Chromatin fiber dynamics under tension and torsion

Abstract

Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.

Keywords: DNA; chromatin; magnetic tweezers; nucleosome; optical tweezers; single molecule.

Figure 1.

Molecular microscopy views of chromatin fibers. (a) Transmission Electron Microscopy (TEM) image of a nucleosomal array extracted from Chinese hamster ovary cells, spread in water and observed in annular darkfield mode after uranyl acetate staining (bar 100 nm); adapted from [7]. Insert: nucleosome crystal structure (from 1kx5 PDB coordinates). (b) Atomic Force Microscopy (AFM) image of unfixed chromatin fibers extracted from chicken erythrocytes and spread on glass in low ionic strength buffer (imaged area 600 × 600 nm); adapted from [8].

Figure 2.

Various methods have been developed to manipulate single nucleosomal arrays. Depending on the setup, these experiments enable the study of nucleosome assembly/disassembly under constraints, to apply force (tension) and/or torque (torsion) on chromatin fibers and measure their mechanical response. These techniques use either OT (a,b), MT (c,d), flow (d,e) or the cantilever of an AFM (f) to apply constraints to a chromatin fiber attached at the other end to the surface of a cover slip (a,c,d,f) or to the extremity of a micropipette (b,e). Chromatin can thus be pulled (a,b,c,d,e,f) and rotated (c). a, b, and f are position clamps, while c, d, and e can be used as both force or position clamps.

Figure 3.

Force/extension curves and their interpretation (see experimental setup Figure 2a). (a) Force versus length response of a single chromatin fiber handled by an OT. The fiber was directly assembled in the flow cell of the instrument from a single λ DNA molecule and Xenopus cell-free extracts: these contain all core histones but lack linker histones. A portion of the representative force curve (upper panel) is enlarged in the lower panel. The discontinuities in the curve correspond to unraveling of individual nucleosomes within the fiber. The blue curve represents the relaxation) response, which exhibited a naked DNA-like behavior [35]. (b) A schematic of the step-wise mechanical disruption of the nucleosomal particle (unpeeling of nucleosomal DNA from the histone core) as suggested by Brower-Toland et al. [37] (see also Table 1).

Figure 4.

Rotation/extension curves and their interpretation (see experimental setup in Figure 2c). (a) Length versus rotation response at 0.35 pN of naked DNA (red), partially reconstituted (green) and saturated (blue) nucleosomal array. (b) Length versus rotation response of a saturated reconstituted nucleosomal array. Hysteresis is observed between the onward (blue) and backward (green) curves when a high positive torsion is applied [up to 70 positive turns, while torsion applied was less than 50 turns in (a)]; the zero-turn rotation reference corresponds to the relaxed state of naked DNA (red dotted curve). (c) The shortening, shifting and flattening of the curves in (a) is interpreted as the consequence of nucleosome reconstitution (each nucleosome wraps ∼50 nm of DNA in one negative superhelical turn) and conformational flexibility (three-state model) [31]. (d) The hysteresis observed at high torsion in (b) is interpreted as the consequence of a transient chiral transition of nucleosomes to an altered right-handed form [53].

Figure 5.

Nucleosome remodeling assessed by OT. (a) The nucleosome construct is attached to the bead by a biotin-streptavidin bond and to the coverslip by a digoxigenin--anti-digoxigenin linkage. The bead is kept fixed by tuning the laser power (optical trap) while coverslip is moving away from the bead, imparting the unzipping of the (in red) part of the dsDNA from the nick. (b) Plotting applied force (calibrated by laser power) versus number of base pairs unzipped provides a mapping of nucleosome position at single base-pair resolution. The three curves are the results obtained with three different constructs: naked DNA (in black), tetrasome (in blue) and nucleosome (in red). After remodeling by SWI/SNF the nucleosome is moved away from its initial position. Repeating the experiment on a sample of ∼150 nucleosomes results in a histogram of displacements, that is symmetrical around zero with a standard deviation of 28 bp.