription spécifiques incluant Ascl1, Olig2 and Sox10 mais le mécanisme impliqué est encore peu connu. Sachant que les facteurs du remodelage de la chromatine sont des régulateurs nécessaires à la formation de la boucle promoter-enhancer permettant l’initiation de la transcription, nous nous sommes focalisé sur Chd7 (Chromodomain-Helicase-DNA-Binding 7), un membre de la famille de protéine CHD. Dans une première étude, nous avons montré que Chd7 est hautement enrichi dans le lignage oligodendroglial avec un pic d’expression pendant la différenciation des OLs. Nous avons également montré que la délétion conditionnelle de Chd7 diminuait la différentiation des OLs pendant la (re)myélinisation. Dans un seconde étude, nous avons utilisé des techniques de génomique sur les OPCs purifiés pour étudier la régulation par Chd7 de gènes impliqués dans la différenciation, la survie et la prolifération des OPCs. Dans ce but, nous avons générer des délétions inductible de Chd7 spécifiquement dans les OPCs (Chd7iKO) et nous avons analysé le transcriptome (RNA-seq) d’OPCs purifiés à partir de cerveaux de souris P7 comparé à des contrôles. Nous avons trouvé que Chd7 activait l’expression des gènes impliqué dans la différenciation des OPCs et la myélinisation et inhibait l’apoptose, sans montré de défaut de prolifération. Pour aller plus loin, nous avons étudié Chd8, un paralogue de Chd7, et nous avons montré qu’il est exprimé dans le lignage oligodendrocytaire avec un pic d’expression dans les OL en différenciation, similairement à Chd7. Les données de fixation (ChIP-seq) de Chd7 et Chd8 indiquent que ces deux facteurs du remodelage de la chromatine se fixent sur des gènes communs reliés au processus de différenciation, de survie et de prolifération des OPCs. Intégrant ces données avec celles de facteurs transcriptionnels clés dans l’oligodendrogenèse (Olig2, Ascl1 et Sox10), nous avons construit un modèle de la régulation de l’expression de gènes contrôlés dans le temps et impliqué dans chacune des étapes de la différenciation des oligodendrocytes
Oligodendrocytes (OLs) are myelin-forming cells of the central nervous system wrapping axons and allowing the saltatory conduction of action potentials. In Multiple sclerosis (MS), myelin sheath is destroyed and effective remyelination by oligodendrocyte precursor cells (OPCs) diminishes with disease progression. Therefore, a better understanding of the mechanisms controlling OPC generation and differentiation is essential to develop efficient remyelinating therapies. Oligodendrogenesis, involving the steps of OPC generation, OPC differentiation and maturation of OLs, is a process controlled by specific transcription factors including Ascl1, Olig2 and Sox10 but the mechanisms involved are poorly understood. As it is known that chromatin remodelers are regulatory factors necessary in the formation of the promoter-enhancer loop prior to transcription, we focused our study on Chd7 (Chromodomain-Helicase-DNA-Binding 7), a member of the CHD protein family. In a first study, we showed that Chd7 is highly enriched in the oligodendroglial lineage cells with a peak of expression during OL differentiation and that Chd7 OPC-conditional deletion impairs OL differentiation during (re)myelination. In a second study, we used unbiased genome wide technics in purified OPCs to study Chd7 regulation of genes involved in OPC differentiation, proliferation and survival. To this aim, we have generated OPC-specific inducible Chd7 knock-out (Chd7-iKO) and analyse the transcriptome (RNA-seq) of purified OPCs from P7 mouse cortices compared to control littermates. We found that Chd7 promote the expression genes involved in OPC differentiation and myelination and inhibits apoptosis, without affecting OPC proliferation. Furthermore, we investigated Chd8, a paralog of Chd7, showing that it is expressed in the oligodendroglial lineage with a peak of expression in differentiating oligodendrocytes, similar to Chd7. Genome wide binding (ChIP-seq) profiling for Chd7 and Chd8 indicate that these two chromatin remodelers bind to common genes related to OPC differentiation, survival and proliferation. Integrating these datasets with other key transcriptional regulators of oligodendrogenesis (Olig2, Ascl1 & Sox10), we have built a model accounting for the time-controlled regulate expression of genes involved in each step of OL differentiation

Les complexes histones méthyltransférases SET1, hautement conservés de la levure aux mammifères, sont ciblés aux régions promotrices par la protéine CFP1/CXXC, résultant en l’implémentation de la méthylation de la lysine 4 de l’histone H3 (H3K4me), modification post-traductionnelle influençant l’expression des gènes selon le contexte chromatinien. La présence de plusieurs complexes SET1 distincts dans différents systèmes modèles eucaryotes a compliqué l’étude de leurs fonctions dans un contexte développemental. Caenorhabditis elegans contient une seule protéine homologue de SET1, SET-2, et d’uniques homologues des autres sous-unités du complexe, RBBP5, ASH2, WDR5, DPY30 et CFP1. Cependant, la composition biochimique du complexe n’a pas été décrite. En couplant des expériences de co-immunoprécipitation avec des analyses de spectrométrie de masse, j’ai identifié le complexe SET-2/SET1 dans les embryons de C. elegans. D’autre part, j’ai montré que le complexe SET-2/SET1 co-immunoprécipite aussi un autre complexe conservé modifiant la chromatine et j’ai mis en évidence les interactions mises en jeu entre ces deux complexes. Mon analyse génétique a démontré que les mutants de perte de fonction des sous-unités des deux complexes partagent des phénotypes communs, en cohérence avec des fonctions développementales communes. Le laboratoire a également entrepris des expériences de transcriptomique et d’immunoprécipitation de la chromatine montrant un nouveau rôle de CFP-1 dans le recrutement de ce complexe au niveau de sites spécifiques de la chromatine
The highly conserved SET1 family complexes are targeted by CFP1/CXXC protein to promoter regions through multivalent interactions to implement methylation of histone H3 Ly4 (H3K4me), a modification that correlates with gene expression depending on the chromatin context. The presence of distinct SET1 complexes in multiple eukaryotic model systems has hampered studies aimed at identifying the complete array of functions of SET1/MLL regulatory networks in a developmental context. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RBBP5, ASH2, WDR5, DPY30 and CFP1. The biochemical composition of the complex however, has not been described. Through the use of co-immunoprecipitation coupled to mass spectrometry-based proteomics, I identified the SET-2/SET1 complex in C. elegans embryos. Most importantly, I showed that the SET-2/SET1 complex also co-immunoprecipitates another conserved chromatin-modifying complex and I highlighted the interactions involved between these two complexes. My genetic analysis revealed that loss of function mutants of the two complex subunits share common phenotypes, consistent with common developmental functions. The laboratory has also undertaken transcriptomic and chromatin immunoprecipitation experiments showing that CFP-1 has a role in the binding of this complex at specific chromatin regions

Expression of milk proteins including β-lactoglobulin is controlled by prolactin activation of the transcription factor Stat5 via the Janus kinase/Signal transducer and activator of transcription (Jak/STAT) pathway. Stat5 has previously been shown to tetramerise where binding sites are tandemly linked and the proximity of these binding sites appears to be important for these interactions. This work and previous large scale mapping of the β-lactoglobulin promoter shows that the dyad of a strongly positioned nucleosome lies at -184 bp from the transcription start on the promoter of the β-lactoglobulin gene. This brings together two binding sites for Stat5, at the points of entry and exit of DNA from the nucleosome that would otherwise be spaced 185 bp apart, an arrangement that could potentially bring bound Stat5 dimers closer enough to facilitate tetramerisation. The chromatin structure over the active and inactive gene promoter is different; there are two alternative nucleosome positions in the active and only one in the inactive promoter. One of these positioning sites would not allow the tetramerisation interaction to take place. In order to understand better the mechanisms by which the expression of β-lactoglobulin is regulated by Stat5 we set out to investigate the role of these positioned nucleosomes in Stat5 binding in vitro. Stat5A and B binding patterns on both naked DNA and on reconstituted chromatin probes are shown by a series of bandshift experiments using purified recombinant Stat5 produced in a baculovirus expression system. Characterisation of Stat5 reveals the protein to be phosphorylated and able to bind DNA. A mutation, W37A, which removes the ability of Stat5 to form dimer-dimer interactions was employed to further investigate a potential role of tetramerisation influencing Stat5 binding in a chromatin context. This architectural feature could act to control the temporal and tissue specific expression of β-lactoglobulin.

