RSC remodeling of oligo-nucleosomes: an atomic force microscopy study

RSC is an essential chromatin remodeling factor that is required for the control of several processes including transcription, repair and replication. The ability of RSC to relocate centrally positioned mononucleosomes at the end of nucleosomal DNA is firmly established, but the data on RSC action on oligo-nucleosomal templates remains still scarce. By using Atomic Force Microscopy (AFM) imaging, we have quantitatively studied the RSC- induced mobilization of positioned di- and trinucleosomes as well as the directionality of mobilization on mononucleosomal template labeled at one end with streptavidin. AFM imaging showed only a limited set of distinct configurational states for the remodeling products. No stepwise or preferred directionality of the nucleosome motion was observed. Analysis of the corresponding reaction pathways allows deciphering the mechanistic features of RSC-induced nucleosome relocation. The final outcome of RSC remodeling of oligosome templates is the packing of the nucleosomes at the edge of the template, providing large stretches of DNA depleted of nucleosomes. This feature of RSC may be used by the cell to overcome the barrier imposed by the presence of nucleosomes.

💡 Research Summary

In this study the authors employed atomic force microscopy (AFM) to directly visualize and quantitatively analyze how the yeast chromatin‑remodeling complex RSC (Remodels Structure of Chromatin) acts on defined oligonucleosomal substrates. Using the high‑affinity 601 positioning sequence, they assembled homogeneous dinucleosome and trinucleosome templates in which the nucleosomes are precisely spaced (58 bp linker for dinucleosomes, 50 bp for trinucleosomes) and flanked by free DNA arms. Biochemical validation by EMSA and DNase I footprinting confirmed that both nucleosomes are correctly positioned and fully occupied by histone octamers.

RSC was added together with ATP and incubated at 29 °C for varying times or concentrations. After stopping the reaction by dilution, the samples were deposited on APTES‑mica and imaged in air in tapping mode. In the absence of ATP no remodeling was observed, whereas the presence of ATP and RSC generated a limited set of five distinct configurational states, designated #1 through #5. State #1 corresponds to the initial arrangement with both nucleosomes occupying their 601 sites. State #2 shows one nucleosome displaced to a DNA end while the other remains at its positioning site. State #3 represents two nucleosomes packed together after one has slid into the other’s vicinity. State #4 has both nucleosomes packed at the same DNA end, and State #5 displays each nucleosome at opposite ends of the DNA fragment. Importantly, no intermediate “stepwise” positions were detected; the system jumps between these quantized configurations.

Quantitative analysis of >1,000 particles per condition revealed that increasing RSC concentration (or reaction time) progressively depletes the initial state #1, transiently populates the intermediate states #2 and #3, and finally enriches the terminal states #4 and #5. This behavior indicates that RSC moves nucleosomes until they encounter a physical barrier – either a neighboring nucleosome or the DNA terminus – at which point sliding stops. The equivalence of RSC concentration and incubation time when plotted as a normalized reaction coordinate suggests Michaelis–Menten‑type kinetics for the remodeling reaction under the experimental conditions.

Additional experiments with mononucleosomes bearing a streptavidin label at one DNA end demonstrated that RSC does not exhibit a preferred direction of movement; nucleosomes slide equally well toward either DNA end. A minor fraction of particles showed complete eviction of one histone octamer, but this occurred at low frequency and was not central to the mechanistic conclusions.

Collectively, the data support a model in which RSC remodels chromatin not by a gradual, incremental sliding of nucleosomes along DNA, but by a “push‑till‑stop” mechanism: a nucleosome is translocated in a processive manner until it reaches an obstacle, leading to a quantized final position. This results in the packing of nucleosomes at DNA termini and the creation of extended nucleosome‑free stretches. In vivo such a mechanism could be exploited to clear nucleosomal barriers ahead of transcription, replication or DNA repair machineries, thereby facilitating access to underlying DNA sequences. The study therefore expands the conceptual framework of chromatin remodeling, positioning RSC as a factor capable of generating large nucleosome‑free domains through directed nucleosome crowding rather than simple repositioning.