RSC remodeling of oligo-nucleosomes: an atomic force microscopy study

RSC is an essential chromatin remodeling factor that is required for the control of several processes including transcription, repair and replication. The ability of RSC to relocate centrally positioned mononucleosomes at the end of nucleosomal DNA is firmly established, but the data on RSC action on oligo-nucleosomal templates remains still scarce. By using Atomic Force Microscopy (AFM) imaging, we have quantitatively studied the RSC- induced mobilization of positioned di- and trinucleosomes as well as the directionality of mobilization on mononucleosomal template labeled at one end with streptavidin. AFM imaging showed only a limited set of distinct configurational states for the remodeling products. No stepwise or preferred directionality of the nucleosome motion was observed. Analysis of the corresponding reaction pathways allows deciphering the mechanistic features of RSC-induced nucleosome relocation. The final outcome of RSC remodeling of oligosome templates is the packing of the nucleosomes at the edge of the template, providing large stretches of DNA depleted of nucleosomes. This feature of RSC may be used by the cell to overcome the barrier imposed by the presence of nucleosomes

Stress Clamp Experiments on Multicellular Tumor Spheroids

The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10 kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression.Comment: 5 pages, 4 figure

The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct AFM visualization of variant chromatin

Chromatin organization and dynamics is studied in this work at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique and numerical modeling. We demonstrate that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by AFM, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show "beads on a string" structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results strongly suggest that higher-order organization of H1-free nucleosomal arrays is mainly determined by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers.Comment: Biophys J. in pres

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a ‘defective’ docking domain may be a primary structural role of H2A.Bbd in chromatin

The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA

AFM Imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding

We propose a combined experimental (Atomic Force Microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows to determine simultaneously the DNA complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleo-proteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the in the length distribution of DNA complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA complexed length, we extract the net wrapping energy of DNA onto the histone octamer, and compare it to previous studies.Comment: 25 pages,5 figures, to appear in Biophysical Journa

Séquençage de l’ADN par nanopores

Après des années de développement, l’utilisation du nanopore comme sonde pour séquencer les molécules d’ADN est maintenant une possibilité viable et prometteuse. La détection d’une seule paire de bases lors du transport de l’ADN permet d’enregistrer de très longs fragments de polynucléotides, avec une parallélisation et des vitesses élevées. Dans cette revue, les méthodologies actuelles fondées sur la détection électrique et les nanopores biologiques seront présentées de même que les nouvelles méthodes utilisant des nanopores à l’état solide, ou la détection optique

Dynamique à l'équilibre et hors d'équilibre de la chromatine visualisée par microscopie de force atomique : effet des variants d'histones et des facteurs de remodelage

The organization of DNA into nucleosome interferes with several cellular processes. To overcome this physical barrier, the cell recruits ATP-remodeling machines and histone variants. In this work, we use Atomic Force Microscopy to visualize isolated mono- and oligo-nucleosomes at equilibrium and out of equilibrium.We show that H2A.Bbd variant incorporation into the nucleosome modifies both the structure and dynamics of the mono-nucleosome and that its presence alters the ability of chromatin to form a higher order structure. Using a polymer model, we quantitatively explain this behavior by the mono-nucleosome flexibility. Then, studying the mechanism of nucleosome remodeling by SWI/SNF and RSC on mono-nucleosomes, we reveal a reaction intermediate visible as an over-complexed nucleosome before the appearance of the final slided state. Focusing on di-nucleosomes we report various slided states and construct a stochastic model showing that RSC is a processive and sequential randomizer.L'organisation de l'ADN sous forme de nucléosome interfère avec différents processus cellulaires. La cellule recrute des facteurs de remodelage et des variants d'histones pour surmonter cette barrière physique. Dans ce travail, nous utilisons la Microscopie à Force Atomique pour visualiser des mono- et des oligo- nucléosomes à l'équilibre et hors-équilibre.Nous montrons que le variant H2A.Bbd modifie la structure et la dynamique du mono-nucléosome et que sa présence altère la faculté de la chromatine à former une structure d'ordre supérieur. En utilisant un modèle physique nous expliquons quantitativement ce comportement par la flexibilité du mono-nucléosome.Nous étudions ensuite le mécanisme du remodelage de mono-nucléosomes par SWI/SNF et RSC. Nous mettons en évidence un intermédiaire réactionnel sous la forme d'un nucléosome sur-complexé apparaissant avant le nucléosome glissé. Enfin au niveau des di-nucléosomes nous montrons que RSC est un ‘randomiseur' processif et séquentiel