Early studies show that nucleosome remodeling occurs without gross changes in either histone protein stoichiometry or configuration,[69–71]
and indeed nucleosome substrates in which histones were cross-linked together appears to be remodeled in a facile manner.[71, 72]
Although it is clear that histone–DNA interactions are altered during remodeling, the exact details remain unclear. A comparison of nucleosomes assembled with end-labeled versus body-labeled DNAs suggested that even though the register of specific DNA contacts to histones is altered, the DNA remains more or less in association with the histone surface.
By using a site-directed histone→DNA crosslinking approach, the Hayes group demonstrated that cross-linking does not significantly inhibit hSWI/SNF remodeling activity as monitored by DNase I footprinting.
However, a single crosslink between the DNA and the histone surface drastically reduced SWI/SNF-induced increases in the accessibility of the nucleosome DNA to restriction enzymes—even at sites distant from the crosslink—this indicates that hSWI/SNF remodeling requires transient global disruption of histone–DNA interactions.
This result is consistent with other in vitro studies that show that SWI/SNF alters nucleosome structure by at least transiently disrupting histone–DNA interactions.[52, 72]
A significant clue as to the mechanism of ATP-dependent remodeling was brought to light in studies by the Cairns, Peterson, and Owen-Hughes’ groups. Because the helicase-related domain of the Snf2 subunit did not exhibit classic helicase strand-displacement activity, these groups suspected that SWI/SNF and related complexes might exhibit a translocase activity, and that such an activity might result in an increase in super-helical torsion in the DNA.
Indeed, the Owen-Hughes group found that purified SWI/SNF complex (as well as several other remodeling complexes) induced detectable torsional stress in naked DNA fragments in an ATP-dependent manner, which was detected as extrusion of cruciform structures. These results imply that not only can SWI/SNF induce torsional stress in DNA, but also that the complex must contact DNA at a distal site from the ATPase domain to create a topologically isolated domain in which the stress can be transiently contained.
In a separate study, the Cairns group showed that the related RSC complex does indeed exhibit DNA translocase activity coupled to ATP hydrolysis, with a processivity limit of about 70 bp.
Interestingly, Peterson and co-workers demonstrated that nucleosomes within topologically constrained minicircles were refractory to remodeling as determined by restriction enzyme accessibility.
Moreover, they found that the disruption of his-tone–DNA interactions is nucleosome-specific; no such disruption was observed when non-specific histone–DNA complexes were confronted with the SWI/SNF complex.
These results imply that SWI/SNF and related complexes specifically bind to nucleosomes and employ torsional stress within the DNA of the nucleosome substrate to remodel nucleosome structure. A model emerges whereby the SWI/SNF complex contacts at least two sites in nucleosomes: a static anchoring interaction, and a site that translocates along the helix, pumping superhelical stress into the DNA to disrupt histone–DNA interactions.
Though the exact manner by which SWI/SNF-dependent torsional stress results in increased access to nucleosomal DNA remains unclear, data suggest that that remodeling results in the generation of a bona fide structurally altered nucleosome intermediate. Quantitative restriction enzyme analysis of remodeling by the BRG1 subunit suggests that short stretches of DNA sites within the nucleosome are transiently released from histone–DNA interactions and available for trans
-acting factor binding.
The data indicate that multiple distinct remodeled species are generated by a mechanism that is not easily explained by simple uncoiling of DNA from the edges of the nucleosome or nucleosome sliding and that these species are in-terconverted by the enzyme. Although the stability and lifetime of these structurally altered states have been the subject of debate,[77, 78]
current data suggest that such intermediates rapidly decay into a bona fide nucleosome that encompasses a different segment of DNA than the original nucleosome, whereas other remodelers appear to produce a “moved” nucleosome with even more fleeting intermediates.
Therefore, a primary outcome of ATP-dependent remodeling by SWI/SNF, RSC and other remodelers is the movement or mobilization of nucleosomes along the DNA.[54, 55]
Such mobilization would stably expose sites previously blocked by association with the histone octamer. Thus, much of the work regarding ATP-dependent remodeling has focused on the mechanism of nucleosome mobilization.
