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Phase Diagram of Nucleosome Core Particles

S. Mangenot1, A. Leforestier1, D. Durand2 and F. Livolant1*

1Laboratoire de Physique desSolides, CNRS UMR 8502, Bat510, Universite Paris-Sud91405 Orsay Cedex, France

2LURE, Universite Paris-SudBat. 209D, BP34, 91898 OrsayCedex, France

We 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 author

Introduction

The 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 – 3 Nevertheless, 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 a

large 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 – 7 Information 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.8 Although 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 – 11 andX-ray diffraction experiments.12 Our goal here is tofocus our interest on the richness of the phasediagram under conditions of concentration and

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: NCP, nucleosome core particle;EM, electron microscopy; PEG, polyethylene glycol.

doi:10.1016/j.jmb.2003.09.015 J. Mol. Biol. (2003) 333, 907–916

salt 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.

Results

Dense phases of NCP formed under lowsalt conditions

Lamello-columnar phase of NCP

The lamello-columnar phase is found formonovalent salt concentrations ranging fromCs ¼ 3.5 mM to Cs ¼ 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 Cs ¼ 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.10 It was further confirmed byX-ray diffraction analysis.12 The 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 normal to the plane of thebilayer. The front sides of the NCP (F, with theDNA ends) are facing the solvent, while the NCPback sides (B) are oriented inwards the bilayer. Asseen on the cryo-section of Figure 1b, the circular(or slightly elliptical) shape of the NCP is observedwhen the column is perfectly seen in top view. Thefact that NCP cannot be recognized everywherealong a given bilayer reveals slight deviations ofthe orientation of the columns. The orientation ofthe columns may also be different in adjacentbilayers. We have discussed how these bilayersare most probably stabilized by attractive inter-actions mediated by the amino terminal tail of his-tone H2B.10 We suspect that the formation of thesetubes and of the more complex textures presented

here originate from the chiral and electrostaticinteractions that come into play between NCPcolumns in the bilayers and between bilayersthemselves (unpublished results).

The lamellar phase was studied by X-ray formonovalent salt concentrations Cs ranging from15 mM to 25 mM and under pressures rangingfrom 5 atm to 25 atm. The scattering profiles(Figure 4, below) provide three types ofinformation. (i) In the small q-range threediffraction peaks are characteristic of the lamellarorganization with an inter-lamellar distancedecreasing from 376 A to 358 A when the osmoticpressure is increased. (ii) Other diffraction peaksobserved at higher q-values sign the existence of abi-dimensional monoclinic ordering within eachlayer of a lamella. (iii) Three broad scatteringmaxima superimposed to the narrow diffractionpeaks indicate that the NCPs are more disorderedin some parts of the sample. From X-ray data theNCP concentration can be estimated to 280–320 mg/ml in the salt and PEG concentrationranges mentioned above.

Inverse columnar hexagonal phase

For higher pressures (above 25 atm), NCPs self-organize in a different way. Freeze-fractureelectron microscopy does not reveal any lamellarstructure any more. Instead, hexagonal patternscan be seen over large domains (Figure 2b0). Inother regions of the same replicas, parallel columnsof stacked NCP can be seen (Figure 2a0). Alignedside-by-side, these columns also form bilayers(Figure 2a0, in the circle) but instead of extendinglaterally over long distances as they did in thelamellar phase, these bilayers form a honeycombhexagonal network of parameter a (sketched inFigure 2a and b). The solvent is located in parallelchannels separated from each other by the bilayers.The volume of these channels varies, as also thehexagonal parameter a to follow any change in theNCP concentration. As a matter of fact, thediameter of the NCP (11 nm), which is large com-pared to the dimensions of the hexagonal network,imposes discrete steps of variation of a. Multiplevalues of a have been measured: a < 38 nm,a < 45 nm and a < 54 nm (^2 nm). Two networksmay coexist under equilibrium conditions. Fromthese different values of a, and from the corre-sponding NCP organization (not shown), the NCPconcentration was calculated to vary from about320 mg/ml to 420 mg/ml in this phase. X-raydiffraction analyses will be necessary to get moreinformation on the fine details of the structure. Wenamed this phase “inverse hexagonal phase” byanalogy with amphiphilic systems in which polarmolecules also form bilayer structures.

