A dynamic view of DNA structure within the nucleosome: Biological implications

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Highlights

  • What are the DNA dynamical properties within a nucleosome?

  • The dynamics of nucleosomal DNA is put in perspective by comparison to free DNA.

  • Essential local dynamical characteristics of bound DNA mirror those of free DNA.

  • The modulation of dynamics by the sequence is a permanent DNA property.

  • Such bound and free DNA similarities may be exploited in chromatin organization.

Abstract

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNANCP) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using CHARMM36 MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNANCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNANCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNANCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties.

These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNANCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP.

Introduction

The nucleosome core particle (NCP) is one of the most notorious DNA-protein complexes, as the fundamental building block of packaged DNA in eukaryotic cells. X-ray structures showed that the DNA in NCP (DNANCP) wraps ~1.7 times around eight histone proteins (two copies of H2A, H2B, H3, and H4) to form a super-helical double helix, as described in numerous reviews (Cutter and Hayes, 2015, Kornberg and Lorch, 1999, Luger, 2001, Luger et al., 1997, Luger and Richmond, 1998, McGinty and Tan, 2015, Zhou et al., 2018). DNANCP of different sequences and lengths (145 to 147 bp) adapts to highly conserved histone binding motifs, regularly positioned at the surface of the histone structured domains. The DNANCP superhelical path and the associated curvature rely on two inter base-pair parameters, roll and slide (Olson and Zhurkin, 2011, Tolstorukov et al., 2007, Xu and Olson, 2010), complemented by twist and rise adjustments (Edayathumangalam et al., 2005, McGinty and Tan, 2015, Muthurajan et al., 2003, Ong et al., 2007, Tan and Davey, 2011). Along the DNA sequences, the values of these inter base-pair parameters and the groove dimensions follow more or less marked sinusoidal profiles, with oscillation period close to 10 base-pairs (bp) (Bishop, 2008, Olson and Zhurkin, 2011, Wu et al., 2010, Xu and Olson, 2010, Yang and Yan, 2011).

The DNA access and readability are often presented as closely related to the in vivo nucleosome positioning that results from complex processes involving a series of trans-acting factors (reviews: (Hughes and Rando, 2014, Lieleg et al., 2015, McGinty and Tan, 2016, Zhou et al., 2018)), but also the intrinsic DNA properties. However, disassembling DNANCP from the histones is not an absolute requirement for binding it and forming NCP-factor complexes. Even when complexed with the histones, DNANCP remains available enough to interact or even operate with chaperones, chromatin remodelers, enzymes or transcription factors (Fernandez Garcia et al., 2019, Kobayashi and Kurumizaka, 2019, Mayanagi et al., 2019, McGinty and Tan, 2016, Speranzini et al., 2016, Volokh et al., 2016, Zhou et al., 2018). The DNase I enzyme is an interesting example, since it has long been exploited in nucleosome studies (Klug and Lutter, 1981, Simpson and Stafford, 1983) and still is (Zhong et al., 2016). Essentially, the catalytic site of DNase I fills the DNA minor groove and cleaves the phosphodiester linkage (Lahm and Suck, 1991, Weston et al., 1992). Applied on NCP, this enzyme targets the wide minor grooves pointing outwards, opposite to the histone octamer, and produces a typical oscillatory cleavage profile (Zhong et al., 2016 and references herein). This property was cleverly exploited to detect transcription factors that bind to DNANCP and consequently disrupt the DNAse I periodic cleavage pattern (Zhong et al., 2016).

The ability of DNANCP to recognize proteins raises in particular the issue of its dynamical behavior. Indeed, it is now accepted that small, frequently subtle motions of free (unbound) DNA affect the assembly of nucleoprotein complexes (Battistini et al., 2019, Rohs et al., 2010). How the local (for instance roll or twist) or semi-local (groove dimensions, curvature) DNA sequence-dependent flexibility influences protein affinities was documented on various systems (Abe et al., 2015, Azad et al., 2018, Djuranovic et al., 2004, Djuranovic and Hartmann, 2005, Heddi et al., 2010a, Heddi et al., 2008, Koudelka and Carlson, 1992, Parvin et al., 1995, Tisné et al., 1999). As typical examples, the dynamics of NF-κB DNA targets is needed to transiently expose the specific base atom pattern recognized by the protein (Tisné et al., 1999, Wecker et al., 2002); also, the DNase I cleavage efficiency is increased by a malleable minor groove that favors the enzyme anchoring (Heddi et al., 2010a).