Several crystal structures of nucleosome core particles suggest that nucleosome movement or mobilization could occur by a “twist-diffusion” mechanism.[79, 80]
Within the nucleosome, strong sites of interactions occur approximately once every 10 bp of DNA, such that essentially each 10 bp stretch is, to an extent, topologically isolated. The twist diffusion mechanism posits that a DNA twist “defect” containing ±1 base pairs compared to the nominal helical twist can be accommodated within any 10 bp stretch of DNA. If the defect diffused through the entire length of the nucleosome, the histone octamer would be advanced along the DNA by one base pair. This model has the attribute that the majority of high-energy his-tone–DNA interactions would be preserved at any one moment in time, while the DNA screws along its long axis, along the ‘grooves’ in the surface of the histone octamer. However, the one-step nature of the advancement would require a complete rotation of the DNA on the nucleosome surface and thus requires passage through a strongly disfavored rotational orientation for nucleosome positioning sequences. Nevertheless, it is likely that the free energy derived from ATP hydrolysis would more than overcome this barrier.
To test whether a twist-diffusion mechanism is responsible for ATP-dependent remodeler-induced nucleosome mobilization, Aoyagi and co-workers placed branched DNA structures in the center of a nucleosome and measured the extent and rate of remodeling by the human SWI/SNF complex and the Xenopus
Mi-2 complex.[81, 82]
If a pure twist diffusion mechanism is used by the remodelers for nucleosome mobilization, it is likely that the steric bulk of the branched DNA structures would interfere or block movement of the histone octamer along the DNA. However, both enzymes were unaffected by either single or double-stranded branched structures in the center of the nucleosome, strongly indicating that this mechanism does not account for mobilization. A similar result was obtained by Längst and co-workers in which the attachment of streptavidin-bound beads to the center of the nucleosome DNA did not inhibit nucleosome mobilization by the ACF complex.
It is interesting to note that branched DNA structures placed outside of the nucleosome can hinder SWI/SNF-dependent mobilization in cis
, perhaps due to steric interference with binding of the enzyme to the DNA.
The dynamic process of spontaneous unwrapping of DNA from the edges of the nucleosome
also has been hypothesized to be related to the ATP-dependent remodeling and nucleosome-mobilization process. Upon uncoiling, rewrapping, or capturing the end of the nucleosome DNA at a position ~10n base pairs beyond the original point would lead to a loop of ~10n bp. This loop could then be propagated through the nucleosome without further net loss of histone–DNA contacts. Whereas a tenable and attractive model, only modest direct evidence for the existence of a DNA loop during nucleosome mobilization has been obtained. By using a single-molecule “molecular tweezers” approach, Bustamante, Peterson, and colleagues showed that RSC and SWI/SNF translocate DNA in a nucleosome-dependent fashion, likely by forming large internal loops within the nucleosomes.
Several labs have attempted to measure the “step size” — the size of one continuous movement of the nucleosome — that might be related to the size of the loop.[83, 85]
Crosslinking results, mentioned above, indicating that remodeling as measured by restriction enzyme accessibility assays requires transient global disruption of his-tone–DNA interactions are also consistent with the loop-recapture mechanism.
In addition, the Längst group employed a novel assay exploiting the preferential intercalation of ethidium bromide to naked DNA to provide evidence for a region free of histone–DNA interactions within the nucleosome during ACF nucleosome remodeling.
Moreover, a recent cryo-electron microscopy and biochemical study of RSC-remodeled mononucleosomes provides evidence for remodeling intermediates containing internal loops.
In addition, these workers found that remodeling intermediates were not yet moved from their original position but contained more DNA than canonical nucleosomes. A similar result was recently obtained by Bartholomew and colleagues by precisely mapping histone–DNA interactions by using a DNA methylase protection assay known as MapIT, which provides single-molecule information
and revealed a subset of SWI/SNF remodeled products that exhibited protection of more than one nucleosome’s worth of DNA.
These intermediates might have been brought about by a loop-recapture mechanism, but other possibilities exist (see below).
A simple model for remodeling would thus be that a translocating enzyme encounters a dynamic nucleosome and peels off DNA as it proceeds around the structure, disrupting his-tone–DNA interactions and exposing sites to trans
-acting factors. However, recent work provides evidence that the primary site of the translocation of SWI/SNF along DNA is actually located within the nucleosome, about two helical turns from the dyad axis. Two studies pinpointed the sites of interaction of SWI/SNF and RSC, respectively, by using gapped DNA substrates, which are known to block progress of translocases along DNA.[55, 56]
These workers showed that translocation is blocked when the gaps reach the site ~two helical turns from the dyad; the effect is directional, such that translocation in the opposite direction is not affected.[55, 56]
These results are consistent with footprinting results and for the smaller ISWI complex, which contacts nucleosome DNA at approximately the same site and also is affected similarly by gaps in the DNA.
