While experiments provide valuable information about biological molecules, current technology cannot yet monitor atomic fluctuations at relevant time scales. Theoretical computational simulations are able to model the appropriate interactions at atomic resolution. Computational techniques have become widely used for identifying interactions in biological systems. Such methods have proven quite accurate in their ability to reproduce experimental data and also in screening and predicting pertinent activities. Molecular modeling employs theoretical and computational techniques to elucidate biologically relevant information from macromolecular structures. Three biological systems, the nucleosome core particle, myoglobin and glycosyl hydrolase family 1 beta-glucosidases will be examined with molecular modeling methods. Results of our analyses provide information about DNA flexibility and packaging, internal migration of ligands in a small protein, and substrate specificity of an enzyme system. / Ph. D.

Properly representing the principle physical interactions of complex biological systems is paramount for building powerful, yet simple models. As an in depth look into different biological systems at different scales, multiple models are presented. At the molecular scale, an analytical solution to the (linearized) Poisson-Boltzmann equation for the electrostatic potential of any size biomolecule is derived using spherical geometry. The solution is tested both on an ideal sphere relative to an exact solution and on a multitude of biomolecules relative to a numerical solution. In all cases, the bulk of the error is within thermal noise. The computational power of the solution is demonstrated by finding the electrostatic potential at the surface of a viral capsid that is nearly half a million atoms in size. Next, a model of the nucleosome using simplified geometry is presented. This system is a complex of protein and DNA and acts as the first level of DNA compaction inside the nucleus of eukaryotes. The analytical model reveals a mechanism for controlling the stability of the nucleosome via changes to the total charge of the protein globular core. The analytical model is verified by a computational study on the stability change when the charge of individual residues is altered. Finally, a multiple model approach is taken to study bacteria that are capable of different responses depending on the size of their surrounding colony. The first model is capable of determining how the system propagates the information about the colony size to those specific genes that control the concentration of a master regulatory protein. A second model is used to analyze the direct RNA interference mechanism the cell employs to tune the available gene transcripts of the master regulatory protein, i.e. small RNA - messenger RNA regulation. This model provides a possible explanation for puzzling experimentally measured phenotypic responses. / Ph. D.

Au sein du noyau, l'ADN est organise en chromatine dont l'unité de base est le nucléosome. La structure de la chromatine est très dynamique, ce qui est nécessaire pour la plupart des opérations qui se produisent dans l'ADN telles que la réplication, la transcription, la réparation et la recombinaison. Le nucléosome est constitué de deux dimères H2A/H2B et deux dimères H3/H4 associés avec 147 paires de bases d'ADN. La protéine Nap1 est un chaperon d'histone H2A/H2B impliquée dans l'assemblage et démontage des nucléosomes. Nap1 protège les interactions non spécifiques entre l'ADN chargé négativement et les dimères H2A/H2B chargés positivement, afin de permettre la formation de la structure ordonnée des nucléosomes. Lors de l'assemblage des nucléosomes, les dimères d'histones H3/H4 sont déposés en premier lieu, suivi par le dépôt de dimères H2A/H2B. Lors du démontage du nucléosome, les dimères H2A/H2B sont retirés avant le retrait des dimères H3/H4. La determination de la structure du complexe Nap1-H2A/H2B pourra permettre une meilleure compréhension du processus d'assemblage du nucléosome. Dans cette étude, nous voulons comprendre comment le chaperon Nap1 cible spécifiquement les dimères d'histones H2A/H2B pour l'assemblage des nucléosomes. Notre objectif est de caractériser la structure et la fonction du complexe de Nap1-H2A/H2B. Ainsi nous nous sommes tout d'abord intéresse à la stoechiometrie de ce complexe. Nous avons trouvé qu'un dimère de Nap1 s'associe à un dimère H2A/H2B (Nap1_2-H2A/H2B). D'autre part, l'analyse par spectrométrie de masse non-dénaturante a montré que ce complexe de base peut s'oligomériser et contenir jusqu'à 6 copies de Nap1_2-H2A/H2B. L'analyse de ce complexe par spectrométrie de masse non-dénaturant a montré que ce complexe peu oligomériser dans un grand complexe contenant jusqu'à 6 copies de Nap1_2-H2A/H2B. Nous avons également obtenu la première structure cristalline à basse résolution de ce complexe. L'analyse du même complexe par microscopie électronique à coloration négative a révélé la présence en solution du même oligomère que dans l'unité asymétrique du cristal, qui contient aussi 6 copies de Nap1_2-H2A/H2B. Ainsi, nous avons pu mettre en évidence de nouvelles interfaces d'interaction entre les différents composants de ce complexe qui nous permettent de mieux comprendre le processus d'assemblage des nucléosomes. Le remodelage de la chromatine permet l'expression des gènes eucaryotes. Ce remodelage nécessite des enzymes telles que des histone acétyltransférases (HAT) et les chaperons d'histones. Les HATs acétylent les chaînes latérales des lysines. Il a été proposé que les HATs et les histones chaperons agissent en synergie pour moduler la structure de la chromatine pendant la transcription. La HAT p300 a été proposé d'interagir avec l'histone chaperon Nap1. Nous avons entrepris de caractériser cette interaction. Malheureusement, nos expériences n'ont pas pu détecter d'interaction directe entre ces protéines.

