The ability to manipulate the coding sequence of histone genes together with the expression and assembly of these proteins in vitro provides an opportunity to monitor the direct effects of these mutations on chromatin dynamics. We have identified differences in the sensitivities of chromatin-remodelling enzymes to mutations affecting different aspects of nucleosome structure. This indicates there are differences in the underlying mechanisms by which even closely related enzymes act.
We observed that remodelling by the enzyme Chd1, was affected very little in terms of either the products generated or the rates at which nucleosomes were repositioned (B; ). Although the initial rate of remodelling by RSC was affected to a greater extent than Chd1 the magnitude of the effects remained low, with the maximum effect being 2.2-fold (E). This indicates that both enzymes were able to engage and reposition nucleosomes efficiently. Consistent with this the binding of RSC to nucleosomes bearing the H3 E50A, I51A and Q55A mutations was equivalent to wild-type nucleosomes (Supplementary Figure 2
). The limited effects of these mutations on the rate of nucleosome sliding by RSC and Chd1 contrasts with the more substantial effects these mutations were observed to have on spontaneous thermal nucleosome repositioning (compare B, E and F). This lack of correlation is likely to reflect significant differences in the mechanisms for thermal and ATP-dependent nucleosome repositioning.
Figure 7. Remodelling enzymes are distinctly affected by histone H3 mutations. (A) The histone H3 αN helical map, introduced in ref. 10. This map allows the impact of each interaction surface to be compared among the different remodellers. (B) The initial (more ...)
In contrast to the limited effects on the initial rates of remodelling, the outcome of RSC remodelling reactions was affected dramatically with many mutations altering the spectrum of products generated (C). A related but less dramatic effect was observed with the SWI/SNF complex (D). While RSC repositions wild-type nucleosomes to a series of locations in which the nucleosome dyad is so close to the edge of the DNA fragment that up to 57 bp of DNA are removed from one edge of the nucleosome, these locations were under-represented when H3 I51A mutant nucleosomes were used as a substrate (). This means that the mechanics of moving nucleosomes to locations in which DNA is unravelled differ from those involved in repositioning within the confines of a DNA fragment.
It is notable that there is a good correlation between residues that have strong effects on thermally driven nucleosome positioning and the inability of RSC to move nucleosomes into positions where DNA is unravelled (compare C and F). At first this appears to be counterintuitive as nucleosomes that are inherently more mobile cannot be repositioned so effectively by RSC. However, it is also true that the H3 E50A, I51A and Q55A mutations reduce the stability of histone octamers (10; data not shown). One model for RSC action involves the RSC complex drawing DNA over the surface of nucleosomes while remaining bound to the histone octamer in a fixed orientation (26
). The reduction to octamer stability resulting from the H3 E50A, I51A and Q55A mutations might result in the octamer rather than the DNA being distorted as a result of ATP hydrolysis. Furthermore, as RSC starts to move nucleosomes to locations in which DNA is unravelled from their surface, the overall loss of histone contacts will require the ATPase motor within the RSC complex to exert greater forces. Indeed, it has been estimated that a force of 3 pN will be required to dissociate each helical contact (27
). The increased forces that are applied during DNA unravelling might drive a reconfiguration of the histone octamer that prevents any further repositioning. This could explain why RSC is able to move nucleosomes to positions where histone DNA contacts are retained, but not to locations where DNA is unravelled. In this case the reason why Chd1 is not affected in the same way could be simply that the repositioning of nucleosomes to more central locations does not involve unravelling DNA to the same extent.
Having observed that mutations to the H3 αN region of the nucleosome can affect the outcome of remodelling reactions, it will be of interest to investigate whether histone modifications exert similar effects. Our own preliminary observations made using mutations designed to mimic a selection of modifications known to occur in this region of the nucleosome revealed only subtle effects on the action of ATP-dependent remodelling enzymes (data not shown). It is possible that the use of amnio acid substitutions is not a sufficiently accurate mimic for post-translational modifications and hopefully the development of new technologies will allow the effect of histone modifications within the globular core of nucleosomes to be tested directly (28
). Nonetheless, our observations illustrate a principle by which alterations to the structure of nucleosomes can selectively affect the outcome of remodelling reactions. It is of special note that the unravelling of DNA is affected as this might be anticipated to affect the ability of enzymes to catalyse octamer removal by collision (26
). As a result these observations expand the repertoire of different means by which post-translational modification of histones can potentially exert effects on chromatin dynamics (3