Eukaryotic genomes are typically organized as chromatin, the complex of DNA and proteins that forms chromosomes within the cell\\\'s nucleus. Chromatin has pivotal roles for a multitude of functions, most of which are carried out by a complex system of covalent chemical modifications of histone proteins. The propagation of patterns of these histone post-translational modifications across cell divisions is particularly important for maintenance of the cell state in general and the transcriptional program in particular. The discovery of epigenetic inheritance phenomena - mitotically and/or meiotically heritable changes in gene function resulting from changes in a chromosome without alterations in the DNA sequence - was remarkable because it disproved the assumption that information is passed to daughter cells exclusively through DNA. However, DNA replication constitutes a dramatic disruption of the chromatin state that effectively amounts to partial erasure of stored information. To preserve its epigenetic state the cell reconstructs (at least part of) the histone post-translational modifications by means of processes that are still very poorly understood. A plausible hypothesis is that the different combinations of reader and writer domains in histone-modifying enzymes implement local rewriting rules that are capable of \\\"recomputing\\\" the desired parental patterns of histone post-translational modifications on the basis of the partial information contained in that half of the nucleosomes that predate replication. It is becoming increasingly clear that both information processing and computation are omnipresent and of fundamental importance in many fields of the natural sciences and the cell in particular. The latter is exemplified by the increasingly popular research areas that focus on computing with DNA and membranes. Recent work suggests that during evolution, chromatin has been converted into a powerful cellular memory device capable of storing and processing large amounts of information. Eukaryotic chromatin may therefore also act as a cellular computational device capable of performing actual computations in a biological context. A recent theoretical study indeed demonstrated that even relatively simple models of chromatin computation are computationally universal and hence conceptually more powerful than gene regulatory networks. In the first part of this thesis, I establish a deeper understanding of the computational capacities and limits of chromatin, which have remained largely unexplored. I analyze selected biological building blocks of the chromatin computer and compare it to system components of general purpose computers, particularly focusing on memory and the logical and arithmetical operations. I argue that it has a massively parallel architecture, a set of read-write rules that operate non-deterministically on chromatin, the capability of self-modification, and more generally striking analogies to amorphous computing. I therefore propose a cellular automata-like 1-D string as its computational paradigm on which sets of local rewriting rules are applied asynchronously with time-dependent probabilities. Its mode of operation is therefore conceptually similar to well-known concepts from the complex systems theory. Furthermore, the chromatin computer provides volatile memory with a massive information content that can be exploited by the cell. I estimate that its memory size lies in the realms of several hundred megabytes of writable information per cell, a value that I compare with DNA itself and cis-regulatory modules. I furthermore show that it has the potential to not only perform computations in a biological context but also in a strict informatics sense. At least theoretically it may therefore be used to calculate any computable function or algorithm more generally. Chromatin is therefore another representative of the growing number of non-standard computing examples. As an example for a biological challenge that may be solved by the \\\"chromatin computer\\\", I formulate epigenetic inheritance as a computational problem and develop a flexible stochastic simulation system for the study of recomputation-based epigenetic inheritance of individual histone post-translational modifications. The implementation uses Gillespie\\\'s stochastic simulation algorithm for exactly simulating the time evolution of the chemical master equation of the underlying stochastic process. Furthermore, it is efficient enough to use an evolutionary algorithm to find a system of enzymes that can stably maintain a particular chromatin state across multiple cell divisions. I find that it is easy to evolve such a system of enzymes even without explicit boundary elements separating differentially modified chromatin domains. However, the success of this task depends on several previously unanticipated factors such as the length of the initial state, the specific pattern that should be maintained, the time between replications, and various chemical parameters. All these factors also influence the accumulation of errors in the wake of cell divisions. Chromatin-regulatory processes and epigenetic (inheritance) mechanisms constitute an intricate and sensitive system, and any misregulation may contribute significantly to various diseases such as Alzheimer\\\'s disease. Intriguingly, the role of epigenetics and chromatin-based processes as well as non-coding RNAs in the etiology of Alzheimer\\\'s disease is increasingly being recognized. In the second part of this thesis, I explicitly and systematically address the two hypotheses that (i) a dysregulated chromatin computer plays important roles in Alzheimer\\\'s disease and (ii) Alzheimer\\\'s disease may be considered as an evolutionarily young disease. In summary, I found support for both hypotheses although for hypothesis 1, it is very difficult to establish causalities due to the complexity of the disease. However, I identify numerous chromatin-associated, differentially expressed loci for histone proteins, chromatin-modifying enzymes or integral parts thereof, non-coding RNAs with guiding functions for chromatin-modifying complexes, and proteins that directly or indirectly influence epigenetic stability (e.g., by altering cell cycle regulation and therefore potentially also the stability of epigenetic states). %Notably, we generally observed enrichment of probes located in non-coding regions, particularly antisense to known annotations (e.g., introns). For the identification of differentially expressed loci in Alzheimer\\\'s disease, I use a custom expression microarray that was constructed with a novel bioinformatics pipeline. Despite the emergence of more advanced high-throughput methods such as RNA-seq, microarrays still offer some advantages and will remain a useful and accurate tool for transcriptome profiling and expression studies. However, it is non-trivial to establish an appropriate probe design strategy for custom expression microarrays because alternative splicing and transcription from non-coding regions are much more pervasive than previously appreciated. To obtain an accurate and complete expression atlas of genomic loci of interest in the post-ENCODE era, this additional transcriptional complexity must be considered during microarray design and requires well-considered probe design strategies that are often neglected. This encompasses, for example, adequate preparation of a set of target sequences and accurate estimation of probe specificity. With the help of this pipeline, two custom-tailored microarrays have been constructed that include a comprehensive collection of non-coding RNAs. Additionally, a user-friendly web server has been set up that makes the developed pipeline publicly available for other researchers
Eukaryotische Genome sind typischerweise in Form von Chromatin organisiert, dem Komplex aus DNA und Proteinen, aus dem die Chromosomen im Zellkern bestehen. Chromatin hat lebenswichtige Funktionen in einer Vielzahl von Prozessen, von denen die meisten durch ein komplexes System von kovalenten Modifikationen an Histon-Proteinen ablaufen. Muster dieser Modifikationen sind wichtige Informationsträger, deren Weitergabe über die Zellteilung hinaus an beide Tochterzellen besonders wichtig für die Aufrechterhaltung des Zellzustandes im Allgemeinen und des Transkriptionsprogrammes im Speziellen ist. Die Entdeckung von epigenetischen Vererbungsphänomenen - mitotisch und/oder meiotisch vererbbare Veränderungen von Genfunktionen, hervorgerufen durch Veränderungen an Chromosomen, die nicht auf Modifikationen der DNA-Sequenz zurückzuführen sind - war bemerkenswert, weil es die Hypothese widerlegt hat, dass Informationen an Tochterzellen ausschließlich durch DNA übertragen werden. Die Replikation der DNA erzeugt eine dramatische Störung des Chromatinzustandes, welche letztendlich ein partielles Löschen der gespeicherten Informationen zur Folge hat. Um den epigenetischen Zustand zu erhalten, muss die Zelle Teile der parentalen Muster der Histonmodifikationen durch Prozesse rekonstruieren, die noch immer sehr wenig verstanden sind. Eine plausible Hypothese postuliert, dass die verschiedenen Kombinationen der Lese- und Schreibdomänen innerhalb von Histon-modifizierenden Enzymen lokale Umschreibregeln implementieren, die letztendlich das parentale Modifikationsmuster der Histone neu errechnen. Dies geschieht auf Basis der partiellen Informationen, die in der Hälfte der vererbten Histone gespeichert sind. Es wird zunehmend klarer, dass sowohl Informationsverarbeitung als auch computerähnliche Berechnungen omnipräsent und in vielen Bereichen der Naturwissenschaften von fundamentaler Bedeutung sind, insbesondere in der Zelle. Dies wird exemplarisch durch die zunehmend populärer werdenden Forschungsbereiche belegt, die sich auf computerähnliche Berechnungen mithilfe von DNA und Membranen konzentrieren. Jüngste Forschungen suggerieren, dass sich Chromatin während der Evolution in eine mächtige zelluläre Speichereinheit entwickelt hat und in der Lage ist, eine große Menge an Informationen zu speichern und zu prozessieren. Eukaryotisches Chromatin könnte also als ein zellulärer Computer agieren, der in der Lage ist, computerähnliche Berechnungen in einem biologischen Kontext auszuführen. Eine theoretische Studie hat kürzlich demonstriert, dass bereits relativ simple Modelle eines Chromatincomputers berechnungsuniversell und damit mächtiger als reine genregulatorische Netzwerke sind. Im ersten Teil meiner Dissertation stelle ich ein tieferes Verständnis des Leistungsvermögens und der Beschränkungen des Chromatincomputers her, welche bisher größtenteils unerforscht waren. Ich analysiere ausgewählte Grundbestandteile des Chromatincomputers und vergleiche sie mit den Komponenten eines klassischen Computers, mit besonderem Fokus auf Speicher sowie logische und arithmetische Operationen. Ich argumentiere, dass Chromatin eine massiv parallele Architektur, eine Menge von Lese-Schreib-Regeln, die nicht-deterministisch auf Chromatin operieren, die Fähigkeit zur Selbstmodifikation, und allgemeine verblüffende Ähnlichkeiten mit amorphen Berechnungsmodellen besitzt. Ich schlage deswegen eine Zellularautomaten-ähnliche eindimensionale Kette als Berechnungsparadigma vor, auf dem lokale Lese-Schreib-Regeln auf asynchrone Weise mit zeitabhängigen Wahrscheinlichkeiten ausgeführt werden. Seine Wirkungsweise ist demzufolge konzeptionell ähnlich zu den wohlbekannten Theorien von komplexen Systemen. Zudem hat der Chromatincomputer volatilen Speicher mit einem massiven Informationsgehalt, der von der Zelle benutzt werden kann. Ich schätze ab, dass die Speicherkapazität im Bereich von mehreren Hundert Megabytes von schreibbarer Information pro Zelle liegt, was ich zudem mit DNA und cis-regulatorischen Modulen vergleiche. Ich zeige weiterhin, dass ein Chromatincomputer nicht nur Berechnungen in einem biologischen Kontext ausführen kann, sondern auch in einem strikt informatischen Sinn. Zumindest theoretisch kann er deswegen für jede berechenbare Funktion benutzt werden. Chromatin ist demzufolge ein weiteres Beispiel für die steigende Anzahl von unkonventionellen Berechnungsmodellen. Als Beispiel für eine biologische Herausforderung, die vom Chromatincomputer gelöst werden kann, formuliere ich die epigenetische Vererbung als rechnergestütztes Problem. Ich entwickle ein flexibles Simulationssystem zur Untersuchung der epigenetische Vererbung von individuellen Histonmodifikationen, welches auf der Neuberechnung der partiell verlorengegangenen Informationen der Histonmodifikationen beruht. Die Implementierung benutzt Gillespies stochastischen Simulationsalgorithmus, um die chemische Mastergleichung der zugrundeliegenden stochastischen Prozesse über die Zeit auf exakte Art und Weise zu modellieren. Der Algorithmus ist zudem effizient genug, um in einen evolutionären Algorithmus eingebettet zu werden. Diese Kombination erlaubt es ein System von Enzymen zu finden, dass einen bestimmten Chromatinstatus über mehrere Zellteilungen hinweg stabil vererben kann. Dabei habe ich festgestellt, dass es relativ einfach ist, ein solches System von Enzymen zu evolvieren, auch ohne explizite Einbindung von Randelementen zur Separierung differentiell modifizierter Chromatindomänen. Dennoch ängt der Erfolg dieser Aufgabe von mehreren bisher unbeachteten Faktoren ab, wie zum Beispiel der Länge der Domäne, dem bestimmten zu vererbenden Muster, der Zeit zwischen Replikationen sowie verschiedenen chemischen Parametern. Alle diese Faktoren beeinflussen die Anhäufung von Fehlern als Folge von Zellteilungen. Chromatin-regulatorische Prozesse und epigenetische Vererbungsmechanismen stellen ein komplexes und sensitives System dar und jede Fehlregulation kann bedeutend zu verschiedenen Krankheiten, wie zum Beispiel der Alzheimerschen Krankheit, beitragen. In der Ätiologie der Alzheimerschen Krankheit wird die Bedeutung von epigenetischen und Chromatin-basierten Prozessen sowie nicht-kodierenden RNAs zunehmend erkannt. Im zweiten Teil der Dissertation adressiere ich explizit und auf systematische Art und Weise die zwei Hypothesen, dass (i) ein fehlregulierter Chromatincomputer eine wichtige Rolle in der Alzheimerschen Krankheit spielt und (ii) die Alzheimersche Krankheit eine evolutionär junge Krankheit darstellt. Zusammenfassend finde ich Belege für beide Hypothesen, obwohl es für erstere schwierig ist, aufgrund der Komplexität der Krankheit Kausalitäten zu etablieren. Dennoch identifiziere ich zahlreiche differentiell exprimierte, Chromatin-assoziierte Bereiche, wie zum Beispiel Histone, Chromatin-modifizierende Enzyme oder deren integrale Bestandteile, nicht-kodierende RNAs mit Führungsfunktionen für Chromatin-modifizierende Komplexe oder Proteine, die direkt oder indirekt epigenetische Stabilität durch veränderte Zellzyklus-Regulation beeinflussen. Zur Identifikation von differentiell exprimierten Bereichen in der Alzheimerschen Krankheit benutze ich einen maßgeschneiderten Expressions-Microarray, der mit Hilfe einer neuartigen Bioinformatik-Pipeline erstellt wurde. Trotz des Aufkommens von weiter fortgeschrittenen Hochdurchsatzmethoden, wie zum Beispiel RNA-seq, haben Microarrays immer noch einige Vorteile und werden ein nützliches und akkurates Werkzeug für Expressionsstudien und Transkriptom-Profiling bleiben. Es ist jedoch nicht trivial eine geeignete Strategie für das Sondendesign von maßgeschneiderten Expressions-Microarrays zu finden, weil alternatives Spleißen und Transkription von nicht-kodierenden Bereichen viel verbreiteter sind als ursprünglich angenommen. Um ein akkurates und vollständiges Bild der Expression von genomischen Bereichen in der Zeit nach dem ENCODE-Projekt zu bekommen, muss diese zusätzliche transkriptionelle Komplexität schon während des Designs eines Microarrays berücksichtigt werden und erfordert daher wohlüberlegte und oft ignorierte Strategien für das Sondendesign. Dies umfasst zum Beispiel eine adäquate Vorbereitung der Zielsequenzen und eine genaue Abschätzung der Sondenspezifität. Mit Hilfe der Pipeline wurden zwei maßgeschneiderte Expressions-Microarrays produziert, die beide eine umfangreiche Sammlung von nicht-kodierenden RNAs beinhalten. Zusätzlich wurde ein nutzerfreundlicher Webserver programmiert, der die entwickelte Pipeline für jeden öffentlich zur Verfügung stellt