Le nucléosome est l’unité élémentaire de la compaction de l’ADN dans les cellules eucaryotes. C’est un complexe composé par un long segment d’ADN enroulé 1.7 fois en super-hélice autour d’un cœur de huit protéines histones. Les nucléosomes contrôlent l’accessibilité de l'ADN en s'associant et se dissociant le long des génomes et, ce faisant, sont directement impliqués dans la plupart des processus nucléaires. Le but principal de ce travail a été de décrire l'interface ADN-histones en solution pour mieux comprendre la stabilité du nucléosome. Nous avons voulu savoir en particulier comment l'ADN est maintenu enroulé autour du cœur d'histone et comment la séquence de l'ADN pourrait éventuellement affecter l'interface ADN-histones. Plusieurs nucléosomes ont été étudiés par dynamique moléculaire en solvant explicite ; ils diffèrent par la taille des queues d'histone et par les séquences d'ADN qui les forment. Pour garantir une analyse objective de la topologie de l’interface ADN-histones, une méthode basée sur les pavages de Delaunay-Laguerre originellement dédiée aux protéines a été adaptée aux acides nucléiques. Nous montrons ainsi que l'interface ADN-histones est constituée d'un réseau d'interaction très dense, caractérisé par des aires de contact électrostatique et hydrophobe équivalentes. Les queues d'histone renforcent significativement l'interface. Le comportement dynamique des arginines des cœurs structurés et des queues d'histone qui interagissent avec les petits sillons de l'ADN a été examiné en détail. Les cations écrantent les répulsions entre les hélices d'ADN juxtaposées l'une au dessus de l'autre du fait de l'enroulement en super-hélice. Enfin, l’interface ADN-histones est globalement retrouvée dans les nucléosomes formés avec des séquences d’ADN défavorables au nucléosome. Ceci suggère qu'une fois le nucléosome formé, il n'y a pas d'effet décisif de la séquence de l'ADN sur l'interface. / The nucleosome is the fundamental unit of DNA compaction in eukaryotic cells. It consists in a long DNA segment (145-147 bp) wrapped in 1.7 left-handed superhelix turns around a histone octamer. Nucleosomes control the DNA accessibility by assembling and disassembling along the genomes and are therefore involved in most nuclear processes.The main aim of the thesis was to describe the DNA-histone interface in solution to better understand the nucleosome stability. We examined in particular how the DNA is maintained wrapped around the histone and how its sequence affects the DNA-histone interface. Several nucleosomes were studied using molecular dynamics in explicit solvent ; they differed by the tail length and the DNA sequences. To ensure an objective analysis of the topology of the DNA-histone interface, a method based on Delaunay-Laguerre tessellations, originally developed for proteins, was adapted to nucleic acids.Our results show that the DNA-histone interface is composed by a dense network of interactions, characterized by equivalent electrostatic and hydrophobic contact area. The histone tails significantly reinforce the interface. The behavior of arginines belonging to the histone structured cores or tails and that interact with the DNA minor groove was scrutinized in detail. Cations shield the repulsive interactions between the two DNA gyres, closely juxtaposed one above the other because of the superhelix wrapping. Finally, the DNA-histone interface is globally not affected in nucleosomes containing DNA sequences known to disfavor nucleosomes. This suggests that, once the nucleosome established, there is no significant effect of the DNA sequence on the interface.