The textures of this phase, observed in polariz-ing microscopy, are consistent with the structuredescribed above. Rigid rods, either isolated ororganized into 3D spiky spheres (Figure 2c), ormore classical hexagonal textures (not shown) can

908 Phase Diagram of Nucleosome Core Particles

be seen. Between crossed polars, the brightness ofeach rod is intense and its extinction occurs atonce for given orientations of the rod, whichreveals a unidirectional orientation of the NCPcolumns in these monodomains.

Dense phases of NCP formed under highsalt conditions

For salt concentrations ranging from 50 mM to160 mM, and under applied pressures varyingfrom 4.7 atm to 13 atm, a 3D crystal forms as

indicated by X-ray diffraction patterns (Figure 3).12

Columns of stacked NCP are aligned and closelypacked to form a columnar quasi-hexagonalstructure, as sketched in Figure 3a–a00. In a firstapproximation the crystalline cell is orthorhombic,like that observed in crystals obtained withreconstituted NCP in the presence of divalentions.4,13,14 Some additional Bragg peaks could resultfrom a superstructure along the column axis orfrom a slight distortion of the orthorhombic unitcell. Increasing the osmotic pressure from 4.7 atmto 13 atm produces similar reductions of all three

Figure 1. Lamello-columnar phase of NCP formed under low salt conditions (Cs , 25 mM). a–a00, Sketch of thelamello-columnar structure. Nucleosomes are first drawn to identify their front (F) and back (B) sides, that correspond,respectively, to the entry and exit sites of their pseudo-dyad axis d. Nucleosomes are then drawn as small cylinders (inperspective view) or circles (in top view) with a black dot that identifies their front side (F) with the two free ends ofDNA. Nucleosomes are stacked on top of each other to form columns and these columns are aligned in parallel toform bilayers (L). The front sides of the NCP, with the two free ends of the DNA strand, face the solvent layer separ-ating the bilayers, and the back sides (B) are oriented inwards to the bilayers. Bilayers themselves form a lamellarstructure with a period dL. b, Cryo EM section normal to the plane of the bilayers (as drawn in a00). Bilayers (L) areseparated by solvent layers (p ). NCPs can be seen in top views in regions where the axis of the columns is perfectlynormal to the section plane. Their shape is slightly elliptical, revealing that they are slightly tilted in the columns. c,A stacked series of superimposed bilayers, with a periodicity dL, is coiled to form a hollow cylinder, seen almostalong its axis on a EM freeze-fracture replica. d–f, Textures of the lamello-columnar phase formed under increasingosmotic pressure conditions (PEG 19–35%): isolated tubes in side view (d), tubes budding new tubes at theirextremities (d and e), and spherulites (f). d, e and f, Interferential Nomarski contrast.

Phase Diagram of Nucleosome Core Particles 909

orthorhombic parameters: a, b and c decrease from115.4 A, 203.6 A and 119.0 A to 110.1 A, 194.0 Aand 112.7 A, respectively. A 2D hexagonal meso-phase may also form under identical pressure andsalt conditions: we found that the initial NCP con-centration before addition of the PEG solutiondetermines the formation of the 2D or 3D columnar

phase (see Mangenot et al.12 for details). The lateralpacking of the NCP columns is then perfectlyhexagonal (Figure 3a and a00), and there is nocorrelation between the longitudinal order of NCPalong the column and the lateral organization ofcolumns. Whatever the 2D or 3D ordering of NCPin these phases, the final NCP concentrations arequite the same, close to 500 mg/ml, under apressure of 4.7 atm. This concentration may riseup to about 610 mg/ml under higher pressures(23.5 atm) at the expense of a good ordering. Theimportant point we raise here is that differentfinal states can be obtained depending on thestory of the sample preparation, while all finalconcentrations are identical.