Chez les eucaryotes, l’ADN s’enroule autour de protéines appelées histones pour former la chromatine. Cette structure permet, d’une part, de compacter le génome dans le noyau, mais également de réguler son expression. En effet, les histones sont porteuses d’information dite « épigénétique » : elles existent sous différentes formes, les variants d’histone, et peuvent porter des modifications post-traductionnelles. La présence de tels variants et modifications organise le génome en domaines au statut transcriptionnel différent.La réplication de l’ADN déstabilise la structure chromatinienne, et représente ainsi un défi pour la cellule qui doit à la fois dupliquer son matériel génétique et transmettre son paysage épigénétique pour garantir le maintien de son identité. Ceci passe par le recyclage des histones parentales, processus essentiel pour transmettre de manière fidèle les variants d’histone et leurs modifications.Au cours de ma thèse, j’ai tenté de répondre à la question : comment les variants d’histone H3.1 et H3.3 sont-ils recyclés au cours de la réplication de l’ADN ? En particulier, je me suis intéressée au rôle du chaperon d’histone Anti-Silencing Function 1 (ASF1) dans ce processus.Mon approche a été de développer une technique de microscopie de super-résolution (STORM) pour visualiser les variants d’histone parentaux précisément aux sites de réplication. Grâce à cette technologie, j’ai pu étudier l’impact de la déplétion d’ASF1 sur le recyclage des histones parentales, apportant ainsi des éléments de compréhension sur les mécanismes fondamentaux qui transmettent l’information épigénétique
In eukaryotes, DNA wraps around proteins called histones to form chromatin. This structure allows, first, the compaction of the genome in the nucleus, but also the regulation of its expression. Indeed, histones can be a source of information referred to as “epigenetic”: they exist under different forms, histone variants, and can have post-translational modifications. The presence of these variants and modifications organizes the genome into domains with different transcriptional status.DNA replication destabilizes chromatin structure and, therefore, represents a challenge for the cell, which must duplicate its genetic material while also transmitting its epigenetic landscape in order to maintain its identity. In this context, recycling parental histones is essential to faithfully transmit histone variants and their modifications.During my PhD, I tried to address the question: how are the histone variants H3.1 and H3.3 recycled during DNA replication? In particular, I investigated the role of the histone chaperone Anti-Silencing Function 1 (ASF1) in this process.My approach was to develop a super-resolution microscopy technique (STORM) to visualize parental histone variants precisely at replication sites. Using this technology, I could study the impact of ASF1 depletion on the recycling of parental histones, and further our understanding of fundamental mechanisms that transmit epigenetic information

In the nucleus of eukaryotic cells, DNA wraps around histone proteins to form nucleosomes, which in turn associate in a compact and dynamic fiber called chromatin. The physical properties of this fiber at different lengthscales, from the DNA double-helix to micrometer-sized chromosomes, are essential to the complex mechanisms of gene expression and its regulation. The present thesis is a contribution to the development of physical models, which are able to link different scales and to interpret and integrate data from a wide range of experimental and computational approaches. In the first part, we use Molecular Dynamics simulations of DNA oligomers to study doublehelical DNA at different temperatures. We estimate the sequence-dependent contribution of entropy to DNA elasticity, in relation with recent experiments on DNA persistence length. In the second part, we model the DNA-histone interactions within the nucleosome core particle,using DNA nanomechanics to extract a force field from a set of crystallographic nucleosome structures and Molecular Dynamics snapshots. In the third part, we consider the softer part of the nucleosome, the linker DNA between coreparticles which transiently associates with the histone H1 to form a "stem".We combine existing structural knowledge with experimental data at two different resolutions (DNA footprints and electro-micrographs) to develop a nanoscale model of the stem.

Comment le zygote acquiert la totipotence à partir de deux cellules complètement différenciées, et comment les décisions du destin cellulaire sont faites plus tard dans le développement sont des questions biologiques essentielles. Les études menées au cours de la première partie de mon doctorat ont contribué à l'annotation de la composition de la chromatine embryonnaire en ce qui concerne les variantes des histones et des modifications post-traductionnelles. L'expression ectopique de H2A.Z après la fécondation réduit la progression du développement, ce qui suggère que l'absence de H2A.Z au début du développement pourrait être importante pour l'organisation de la chromatine embryonnaire nouvellement formée. Deuxièmement, j'ai étudié la dynamique des histones dans l'embryon de souris en développement. La reprogrammation épigénétique après la fécondation est accompagnée par une étonnante forte mobilité des histones dans le noyau. Ma thèse a contribué à la compréhension des événements dynamiques affectant la chromatine embryonnaire pendant le remodelage épigénétique après la fécondation
How the zygote acquires totipotency from two differentiated cells, and how cell fate decisions are made later in development is a pivotal biological question. The studies conducted during the first part of my doctorate contributed to the annotation of embryonic chromatin composition with regards to histone variants and PTMs, and more specifically those correlated with active chromatin regions. The histone variant H2A.Z was shown to be present on embryonic chromatin in a stage-specific manner. Ectopic expression of H2A.Z after fertilization reduced developmental progression, suggesting that absence of H2A.Z at the onset of development might be important for the organization of the newly formed embryonic chromatin. Secondly, I investigated histone dynamics in the developing mouse embryo. Our work represents the first report on histone mobility during early mouse embryogenesis. My thesis contributed to the understanding of the dynamic events affecting embryonic chromatin during epigenetic remodeling after fertilization