FACT est une protéine clé, qui joue de multiples rôles, y compris dans la transcription et la réparation de l'ADN endommagé. Néanmoins, comment FACT participe à la réparation et à la transcription de la chromatine n'est pas élucidé. Dans ce travail nous avons tout d'abord étudié le rôle de FACT dans le processus de réparation par excision de base (BER). Nous avons utilisé des nucléosomes reconstitués avec de l'ADN à uracile incorporé au hasard. Nous avons trouvé que l'enzyme UDG est capable d'enlever les uraciles localisés du côté de la solution et pas les uraciles se trouvant en face de l'octamère d'histone. La présence simultanée de FACT et de RSC (facteur de remodelage de la chromatine, impliqué dans la réparation) permet un enlèvement efficace des uraciles localisés du côté de l'octamère d'histone par l'UDG. De plus, l'action concertée de FACT et RSC contribue à l'enlèvement de la lésion oxidative 8-oxoG, autrement inaccessible, de la matrice nucléosomale par l'enzyme OGG1. Ce résultat est obtenu grâce à une activité « co-remodelatrice » de la protéine FACT. Dans ce travail nous décrivons pour la première fois cette nouvelle propriété de FACT et nous montrons par une série d'expériences biochimiques que FACT est capable de stimuler l'activité de remodelage du RSC. Nos expériences montrent que la présence de FACT augmente l'efficacité de RSC à transformer l'énergie libérée par l'hydrolyse de l'ATP en travail « mécanique ». Les données obtenues suggèrent une nature stochastique du BER in vivo, FACT étant un facteur clé dans le processus de réparation. Nous avons également investigué l'implication de l'activité co-remodelatrice de FACT dans la fixation de NF-kB aux matrices nucléosomales. La production de nucléosomes remodelés, mais non - mobilisés (remosomes) n'est pas suffisante pour promouvoir la fixation de NF-kB. Pourtant, la mobilisation des nucléosomes par l'intermédiaire de RSC permet une interaction efficace entre NF-kB et l'ADN nucléosomal. Toutes ces données sont essentielles pour le décryptage du mécanisme moléculaire par lequel FACT agit dans le BER et dans la transcription médiée par NF-kB. / FACT is a vital protein which has multiple roles including one in transcription and repair of damaged DNA. However, how FACT assists repair and transcription remains elusive. In this work, we have first studied the role of FACT in Base Excision Repair (BER). We used nucleosomes containing DNA with randomly incorporated uracil. We found that the enzyme UDG is able to remove uracils facing the solution and not the uracils facing the histone octamer. The simultaneous presence of FACT and RSC (a chromatin remodeler involved in repair) allows, however, a very efficient removal of uracil facing the histone octamer by UDG. In addition, the concerted action of FACT and RSC permits the removal of the otherwise un-accessible oxidative lesion 8-oxoG from nucleosomal templates by OGG1. This was achieved thanks to the co-remodeling activity of FACT. Here we described for the first time this novel property of FACT and we show in a series of biochemical experiments that FACT is able to boost the remodeling activity of RSC. The experiments reveal that the presence of FACT increases the efficiency of RSC to transform the energy freed by ATP hydrolysis into “mechanical” work. The presented data suggest a stochastic nature of BER functioning in vivo, FACT being a key factor in the repair process. The implication of the co-remodeling activity of FACT in NF-kB factor binding to nucleosomal templates was also investigated. The generation of remodeled, but not mobilized nucleosomes (remosomes), was not sufficient to promote NF-kB binding. However, the RSC-induced nucleosome mobilization allows efficient NF-kB interaction with nucleosomal DNA. Our data are instrumental in deciphering the molecular mechanism of FACT implication in BER and NF-kB mediated transcription.

No description available.

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.