However, to which extent are structural fluctuations, potentially functionally relevant, preserved, damped or even suppressed in the case of a bound, and therefore constrained, DNA? For such a question, DNANCP is a paradigm: on one hand, at least a residual malleability should be preserved to ensure the structural fit with external partners; on the other hand, DNANCP seems robustly constrained since its super helix path is maintained by an especially dense interaction network involving both the histone structured domains and specific N-terminal regions along the two DNA strands, as recently observed on a nucleosome simulated in solution (Elbahnsi et al., 2018). Despite this, studies suggest that at least some DNANCP properties are reminiscent of those of free DNA. Indeed, free and DNANCP profiles are qualitatively parallel for i) roll and twist (Chua et al., 2012, Richmond and Davey, 2003), ii) roll, twist and phosphate group conformers (Heddi et al., 2010b), iii) roll, twist, and slide (Marathe and Bansal, 2011, Wu et al., 2010), or iv) roll, twist and groove width (Xu and Olson, 2010). This is probably indicative of coordinated motions at the dinucleotide level as observed in free DNA (Dans et al., 2019, Dršata et al., 2013, Heddi et al., 2010b, Heddi et al., 2006, Imeddourene et al., 2016). Concerning the dispersion of DNANCP helical parameter values, a direct way to characterize the DNANCP malleability, studies based on X-ray (Dlakić et al., 2005) or simulations (Roccatano et al., 2007, Sun et al., 2019) mentioned that the ranges of thermal motion of DNANCP and free DNA were similar.

The scarcity of insights on DNANCP dynamics reflects the notorious difficulty to decipher dynamical properties experimentally, in particular DNA flexibility, a situation further aggravated in the case of nucleoprotein complex as large as NCP. While potentially influenced by biases such as intermolecular NCP contacts and crystal packing (Harp et al., 2000, McGinty and Tan, 2015, Tsunaka et al., 2005), examining X-ray structures is of course helpful, but the essentially static character of solid state models limits the information about dynamics. Solution or solid-state NMR is potentially of great interest to approach DNANCP dynamics but remains impracticable because the combination of the DNANCP molecular size and the limited dispersion of chemical shifts causes uninterpretable broadened and overlapping signals (van Emmerik and van Ingen, 2019). In this situation, molecular simulations are a promising alternative, especially considering the improvements of DNA force fields (Ben Imeddourene et al., 2015, Dans et al., 2017, Hart et al., 2012, Zgarbová et al., 2017, Zgarbová et al., 2013). Two among these force fields, Parmbsc0εςOLI (Zgarbová et al., 2013) and CHARMM36 (Hart et al., 2012), were tested on a series of simulations of 1 μs each on four free DNA dodecamers related to the 601 sequence (Ben Imeddourene et al., 2015). We recall that the 601 sequence, also called Widom sequence, is widely used for positioning nucleosomes in in vitro and in vivo experiments because of its high affinity for the histone octamer (Thåström et al., 2004). The MDs of dodecamers yielded output in reasonable agreement with NMR-inferred data, despite being intentionally carried out without experimental structural restraints, and in spite of strong differences in the conception and parametrization of the two underlying force fields. CHARMM36 in particular provided a satisfactory representation of the backbone dihedral transitions and their modulation by the dinucleotide sequence, features that had long been defective in prior simulations.

Given the force field progress, we decided to carry out MD simulations of a nucleosome formed with the 601 sequence in explicit solvent using the CHARMM36 force field (Hart et al., 2012), for a total duration of more than 1 μs. Our aim in this previous work was to describe the DNA/histone interface (Elbahnsi et al., 2018) in conjunction with VLDM, a Voronoi tessellation-based method analyzing the topology of interacting elements without any empirical or subjective adjustment (Elbahnsi et al., 2018, Esque et al., 2013, Retureau et al., 2019). Here, we exploited the same simulations to gain a direct view of the behavior of DNANCP in solution and highlight the main features of the DNANCP dynamics. To obtain a comparison between free and histone bound DNA, we also draw on MDs of free dodecamer segments that together cover 39 base pairs of the 5′ half of 601 sequence (Fig. 1). As implied above, these dodecamer simulations were initially performed for force field tests (Ben Imeddourene et al., 2015), with protocol and force-field consistent with the MDs of NCP.