L’héritage épigénétique transgénérationnelle est un phénomène très controversé, selon lequel un phénotype non-génétiquement déterminé peut être transmis à la génération suivante. Jusqu'à présent, ce mode de transmission a été décrit dans quelques cas et il a été suggéré que les composants de la chromatine peuvent être impliqués, y compris des protéines du groupe Polycomb, qui agissent comme des répresseurs de gènes clés du développement et coordonnent la différenciation cellulaire et la prolifération. Les mécanismes moléculaires à la base du rôle de la répression génique Polycomb-dépendante à hérédité épigénétique transgénérationnelle sont loin d'être compris. Par conséquent, j’ai développé un système expérimental chez Drosophila melanogaster pour induire un héritage épigénétique transgénérationnelle stable, dans lequel des états d'expression génique alternatifs peuvent être transmis en présence de la même séquence d'ADN. A partir de ces « épilignes » stables, j’ai pu disséquer certaines des propriétés génétiques des épiallèles induits, tels que leur héritage quantitatif et leur capacité à communiquer à longue distance. En outre, les épiallèles montrent une synergie dans leur expression et transmission héréditaire. L'une des signatures moléculaires des épiallèles est une différence de répression médiée par les complexes Polycomb et par leur marque d’histone caractéristique. Cette distribution différente est indépendante de l’activité transcriptionnelles des gènes en aval, au moins dans un stade de développement précoce, et pourrait influer l'organisation tridimensionnelle du locus impliqué. Curieusement Ago2, un composant de la voie ARNi, a été montré interagir avec les épiallèles génétiquement et la protéine Ago2 se fixe directement à leur chromatine, ce qui indique un rôle possible pour le ncRNAs dans l'expression des épiallèles et éventuellement dans leur transmission. Ces résultats plaident en faveur e l’existence d’une hérédité épigénétique transgénérationnelle stable chez les métazoaires et fournissent un modèle qui se prête à une dissection moléculaire de ce phénomène
Transgenerational epigenetic inheritance is a hotly debated phenomenon whereby a non-genetically determined phenotype can be transmitted to the next generation. So far, this mode of inheritance has been described in few cases and it was suggested that chromatin components might be involved, including Polycomb group proteins, which act as repressors of key developmental genes and coordinate cell differentiation and proliferation. The molecular mechanisms linking Polycomb-mediated silencing to transgenerational epigenetic inheritance are far from being understood. Therefore, I developed an experimental system in Drosophila melanogaster to induce stable transgenerational epigenetic inheritance, in which alternative gene expression states can be transmitted in the presence of the same DNA sequence. Starting from these highly stable “epilines”, I could dissect some of the genetic properties of the induced epialleles, such as their quantitative inheritance and their ability to trans-communicate. Moreover, the epialleles displayed synergy in their expression and transmission. One of the molecular signatures of the epialleles is the differential presence of the Polycomb repressive complexes and their related epigenetic marks. This different distribution is independent of the transcriptional activity of the downstream genes, at least in an early developmental stage, and could influence the three-dimensional organization of the locus involved. Intriguingly Ago2, an RNAi pathway component, has been found to genetically interact with the epialleles and to be directly bound on their chromatin, indicating a possible role for the ncRNAs in the expression of the epialleles and possibly in their transmission. These results make a case for strong and stable transgenerational epigenetic inheritance in metazoan and provide a model that is amenable for the molecular dissection of this phenomenon

Chaque division cellulaire requiert une duplication précise du génome. Des dizaines de milliers de sites d’initiation de la réplication d’ADN (origines de réplication) sont impliqués dans la réplication complète du génome humain. L’activation des origines de réplication est régulée précisément et des études génomiques extensives ont démontré la présence de caractéristiques génomiques associées à l’activation des origines de réplication. Le complexe de pré-réplication (pre-RC) est la base de l’initiation de la réplication et consiste en deux sous-complexes majeurs : l’ « origin recognition complex » (ORC) qui interagit directement avec l'ADN et est nécessaire pour recruter le second sous-complexe, les hélicases Mcm2 7, qui sont responsables de l'initiation de la réplication. La régulation de l’assemblage du pre-RC est bien étudiée, mais les caractéristiques de la chromatine qui déterminent le positionnement du pre-RC sur le génome restent peu connues. Les études génomiques par immuno-précipitation de la chromatine et séquençage à haut débit (ChIP-seq) des pre-RCs sont rares et jusqu’à aujourd’hui seulement disponibles pour ORC. Du fait que Mcm2-7 migre de son site de chargement initial, il est crucial d'obtenir des informations sur le positionnement des Mcm2-7 pour la compréhension complète de la régulation de la réplication. Ce travail présente la première analyse génomique par méthode ChIP-seq des deux sous-unités majeures du pre-RC, ORC et Mcm2-7, dans la lignée cellulaire de lymphome de Burkitt Raji infectée par le virus d’Epstein-Barr (EBV). La présence du génome d’EBV permet d'avoir un contrôle interne de la qualité de nos expériences, en comparant les positions de pre-RC déterminées avec des positions du pre-RC précédemment publiées. Sur le génome humain, les résultats de séquençage du pre-RC corrèlent bien avec des zones de réplication active. De façon intéressante, les zones de terminaison de la réplication étaient spécifiquement bas en pre-RC, spécialement en Mcm2-7. La localisation des sites d'initiation de la réplication identifiés est généralement bien corrélée avec les sites de transcription active. En effet, des sites d’assemblage du pre-RC de haute affinité sont localisés préférentiellement en voisinage de sites de transcription active, ce qui est possiblement dû à l’accessibilité de la chromatine dans ces régions. La fixation de Mcm2-7 fluctue de façon dépendante du cycle cellulaire, ce qui suggère des translocations de Mcm2 7 en G1, probablement dépendantes de la machinerie active de la transcription. Ces résultats indiquent que les positions de ORC et Mcm2-7 sont principalement dépendantes de l’accessibilité de la chromatine avec un accès privilégié dans la chromatine active et Mcm2 7 étant le déterminant majeur de l’initiation de la réplication. Au sein de l'hétérochromatine, ORC est enrichi dans des zones associées avec l'histone modifié H4K20me3. Cependant, cet enrichissement est moins important pour les Mcm2-7. En utilisant un système de réplication basé sur des plasmides, nous avons démontré que l’association d'ORC et H4K20me3 favorise l’assemblage du pre-RC et l’initiation de la réplication. Cette observation suggère que l’interaction ORC-chromatine est le déterminant majeur de la régulation de la réplication d’ADN au sein de l’hétérochromatine. En conclusion, cette étude propose deux mécanismes différents de la régulation de l'assemblage du pre-RC dépendants de l’environnement de la chromatine
With every cell division, the genome needs to be faithfully duplicated. Tens of thousands of DNA replication initiation sites (origins of replication) are involved in replicating the human genome. Origin activation is precisely regulated and extensive genome-wide studies found association of origin activation to several different genomic features. The pre-replication complex (pre RC) is the basis for replication initiation and consists of two major subcomponents: the origin recognition complex (ORC) binds DNA and is required for loading of the second component, Mcm2-7 helicases, which initiate DNA replication. Regulation of pre-RC assembly is well studied, however, chromatin features driving pre RC positioning on the human genome remain largely unknown. Genome-wide pre-RC chromatin immunoprecipitation experiments followed by sequencing (ChIP-seq) studies are rare and so far only performed for ORC. As Mcm2-7 can translocate from their initial loading site, information about Mcm2-7 positioning are required for full understanding of DNA replication regulation.This work presents the first genome-wide ChIP-seq analysis of the two major pre-RC subcomponents ORC and Mcm2-7 in the Epstein-Barr virus (EBV) infected Burkitt’s lymphoma cell line Raji. Successful ChIPs were validated on the EBV genome by comparing obtained pre RC positions with already existing pre-RC ChIP-on chip data. On the human genome, pre-RC sequencing results nicely correlated with zones of active replication. Interestingly, zones of replication termination were specifically depleted from pre-RC components, especially from Mcm2 7. Active DNA replication is known to correlate with active transcription. Indeed, strong pre-RC assembly preferentially occurred at sites of active transcriptional regulation, presumably determined by chromatin accessibility. Strong Mcm2-7 binding thereby fluctuated cell cycle-dependently, arguing for Mcm2-7 translocations during G1, possibly depending on the active transcriptional machinery. These results indicate ORC and Mcm2-7 positions being mainly dependent on chromatin accessibility in active chromatin, with Mcm2-7 being the major determinant of replication initiation. In heterochromatin, ORC was enriched at H4K20me3 sites, while Mcm2-7 enrichment was less prominent. Employing a plasmid-based replication system, ORC association to H4K20me3 was proven to promote successful pre-RC assembly and replication initiation, situating direct ORC-chromatin interactions being the major determinant for DNA replication regulation in heterochromatin. Taken together, this study proposes two different modes of pre-RC assembly regulation depending on chromatin environment