Background: In all higher organisms, the nuclear DNA is condensed into nucleosomes that consist of DNA wrapped around a core of highly conserved histone proteins. DNA bound to histones and other structural proteins form the chromatin. Generally, only few regions of DNA are accessible and most of the time RNA polymerase and other DNA binding proteins have to overcome this compaction to initiate transcription. Several proteins are involved in making the chromatin more compact or open. Such chromatin-modifying proteins make distinct post-translational modifications of histones – especially in the histone tails – to alter their affinity to DNA. Aim: The main aim of my thesis work is to study the targeting of chromatin modifiers important for correct gene expression in Drosophila melanogaster (fruit flies). Primary DNA sequences, chromatin associated proteins, transcription, and non-coding RNAs are all likely to be involved in targeting mechanisms. This thesis work involves the development of new computational methods for identification of DNA motifs and protein factors involved in the targeting of chromatin modifiers. Targeting and functional analysis of two chromatin modifiers, namely male-specific lethal (MSL) complex and CREB-binding protein (CBP) are specifically studied. The MSL complex is a protein complex that mediates dosage compensation in flies. CBP protein is known as a transcriptional co-regulator in metazoans and it has histone acetyl transferase activity and CBP has been used to predict novel enhancers. Results: My studies of the binding sites of MSL complex shows that promoters and coding sequences of MSL-bound genes on the X-chromosome of Drosophila melanogaster can influence the spreading of the complex along the X-chromosome. Analysis of MSL binding sites when two non-coding roX RNAs are mutated shows that MSL-complex recruitment to high-affinity sites on the Xchromosome is independent of roX, and the role of roX RNAs is to prevent binding to repeats in autosomal sites. Functional analysis of MSL-bound genes using their dosage compensation status shows that the function of the MSL complex is to enhance the expression of short housekeeping genes, but MSL-independent mechanisms exist to achieve complete dosage compensation. Studies of the binding sites of the CBP protein show that, in early embryos, Dorsal in cooperation with GAGA factor (GAF) and factors like Medea and Dichaete target CBP to its binding sites. In the S2 cell line, GAF is identified as the targeting factor of CBP at promoters and enhancers, and GAF and CBP together are found to induce high levels of polymerase II pausing at promoters. In another study using integrated data analysis, CBP binding sites could be classified into polycomb protein binding sites, repressed enhancers, insulator protein-bound regions, active promoters, and active enhancers, and this suggested different potential roles for CBP. A new approach was also developed to eliminate technical bias in skewed experiments. Our study shows that in the case of skewed datasets it is always better to identify non-altered variables and to normalize the data using only such variables.

The human genome consists of over 3 billion nucleotides and would be around 2 meters long if uncoiled and laid out. Each human somatic cell contains all this in their nucleus which is only around 5 µm across. This extreme compaction is largely achieved by wrapping the DNA around a histone octamer, the nucleosome. Still, the DNA is accessible to the transcriptional machinery and this regulation is highly dynamic and change rapidly with, e.g. exposure to drugs. The individual histone proteins can carry specific modifications such as methylations and acetylations. These modifications are a major part of the epigenetic status of the DNA which contributes significantly to the transcriptional status of a gene - certain modifications repress transcription and others are necessary for transcription to occur. Specific histone methylations and acetylations have also been implicated in more detailed regulation such as inclusion/exclusion of individual exons, i.e. splicing. Thus, the nucleosome is involved in chromatin remodeling and transcriptional regulation – both directly from steric hindrance but also as a signaling platform via the epigenetic modifications. In this work, we have developed tools for storage (Paper I) and normalization (Paper II) of next generation sequencing data in general, and analyzed nucleosome locations and histone modification in particular (Paper I, III and IV). The computational tools developed allowed us as one of the first groups to discover well positioned nucleosomes over internal exons in such wide spread organisms as worm, mouse and human. We have also provided biological insight into how the epigenetic histone modifications can control exon expression in a combinatorial way. This was achieved by applying a Monte Carlo feature selection system in combination with rule based modeling of exon expression. The constructed model was validated on data generated in three additional cell types suggesting a general mechanism.

Epigenetics is the study of heritable changes in gene expression that do not involve changes in the underlying DNA sequence. Epigenetic pathways appeared in eukaryotes where multicellular organisms differentiate into different cell types, associated with different phenotypes. The differentiation process is achieved by modifications of the chromatin structure which, by altering the access of trans-factors to the DNA, result in gene activation and repression. Epigenetic mechanisms are therefore viewed as an "extra" layer of information that modulate the genetic information in time and space, necessary for the development and the response to environment stimuli. Although the recent development of high-throughput sequencing technologies has provided an unprecedented insight into epigenetic pathways, the mechanisms controlling the chromatin dynamic as well as their downstream effects on cellular processes are far from being fully understood. In this thesis, our focus will be restricted to mechanisms acting on the nucleosome level. The first chapter will present the factors known to influence nucleosome positioning as well as the challenges related to the measurement of the nucleosome architecture. The second chapter will introduce a statistical approach, NucleoFinder, capable of identifying nucleosomes consistently positioned in a population of cells. In chapter three, we will make use of NucleoFinder to investigate the importance of cis and trans-factors on the nucleosome architecture in human and show that, despite variation across functional regions, cis-factors have a very modest in influence on nucleosome positioning. Finally, in chapter four, we will aim to identify clusters of histone modifications specific to functionally distinct regions, characterize their function and their association with gene expression level.