Single chromatin fibers were reconstituted in vitro by salt dialysis from purified histone octamers and 2×18 tandem repeats of the 5S DNA positioning sequence. The fibers were flanked by naked ∼600 bp DNA spacers and ∼500 bp DNA stickers modified with digoxigenin and biotin destined to be linked respectively to the coated bottom of the flow cell and to the paramagnetic bead. This construction is then placed under the rotating magnet of a magnetic tweezers set-up to exert a torque and a pulling force on the fiber. The fiber stretching (force-vs.-extension) and torsional (extension-vs.-rotation) behaviors were then recorded. Whereas the stretching behavior is similar to that previously observed by other authors using e. g. optical tweezers, the torsional behavior shows a bell-shaped curve with a breath much larger than obtained with naked DNA of the same length. These curves were fitted with the worm-like rope model widely used for DNA, which represents the molecule (here the chromatin fiber) as an isotropic elastic rod of defined bending, stretching and twisting moduli. Good fittings were obtained with a fiber bending persistence length of 28 nm and a stretching modulus of 8 pN, in agreement with previous studies. In contrast, the twisting persistence length, obtained here for the fist time, was exceptionally low, ∼5 nm against ∼80 nm for DNA. Such a fiber high torsional resilience was inconsistent with the existence of nucleosomes locked in their canonical negatively-crossed conformation. In contrast, it could be described by a molecular model of the fiber architecture in which nucleosomes are in a thermodynamic equilibrium between the three conformational states initially identified for single nucleosomes on DNA minicircles (reviewed in ref. 1), depending on the crossing statuses of entry-exit DNAs (negative, null or positive). Beyond applied torsions at which all nucleosomes are forced to cross either negatively or positively, the fiber length decreases rapidly and linearly. By analogy to the torsional behavior of DNA (ref. 2), this was attributed to the formation of plectonemes, in which nucleosomes are extruded to the outside (ref. 3). When fibers were submitted to large positive torsions beyond their maximal compaction, the backward curves obtained upon reversing the torsion depart from the onward curves. A positive shift is observed at positive torsions which progressively disappears at negative torsions. This reversible hysteresis corresponds to the transition of the nucleosomes to a transient altered state which traps one positive turn. Comparison with the response of fibers of tetrasomes obtained through depletion of H2A-H2B dimers using NAP-1, heparin or salt led us to conclude that the transition involves three main steps : 1) a breaking of the docking of the dimers on the (H3-H4)2 tetramer; 2) a switching of the tetramer from its left-handed to the right-handed chiral conformation previously described (reviewed in ref. 4); and 3) a undetermined rearrangement of the dimers insuring that the overall compaction of the resulting “reversomes” (for reverse nucleosomes) is similar to that of the starting nucleosomes (ref. 5) This dynamics of the nucleosomes at the level of the entry-exit DNAs, which leads to a large reorganization of the three-dimensional fiber architecture, may affect DNA binding of regulating proteins in vivo, as all tracking processes, e. g. replication and transcription, involve the generation of torsional stress. The nucleosome-reversome transition is likely to serve to relieve the almost insurmountable block against transcription by the main RNA polymerase otherwise exerted by H2A-H2B dimers in the absence of intervening factors. References 1) Prunell A. & Sivolob A. (2004) . "Paradox lost : Nucleosome structure and dynamics by the DNA minicircle approach" In Chromatin structure and dynamics : state-of-the-art. (Zlatanova, J. & Leuba, S. H., eds), Elsevier Science, Amsterdam. New Comprehensive Biochemistry, 39, 45-73. 2) Strick, T. R., Allemand, J. F., Bensimon, D., Bensimon, A. and Croquette, V. (1996). The elasticity of a single supercoiled DNA molecule. Science 271, 1835-1837. 3) Bancaud A., Conde e Silva N., Barbi M., Wagner G., Allemand J.-F., Mozziconacci J., Lavelle C., Croquette V., Victor J.-M., Prunell A. & Viovy J.-L. (2006). Structural plasticity of single chromatin fibers revealed by torsional manipulation. Nat. Struct. Mol. Biol. 13, 444-450. 4) Sivolob A. & Prunell A. (2004). Nucleosome conformational flexibility and implications for chromatin dynamics. Phil. Trans. Roy. Soc. A. 362, 1519 - 1547. 5) Bancaud A., Wagner G., Conde e Silva N., Lavelle C., Wong H., Mozziconacci J., Barbi M., Sivolob A., Le Cam E., Mouawad L., Viovy J.-L., Victor J.-M. & Prunell A. (2007). Nucleosome chiral transition under positive torsional stress in single chromatin fibers. Mol. Cell 27, 135-147.