Dynamics of nucleosomes revealed by time-lapse atomic force microscopy

Abstract

The dynamics of chromatin provides the access to DNA within nucleosomes, and therefore, this process is critically involved in the regulation of chromatin function. However, our knowledge of the large-range dynamics of nucleosomes is limited. Answers to the questions, such as the range of opening of the nucleosome and the mechanism via which the opening occurs and propagates, remain unknown. Here we applied single-molecule time-lapse atomic force microscopy (AFM) imaging to directly visualize the dynamics of nucleosomes and identify the mechanism of the large range DNA exposure. With this technique, we are able to observe the process of unwrapping of nucleosomes. The unwrapping of nucleosomes proceeds from the ends of the particles, allowing for the unwrapping of DNA regions as large as dozens of base pairs. This process may lead to a complete unfolding of nucleosomes and dissociation of the histone core from the complex. The unwrapping occurs in the absence of proteins involved in the chromatin remodeling that require ATP hydrolysis for their function, suggesting that the inherent dynamics of nucleosomes can contribute to the chromatin unwrapping process. These findings shed a new light on molecular mechanisms of nucleosome dynamics and provide novel hypotheses about the understanding of the action of remodeling proteins as well as other intracellular systems in chromatin dynamics.

Figure 1

Schematics for nucleosome particles with different number of DNA supercoiling turns around the histone core.

Figure 2

AFM image taken in air of the nucleosome samples. Nucleosomes marked with numbers 1.7, 1.4 and 1.0 correspond to NCPs with ∼1.7, ∼1.4 and ∼1.0 turns of DNA wrapped around the core particle.

Figure 3

Two step unwrapping process. (A) Consecutive AFM images of nucleosomes with two step unwrapping process, taken during continuous scanning in the buffer. Each frame is 200 nm. (B) Dependence of arm lengths on the frame number. (C) The dependence of number of DNA turns around the core calculated from arm lengths (black) and from the angle between DNA arms (red) on the frame number. The dependence of nucleosome volume on the frame number is shown in blue (right Y-axis). Each frame takes about 170 second to scan.

Figure 4

One step unwrapping process. (A) Consecutive AFM images of nucleosomes with one step unwrapping, taken during continuous scanning in the buffer. Scan size is 100 nm. (B) Dependence of arm lengths (in nm) on the frame number. (C) The dependence of number of DNA turns around the core calculated from arm lengths (black) and from the angle between DNA arms (red) on the frame number. The dependence of nucleosome volume on the frame number is shown in blue (right Y-axis). Each frame takes about 170 second to scan.

Figure 5

The dependence of the number of DNA turns around the core calculated from the arm lengths (black) and from the angle between the DNA arms (red) on the frame number for two sets of the data shown in Figs. S1 and S2 respectively. The dependence of nucleosome volume on the frame number is shown in blue (right Y-axis).