Structural plasticity of single chromatin fibers revealed by torsional manipulation
📝 Abstract
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.
💡 Analysis
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.
📄 Content
time. Force micromanipulation has revealed the existence of an internucleosomal attraction that maintains the higher-order chromatin structure in physiological conditions 5 and a reversible peeling of ~80 bp of nucleosomal DNA below 15 pN 6 , presumably accompanied by the destabilization of H2A-H2B dimers. Above this force, discrete disruption events of 25 nm each were observed, which were attributed to tetrasome ((H3-H4) 2 -DNA complex) dissociation 6,7 .
Here, we report the first investigation of the torsional response of single chromatin fibers using magnetic tweezers 8 . Nucleosome arrays reconstituted on 5S tandemly repeated positioning sequences were found to be able to accommodate large amounts of negative or positive supercoiling without much change in their length. A quantitative model is proposed, based on a dynamic equilibrium between the three conformations of the nucleosome previously identified through the minicircle approach (a single nucleosome reconstituted on a DNA minicircle) 9 . In these states, the nucleosome entry/exit DNAs can cross negatively (as in the canonical structure 2 ), positively, or do not cross at all. The model fits the chromatin length-vs.-torsion response at different levels of compaction. It also shows how the torsional constraint, depending on its amplitude and sign, can force nucleosomes to switch conformation, and induce a large reorganization of the fiber architecture. These findings provide simple answers to long-standing topological mysteries of DNA in chromatin. Moreover, the dynamic chromatin it describes may underlie the dynamic nature of the binding of most chromatin-associated proteins 10,11 .
Nucleosome arrays were reconstituted by stepwise dilution using a linear DNA containing 36 tandemly repeated 208 bp 5S positioning sequences 12 , and core histones purified from chicken erythrocytes. These fibers were then flanked by two naked DNA spacers, to avoid histone-mediated hydrophobic interaction with the surfaces, and by two “stickers” that link the fiber to the coated bottom of the flow cell and to the paramagnetic bead (blue and orange segments in Fig. 1). A pair of magnets was placed above this construction, and different torsions were applied by rotating the magnets about the vertical axis. The magnets’ vertical position specifies the stretching force, i.e. the fiber extension, which was measured by recording the 3D-position of the bead 8 .
The typical torsional behavior of a single chromatin fiber in low salt buffer B 0 (see Methods) is shown in Fig. 2a at 0.34 pN (blue curve). Following chemical dissociation of the nucleosomes, the response of the corresponding naked DNA was obtained (red). This latter curve displays a mechanical effect of torsion and an asymmetry for negative supercoiling, which are signatures of an unnicked single duplex DNA 8 . Compared to naked DNA, chromatin is shorter by ~1.35 µm, and its centre of rotation is shifted by -24±2 turns. This corresponds to a shortening of ~-55 nm per negative turn, as expected for one nucleosome, since 50 nm correspond to 150 bp.
Nucleosomes were also disrupted mechanically by increasing the tension, after supplementing B 0 with 50 mM NaCl and 2×10 -3 % NAP-1 (Nucleosome Assembly Protein-1, gift from S. Leuba). At 7.7 pN, 14 individual lengthening steps with an average height of 24.2±1.9 nm were detected (Fig. 2b), in agreement with 6 . This process occurred at a lower force than in 6 , presumably because NAP-1 interacts with core histones in vitro 13 and favors their release. Interestingly, it was partially reversible, as also reported in 6 . In the course of two successive pulling phases at 7.7 pN, separated by a 50 sec pause at 0.67 pN, the fiber contracted by -28.5 nm during the pause, roughly corresponding to one individual reassociation.
The response in torsion of the partially disrupted fiber was subsequently probed in B 0 at low force and, as expected, found to be intermediate, in both rotation and length, between the responses of the original fiber and of DNA (Fig. 2a, green). The shifts in length and in topology between the new fiber and DNA were respectively ~700 nm and -13±1.5 turns, or ~54 nm per turn, identical to the above value. Assuming that each step corresponds to the dissociation of one nucleosome, then the topological deformation per nucleosome can be estimated to -(11±1.5)/14=-0.8±0.1 turn.
The rotational behavior of ten fibers was subsequently compared by plotting their maximal length at 0.3 ± 0.07 pN versus the rotational shift of those maxima relative to their corresponding naked DNA (Fig. 2c). A linear trend was observed, with most data points well-aligned and a rate close to 55 nm/turn. This is the expected behavior for regular nucleosome arrays with a variable number of nucleosomes. The corresponding nucleosome arrays were thus referred to as regular. A few fibers, however, deviated from this linear trend. We show in the Supplementary (Fig. S1) that these deviations
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