Several analyses presented here address DNA backbone motions, for the following reasons. Backbone motions in free B-DNA consist in coordinated conformational transitions of ε and ζ dihedral angles between two states called BI and BII. BI and BII populations on oligonucleotides are extrapolated from 31P NMR chemical shifts (Heddi et al., 2006, Tian et al., 2009), relatively easily measured (Gorenstein, 1992). NMR-based studies have provided a general framework to understand the BI ↔ BII equilibrium and its structural effect on the double helix (Heddi et al., 2010b, Heddi et al., 2006, Imeddourene et al., 2016, Isaacs and Spielmann, 2001, Schwieters and Clore, 2007, Tian et al., 2009). Combining experimental data and modelling established that, in NpN·NpN steps (N for any nucleotide), motions of the two facing phosphodiester linkages are concomitant with variations of the relative positions of two successive bases (Dans et al., 2019, Dršata et al., 2013, Heddi et al., 2010b, Heddi et al., 2006, Imeddourene et al., 2016). This reciprocal dependence involves the BI/BII states, the slide, roll, twist (Ben Imeddourene et al., 2015, Heddi et al., 2010b, Imeddourene et al., 2016) and, to a lesser extent, the rise, tilt and shift (Dršata et al., 2013). The BI or BII density per 4–5 bp segments and the groove dimensions are also coupled (Oguey et al., 2010, Xu et al., 2014). Another key point is that NMR data ascertained that the BI/BII propensity is highly sensitive to the DNA sequence (Heddi et al., 2010b, Heddi et al., 2006, Schwieters and Clore, 2007, Tian et al., 2009, Xu et al., 2014). Owing to the structural couplings, the BI/BII populations of each dinucleotide reflect the conformational landscape that this dinucleotide explores. So, in free DNA, ApA/G·T/CpT and ApT/C·A/GpT have only access to a restricted conformational region characterized by predominant BI conformer, negative slide, null or positive roll and low or moderate twist. On the other hand, GpG·CpC, CpG·CpG, GpC·GpC and CpA·TpG are more flexible, with BI ↔ BII oscillations associated to the broadening of slide, roll and twist ranges towards positive, negative and high values, respectively. In sum, the phosphate group motion is an effective reporter of the sequence-dependent local deformability of DNA, and its intramolecular energetics. This is the reason why the backbone states deserve such attention.

This study presents extensive analyzes from careful simulations carried out on DNANCP, and relevant free oligomers, with a state-of-the-art protocol and validated energy model. It documents multiple aspects of DNANCP dynamics including atomic fluctuations, motions of backbone, variability of inter base pair parameters and couplings involving helical descriptors. The similarities or differences between DNANCP and free DNA are systematically surveyed. Thanks to the exhaustive dataset collected on the DNA-histone interface, we also examine the relation between the DNANCP dynamics and the contacts with the histones. Then, the role of the different dinucleotide intrinsic properties on the propensity of a DNA sequence to assemble into a nucleosome is considered in the light of the above properties of DNANCP. Taken together, those observations provide a clearer picture of nucleosomal DNA when considering its biological functions.

Section snippets

Materials and methods

The details of our Molecular Dynamics (MD) setup were presented in two previous papers, one on the nucleosome (Elbahnsi et al., 2018) and the other on the free DNA dodecamers (Ben Imeddourene et al., 2015). We therefore only summarize here the main aspects of the protocols.

Overview of the studied trajectories

Four nucleosome systems (SYS1, SYS1-bis, SYS2 and SYS2-bis) were considered, which differed initially by the conformations of the histone unstructured domains, called tails (see Fig. S1, Table S1 and Materials and Methods). As explained in Introduction, the four simulations were recently used to provide a detailed but focused description of the DNA-histone interface in solution (Elbahnsi et al., 2018). In this previous work, the integrity and stability of the nucleosome was checked following

Conclusions

The widespread depictions of NCPs have popularized the view of a packed DNA appearing quite different from its free state, because of its super-helical path wrapped around the histone structured domains. Such tight bending around the histones could suggest a regime for DNANCP with dramatically altered properties compared to those of free DNA, including highly restricted dynamics. The DNA-histone interface seems a priori to corroborate this view, considering the dense interaction network that

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The simulations were carried out on the GENCI-CEA platform.

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