Nucleosome hopping and sliding kinetics determined from dynamics of single chromatin fibers in Xenopus egg extracts

Abstract

Chromatin function in vivo is intimately connected with changes in its structure: a prime example is occlusion or exposure of regulatory sequences via repositioning of nucleosomes. Cell extracts used in concert with single-DNA micromanipulation can control and monitor these dynamics under in vivo-like conditions. We analyze a theory of the assembly-disassembly dynamics of chromatin fiber in such experiments, including effects of lateral nucleosome diffusion (''sliding'') and sequence positioning. Experimental data determine the force-dependent on-and off-rates as well as the nucleosome sliding diffusion rate. The resulting theory simply explains the very different nucleosome displacement kinetics observed in constant-force and constant-pulling velocity experiments. We also show that few-piconewton tensions comparable to those generated by polymerases and helicases drastically affect nucleosome positions in a sequence-dependent manner and that there is a long-lived structural ''memory'' of force-driven nucleosome rearrangement events.chromatin assembly ͉ chromatin disassembly I n the nucleus, chromatin undergoes continual structural rearrangement. Chromatin fibers have been observed to undergo large-scale diffusion-like motions (1, 2) and rapid local motions (3), possibly caused by the action of processive enzymes such as nucleic acid polymerases and helicases. At smaller scales, fluorescence recovery after photobleaching studies in vivo have shown histones to be mobile to some degree (4). Both large-scale conformational and nucleosomal rearrangements are biologically important, e.g., through their influence on gene regulation (5, 6).These in vivo results are complemented by biochemical experiments indicating that DNA can be transiently released from nucleosomes (7,8) and recent DNA-pulling experiments that show nucleosome disruption by forces ranging from a few to tens of piconewtons (9-12). Single-nucleosome or single-chromatinfiber experiments carried out in protein-free buffers provide useful quantifications of histone-DNA interaction strengths and force-driven opening rates. However, affinities and rates obtained from studies of isolated fibers may be very different relative from those occurring in vivo because of the very high levels of chromatin-acting enzymes found in the cell. This issue can be addressed by combining cell extracts with single-molecule methods for reading out folding and unfolding of protein-DNA complexes in real time, which permits observation of singlechromatin fiber dynamics under conditions close to those found in vivo (10,(13)(14)(15)(16).Here, we present a theory of chromatin fiber dynamics in Xenopus egg extracts, based on assembly, displacement, and lateral diffusion (''sliding'') of nucleosome units. Recent experimental data for sequence dependence of nucleosome affinities (17), combined with measurements of single-chromatin fiber assembly and disassembly in Xenopus egg extracts, constrain the theory sufficiently to determine force-dependent nucleosome on-and off-rates, the seque...

Nucleosome positioning in a model of active chromatin remodeling enzymes

“…To examine the ATP-independent case we computed the dynamics with two different thermal nucleosome diffusion rates estimated previously: D ¼ 10 −17 cm 2 ∕s (0.01 bp 2 ∕s) estimated theoretically (24,25) and D ¼ 10 −15 cm 2 ∕s as estimated experimentally for ATP-depleted Xenopus egg extracts (19). As shown by red and green curves in Fig.…”

“…The on-rate r on ¼ 12 s −1 determined experimentally in cell-extract experiments (19) was used. We take the "hard core" size of nucleosomes to be k ¼ 147 bp.…”

“…19. In brief, at each step of the computation we use the rates of the events that are possible to compute the time interval until the next stochastically determined on-, off-, or slide event.…”

“…The probability P i at low nucleosome density determines the potential V i ¼ −k B T ln P i , up to an overall constant. To determine the absolute value of V i under cellular conditions, we use the value hVi ¼ 1 N ∑ N i¼0 V i ¼ −42 k B T determined experimentally from nucleosome assembly experiments in ATP-depleted Xenopus egg extracts (19).…”

Shape Universality Classes in the Random Sequential Adsorption of Nonspherical Particles

“…Such an insight is important, e.g., to improve the modelling of nucleosome adsorption on DNA, which is described by variants of RSA processes on a 1d line [45][46][47]. Nucleosomes indeed have non-spherical shapes and can adsorb in variable orientations [48,49].…”