We reported previously the formation of acolumnar phase under high salt conditions, basedupon optical and electron microscopy obser-vations.9,11 Spectacular macroscopic hexagonaldomains form, that further divide into six branches(Figure 3b), as a consequence of the chiral structureof the NCP.11 We suspect that these germs are 3Dquasi-hexagonal crystals because their texturesand defects are not characteristic of 2D meso-phases. They also lack any fluidity and never fusetogether. More rarely, more classical hexagonaltextures are also observed (Figure 3c). We assumethat these latter textures correspond to the 2Dmesophase. Micro-focus diffraction experimentson selected regions of both samples would benecessary to assign a structure to each textureunambiguously.

Figure 3. Dense phases of NCP prepared under highsalt conditions (Cs . 50 mM NaCl). a, Columns align inparallel in a close-packing arrangement, either hexagonal(a00) or orthorhombic (quasi-hexagonal) (a0). b and ctextures observed in polarizing microscopy: typicalhexagonal germs with their six branches (noted 1–6) (b)or hexagonal textures (c).

Figure 2. Inverse hexagonal phase, found under highpressure, and under low salt conditions (Cs , 25 mM). aand b, Schematic views of the hexagonal structure ofparameter a with the corresponding patterns observedin freeze-fracture EM in planes either parallel (a0) ornormal (b0) to the orientation of the columns. a0, Onecan recognize the stacking of NCP in the columns(arrow), and the association of two series of columns toform one bilayer (in the circle). The structure of eachbilayer is similar to that described in Figure 1a and b,but it does not extend laterally over large distances. Thebilayers separate the solvent channels. In b0 the solventchannels (red dots) form an hexagonal network, thatextends over large distances. The bilayer structure ofthe walls that separate these channels (drawn in b) isnot resolved here. This inverse hexagonal phase mayappear in the form of rigid birefringent rods in polariz-ing microscopy (c).


Phases formed at intermediatesalt concentrations

The organization of NCP is more complex in theintermediate range of salt concentration(25 , Cs , 50 mM). From X-ray diffractionexperiments (Figure 4), we observed that a densephase of columns forms progressively without anylong-range ordering between columns and withNCPs irregularly piled along a column. Furtheron, de-mixing progressively takes place andseveral months later the lamello-columnar phaseand the 2D hexagonal (or 3D quasi-hexagonal)phases, described above under low salt and highsalt conditions, respectively, are found to coexist.12

Samples prepared under similar conditions andobserved in the polarizing microscope revealunexpected textures due to microphase separationeffects. After a complex process of organization(unpublished results), a superimposed series oflayers forms parallel to the observation plane.They are made of platelets with a puzzle-pieceshape (Figure 5b and b0). It is remarkable that thelimits of the domains do not superimpose but arequite systematically out of phase in the successivelayers. At a higher magnification, thin tubes (thesame as described in the low salt lamello-columnarphase) can be seen on both surfaces of the layers.

Their density may be rather low, or quite high asshown in Figure 5c. The tubes do correspondunambiguously to the lamello-columnar structureand we suppose from the X-ray data12 that thelayers correspond to the (quasi) hexagonal phase,although they are not characteristic of hexagonaltextures. This organization of these two phasesinto micro domains is sketched in Figure 5a.

Isotropic to ordered phases transition

In a previous work10 we described, by cryoEM ofthin films, how NCPs stack to form isolatedcolumns which themselves form an isotropicsolution, under low salt conditions (,25 mM) at aconcentration just below the transition to thelamello-columnar phase. NCP solutions preparedfor Cs ¼ 15 mM and for NCP concentrationsranging from 5 mg/ml to 250 mg/ml (in theabsence of PEG) give IðqÞ profiles slightly differentfor q , 0:05 A21 that reflect the evolution of thestructure factor with NCP concentration. ForCNCP ¼ 250 mg/ml the curve is also slightlydifferent around q ¼ 0.11 A21 (Figure 6), which

Figure 4. Profiles of the integrated intensities IðqÞ fordifferent salt concentrations: 25 mM (lamello-columnarphase), 160 mM (3D orthorhombic crystal), 50 mM (2Dhexagonal phase) and 37 mM (biphasic sample beforeand after phase separation).

