It has long been thought that SWI/SNF primarily moves nucleosomes along DNA, but is unable on its own to disassemble nucleosomes based on studies using mononucleosomes as substrates. There have been hints over the years that SWI/SNF may have the ability to partially disassemble nucleosomes as it can inefficiently transfer histone octamers to free DNA and exchange histone H2A/H2B dimers (Bruno et al., 2003
; Lorch et al., 1999
). The efficiency of these reactions however did not suggest that they are the primary activities of SWI/SNF. It has become a point of debate whether SWI/SNF or RSC have the intrinsic ability to disassemble nucleosomes alone or instead require histone chaperones such as Asf1 and Nap1 or free DNA as histone acceptors for efficient nucleosome disassembly (Adkins et al., 2004
; Adkins et al., 2007
; Boeger et al., 2003
; Boeger et al., 2004
; Korber et al., 2006
; Reinke and Hörz, 2003
We have shown that when SWI/SNF remodels a dinucleosome first one nucleosome is moved towards an adjoining nucleosome with one H2A/H2B dimer being displaced (). In a subsequent and slower step, an H3/H4 tetramer is displaced resulting in the entire disassembly of one nucleosome and ultimately a lone nucleosome remaining on the DNA template. These data also demonstrate that nucleosome disassembly by SWI/SNF does not require free DNA or histone chaperones as histone acceptors, but instead can proceed efficiently in their absence. In vivo data had suggested that chromatin remodeling at the PHO5
promoter acts in this manner to create a nucleosome free region (Boeger et al., 2008
). Our data point to efficient displacement of H2A/H2B and nucleosome disassembly by SWI/SNF requiring an adjacent nucleosome. Why then does SWI/SNF require two nucleosomes in order to displace one? A recent model was proposed for SWI/SNF and RSC colliding nucleosomes together to form overlapping dinucleosomes (Engeholm et al., 2009
). This model predicts the loss of H2A/H2B dimers, but falls short of explaining how complete nucleosome disassembly might occur. Also our data shows that SWI/SNF is unable to move two nucleosomes together with ≤6 bp of linker DNA. It is expected that the narrow, enclosed nucleosome binding trough constituting the SWI/SNF active site (Dechassa et al., 2008
) would preclude accommodating one octamer being actively moved along DNA and a second encountered nucleosome to form an overlapping dinucleosome. These same constraints are also probably true for the RSC complex (Chaban et al., 2008
). Therefore it is more likely that SWI/SNF itself rather than the nucleosome on which it is translocating directly collides with and preferentially destabilizes an adjoining, distal nucleosome.
Model for SWI/SNF-Mediated Disassembly of Nucleosomes in Arrays
We propose a different model based on our data and the key observation of the ordered nucleosome disassembly of SWI/SNF resembling that observed by RNA polymerase II (Kireeva et al., 2005
; Kulaeva et al., 2007
; Studitsky et al., 2004
). The nucleosome collision model assumes that the role of SWI/SNF is merely to mobilize nucleosomes and discounts its ability to translocate and track along DNA to directly affect an adjacent nucleosome. Both SWI/SNF and RNA polymerase II are large molecular machines that in translocating along DNA dissociate it from the histone octamer surface in nucleosomes they encounter and trap or prevent DNA from re-associating with the histone octamer. The observed sequential loss of an H2A/H2B dimer followed by the displacement of an H3/H4 tetramer is the expected outcome from this kind of mechanism. Evidence for SWI/SNF mobilizing a single nucleosome bound in its active site while invading a second adjacent nucleosome comes from mapping single dinucleosomes remodeled under recruitment conditions (). DNA protection shows that, as the nucleosome proximal to the Gal4 binding site moves towards the distal nucleosome, the distal nucleosome can become more accessible and generally lacks a canonical 147 bp size protection (, Group II). These changes in the nucleosome distal to the bound transcription activator all occur without the linker DNA separating the two nucleosomes being protected once SWI/SNF is removed. The model in shows SWI/SNF competing for DNA bound to the octamer in the distal nucleosome (D) while it pulls DNA into and mobilizes the proximal nucleosome (P).
