Phase Diagram of Nucleosome Core ParticlesIntroductionResultsDense phases of NCP formed under low salt conditionsDense phases of NCP formed under high salt conditionsPhases formed at intermediate salt concentrationsIsotropic to ordered phases transitionPhase diagramDiscussionPhase diagrams and kinetic effectsBiological relevanceMaterials and MethodsNCPSample preparationMicroscopyX-ray diffractionAcknowledgementsReferencesPhase Diagram of Nucleosome Core ParticlesS. Mangenot1, A. Leforestier1, D. Durand2and F. Livolant1*1Laboratoire de Physique desSolides, CNRS UMR 8502, Baˆt510, Universite´Paris-Sud91405 Orsay Cedex, France2LURE, Universite´Paris-SudBat. 209D, BP34, 91898 OrsayCedex, FranceWe present a phase diagram of the nucleosome core particle (NCP) as afunction of the monovalent salt concentration and applied osmoticpressure. Above a critical pressure, NCPs stack on top of each other toform columns that further organize into multiple columnar phases. Anisotropic (and in some cases a nematic) phase of columns is observed inthe moderate pressure range. Under higher pressure conditions, alamello-columnar phase and an inverse hexagonal phase form under lowsalt conditions, whereas a 2D hexagonal phase or a 3D orthorhombicphase is found at higher salt concentration. For intermediate salt concen-trations, microphase separation occurs. The richness of the phase diagramoriginates from the heterogeneous distribution of charges at the surface ofthe NCP, which makes the particles extremely sensitive to small ionicvariations of their environment, with consequences on their interactionsand supramolecular organization. We discuss how the polymorphism ofNCP supramolecular organization may be involved in chromatin changesin the cellular context.q 2003 Elsevier Ltd. All rights reserved.Keywords: nucleosome; phase diagram; ordered phases; supramolecularorganization; chromatin*Corresponding authorIntroductionThe eukaryotic genome is packaged intochromatin. Recent advances have revealed thatchromatin structure is highly dynamic and subjectto reversible changes in higher-order folding andnucleosome positioning. The structural changesare largely mediated by enzymatic covalent modi-fications of DNA and of the flexible N-terminalamino acid residues of the core histones and bynon-covalent alterations of nucleosome archi-tecture driven by ATP-dependent chromatinremodeling enzymes.1–3Nevertheless, we seriouslylack structural data about these multiple localchanges of chromatin organization that occurlocally (at the scale of a gene or a group of genes)inside the living cell. To overcome the difficulty ofanalyzing the structural details of chromatinorganization and their changes in situ, simplifiedexperimental models can be used to explore themultiple interactions and possible supramolecularorganizations that chromatin elementary units, thenucleosome core particles (NCPs), can form over alarge range of experimental conditions. 3D crystalsobtained with NCP reconstituted from recombi-nant DNA and histones were used to determinethe atomic structure of the NCP.4–7Information onthe interactions between the particles has alsobeen collected by the analysis of the contactsbetween NCPs inside these crystals. It was shownthat these interactions depend highly on slightchanges in the charges carried by the histonetails.8Although interactions between NCPs insidethe crystals may differ from the interactions thatcome into play inside the nucleus, they give usinformation about possible relative positioning ofnucleosomes inside chromatin. However, thedramatic limitation of these crystallographicstudies comes from the limited sets of experi-mental conditions that can be explored. To bypassthis limitation, some years ago we began asystematic survey of the phases formed by theNCP in changing conditions of ionic strength andosmotic pressure. Multiple phases have beenobserved that cover a large range of monovalentsalt and NCP concentrations. Most of them havebeen characterized precisely by combining opticaland electron microscopy observations9–11andX-ray diffraction experiments.12Our goal here is tofocus our interest on the richness of the phasediagram under conditions of concentration and0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.E-mail address of the corresponding author:[email protected] used: NCP, nucleosome core particle;EM, electron microscopy; PEG, polyethylene glycol.doi:10.1016/j.jmb.2003.09.015 J. Mol. Biol. (2003) 333, 907–916salt that may be interesting from a biological pointof view. We set out to determine the broad con-ditions under which each phase forms, and wherepossible to follow interconversions between them.We show how slight changes in the ionic con-ditions may have tremendous effects on the inter-actions between particles and produce largechanges in their supramolecular organization.ResultsDense phases of NCP formed under lowsalt conditionsLamello-columnar phase of NCPThe lamello-columnar phase is found formonovalent salt concentrations ranging fromCs¼ 3.5 mM to Cs¼ 25 mM and for pressuresranging from about 3 atm to 25 atm. Within thisrange of experimental conditions, multiple texturesare observed in optical microscopy (Figure 1d–f).The evolution of these textures was followed byincreasing the applied osmotic pressures at a saltconcentration Cs¼ 15 mM. Isolated tubes formfirst, under pressures ranging from 3 atm to 5 atm(Figure 1d). They progressively shorten and newconnected tubes form at their extremities(5–10 atm) leading to the formation of densespherulites (15–25 atm) (Figure 1e and f). Thewalls of the tubes are formed by coiling of a seriesof stacked layers, as seen in freeze-fracture EMwhen the fracture plane is normal to the axis ofthe tube (Figure 1c). These various textures arealso observed independently of the presence of adialysis membrane separating the NCP from thePEG. Whatever the observed textures, the phase islamello-columnar. A first description of this phasewas given using cryo-sections of vitrified materialobserved in cryoEM.10It was further confirmed byX-ray diffraction analysis.12The structure of thisphase is sketched in Figure 1, in a perspectiveview (Figure 1a0) and in a section plane, normal tothe plane of the layers (Figure 1a00). Bilayers ofNCP columns (L) alternate with layers of solvent(stars in Figure 1b). NCPs are stacked on top ofeach other in the columns and oriented with theirdyad axis more or less
View Full Document
Unlocking...