Les plantes sont caractérisées par une remarquable plasticité développementale. En vertu de leur mode de vie sessile, elles sont capables d'adaptations phénotypiques rapides en réponse à des signaux environnementaux. En particulier, les plantes ont la capacité de détecter les conditions de lumière par de multiples photorécepteurs et utilisent cette information pour adapter leur morphologie et leur physiologie à un environnement changeant. Par exemple, la première perception de la lumière par des jeunes plantules, émergeant du sol, induit des changements profonds dans l'expression des gènes qui lancent la croissance et l'activité photosynthétique. Au cours de cette transition, la reprogrammation de l'expression du génome s'accompagne de réorganisations massives de l'organisation de la chromatine dans la grande majorité des cellules des feuilles embryonnaires, les cotylédons. Ainsi, chez la plante Arabidopsis thaliana, le dé-étiolement des cotylédons est associé à une condensation rapide des principaux domaines hétérochromatiniens en 8 à 10 "chromocentres" qui se forment autour des centromères. L’étude des voies de signalisation contrôlant ce processus nous a conduit à l'identification d’un acteur moléculaire clé pour la dynamique d'organisation des chromocentres en réponse à la lumière, les histones H1. Ces histones de liaison inter-nucléosomiques sont des composants structurels conservés qui contribuent à réguler l'organisation et la condensation locale et a grande échelle de la chromatine en limitant l'accessibilité à l'ADN pour de nombreux facteurs telles que les ARN polymérases. Cette thèse porte sur la caractérisation des réarrangements chromatiniens médiés par des variants d'histone H1. Des approches cytologiques et génomiques ont permis d’appréhender l'influence des trois différents variants H1 d'Arabidopsis thaliana dans la définition des structures 3D du génome et de la chromatine des cellules de cotylédons. La combinaison de tests d’accessibilité à la transposase Tn5 (ATAC) et de capture de conformation chromosomique (Hi-C) permet également de disséquer comment les histone H1 impactent la topologie du génome et l'adaptation du paysage chromatinien pour un nouveau programme de transcription. L'analyse de l'abondance et le profilage par immuno-précipitation quantitative de marques d'histones (ChIP-Rx) a également permis d'identifier le rôle joué par les histones H1 sur le paysage chromatinien répressif ainsi que son impact fonctionnel sur de nombreux gènes et éléments répétés du génome, potentiellement en restreignant l'accès à des facteurs de transcription sur des motifs de séquence spécifiques. Collectivement, ce travail a permis de disséquer les spécificités et les redondances fonctionnelles des variants d'histones de liaison en tant que régulateurs moléculaires du paysage chromatinien chez les plantes
Being capable of rapid phenotypic adaptations in response to environmental cues, plants are characterized by a remarkable developmental plasticity. Specifically, plants have the ability to sense light conditions by multiple photosensory receptors and to use this crucial information to adapt their morphology and physiology to a changing environment. For example, the first perception of light by young plantlets emerging from the soil induces deep changes in gene expression that launch growth and photosynthetic activity. During this transition, genome expression reprogramming is accompanied by massive rearrangements of chromatin sub-nuclear organization. In the Arabidopsis thaliana plant species, a large part of heterochromatin containing silent and condensed repeated elements is scattered within multiple foci in the nucleoplasm of most cotyledon cells when grown in darkness. Upon exposure to light, cotyledon de-etiolation triggers the rapid condensation of heterochromatic domains into 8-to-10 large chromocenters that form around centromeres. This phenomenology has led us to the identification of histone H1 variants as key molecular players in triggering chromocenter dynamics. These inter-nucleosomal linker histones are conserved structural components of eukaryotic chromatin that contribute to both local and higher-order chromatin organization and condensation, notably restricting DNA accessibility to multiple factors such as RNA polymerases. In this thesis cytological and genomic approaches were used to investigate the influence of the three Arabidopsis thaliana H1 variants in the definition of the genome and the 3D chromatin structure in cotyledon cells. The combination of Assay for Transposase-Accessible Chromatin (ATAC) and Chromosome Conformation Capture (Hi-C) allowed dissecting how H1 histones impact genome topology and the adaptation of the chromatin landscape for a new transcriptional program. The analysis of histone marks abundance and their genome-wide profiling using quantitative chromatin immunoprecipitation (ChIP-Rx) further enhances current knowledge. We uncovered the functional impact of histones H1 in defining chromatin repressive landscape on many genes and repeated elements, potentially by restricting access to transcription factors on specific sequence motifs. Collectively, this work has allowed deciphering the specific and redundant functional implications of histone H1 variants as key molecular regulators of the chromatin landscape in plants

Chromatin compaction is regulated by different factors, among them histone posttranslational modifications. There are different histone modifications, and among them, acetylation and methylation of lysine residues. Acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), enzymes that catalyze addition and removal, respectively, of acetyl groups from histone lysine residues. Among the four classes of HDACs, the class III which correspond to the members of the Sir2 family or Sirtuins are quite unique. They participate in the response to a wide variety of stress stimuli through their requirement of NAD+ as a cofactor in their enzymatic activity. Mammals harbor seven Sirtuins (SirT1-SirT7). Among them, SirT6 is a nuclear protein involved in both genomic stability and metabolic homeostasis. Interestingly, SirT6 regulates the majority of these processes through an effect on chromatin, based on deacetylation of a histone mark, H3K9ac. However, the mechanism involved has not been fully characterized. In this work, we aim to study the consequences of SirT6 function in chromatin organization. We show that SirT6 overexpression induces gene silencing, which fits with the role of SirT6 as a repressor, shown by previous reports. Our studies show that SirT6 interacts with proteins or multi-protein complexes also involved in gene silencing, such as components of NuRD complex, or the HMTs EZH2, Suv39h1. However, we have focused the project in understanding the functional relationship between SirT6 and a H3K9-specific methyltransferases that we have identified as Suv39h1 and G9a. Suv39h1 trimethylates H3K9 and is essential in the establishment and maintenance of pericentromeric and telomeric constitutive heterochromatin. Interestingly, Suv39h1 was previously found to interact with SirT1 in the context of constitutive and facultative heterochromatin formation. Our work shows that the functional relationship between Suv39h1 and SirT6 is quite different from the already described between SirT1 and Suv39h1. SirT6 mediates a non canonical monoubiquitination in Suv39h1 in three conserved cysteines of the pre-SET domain. We have also identified SKP2 as the E3 ubiquitin ligase responsible of this monoubiquitination. SKP2 has a well-known role in promoting poliubiquitination and degradation of protein involved in G1/S checkpoint such as p21 and p27 and its levels are regulated during cell cycle progression. Our data show that SKP2 levels are also regulated by SirT6 through deacetylation, which in turn induce a double phosphorylation that prevents its degradation. Suv39h1 monoubiquitination is induced by double thymidine block and nocodazole treatments that arrest cells in G1/S phase and early mitosis, respectively. Furthermore, Suv39h1 monoubiquitination is induced by NF-kB pathway activation such as TNFa treatment, the overexpression of the NF-kB transcription factor RelA, or of the activator IKKa. Moreover, the promoter of the NF-kB global repressor, IkBa, is regulated by the interplay between Suv39h1, SKP2 and SirT6. Thus, upon activation of the pathway by TNFa treatment, SirT6 induces the Suv39h1 monoubiquitination through SKP2 activation and monoubiquitinated Suv39h1 is removed from the promoter allowing the transcription activation of IkBa by RelA. This novel model provides not only new insights in the regulation of NFkB pathway but also unveils new roles for SirT6, SKP2 and Suv39h1.
La compactación de la cromatina es regulada por diferentes factores entre los cuales destacan las modificaciones post-traduccionales de las histonas. Hay diferentes modificaciones de histonas entre ellas las desacetilationes y las metilaciones. Los niveles de acetilación están regulados por las histonas acetiltransferasas (HATs) y las histonas deacetilasas (HDACs), proteínas que añaden o quitan grupos acetil a las lisinas de las histonas, respectivamente. Los miembros de la familia Sir2 o de las sirtuínas constituyen la clase III de las HDACs y participan en respuesta a muchas formas de estrés. Entre ellas, está SirT6 que desacetila H3K9 acetilado para promover silenciamiento. En este proyecto mostramos que la sobreexpresión de SirT6 produce silenciamiento génico tal y como se había descrito previamente. Además, SirT6 interacciona con proteínas involucradas en silenciamiento génico como es el complejo NuRD y EZH2 y la metiltransferases de H3K9, G9a y Suv39h1. Suv39h1 trimetila H3K9 necesario para la estructura de la heterocromatina. Está caracterizada la interacción entre Suv39h1 y SirT1 en el contexto de formación de la formación de heterocromatina. La relación entre Suv39h1 y SirT6 es bastante diferente a la de Suv39h1 y SirT1. SirT6 media una monoubiquitinación no canónica en tres cisteínas conservadas del dominio pre-SET de Suv39h1. Entre las E3 ubiquitina ligasas que interaccionan con Suv39h1 y SirT6 como CHIP y CHFR, encontramos que la responsable de la monoubiquitinación de Suv39h1 es SKP2. Mostramos que los niveles de SKP2 están regulados también por SirT6 la cual desacetila SKP2 e induce su fosforilación evitando así su degradación. La monoubiquitinación de Suv39h1 es inducida con tratamiento de doble bloqueo y timidina, que paran las células en G1/S y mitosis temprana, respectivamente. Además, la activación de la vía de NFkB induce la monoubiquitinación de Suv39h1. La expresión del regulador negativo de la vía de NFkB, IkBa, está regulada por la interacción entre Suv39h1, SirT6 y SKP2 y bajo tratamiento con TNFa. SirT6 induce la monoubiquitinación de Suv39h1 a través de SKP2 y Suv39h1 monoubiquitinizado es desplazado del promotor permitiendo la activación de la transcripción de IkBa por RelA. Este modelo proporciona nuevas perspectivas de la regulación de la vía NFkB.

Successful chromosome segregation is crucial for the survival of a cell and to avoid diseases such as cancer. Anaphase bridges are a type of segregation defect that can arises from structurally compromised chromosomes. Little is known about the mechanisms that normally prevent them. In this study I screened for genes that normally prevent anaphase bridges in order to learn more about their origin. I found anaphase bridges to arise in replication mutants and it is possible to trigger these bridges by exposing cells to replication stress. Thus, impaired replication is one cause for anaphase bridges. Further I identified a role for the mitotic exit network (MEN) in chromosome segregation. MEN mutants display anaphase bridges and I present evidence that these bridges arise from telomeric regions and may involve un-replicated DNA.
La correcta segregació dels cromosomes és esencial per la supervivencia de la cèl·lula i per evitar l’aparició de certes malalties com el càncer. Els ponts anafàsics són un tipus d’error de segregació que pot ser originat per defectes estructurals dels cromosomes. Es coneix molt poc sobre els mecanismes que eviten la formació d’aquests ponts anafàsics. En aquest estudi he fet un análisis global dels diferents gens que normalment eviten la formació d’aquests ponts, per abançar en la comprensió del seu origen. He vist que el ponts anaphasics es formen en mutants que tenen afectat el proces de replicació i que és posible de provocar la formació d’aquests ponts exposant les cèl·lules a estrés replicatiu. Per tant, els problemes en la replicació són una de les causes dels ponts d’anafase. He identificat el rol de “mitotic exit network (MEN)” en la segregació cromosómica. Els mutants per MEN formen ponts anafàsics i mostren evidències que aquests ponts probenen de regions telomèriques i podrien incloure DNA no replicat.

