Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.Comment: 18 pages, 7 figures, Supplementary information available at http://www.nature.com/nsmb/journal/v13/n5/suppinfo/nsmb1087_S1.htm

Nucleosome Chiral Transition under Positive Torsional Stress in Single Chromatin Fibers

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns, at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the (H3-H4)2 tetramer bound with ~50 bp of DNA) obtained by depletion of H2A-H2B dimers, suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented righthanded tetrasome. In view of its low energy, <8 kT, we propose this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer which in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.Comment: 33 pages (double spacing), 7 figure

Chromatin polymorphism and the nucleosome superfamily: a genealogy.

International audienceNucleosomes were discovered more than thirty years ago as the basic repeating units of chromatin. Since then, nucleosomes have progressively revealed their taste to come in many appearances, upon either adjunction of other proteins (e.g., a fifth histone or a nonhistone protein, HMG-N), histone substitution for isoforms (histone variants), depletion of one or the two H2A-H2B dimers (sub-nucleosomes), intimate two-particle association, or isomeric structural alterations. The resulting entities, some of them are only transient, acquire new properties useful for their specific roles in chromatin function. These structures are presented here in the chronological order of their identification, from the chromatosome to the sub-nucleosomal hexasome and tetrasome, and from the dinucleosomal altosome and nucleodisome to the nucleosome variants and altered forms: the old lexosome and the most recent reversome

Polymorphisme structural et dynamique du nucléosome (influence de la séquence d'ADN)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Le nucléosome centromérique et la fibre de chromatine

Ce mémoire présente les résultats que j ai obtenus pendant ces années passées au laboratoire de Biochimie de la Chromatine, le plus souvent en collaboration avec d autres laboratoires en France, en Ukraine et aux Etats-Unis. Deux questions m ont particulièrement intéressée : Structure et dynamique du nucléosome centromérique. Le centromère, qui permet la ségrégation des chromatides sœurs pendant la mitose, est un des plus gros complexes nucléaires. Alors qu on connaît maintenant beaucoup des protéines qui le constituent, on ne savait pratiquement rien sur la structure et la dynamique du nucléosome centromérique, qui a la particularité de contenir un variant de H3 : CENP-A. La technique originale des minicercles, développée au laboratoire depuis de nombreuses années, a été ici irremplaçable. Les résultats, inattendus, nous ont permis de donner un élément de réponse à la question centrale du mécanisme de l adressage de ce variant d histone au centromère (article I). Réponse en rotation d une fibre de chromatine. La réponse en traction d une fibre de chromatine est relativement bien connue. Mais nous sommes les premiers à avoir étudié sa réponse en rotation, grâce à un système de pinces magnétiques. Les résultats ont permis de généraliser à la fibre de chromatine les notions d états conformationnels du nucléosome (article II) et de transition chirale du tétrasome (un constituant du nucléosome), initialement mises au jour grâce au système des minicercles. On a ainsi montré que la transition chirale pouvait concerner le nucléosome entier, et pas seulement le tétrasome (article III).PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

CENP-A-containing Nucleosomes: Easier Disassembly versus Exclusive Centromeric Localization.

International audienceCENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the alpha-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(+/-2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)(2) tetramer was also more readily destabilized with heparin than the (H3-H4)(2) tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression

Structure et fonction de la chromatine

SIGLECNRS-CDST / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc