Based on our results, we view the nucleosomal stem as a dynamic, polymorphic, hierarchically organized structure composed of several parts:
comprising the globular part of H1 and the first 10
bp of the linkers, where gH1 preferentially establishes three contacts: one at the dyad of the histone octamer and two with the linkers, which remain on average straight and symmetric up to bp 8. The formation of the root considerably reduces the range of fluctuations of the linkers and suppresses unwrapping from the histone core.
or relatively rigid inner part of the stem, comprising the linkers up to bp 20±2, in direct contact with the cationic amino
acids of the C-terminus of H1. It is in this region, that the DNA is deformed to partially align the two linkers. In our nanomechanical model, 90% of the elastic energy of the fully-protected stem structure of ~2kBT
are located in the trunk. In our experiments, the formation of the trunk required a tail length of at least 15 AA (H1-127), indicating a biochemical control mechanism for this step.
A flexible crown
or outer stem where the branching linkers exhibit substantial fluctuations, while preserving well-defined preferential contacts. We note that by imposing a boundary condition on the linker conformation, the influence of the trunk structure may extend beyond the region of direct interactions between H1 and the linker DNA. In particular, the linkers might appear connected without there being strong direct interactions. In our experiments, this influence extends to at least 40
bp away from the NCP, reaching the typical linker lengths of native fibers (40
bp for a half-linker
). Our experiments (23
) also suggest that the full C-terminus, while possibly stabilizing further the stem, does not qualitatively modify its structure. As a natural explanation for this effect, we suggest that the terminus may remain confined in the trunk region.
The hierarchical organization implies that the stem opens and closes in a zipper-like fashion. Under our experimental conditions, thermal fluctuations mainly affect the branching linkers in the crown. The response to external forces depends on their magnitude. While weak forces should essentially deform the crown, larger forces should disrupt the trunk and eventually the root before unwrapping the core particle (51
). Such forces may be exerted in a controlled manner in single-molecule experiments (52
). They also arise during the condensation of the chromatin fiber, where H1 might accommodate a fairly large range of linker conformations, if other interactions (18
) compensate the free energy cost of a stem deformation or a partial stem disruption. We note, that this view of the stem is a natural extension of the current, dynamic picture of the nucleosome core particle: instead of a passive and rigid wrapping of 147
bp of DNA in a conformation resembling crystal structure(s) (7
), DNA and histone octamers form a highly dynamic complex, where DNA spontaneously unwraps and rewraps from the ends (48
) with actively created (55
), diffusing (56
) defects ensuring mobility (58
How do our results match those of previous studies? There is a remarkable diversity of experimental (25
) and modeling (24
) results for the mode in which gH1 binds in what we now refer to as the ‘root’ of the stem. The structures presented here are derived from experiments where mono-, di- and tri-nucleosomes were reconstituted following a carefully elaborated protocol recreating in vivo
conditions such as the presence of the chaperone NAP-1. If we believe them to represent a free energy minimum for systems dominated by intra-stem interactions, it is an interesting question, whether other binding modes might serve to stabilize alternate structures in nucleosomes under external constraints. If one interprets the multitude of predicted gH1 binding modes in the root as a feature
of the molecule and not as a failure of the employed modeling schemes, then the ability of the stem to adapt to external constraints might be even larger than apparent from our experiments.
Little was known about the trunk region. Neglecting fluctuations, Bharath et al.
) used structural analogies and bioinformatics methods to predict a placement of gH1 close to the two-contacts model of Zhoul et al.
). For the trunk, they proposed a particular conformation of the C-terminus making contact with the linker DNA up to 24
bp away from the NCP, beyond which the sharply bent linkers were supposed to diverge in the crown. While the predicted extension of the H1-DNA contacts is rather close to that of our trunk, the details of the proposed stem structure are incompatible with our experimental results. This hold for the resulting protection pattern close to the DNA dyad as well as for the predicted divergence of the two linkers beyond the contact zone with the H1 tail, which is difficult to reconcile with the experimentally observed protections P3 and P4 (see ).
Interestingly, our results shed some new light on the question, which factors determine the structure of dense chromatin fibers. Is it the structure and elasticity of the stem, which discriminates between the possible helical arrangements of nucleosomes? Or do the linkers simply adapt to whatever constraints follow from the packing and the interactions of the core particles (63
), thereby possibly frustrating the formation of a proper stem? Robinson et al.
) reported the properties of well-defined chromatin fibers reconstituted in vitro
from poly-601 templates in presence of the linker histone H5. The templates were prepared for a range of well-controlled linker lengths (multiples of 10, from 30 to 90
bp). Based on these experiments, Wong et al.
) used DNA elastic models to infer the linker DNA paths in the Robinson et al.
) reconstituted fibers. The resulting Wong et al.
) ‘most favorable’ structures shown in (A and C) belong to a remarkable variety of helix families. Nevertheless, the authors argued that (in our language) the DNA conformation in the root is approximately conserved among the structures: a posteriori
, this feature allows for a common asymmetric three-contact binding mode of gH1/5, rather similar to the one inferred from our experiments. The only exception is the most favorable structure for the shortest linkers (30
bp or 15
bp for a half-linker
), where the steric constraints seem indeed too strong to allow for the formation of the structure preferred by gH1 and where Wong et al.
) propose an alternative binding mode. Otherwise, it is striking to see on A, how, with increasing linker length, the Wong et al.
) conformations reproduce larger and larger parts of the mono-nucleosomal stem shown in B. Linker lengths 40
bp and larger thus possibly allow for the formation of a (deformed) trunk and further stabilization by the H1 tail, whereas they exhibit substantial variations in the external part corresponding to our crown (C). At least qualitatively, the comparison of the two completely independent studies suggests a remarkable agreement between linker conformations required for achieving nucleosome packing in dense chromatin fibers and those stabilized by intra-stem interactions. Furthermore, the comparison illustrates the role of the polymorphic structure and hierarchic organization of the stem in stabilizing a wide variety of fibers. From a kinetic point of view, the emerging picture is that of a folding funnel (64
), where the local stem formation helps to achieve the cooperative fiber folding. Some of these features may already be included in a coarse-grain model of the chromatin fiber (14
), which is compatible with our description of the root of the stem. Quantitatively, we crucially miss experimental or computational estimates of the orientation-dependent interactions between the core particles and of the (deformation-dependent) stem formation and interaction free energies to be able to embark on the systematic modeling of fiber conformations.
Figure 9. Comparison of our mono-nucleosome stem structures to the inferred (59) linker conformations in model chromatin fibers reconstituted from poly-601 templates (35). View along the superhelical axis (top) and perpendicular (bottom). For the fiber conformations, (more ...)