Dynamic Properties of Nucleosomes during Thermal and ATP-Driven Mobilization

Abstract

The fundamental subunit of chromatin, the nucleosome, is not a static entity but can move along DNA via either thermal or enzyme-driven movements. Here we have monitored the movements of nucleosomes following deposition at well-defined locations on mouse mammary tumor virus promoter DNA. We found that the sites to which nucleosomes are deposited during chromatin assembly differ from those favored during thermal equilibration. Taking advantage of this, we were able to track the movement of nucleosomes over 156 bp and found that this proceeds via intermediate positions spaced between 46 and 62 bp. The remodeling enzyme ISWI was found to direct the movement of nucleosomes to sites related to those observed during thermal mobilization. In contrast, nucleosome mobilization driven by the SWI/SNF and RSC complexes were found to drive nucleosomes towards sites up to 51 bp beyond DNA ends, with little respect for the sites favored during thermal repositioning. The dynamic properties of nucleosomes we describe are likely to influence their role in gene regulation.The bulk of eukaryotic genomes do not exist as free DNA, but are associated with histones to form nucleosomes. The structure of nucleosomes has been studied intensively since they were first identified in the 1970s (39). Now several highresolution crystal structures provide detailed information describing how the four histone proteins are folded to form a histone octamer (11,28,46,62,66). DNA is bound to the surface of the histone octamer by over 140 atomic interactions. The most significant of these are hydrogen bonds between the phosphate backbone of the DNA and main chain atoms arranged in a helical path around the octamer surface. This large number of contacts contributes to the high resistance of nucleosomes to thermal and salt-induced dissociation. Given this stability, it is not surprising that the presence of nucleosomes on gene control elements can impede the function of regulatory proteins (59, 69). Indeed, alterations to chromatin structure are being found to play important roles in the regulation of many genes. Despite their resistance to dissociation, nucleosomes are not static entities but can move along DNA. As the nucleosomes of higher eukaryotes are separated by an average of 50 bp of DNA, the ability of nucleosomes to move along DNA provides a potent means by which access to regulatory elements might be altered without requiring the complete dissociation of the histone octamer.Among the first evidence that nucleosomes can move along DNA was the observation that they can migrate off simian virus 40 minichromosomes onto DNA fragments in a slow reaction that is dependent on the physical association of the acceptor DNA (6). Observations consistent with salt-dependent nucleosome sliding were also made by Weischet et al. (65). It was not until the 1990s that this phenomenon was characterized more thoroughly by Bradbury and coworkers, who exploited the fact that nucleosomes located at different positions had altered mobilities during native polyacrylam...

Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy

“…This implies that in vivo nucleosome occupancy is determined by the DNA sequence that wraps the full octamer rather than the ðH3∕H4Þ 2 tetramer. Such behavior is much like the shifted positions that are preferred after thermal or enzymatic remodeling of in vitro reconstituted chromatin fragments (5,41) and emphasizes the central role of chromatin remodelers in in Local Deviations from the Predicted Nucleosome Occupancy. Above we maintained a strict statistical mechanics description of nucleosome positioning with a minimal number of free parameters and assumed that the thermodynamic properties of all nucleosomes were identical.…”

“…2B) (30). The fact that in vitro reconstitution does not yield nucleosomes at their thermodynamically most favorable positions has been observed by Flaus and Owen-Hughes (41). They showed that sites to which nucleosomes are deposited during chromatin assembly on a mouse mammary tumor virus substrate differ from those favored during thermal equilibration or enzymatic remodeling Fig.…”

Chromatin remodeling: insights and intrigue from single-molecule studies

“…Furthermore, during remodeling by either SWI/SNF or the SWI/SNF-family remodeler RSC, a small gap placed in various locations on the nucleosome will move from its initial position to a defined final position about two turns from the dyad 38,41 . Finally, mononucleosomes remodeled by SWI/SNF or RSC move the free DNA end to a final position about two turns from the dyad 28,38,41,45 . Together, these observations strongly suggest that the translocase domain pumps DNA from this internal position on the nucleosome and that the arrival of a DNA gap or end at this position stops translocation 38,41,43,44 .…”

“…These thermodynamic properties underlie the very slow translational movement of nucleosomes in vitro by thermal diffusion 28 and their extremely slow rates of spontaneous disassembly and ejection. These properties also establish the energetic obstacles that remodelers must overcome and are important for interpreting the measurements obtained in single-molecule experiments (Fig.…”

Human SWI/SNF Drives Sequence-Directed Repositioning of Nucleosomes on C-myc Promoter DNA Minicircles

“…However, nucleosome positions established during salt-dialysis assembly can be altered by treatment at elevated temperatures (e.g. (24,25,45,(47)(48)(49)(50)), suggesting that mammalian body temperature might promote some level of nucleosome redistribution in vivo. Thermal repositioning is most rapid at high temperatures (up to 65°C) and in the absence of divalent cations or linker histones (45,48,51,52)).…”