Nucleosomes in Solution Exist as a Mixture of Twist-defect States

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The 2.0 Å crystal structure of a nucleosome core particle in complex with a bivalent pyrrole-imidazole polyamide reveals that this “clamp” effectively crossbraces the two gyres of the DNA superhelix, thereby stabilizing the nucleosome against dissociation. Using X-ray crystallography and footprinting techniques, we show that the clamp preferentially binds nucleosomes over free DNA, and that nucleosomal DNA exists as a mixture of multiple twist-defect intermediates in solution. The nucleosomes exist in one of two different conformations in various crystal structures that trap twist-defect intermediates, even on a strong positioning sequence. Evidence has been obtained supporting the existence of twist-defect states in nucleosomal DNA in solution that are similar to those obtained in crystal structures. Our results also substantiate the idea that twist diffusion may represent an important means of altering the accessibility of nucleosomal DNA both in the presence and in the absence of ATP-dependent chromatin-remodelling enzymes.

Introduction

The three-dimensional structure of the nucleosome core particle (NCP), the fundamental structural unit of chromatin, has previously been determined to high resolution.1, 2, 3, 4, 5 These structures present a detailed view of DNA in a physiologically relevant context,6 and provide insights into the intricacies of DNA compaction in the eukaryotic cell. The nucleosome has long been viewed as an “immovable object” that represents a formidable obstacle to all processes that require access to the DNA substrate.7 This picture is rapidly changing. Despite over 120 direct contacts (and roughly the same number of water-mediated interactions) made by the histone octamer with the DNA at 14 independent contact points over its entire length,1 nucleosomes are highly dynamic and malleable structures that undergo histone exchange, chromatin remodelling, and reversible post-translational modifications.8, 9, 10, 11, 12, 13, 14, 15, 16

Approximately 95% of genomic DNA sequences do not appear to be capable of pronounced sequence-specific nucleosome positioning.17, 18 Positioning is likely to be governed by a combination of DNA bendability and curvature mainly over the central 80 base-pairs of DNA, and is likely to represent a compromise between several competing features that are embedded in the DNA sequence. Whether uniquely, loosely, or not at all positioned, nucleosomes are dynamic and are acted upon by ATP-dependent chromatin-remodelling factors in order to regulate nucleosomal DNA accessibility9, 10, 12 and to bring about subsequent gene activation or repression. Although many ATP-dependent chromatin-remodelling factors have been shown to enhance the mobility of nucleosomes and alleviate the repressive effects of chromatin on transcription,12, 19, 20, 21, 22 the molecular mechanism by which nucleosome repositioning occurs is still controversial.9, 10, 12 Two current models for nucleosome remodelling are based on twist diffusion or propagation of a DNA bulge on the surface of the histone octamer. It has also been recently demonstrated that ATP-dependent chromatin remodelling can exchange or remove histone dimers from nucleosomes.10 One concept that has emerged from these studies is that the nucleosome is a dynamic entity, and further knowledge concerning DNA conformational flexibility in the nucleosome is key to understanding these vital processes.

Here, we investigate the different possible states of the nucleosome in solution and in the crystal lattice using previously described bivalent pyrrole-imidazole polyamides (Py-Im PAs) that target the DNA “supergrooves” within the nucleosome. DNA supergrooves have recently been reported as non-contiguous minor grooves (or major grooves) that are brought into structural alignment across the two gyres of superhelical DNA within the NCP.23 These ligands effectively crossbrace the two gyres of nucleosomal DNA in a site-specific manner, and thus are likely to capture different structural states of the nucleosome in solution. Py-Im PAs bind predetermined DNA sequences with affinities in the nanomolar to the subnanomolar range, and have been shown to modulate specific gene expression.24 Using both DNase I footprinting and affinity cleavage techniques, we have shown previously that Py-Im PAs bind nucleosomes in vitro, demonstrating that much of the DNA in the context of the NCP is readily accessible for molecular recognition.25