Notre génome est continuellement endommagé par des agents provoquant des lésions de l’ADN. Les cassures doubles brins de l’ADN (CDBs) sont les lésions les plus dangereuses. En effet, une CDB mal réparée peut mener à des aberrations de l’ADN pouvant conduire à l’apparition d’un cancer. Dans le but d’éviter les effets délétères des CDBs, nos cellules ont développé une voie de signalisation, nommée réponse aux dommages de l’ADN (RDA), permettant la détection des cassures et l’activation des points de contrôle du cycle cellulaire afin d’arrêter le cycle pendant la réparation des CDBs. Une des caractéristiques principales de la RDA est l’accumulation d’un grand nombre de facteurs sur l’ADN autour de la cassure, formant un foyer visible en microscopie. Cependant, l’efficacité de réparation de l’ADN est entravée par la structure condensée de la chromatine environnante. Les mécanismes de réparation de l’ADN surmontent ce problème en recrutant de nombreuses protéines permettant le réarrangement de la chromatine afin de faciliter la réparation. Le but de mon travail de thèse est d’identifier de nouvelles protéines impliquées dans le remodelage de la chromatine autour des CDBs. D’une part nous avons pour but d’identifier le protéome complet d’un foyer de réparation de l’ADN grâce à la technique PICh (Proteomics of Isolated Chromatin loci). D’autre part, nous étudions le rôle de l’oncoprotéine SET/TAF-1β, que nous avons identifié lors d’un criblage siRNA réalisé dans le but de découvrir de nouveaux facteurs chromatiniens impliqués dans la réparation des CDBs
Various DNA damaging agents, that can cause DNA lesions, assault constantly our genome. The most deleterious DNA lesions are the breaks occurring in both strands of DNA (Double stand breaks: DSBs). Inefficient repair of DSBs can lead to aberrations that may induce cancer. To avoid these deleterious effects of DSBs, cells have developed signalling cascades which entail detection of the lesions and spreading of the signal that leads to arrest in cell cycle progression and efficient repair. A major characteristic of DNA damage response (DDR) is the accumulation of a vast amount of proteins around the DSBs that are visible in the cell as DNA damage foci. However, efficient DNA repair is hampered by the fact that genomic DNA is packaged into chromatin. The DNA repair machinery overcomes this condensed structure to access damaged DNA by recruiting many proteins that remodel chromatin to facilitate efficient repair. The aim of my PhD work is to identify novel proteinsinvolved in the DDR and/or the remodelling of chromatin surrounding DSBs. On one hand, we take advantage of the PICh (Proteomics of Isolated Chromatin loci) technique and we aim to identify the entire proteome of DNA repair foci. On the other hand, we study the role of the oncogene SET/TAFIβ, a major hit of a siRNA screen performed to identify novel chromatin related proteins that play role in repair of DSBs

The aim of my work was to investigate enzymes that potentially modify chromatin and identify possible functions. SET domain containing proteins can catalyse histone lysine methylation. Riz and Blimp are members of the PRDM subfamily of the SET-domain protein group. PRDM proteins contain amino acid substitutions at conserved sites within the SET domain and it is thought that these mutations may inactivate histone methyltransferase (HMT) activity. I have investigated potential functions for this subfamily. My results suggest that the Riz and Blimp bind and demethylate histones in vitro. Set8 is another SET-domain containing protein, but it is catalytically active. Set8 is a member of the SET1 subfamily and it methylates histone H4 at amino acid lysine 20 (H4K20). I generated stable cell lines that express increased levels of Set8 to investigate the physiological role of this enzyme. Overexpression of the enzyme decreases the rate of cellular proliferation and partially reserves a transformed phenotype. Furthermore, increased levels of Set8 affect the cell cycle and my results suggest this could be due to misregulation of mitosis. AKT and S6K are kinases that phosphorylate serine and threonine residues that reside within similar amino acid consensus sequences. I demonstrate that these enzymes phosphorylate histones in vitro, and identify H2AT16 and H3T45 as two phosphorylation sites. I provide evidence for their existence in vivo and in addition, my data implicates AKT as the enzyme responsible for H2AT16ph in vivo. H2AT16ph is associated with mitosis, localising to mitotic chromosomes. In contrast H3T45ph is upregulated during apoptosis in HL-60 cells. Many histone-modifying enzymes have now been linked to a variety of diseases. Analyses of these enzymes should assist with the search for treatments. My results provide additional information into the function of these enzymes. Such data is important, as unlike genetic mutations, aberrant epigenetic modifications are potentially reversible. Therefore, enzymes that catalyse these marks provide potential targets for therapeutic purposes.

The aim of my work was to explore the possibility that the mammalian JAK2 signalling pathway influences the structure and function of chromatin. I have demonstrated that JAK2 is present in the nucleus of both human haematopoietic cell lines and primary cells. My results suggest that JAK2 functions as a histone tyrosine kinase and phosphorylates histone H3 at tyrosine-41 (H3Y41). This novel histone modification, the first described tyrosine phosphorylation on any of the non-variant histones, regulates the binding of heterochromatin protein 1-alpha (HP1α) at a new binding site on chromatin. HP1α uses its chromo-shadow domain to bind the H3Y41 region. Phosphorylation of H3Y41 by JAK2 reduces its affinity for chromatin. This reciprocal relationship was given a functional context by demonstrating its relationship to the expression of a key haematopoietic oncogene Imo2. Genome-wide studies demonstrate that H3Y41ph is present at the 5’ end of genes and is highly correlated with active transcription. This is the first comprehensive genome wide mapping of a histone phosphorylation mark and potentially highlights a role for this novel modification in the regulation of transcription. H3Y41ph was also present at specific cis-regulatory elements on JAK2-STAT5 target genes and genome-wide mapping of STAT5 binding confirmed that STAT5 binding and H3Y41ph was coincident at a significant number of sites within the human genome. This interesting observation suggests that canonical JAK2-STAT5 signalling is not confined to the cytoplasma but also occurs at chromatin. These findings extend the existing paradigm of JAK-STAT signalling and provide a platform for a better understanding of this critical signalling pathway, which is important in both normal development and oncogenesis.

Plants selectively deplete incident light of red (R) wavelength, relative to far-red (FR) wavelength light. Consequently, the relative proportions of R and FR (R:FR ratio) act as an indicator of surrounding vegetation and plants, via the phytochrome photoreceptors, are capable of detecting this. Low R:FR ratio is interpreted as surrounding competition and results in plants eliciting, what is known as, the shade avoidance response. This involves a host of both phenotypic and molecular changes, including increased hypocotyl growth, earlier flowering time and changes in gene expression. The fundamental mechanisms underlying R:FR-mediated changes in gene expression are not fully characterised. Given that increasing evidence in Arabidopsis suggests that the structure of chromatin is integral in the regulation of gene expression in response to environmental stimuli, it was investigated to see if this was the case in R:FR ratio signalling. Here, DNaseI sensitivity assays demonstrate that the shade avoidance genes ATHB2, PIL1 and XTH15 all undergo gross changes in chromatin structure in plants grown in light/dark cycles and treated with low R:FR ratio. These low R:FR induced changes in DNaseI sensitivity are conspicuously absent when plants are grown in continuous light, suggesting an involvement of the circadian clock. Complementary to this, the use of ChIP has identified the coding region of PIL1 to show increased association with hyper-acetylated H3K9 and possibly H3K14 in response to low R:FR ratio. Together, this work demonstrates that changes in R:FR ratio induce changes in gene expression that are correlated with changes in chromatin structure and histone modifications and that these changes could be regulated by the circadian clock. In addition, the identification and construction of multiple mutant and transgenic lines expressing altered levels of chromatin modifying protein was attempted.

The primary level of chromatin organisation consists of arrays of nucleosomes that are present across the genetic template. Advances in the post genomics era have made it possible to determine the positions of nucleosomes genome-wide where it has been observed that nucleosomes adopt a distinct organisation with respect to genetic and trans-binding elements. Amongst the best studied of these is the transcription start site where it has been observed that genic nucleosome locations are well maintained with respect to promoters. DNA and chromatin replication are coupled processes whereby chromatin is disrupted ahead of the replication fork and nucleosomes are rapidly assembled on the nascent DNA template. Classically it has been observed that nascent chromatin is more susceptible to digestion, prompting the possibility of an “immature” chromatin organisation post assembly. However it has not been investigated genome-wide if nucleosomes are initially assembled in phase or must be reorganised post assembly to canonical locations. We have developed two methods of isolating nascent DNA fragments representative of nucleosome positions from synchronised and asynchronous populations of the budding yeast S. cerevesiae. High throughput sequencing has revealed that chromatin is assembled and organised rapidly behind the replication fork. The most nascent chromatin isolated displays typical patterns of nucleosome organisation suggesting that reorganisation of nucleosomes on the nascent template is replication coupled. Deletion of specific histone chaperones and chromatin remodelers perturbs this pathway. However, this can be compensated for by a transcription directed reorganisation of nascent chromatin. Our analysis of nascent chromatin has allowed us to investigate the mechanisms that act to direct chromatin organisation in addition to evaluation of models that describe nucleosome organisation genome-wide.

The work presented in this thesis aims at unravelling human chromatin composition by quantitative proteomics to outline the functional and structural changes occurring during the life of human cells. Chromatin is the structure formed by proteins and RNAs interacting with the genetic material. At present, chromatin is not well defined. It is not easy to investigate either the composition of its constituent proteins or how this arrangement changes. We set out to analyse the chromatin composition changes occurring during the cell cycle. Our procedure couples a SILAC mass spectrometry-based approach with a newly developed biochemical chromatin purification method, which involves fixation of proteins to DNA. By testing two different fixation times (5 and 10 minutes) and three phases of the cell cycle (G1/S, G2, M), we quantified ~3000 proteins providing a broad picture of the global changes on chromatin protein composition. Surprisingly, chromatin seems to be occupied by many unexpected proteins (40%) that appear to be increased during mitosis. We hypothesized that these unexpected proteins come into contact with DNA during mitosis when the nuclear envelope breaks down and the highly negatively charged DNA can be found in proximity to extra nuclear proteins. We used Pulse-SILAC technique that allows to distinguish newly synthesized proteins to test this possibility. By comparing in a single cell cycle and during G0 arrest the incorporation of new proteins into chromatin with their synthesis in the cytoplasm and in the whole cell, we could not find a different behaviour for the unexpected proteins as result of mitosis. Despite the efforts in tracking down the origin of these unexpected proteins, it is still uncertain whether their presence on chromatin is the result of a biological process or, in part, a drawback of the purification methods adopted. However, proving their genuine presence on chromatin will be important to elucidate how chromatin functions.