Thus a model emerges whereby DNA is drawn into the nucleosome by the translocase activity, which engages the nucleosome internally, to form some sort of distorted structure, perhaps with an internal bulge or loop induced by movement of the DNA-binding domain (). The loop could form stochastically, being trapped by binding of the enzyme, or perhaps the translocase activity draws DNA in from the nucleosome edge by forcing movement of the DNA-binding domain (, steps A and B). The loop is then free to translocate around the perimeter of the nucleosome to advance the DNA (steps C, D and F) or the loop DNA might be drawn further into the nucleosome by the translocase domain (steps E and F). Interestingly, very recent results from the Dimitrov group have documented an intermediate that arises during nucleosome remodeling by RSC, which they have dubbed the “Remo-some” (, inset). In this intermediate the histone octa-mer is not yet moved along the DNA but AFM and cryo-EM work clearly show that the remosomes contain apparently randomly oriented internal loops and harbor more DNA than a typical nucleosome.
A basis for leverage against which the translocase activity works is provided by the ultrastructural results, which indicate that a significant portion of the remodeled nucleosomes is grasped by the remodeling complex. Interestingly, a requirement for DNA bulges in nucleosome remodeling or mobilization has not yet been established. Hopefully, in the future, probes capable of detecting internal DNA loops in nucleosomes or bulged regions will be devised, allowing the role of bulges to be investigated. It should be noted that exactly how the translocase activity imparts bulges or distortions in general remains unclear and likely awaits atomic-level structural analysis of the interaction between the ATPase domain and nucleosome.
Figure 2 Model for the mechanism of SWI/SNF induced nucleosome mobilization. The enzyme interacts with the nucleosome DNA from about two turns from the dyad (horizontal line) to well into the linker DNA through the DNA-binding domain (DBD). A translocase domain (more ...)
Recent results also have shed light on a potential mechanism for nucleosome displacement by SWI/SNF. Previous work had shown that SWI/SNF is required to evict nucleosomes upon activation of specific promoters in vivo, with histone chaperones such as Asf-1.[87–89]
Yet, the mechanism of eviction is poorly understood. As mentioned above, in vitro studies show that the SWI/SNF complex or paralogues, such as the RSC complex, can displace H2A/H2B dimers as well as the complete histone octamer, resulting in naked DNA after remodeling activity.
However, the efficiency of histone displacement is relatively low with the mononucleosomes substrates typically employed in such studies and observable displacement requires the presence of large amounts of acceptor or competitor species.
In recent work, Bartholomew and colleagues discovered that remodeling of di- or trinucleosomes resulted in significant amounts of species with reduced histone content consistent with the loss of entire nucleosomes.
Remodeling of these small oligonucleosome substrates resulted in more rapidly migrating products, which previous workers might have assumed were due to nucleosome mobilization and altered nucleosome translational positions.
However, by using a quantitative double-label technique, Bartholomew and colleagues demonstrated that an entire core histone octamer was lost in the products of dinucleosome remodeling, whereas little or no loss is observed with mononucleosomes. Histone loss occurs in stages, beginning with the loss of one H2A/H2B dimer, whereas an entire histone octamer is lost in a second major product. Interestingly, histone displacement occurs in the absence of chaperones or naked DNA as acceptors. Thus, in contrast to the remodeling of mononucleosomes, the data indicate SWI/SNF remodeling of dinucleosomes results in efficient eviction of one of the two original nucleosomes. Precise mapping of nucleosome positions during remodeling indicated nucleosomes are primarily moved to one end of the DNA fragment and that one of the two nucleosomes is evicted during the process, which is consistent with the histone content data. Moreover, by recruiting SWI/SNF to one end of the dinucleosome template through the binding of the transcriptional activator Gal4–VP16, the mapping evidence suggests that the nearby (proximal) nucleosome is moved downstream and eventually runs into and displaces the distal nucleosome.
These results correlate well with a recent report from the Owen-Hughes laboratory that remodeling can result in one nucleosome invading the region of DNA occupied by a neighbor.
However, the Bartholomew group finds that no remodeling occurs if the linker DNA between the two nucleosomes is too short.
Moreover, mapping data indicate that SWI/SNF appears to contact DNA ahead of the advancing nucleosome and thereby encounters the evicted nucleosome itself. A model then emerges whereby one nucleosome occupies a large pocket on the surface of the SWI/SNF complex and stimulates its ATPase-driven DNA translocase activity. The nucleosome in the pocket retains all of its histones, although its structure might be drastically altered, whereas a neighboring nucleosome in the path of the mobilized nucleosome–SWI/SNF complex is evicted from the DNA.