Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

Abstract

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

Figure 1.

Crude sketch of force-controlled nucleosome assembly/disassembly. For naked DNA (a) under tension f, wrapping of a length l of DNA around histones is necessary to assemble a nucleosome (b), doing mechanical work against the applied force of fl. When wrapping and unwrapping occur at equal rates, no net assembly or disassembly will occur, corresponding to assembled nucleosomes and their diassembled components being in chemical equilibrium and therefore equal in free energy. The force at which assembly and disassembly are balanced can therefore be used to compute this free energy difference, via ΔG = fl.

Figure 2.

MT setups. (a) Vertical MT system. A DNA molecule is suspended from the top surface of a flow cell by one end, with its other end attached to a 3-μm-diameter paramagnetic particle that acts as a “handle” to which controlled forces can be applied, by using a permanent magnet below the flow cell. The bead is observed using a microscope objective; a piezoelectric focuser is used to locate the bead in the vertical (z) direction. Illumination of the bead is done through the objective and therefore a reflected-light (dark-field) image is obtained (bottom). Buffer exchanges are carried out using flow through the sample cell. (b) Transverse MT system. The principle is the same as for the vertical tweezer, but with the modifications that in addition to the paramagnetic particle at the right end of the molecule, a nonmagnetic particle is attached at its left end, held using suction on a thin glass capillary. By using a small magnet that can be positioned in the same plane or even below the end of the pipette, forces on the paramagnetic bead can be applied with direction in the plane of focus. The result is that both ends of the DNA molecule can be imaged simultaneously (bottom). An open sample cell is used allowing straightforward removal and replacement of buffer.

Figure 3.

Assembly of chromatin using diluted Xenopus egg extract, against constant forces, for −ATP (apyrase-treated) conditions. (a) Extension of a 97-kb DNA versus time measured by the vertical MT system, for most of the assembly reaction against a 1-pN force. The initial rapid contraction of the molecule during the early stage of assembly starting at the initial extension of 28 μm was not recorded. Boxes are drawn showing the two regions where exponential fits to the data can be made: the initial time course (top box) fits an exponential with a relaxation time of 10 min; later data in the bottom right box fit a slower relaxation time of 35 min. (b) Extension divided by naked DNA contour length l₀ (reduced extension) for different-length assembly runs against forces of 1 pN (filled squares, 97 kb; open circles, 64 kb). Collapse of the curves onto one exponential demonstrates the simple scaling found for the −ATP assembly reaction. (c) Chromatin assembly and disassembly speeds for −ATP conditions show the force dependence expected in a simple force-controlled chemical reaction; figure shows “reduced velocities” (bead velocities divided by naked DNA contour length) of chromatin assembly (negative) and disassembly (positive). A smooth force–velocity relation is obtained, with a zero velocity or stall force of 3.5 pN (dashed line). At this point, assembly and disassembly processes compensate for one another, resulting in no net change in DNA compaction.

Figure 4.

Transverse MT results giving high-resolution DNA extension during chromatin assembly/disassembly, under −ATP (apyrase-treated) conditions. Each panel shows a time series of extension measurements at constant force as indicated. (a) Naked 79-kb DNA under a constant tension of 2.8 pN. The extension shows thermal fluctuations ∼100 nm around a fixed extension. (b) Same DNA as in a, following injection of −ATP extract. Assembly occurs as in Figure 3, but at the higher resolution of the transverse MT, step events of roughly 100-nm amplitude can be observed. Note that assembly and disassembly steps occur, but net assembly occurs due to a larger number of assembly steps. (c) Same DNA as a and b, but now at 3.5 pN, the stall force of the assembly reaction. Note the fluctuations of extension around its roughly constant value; by comparison with a, in addition to Brownian fluctuations, there are step-like fluctuations, of amplitude 50 and 100 nm. (d) Disassembly time series for 4.5 pN. After assembly of a fiber onto a 49-kb DNA at 1 pN, force was increased to 4.5 pN, leading to the disassembly trace shown here. The thermal fluctuations are slightly reduced by both the shorter DNA and the higher force. Step events of 50 and 100 nm are observed. (e) Same DNA as in d but now with force increased to 15 pN. Disassembly occurs more rapidly, and the steps are even more visible. Again, 50- and 100-nm steps are observed. (f) Disassembly time series of a chromatin fiber assembled onto a 15-kb DNA at 9.6 pN. Besides reducing the noise by the shorter molecule, plateau durations are increased due to the smaller number of nucleosomes along the DNA. Extension averages during the plateaus (white lines) allow step size measurement. A 25-nm step, a 50-nm step, and a 75-nm step can be seen. (g) Histogram of the step sizes for 188 steps collected from disassembly runs of 49-kb DNAs by using more than 9 pN. A well-defined peak occurs at ∼50 nm.

Figure 5.

Effect of ATP addition on a chromatin fiber initially assembled with no ATP. Time series of extension measurements by using the transverse MT are shown, for a 49-kb DNA template. Before 180 s: After assembly of chromatin fiber at 1 pN by using untreated extracts, force was increased to 3.5 pN, the stall force for the −ATP reaction. As expected, the behavior of the fiber was similar to that observed under ATP-depleted conditions, with small step-like fluctuations plus thermal extension fluctuations. No net assembly or disassembly occurs. The behavior of the fiber is essentially the same as that observed for the ATP-depleted extract product (Figure 4C). After 300 s: After addition of +ATP extract solution into the experimental chamber, force still held at 3.5 pN. There are two main effects: the fiber tends to disassemble, and large-amplitude, apparently processive runs of extension are observed, in both assembly and disassembly directions.

Figure 6.

Extension dynamics during chromatin assembly/disassembly using extracts with added ATP. Panels show time series of extension for a 97-kb molecule measured using the transverse MT. (a) At a force of 0.7 pN, assembly gradually compacts the DNA. The initial DNA extension of roughly 28 μm is reduced by assembly. The shape of the assembly curve is rather linear apart from some large-scale variations, distinct from the nearly exponential assemblies observed for the −ATP case. (b) Higher resolution plot of extension variations for the fiber of a, for a time window 3 min after the end of a. Large extension Λ and V events are observed, characterized by a well-defined velocity which is the same for the opening and closing legs. Extension remains near 12 μm. (c and d) After increasing force on the fiber of b to 1.2 and 1.7 pN, extension gradually increases, with continuation of the Λ and V events. (e) After increase of force on the fiber of c to 2.7 pN, further extension occurs, again with large Λ and V events. (f) After increasing force to 4.2 pN, further extension occurs without Λ and V events.