Eukaryotic promoters are inherently bidirectional and allow RNA Polymerase II to transcribe both coding and noncoding RNAs. Dynamic disassembly and reassembly is a prominent feature of nucleosomes around eukaryotic promoters. While H3K56 acetylation (H3K56Ac) enhances turnover events of these promoter-proximal nucleosomes, the chromatin remodeler INO80C ensures their proper positioning. In my dissertation, I explore how chromatin dynamics regulate transcriptional homeostasis. In the first part, I investigate the role of H3K56Ac on the nascent transcriptome throughout the eukaryotic cell cycle. I find that H3K56Ac is a global, positive regulator for coding and noncoding transcription by promoting both initiation and elongation/termination. On the contrary, I find that H3K56Ac represses promiscuous transcription following replication fork passage by ensuring efficient nucleosome assembly during S-phase. In addition, I show that there is a stepwise increase in transcription in the S-G2 transition, and this response to gene dosage imbalance does not require H3K56Ac. This study clearly shows that a single histone modification, H3K56Ac can exert both positive and negative effects on transcription at different cell cycle stages. In the second part, I investigate the role of the chromatin remodeler INO80C on the nascent transcription around replication origins. I show that INO80C, together with the transcription factor Mot1, prevents cryptic transcription around yeast replication origins, and the loss of these proteins lead to an increase in DNA double strand breaks. I hypothesize that recruitment of INO80C ensures proper positioning of nucleosomes around origins and the exclusion of RNA Pol II to prevent cryptic initiation. Together these findings indicate that H3K56Ac regulates transcription globally by enhancing nucleosome turnover, and it prevents cryptic transcription and reinforces transcriptional fidelity by promoting efficient nucleosome assembly in the S-phase. In addition, INO80C maintains genome stability by preventing cryptic transcription around the origins.

Eukaryotic organisms contain their entire genome in the nucleus of their cells. In order to fit within the nucleus, genomic DNA wraps into nucleosomes, the basic, repeating unit of chromatin. Nucleosomes wrap around each other to form higher order chromatin structures. Here we study many factors that affect, or are effected by, chromatin structure including: (1) how low-dose inorganic arsenic (iAs) changes chromatin structures and their relation to global transcription and splicing patterns, and (2) how chromatin architectural proteins (CAPs) bind to and change nucleosome dynamics and DNA target site accessibility. Despite iAs’s non-mutagenic nature, chronic exposure to low doses of iAs is associated with a higher risk of skin, lung, and bladder cancers. We sought to identify the genome-wide changes to chromatin structure and splicing profiles behind the cell’s adaptive response to iAs and its removal. Furthermore, we extended our investigation into cells that had the iAs insult removed. Our results show that the iAs-induced epithelial to mesenchymal transition and changes to the transcriptome are coupled with changes to the higher order chromatin structure and CAP binding patterns. We hypothesize that CAPs, which bind the entry/exit and linker DNA of nucleosomes, regulate DNA target site accessibility by altering of the rate of spontaneous dissociation of DNA from nucleosome. Therefore, we investigated the effects of the repressive CAP histone H1, the activating CAP high mobility group D1 (HMGD1), and the neural CAP methyl CpG binding protein 2 (MeCP2) on the dynamics of short chromatin arrays and mononucleosomes and their effect on nucleosomal DNA accessibility. Using biochemical and biophysical analyses we show that all CAP-chromatin structures tested were susceptible to chromatin remodeling by ISWI and created more stable higher order structures than if CAPs were absent. Additionally, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding its cognate DNA site within nucleosomal DNA. Overall, we show that chromatin structure is dynamic and changes in response to environmental signals and that CAPs change nucleosome dynamics that help to regulate chromatin structures and impact transcriptional profiles.

Epigenetic misregulation of gene expression is known to be an important feature in cancer. This has mainly been studied at the level of changes in DNA methylation and histone modifications at individual genes. In this thesis I have set out to investigate whether there are long-range changes in chromatin structure linked to altered gene expression in breast cancer. From large published datasets, I used a computational approach to identify large genomic regions which are coordinately misregulated in breast cancer independent of copy number aberrations (genomic effects). I found 26 regions of co-ordinate regulation of neighbouring genes that are consistent between breast tumours and breast cancer cell lines. These regions had different expression phenotypes (activation, repression, no change) compared to normal breast and also with tumour subtype (luminal vs basal and ER status). The regions of epigenetic regulation (RER) identified in breast cancer were mostly cancer type specific. I investigated the mechanism of long-range misregulation at one such region on chromosome 16p11.2 which is aberrantly activated in breast cancer. Interestingly, in estrogen-receptor positive (ER+ve) cells, genes in this region are upregulated relative to estrogen receptor negative (ER-ve) cells. Using fluorescence in situ hybridisation (FISH) I found that in ER+ve breast cancer cell lines and tumour tissue this region is in a more decondensed chromatin architecture than in ER-ve cell lines and tumour tissue. Furthermore this region was very compact in a normal breast epithelial cell line and breast tissue corresponding to what would be expected from the expression data. Estrogen was found to play a key role in maintaining the aberrant decondensation of chromatin at this locus on chr16p11.2, as shown by compaction of the region by starving ER+ve cells of estrogen and decompaction upon subsequent estrogen treatment. Interestingly there was also an estrogen mediated repositioning of the 16p11.2 RER domain away from the nuclear centre in hormone starved conditions and towards the centre upon estrogen stimulation. Together these results show that estrogen is key to regulating the changes in nuclear organisation and chromatin decompaction at this locus, which are associated with aberrant patterns of gene expression in ER+ve breast cancer.

La complexe protéique Sir (Silent Information Regulator) de la levure bourgeonnante est l’acteur principal dans la formation de l’hétérochromatine, qui provoque l’atténuation de l’expression génique par un mécanisme épigénétique. Le complexe Sir lié à la chromatine maternelle est démonté lors de la réplication génomique et puis réformé sur les deux brins nouvellement répliqué. La dynamique de maintien de Sir sur la chromatine pendant le cycle cellulaire et dans de variables conditions de croissance n’est pas bien comprise. Pour comprendre comment la structure chromatinienne telle que l’hétérochromatine peut être héritée et par conséquent comment les structures épigénétiques sont transmises d’une génération cellulaire à l’autre, nous avons besoin de mesurer la vitesse d’échange des sous-unités du complexe Sir au cours du cycle cellulaire dans différentes conditions de croissance. Nous avons donc utilisé le système RITE qui permet d’échanger deux épitopes attachés à Sir3 (une des sous-unités de Sir) et par la suite mesurer la cinétique de remplacement de Sir3. Nous avons constaté que la Sir3 maternelle est complètement remplacée par la Sir3 nouvellement synthétisées dans les régions télomériques durant le premier cycle cellulaire après la sortie de la phase stationnaire. Nous proposons que cette reprogrammation du complexe hétérochromatique est un mécanisme d’adaptation qui assure l’activation des gènes de réponse au stress par la déstabilisation transitoire de la structure hétérochromatinienne
The budding yeast SIR complex (Silent Information Regulator) is the principal actor in heterochromatin formation, which causes epigenetically regulated gene silencing phenotypes. The maternal chromatin bound SIR complex is disassembled during replication and then, if heterochromatin is to be restored on both daughter strands, the SIR complex has to be reformed on both strands to pre-replication levels. The dynamics of SIR complex maintenance and re-formation during the cell-cycle and in different growth conditions are however not clear. Understanding exchange rates of SIR subunits during the cell cycle and their distribution pattern to daughter chromatids after replication has important implications for how heterochromatic states may be inherited and therefore how epigenetic states are maintained from one cellular generation to the next. We therefore used the tag switch RITE system to measure genome wide turnover rates of the SIR subunit Sir3 before and after exit from stationary phase and show that maternal Sir3 subunits are completely replaced with newly synthesized Sir3 at subtelomeric regions during the first cell cycle after release from stationary phase. We propose that the observed “reset” of the heterochromatic complex is an adaptive mechanism that ensures the activation of subtelomeric stress response genes by transiently destabilizing heterochromatin structure

Nucleosomes comprise the most basic repeating unit of chromatin and provide hubs for the regulation of DNA transcription, replication and repair. ATPase chromatin remodelling complexes establish nucleosome occupancy, positioning and structure in a dynamic fashion to allow fine-tuning of protein-DNA interactions. The ISWI and CHD families of remodelers possess the ability to sample DNA linker length between nucleosomes and space nucleosomes evenly. How these spacing remodelers combine their functions to maintain phasing of nucleosomal arrays, and re-organise these arrays during development remains poorly understood. Furthermore the relationship between nucleosomal array structure and transcriptional regulation is unclear. Dictyostelium discoideum provides a complex chromatin environment and remodeler repertoire, while retaining a compact genome and ease of genetic manipulation. Thus we have utilized this model to generate remodeler null mutants, and double mutants to observe phenotypic effects and interactions. We further compiled comprehensive nucleosome mapping and RNA sequencing data sets for all spacing remodelers in Dictyostelium. Bioinformatic analysis of these data provide novel insights into remodeler functions, and help to establish a paradigm to explain the relationship between remodeler-mediated chromatin organisation and transcriptional regulation.