There are two differences between SWI/SNF and RNA polymerase. First, while mobilizing one nucleosome deep in its active site trough, SWI/SNF itself rather than the nucleosome invades a second nucleosome by moving DNA from it into the SWI/SNF-bound nucleosome (). The other difference is that SWI/SNF does not transfer histone octamers to DNA immediately behind it as is observed for RNA polymerase. SWI/SNF can therefore start with a completely assembled nucleosomal array and remove some or most of the nucleosomes from the template, while RNA polymerase would require a sufficient stretch of nucleosome-free DNA to begin translocation and then only shuttles nucleosomes on DNA rather than evicting them.
Another observation that is consistent with the SWI/SNF collision with distal nucleosomes model rather than the two nucleosome collision is retention of a particular H2A/H2B dimer after remodeling. In the overlapping dinucleosome model, loss of an H2A/H2B dimer is expected to occur with the same frequency from either of the two colliding nucleosomes. We find instead that the H2A/H2B dimer at the leading edge of the mobilized proximal nucleosome is well retained and an H2A/H2B dimer is likely lost from the distal nucleosome. Retention of the H2A/H2B dimer in the nucleosome bound to SWI/SNF is consistent with this nucleosome being unchanged in histone content and agrees with findings that remodeled mononucleosomes do not lose an H2A/H2B dimer. It also suggests that the displacement of an H2A/H2B dimer (and subsequently an H3/H4 tetramer) is not due to the collision of nucleosomes, but rather the collision of SWI/SNF itself with an adjoining nucleosome.
Although in more than half of the single remodeled dinucleosomes mapped the linker DNA remains accessible as one nucleosome is moved towards the other, what about those that resemble overlapping dinucleosomes (, group I)? Dinucleosomes in both groups I and II are most plausibly due to the initial remodeled dinucleosomes collapsing into one of these two structural classes after removal of SWI/SNF. By our model, the proposed loop extruded by SWI/SNF (, DNA in red) is resolved by migration around the octamer away from the distal nucleosome, advancing the proximal towards the distal nucleosome and yielding group I. The two nucleosomes may also be brought closer together in group I molecules when DNA displaced from distal nucleosome (, DNA in green) is rewrapped onto the octamer surface. Overlapping dinucleosomes may only form after displacement of SWI/SNF and therefore not be an accurate representation of the true state of a remodeled dinucleosome.
Previously it had been suggested that the same Gal4-VP16 transcription factor can promote nucleosome disassembly by directing SWI/SNF to move nucleosomes towards the bound Gal4 site (Gutierrez et al., 2007
). We found however that nucleosome movement in a dinucleosome context is away from the Gal4 binding site consistent with other published data (Dechassa et al., 2008
; Zofall et al., 2006
). DNA footprinting found that SWI/SNF recruited by an activator contacted the DNA gyre of the nucleosome opposite from the side of the Gal4 binding site (Dechassa et al., 2008
). In this orientation the nucleosome should move away from the Gal4 binding site based on mapping the binding and translocation site of SWI/SNF with DNA gaps that block nucleosome movement (Zofall et al., 2006
). The direction of nucleosome movement by SWI/SNF in regard to the transcription activator is important because if nucleosome movement is towards the transcription factor then SWI/SNF would cause efficient displacement of the transcription factor from DNA (Fletcher et al., 2002
). SWI/SNF would be less likely to remain at this site once the transcription factor is released. If instead the direction is as observed in this study it could explain how SWI/SNF might be retained at the promoter site and its remodeling activity restricted to a region encompassing a few nucleosomes (Almer et al., 1986
; Jessen et al., 2006
). Targeting SWI/SNF tethered by transcription factors to proximal nucleosomes would be consistent with their preferential clearing as observed for the PHO5
promoter region (Almer et al., 1986
; Jessen et al., 2006
). Our in vitro data correlates well with that observed by Kornberg and colleagues when modeling the stochastic nature of the PHO5
promoter region in which one nucleosome is retained at the promoter (Boeger et al., 2008
). The well-positioned dinucleosome substrate bound by Gal4 has proven to be an effective test system for examining the properties of SWI/SNF in unprecedented detail that appear to reflect and explain several of its in vivo properties.