55 research outputs found
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 âŒ10âbp 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 âdefectiveâ 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
SĂ©quençage de lâADN par nanopores
AprĂšs des annĂ©es de dĂ©veloppement, lâutilisation du nanopore comme sonde pour sĂ©quencer les molĂ©cules dâADN est maintenant une possibilitĂ© viable et prometteuse. La dĂ©tection dâune seule paire de bases lors du transport de lâADN permet dâenregistrer de trĂšs longs fragments de polynuclĂ©otides, avec une parallĂ©lisation et des vitesses Ă©levĂ©es. Dans cette revue, les mĂ©thodologies actuelles fondĂ©es sur la dĂ©tection Ă©lectrique et les nanopores biologiques seront prĂ©sentĂ©es de mĂȘme que les nouvelles mĂ©thodes utilisant des nanopores Ă lâĂ©tat solide, ou la dĂ©tection 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 nuclĂ©osome interfĂšre avec diffĂ©rents processus cellulaires. La cellule recrute des facteurs de remodelage et des variants d'histones pour surmonter cette barriĂšre physique. Dans ce travail, nous utilisons la Microscopie Ă Force Atomique pour visualiser des mono- et des oligo- nuclĂ©osomes Ă l'Ă©quilibre et hors-Ă©quilibre.Nous montrons que le variant H2A.Bbd modifie la structure et la dynamique du mono-nuclĂ©osome et que sa prĂ©sence altĂšre la facultĂ© de la chromatine Ă former une structure d'ordre supĂ©rieur. En utilisant un modĂšle physique nous expliquons quantitativement ce comportement par la flexibilitĂ© du mono-nuclĂ©osome.Nous Ă©tudions ensuite le mĂ©canisme du remodelage de mono-nuclĂ©osomes par SWI/SNF et RSC. Nous mettons en Ă©vidence un intermĂ©diaire rĂ©actionnel sous la forme d'un nuclĂ©osome sur-complexĂ© apparaissant avant le nuclĂ©osome glissĂ©. Enfin au niveau des di-nuclĂ©osomes nous montrons que RSC est un ârandomiseur' processif et sĂ©quentiel
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