X-ray crystallographic studies of NCP–polyamide complexes reveal the nature of structural changes in the nucleosome upon polyamide binding at molecular detail.26 These changes occur exclusively in the nucleosomal DNA without altering the underlying protein–DNA interactions. Due to the palindromic nature of the 146 bp human α-satellite DNA used in our studies, and due to the fortuitous location of the binding sites for the parent polyamide PA1 in NCP reconstituted with this particular DNA fragment (NCP146), two molecules of PA1 bind match sites that are immediately opposed across the two gyres of DNA in NCP146.26 Based on these results, we designed and characterized a bivalent Py-Im PA “clamp” that binds an entire supergroove of nucleosomal DNA in a site-specific manner and effectively crossbraces the two gyres of nucleosomal DNA.23 The bivalent clamp was synthesized as a PA1 dimer connected by an ethylene glycol (PEG) linker. Using X-ray crystallography and footprinting techniques, we have demonstrated that the clamp binds to NCP146 across the supergroove as designed.23 The significantly improved diffraction quality of the NCP146–clamp complex, compared to the unliganded NCP146, suggests that the clamp improves DNA positioning with respect to the histone octamer. Biochemical assays reveal that the clamp binds with very high affinity and specificity, and has a dramatic effect on the in vitro stability of nucleosomes against dilution-induced dissociation.23

Here, we present a more detailed analysis of the NCP146–clamp complex, and compare quantitative footprinting data of a clamp bound to nucleosomes reconstituted with two DNA fragments of almost identical sequence but whose lengths differ by one base-pair. We further show that the DNA in NCP146 continuously samples several conformations in solution, and that the binding of the polyamide clamp favours and stabilizes some conformations in preference to others. The multiple conformations observed in solution appear to be “twist-defect” intermediates, akin to those trapped in the crystal structures. We propose that “twist diffusion” processes may be involved in the redistribution of twist-defect states of the nucleosomes in solution. The occurrence of such dynamic motions in the nucleosomal DNA could help optimize DNA positioning signals with respect to the octamer, could assist ATP-dependent nucleosome sliding, and regulate DNA accessibility at the level of a mononucleosome. The use of a highly positioned DNA sequence in our studies has allowed us to capture subtle perturbations in the dynamic states of nucleosomal DNA in solution. Our studies suggest that conformational flexibility is an attribute of the nucleosomal DNA regardless of whether or not nucleosomes are uniquely positioned.

Section snippets

The polyamide clamp alters the dynamics of DNA “stretching” in NCP146

The 2.0 Å crystal structure of NCP146 in complex with the bivalent clamp polyamide PW12 (NCP146–clamp, PDB accession code 1S32) has been recently reported.23 A comparison with the structure of NCP146 in complex with the unlinked parent molecule, PA1 (NCP146–PA1;26 PDB accession code 1M18) by least-squares superposition reveals that the structure of the polyamide itself remains largely unaffected by the presence of the PEG linker. Local DNA distortions due to ligand binding, as described earlier

Discussion

The energetic stability and position of a nucleosome depend on how well individual nucleosome positioning signals in the DNA sequence are accommodated on the surface of the histone octamer.29 With the exception of simple repeating DNA sequences, unique nucleosome positioning is likely to be a compromise to accommodate several “conflicting interests”, since it is unlikely that any given nucleosome position would place all positioning signals in a perfect orientation. Here, we provide evidence

Reconstitution of NCP

Previously established protocols were used to reconstitute NCP146 and NCP147 from recombinant Xenopus laevis histones and a 146 bp or 147 bp palindromic DNA fragment derived from human α-satellite DNA.1, 31, 32

DNase I, hydroxyl radical footprinting and Kd measurements

Previously published protocols were followed to perform DNase I and hydroxyl radical footprinting experiments, for the measurements of dissociation constants, and the estimation of half-lives of the polyamide PA1 and the clamp polyamide for the 146 bp DNA, NCP146 and NCP147.25, 33

Acknowledgements

We thank Kevin Sullivan for joining us in developing the original idea of the nucleosome clamp; Pamela Dyer for help with reagents; and all Luger laboratory members for help and discussion. We also thank Andrew Flaus for critical reading of the manuscript and for helpful suggestions. This work was supported by a grant supplement GM57148 from the US National Institutes of Health (NIH) to K.L., J.M.G., and P.B.D., and by NIH grant GM61909 to K.L.

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