Chromatin is a complex of proteins and DNA found in the nuclei of eukaryotic cells. It reinforces the DNA and its topology tunes DNA transcription and gene expression. It is formed by nucleosomes, structures composed of an octameric protein core and approximately 147 base pairs of DNA. Chromatin is an extremely complex system, the behaviour of which is ruled by both mechanical and electrostatic factors that are depend on its structure, and biomolecular interactions occurring in the cell nucleus. In this thesis, I analyse chromatin compaction from an electrostatic perspective and focus on the role of electrostatics and solvation as determinants of the topology of chromatin. I examine the effect of the histone tails and propose a methodology to connect electrostatic calculations to the structural and functional features of protein-DNA systems. This methodology can also be combined with coarse-grained representations. I study the electrostatic forces acting on the phosphate atoms of the DNA backbone. I investigate the electrostatic origins of effects such as different stages in DNA unwrapping, nucleosome destabilisation upon histone tail truncation, and the role of specific arginines and lysines undergoing Post - Translational Modifications. I find that the positioning of the histone tails can oppose the attractive pull of the histone core, locally deform the DNA, and tune DNA unwrapping. I conduct an analysis of the porosity of nucleosomes and related to the importance of solvation phenomena. I complement and support my computational findings on nucleosome electrostatic interactions experimental Zeta Potential and Dynamic Light Scattering measurements on single nucleosomes under varying ionic concentrations, providing information on the surface charge and the size of nucleosomes. I present a comprehensive study of the electrostatic interactions between nucleosome pairs sampling different translations and rotations. My analysis aims to provide a cohesive description of nucleosome electrostatic interactions in the chromatin fibre, combining information on the energetics of different relative positions of nucleosomes, especially in very tight packing situations. In addition to numerical estimates of electrostatic interaction energy of nucleosomes at different relative distances and orientations, obtained within the Poisson-Boltzmann framework, I present their approximation by analytical asymptotic expressions, where nucleosomes are approximated as monopoles and dipoles centred in dielectric spheres immersed in an electrolytic solution. I am able to identify a non-linearity region around the nucleosomes, and to exploit the fact that that in points outside that region the electrostatic potential can be described by the linearised Poisson-Boltzmann Equation.

La libération d’hormone glucocorticoïde (GC) est une réponse physiologique à l’exposition au stress qui permet à l’organisme de faire face aux défis environnementaux. Bénéfique lors du travail, un dysfonctionnement de cette réponse adaptative est associé à de multiples pathologies, y compris les troubles psychiatriques. Le CG agit par la liaison avec leur récepteur glucocorticoïde (GR). Il a été démontré que GRD1Cre souris montaient réduit l’activité des neurones dopamines, diminue les réponses à la cocaïne et bloque l’aversion sociale induite par la défaite sociale répétée. GR peut contrôler l’expression des gènes à interagir avec les complexes de remodelage de la chromatine SWI/SNF soit Brahma (brm) soit Brahma-related gene 1 (Brg1) en tant que sous-unité du cœur catalytique ATP. Nous avons constaté que Brg1D1Cre et brm-/- souris montraient une résilience complète a la défaite sociale répétée. De plus, le Brg1D1Cre et la brm-/- souris ont montré une diminution des réponses à la cocaïne dans la sensibilisation comportementale. Les souris Brg1D1Cre au contraire de GRD1Cre ont montré une augmentation normale des tirs de neurones de dopamine après la défaite sociale malgré leur résilience comportementale. Nous avons donc examiné la signalisation cellulaire et l’induction immédiate des gènes précoces dans les zones mutées du cerveau de nos modèles et montre que si la voie de signalisation ERK est normalement induite par une défaite aigue, l’induction de l’expression des gènes c-Fos et Egr1 est réduite dans le striatum dorsal et le Nac. Au total, ces résultats amènent d’autres preuves d’une implication des remodeleurs de la chromatine dans des comportements lies au stress
Glucocorticoid (GC) hormone release is a key physiological response to stress exposure enabling the organism to cope with environmental challenges. Beneficial when working, a dysfunction of this adaptive response is associated to multiples pathologies including psychiatric disorders. GC act through the binding to their receptor, glucocorticoid receptor (GR). It has been shown that GR gene inactivation in dopaminoceptive neurons (GRD1Cre mice) reduces dopamine neurons activity, decreases responses to cocaine and blocks social aversion induced by repeated social defeat. GR can control genes expression through different mechanisms. Among others, it can interact with SWI/SNF chromatin remodeling complexes either Brahma (Brm) or Brahma-related gene 1 (Brg1) as an ATP catalytic core subunit, which can move the DNA along the nucleosomes thereby opening the chromatin and favoring gene transcription. We found that both Brg1D1Cre and Brm-/- mice showed a complete resilience to repeated social defeat. Moreover, both Brg1D1Cre and Brm-/- mice showed decreased responses to cocaine in behavioral sensitization. Brg1D1Cre mice on the contrary to GRD1Cre showed a normal increase of dopamine neuron firing after social defeat despite their behavioral resilience. We therefore examined cell-signaling and immediate early genes induction in the mutated brain areas of our models and showed that while ERK signaling pathway is normally induced by an acute defeat, the induction of c-Fos and Egr1 genes expression are reduced in the dorsal striatum and the NAc. Altogether, these results lead to further evidences for an involvement of chromatin remodelers in stress-related behaviors

La dynamique de la chromatine est affectée par les processus biologiques. Pour comprendre comment la physique de la chromatine et la biologie se repondent, nous avons besoin d'outils pour analyser la dynamique de la chromatine in vivo. Plusieurs systèmes existent pour marquer les loci d'ADN et déterminer efficacement leur position dans le noyau, mais ils présentent des inconvénients dans les cellules de mammifères lorsqu'il s'agit d'étudier le mouvement de la chromatine dans le contexte de processus biologiques. Cela est particulièrement vrai lorsqu'il s'agit de mécanismes où l'ADN doit être lu à proximité du marquage. Pour étudier la dynamique de la chromatine in cellulo, l'équipe Bystricky a développé ANCHOR. ANCHOR repose sur l'insertion d'une séquence courte et non répétitive (ANCH) dans le génome de l'hôte. Cette séquence contient des sites de liaison pour une protéine (OR) qui, une fois liée, s'oligomérise et permet la visualisation du locus marqué. ANCHOR est dérivé des systèmes de partitionnement des chromosomes bactériens. L'outil a été implémenté avec succès dans la levure bourgeonnante (Saad et al. 2014) et plus récemment dans la drosophile (H. Chen, Fujioka, et Gregor 2017 ; Gomez-Lamarca et al. 2018). L'un de mes projets de thèse consistait à appliquer le système ANCHOR dans des cellules humaines. La séquence ANCH3 a été insérée de manière aléatoire et en une seule copie dans le génome de la lignée cellulaire de cancer du sein MCF7 par Hafida Sellou (étudiante en M2) et Fatima Moutahir (technicienne). Pour insérer la séquence ANCH3, les cellules MCF7 ont d'abord été modifiées pour insérer un site FRT dans le génome. Ensuite, un plasmide contenant l'ANCH3 couplé au transgène Cyclin D1 et un site FRT a été transfecté. La recombinaison entre les deux sites FRT a été favorisée par la Flipase. La protéine OR3 étiquetée par fluorescence a été exprimée de manière stable ou transitoire pour permettre l'imagerie du gène CCND1 (voir (Germier et al. 2017, 2018) pour plus de détails). Nous avons voulu établir une preuve de principe pour l'utilisation d'ANCHOR dans des cellules de mammifères. Des cellules MCF7 contenant un transgène CCND1, appelé G7-CCND1 (Germier et al. 2017) ont été transfectées de manière stable avec OR3-Santaka et le locus CCND1 a été suivi sur 24 heures à travers une division cellulaire. Nous avons pu suivre efficacement le locus du transgène sans photoblanchiment. La présence de la protéine OR3-Santaka sur le locus chromatinien n'a pas perturbé la réplication et deux loci ont été effectivement observés dans les deux cellules filles (Germier et al. 2018). En utilisant le système ANCHOR3, nous avons donc développé un outil puissant pour étudier à la fois des événements rapides et courts comme la transcription et des événements à long terme se déroulant sur plusieurs jours, comme la division ou la différenciation cellulaire
Chromatin dynamics are affected by biological processes. To understand how physical behaviour of chromatin and biology work together, we need tools to analyse chromatin motion in living cells. Several systems exist to fluorescently label DNA loci and to effectively determine their position within the nucleus, but they have drawbacks in mammalian cells when it comes to studying chromatin motion in the context of biological processes. This is especially true when it comes to mechanisms where DNA needs to be processed in the vicinity of the labeling. To study chromatin dynamics in cellulo, the Bystricky group developed the ANCHOR DNA labelling system. ANCHOR relies on the insertion of a short, non-repetitive sequence (ANCH) in the host genome. This sequence contains binding sites for a protein (OR) which once bound, oligomerize and allow visualization of the tagged locus. ANCHOR is derived from the bacterial chromosome partitioning systems. The tool was successfully implemented in budding yeast (Saad et al. 2014) and more recently in Drosophila (H. Chen, Fujioka, and Gregor 2017; Gomez-Lamarca et al. 2018). One of my thesis projects was to apply the ANCHOR system in human cells. The ANCH3 sequence was inserted randomly and in one copy in the genome of breast cancer cell line MCF7 by Hafida Sellou (M2 student) and Fatima Moutahir (technician). To insert the ANCH3 sequence, MCF7 cells were first modified to insert a FRT site in the genome. Then, a plasmid containing ANCH3 coupled to Cyclin D1 transgene and a FRT site was transfected. Recombination between the two FRT site was promoted by Flipase. The fluorescently-tagged OR3 protein was either stably or transiently expressed to allow imaging of the CCND1 gene (see (Germier et al. 2017, 2018) for details). We wanted to establish a proof of principle for the use of ANCHOR in mammalian cells. MCF7 cells containing a CCND1 transgene, called G7-CCND1 (Germier et al. 2017) were stably transfected with OR3-Santaka and the CCND1 locus was followed using fast- time lapse microscopy over 24 h through one cell division in a single cell. We could effectively follow the transgene locus without much photobleaching. The presence of OR3-Santaka protein on the chromatin locus did not disturb replication and two loci were effectively observed in the two daughter cells (Germier et al. 2018). Using the ANCHOR3 system, we hence developed a powerful tool to study both rapid, short events such as transcription and long-term events taking place over days, such as cell division or differentiation