Sequence-dependent Nucleosome Structural and Dynamic Polymorphism. Potential Involvement of Histone H2B N-Terminal Tail Proximal Domain

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Abstract

Relaxation of nucleosomes on an homologous series (pBR) of ca 350–370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also “closed”, but with these DNAs positively crossed; and the third was “open”, with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by ∼0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a ∼1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance (∼20 bp) on H3-tail interactions with the small groove at the nucleosome entry–exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them.

Introduction

New features of nucleosome dynamics have recently been revealed, using as a tool topoisomerase I relaxation of mononucleosomes reconstituted on an homologous series of DNA minicircles of unique sequence (the 351–366 bp pBR series, originating from plasmid pBR322).1 In this analysis, the relative amounts of the two, sometimes three, adjacent topoisomers observed in the equilibrium distributions produced by relaxation were plotted as a function of their linking difference, ΔLk. Such a plot (see Figure 3(d), below) showed shoulders or peaks centered at ΔLk values around −1.7, −1 and −0.5, which corresponded to discrete DNA conformational states of specific linking differences, ΔLkn, close to these values. These peaks or shoulders result from the relative energy benefit of relaxing into these particular topoisomers of ΔLkLkn, because only these topoisomers can provide a relaxed loop to the nucleosomes in these particular conformations. This requires the proper minicircle size in order for the topoisomer ΔLk to coincide with the conformational ΔLkn, a condition necessarily met by one minicircle or the other when using a series. Wrapping is normal (∼1.7 turns) in the closed conformations, with DNAs crossing either negatively (ΔLkn ∼−1.7; state s−) or positively (ΔLkn ∼−0.5; state s+), but is lower (∼1.4 turns) in the open, uncrossed, conformation (ΔLkn ∼−1.0; state sO). The breaking of histone–DNA interactions at the nucleosome edges in the sO conformation is consistent with histone–DNA binding interactions being the weakest at superhelix locations (SHL) ±6.5.2 A thermodynamic analysis of this plot confirmed that the s+ conformation is energetically less favorable than the s− conformation (by ∼2kT), with sO lying in between (∼1kT above the “negative”). These relative energies, however, rely on the effective interactions of the histone N-terminal tails with entering and exiting DNAs. Tail destabilization (through their acetylation and/or the presence of phosphate) indeed shifted sO to almost 1kT below s−, making it the energetically most favorable conformation.1

The present work describes results obtained with a new 349–363 bp 5S DNA minicircle series containing the 256 bp Lytechinus variegatus 5S nucleosome positioning sequence.3 When compared to DNA in 5S nucleosomes, DNA in pBR nucleosomes was untwisted by ∼0.2 turn, although naked pBR DNA was slightly more twisted than naked 5S DNA. This result was confirmed and extended by DNase I footprinting, which revealed a ∼1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the gyres through the channel formed by the two aligned small grooves. In contrast, both nucleosomes showed untwistings at the proximal sites, i.e. on the opposite gyre, as also observed in the high-resolution crystal structure.4

The 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the s+ conformation, although their access to s− was easier. The energetics of the system were explored by calculating the loop most probable conformation in the three states using the explicit solutions to the equations of the equilibrium in the theory of the elastic rod model for DNA (referred to below as the “explicit solutions” theory). (The theory5., 6., 7. has been used in this laboratory to simulate tetrasome chiral transition.8., 9.) The most probable conformations strictly depend on the loop end conditions, and hence on the parameters of the DNA superhelix. Results showed that the free energies of s− and s+ states could be affected in opposite directions, as observed experimentally, by modulating the trajectories of entering and exiting DNAs so as to make them more or less divergent when projected onto the superhelix axis. We present a model in which this is accomplished by H2B tail untwisting at the distal sites through action at a distance (∼20 bp) on H3-tail interactions with the small groove at the nucleosome entry–exit.

Section snippets

pBR versus 5S DNAs. Helical periodicity and other parameters

Figure 1(a) shows the electrophoretic profiles of relaxation products, obtained at different temperatures, of the Lk=31 topoisomer of two DNA minicircles of the pBR series. Sizes (360 bp and 362 bp) were chosen to be close to an integral multiple (34) of the helical repeat plus 5 bp. Only within a narrow size interval around 34×10.5+5=362 bp, does relaxation produce two topoisomers, against one outside that interval (a result of DNA rigidity1). The logarithm of the topoisomer ratio in each

Potential influence of DNA bends and/or anisotropic bendability on relaxation data

Permanent bends in, or an anisotropic bendability of, the loop have the potential to bias the relaxation equilibria. This conclusion stands from the experimentally observed and theoretically predicted influence of sequence-directed bending on DNA supercoiling,11., 12., 13. and from the suggestion that bends generally favor writhing over twist.14 Indeed, trajectories of 5S and pBR linear DNAs, as constructed by using roll, tilt and twist parameters from the literature (including references cited

Conclusions

Results of this work open the way to the notion of sequence-dependent nucleosome structural and dynamic polymorphism. This polymorphism may be mediated by H2B N-terminal tail proximal domains, through untwistings of the double helix near SHL±5 where they traverse the superhelix between the two gyres. We propose that the subsequent rotation of the distal DNAs beyond SHL±5 modifies the interactions of H3 tail proximal domains with the small groove near SHL±7 in such a way as to alter the

DNAs

DNA fragments of the pBR series, 351, 353, 354, 355, 356, 358, 360, 361, 362, 363 and 366 bp long, originate from plasmid pBR322, and have been described.1., 9. The 5S series, made of 349, 351, 353, 354, 355, 356, 357, 359, 361 and 363 bp fragments, is new, and originates from BamHI digests of plasmids pUC(349)-pUC(363). The fragments were derived from L. variegatus 5 S rDNA-containing 357 bp fragment described,15 by filling-in at the unique TaqI site (359 bp20), and by either filling-in or Bal31

Acknowledgements

A.P. and A.S. thank NATO for a Collaborative Linkage grant. In the later part of this work, A.S. was the recipient of a temporary Research Associate position from CNRS. C.L. acknowledges the award of a predoctoral fellowship from the French Ministry of Research and Technology.

References (43)

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