Figure 5. Microphase separation effect observed in theintermediate salt range (Cs ¼ 37.5 mM) in flat capillaries(0.2–0.4 mm thick) and observed with interferentialNomarski contrast as sketched in a. b, b0, The sameregion of the preparation observed at two different foci,showing puzzle-pieces of the hexagonal phase separatedfrom one another by a layer of solvent. An enlarged viewof the surface of these plates reveals a network of tubesof the lamello-columnar phase (c).


suggest that a fraction of NCP self-assembled intoshort columns. At CNCP < 270 mg/ml, the lamello-columnar phase is already formed. Therefore,isolated columns may exist under low salt con-ditions over a very narrow concentration range(between 250 mg/ml and 270 mg/ml).

Under high salt conditions (Cs ¼ 160 mM), theformation of the columns was not seen forCNCP # 250 mg/ml (Figure 6). They are clearly vis-ible in solutions to which a pressure of 1.6 atm(12% (w/v) PEG) was applied. The X-ray profileexhibits three broad maxima that are characteristicof a disordered phase of columns, with some dis-order along the columns. The NCP concentrationevaluated from the position of the first maximumis higher than 450 mg/ml (Figure 6). In theabsence of samples prepared at intermediate NCPconcentrations, we cannot predict the range ofconcentrations where the columns form. From thespectra analysis, we cannot predict whether thecolumns form an isotropic or a nematic phase.Optical microscopy observations show that bothcan exist. The isotropic phase turns birefringentunder flow, which is further evidence of theformation of the columns, and the nematicphase is observed for slightly higher NCPconcentrations, in a narrow domain of the phasediagram.

Phase diagram

The combination of experimental data comingfrom X-ray diffraction, optical and electronmicroscopy lead us to present the tentative NCPphase diagram given in Figure 7. More than 30different experimental conditions (PEG and salt)have been analyzed. A larger range of saltconditions has been explored more qualitatively inthe absence of applied pressure. The samestructures were found using both methods. Aremarkable self-assembling property of NCP is toform isolated columns, by stacking on top of eachother, in the absence of any divalent cation and ofDNA linking the NCPs together. These columnsform before any lateral organization is set betweenNCPs and whatever the ionic conditions are. Theyare found in a narrow concentration range(250 , CNCP , 270 mg/ml) at low salt. We suspectthis range to be larger under high salt conditions,although NCP concentrations were not determinedprecisely. These columns further align to form acolumnar nematic phase. Under an osmoticpressure fixed to 4.7 atm, a series of ordered 2D or3D-ordered phases are found: a lamello-columnarphase below 25 mM monovalent salt, a 3Dorthorhombic crystal or a 2D columnar hexagonalphase above 50 mM. The lamello-columnarphase and the high salt phase (2D or 3D) coexistsin the intermediate salt range, and a microphaseseparation occurs. An inverse hexagonal phase isfound in the low salt range for pressures above23 atm.

We observed the formation of 3D crystals usingNCP carrying DNA fragments with some poly-dispersity and longer than 146 bp. The averagedistance between columns is just slightly largerthan in 3D crystals diffracting at highresolution.4,5,13 Nevertheless, the 3D packing is lostwhen DNA fragments become too long (170 bp) ortoo polydisperse. On the contrary, the lamello-columnar and the inverse hexagonal phases affordsignificant variations of the DNA length carriedby the NCP (at least 146–170 bp). This is notsurprising, since the free ends of the DNA mayextend on both external faces of the bilayerswithout any geometrical constraint. We observethat the boundaries between these different phases,determined by ionic and pressures conditions,move with the DNA fragment length. This is dueto the increase in the negative phosphate chargesthat changes the net charge of the NCP. For thesame reasons, displacements of the phaseboundaries are also observed when the histonetails of the NCP are partly removed. As aconsequence, in samples containing NCP with awell-defined DNA length, half intact and halfdeleted of their H3 and H2B tails, a segregationoccurs under low salt conditions: intact NCP formmicrodomains of the lamello-columnar phasewhile the other NCPs pack into 3D hexagonalcrystals.

Figure 6. Scattered intensity IðqÞ of NCP solutions ofconcentration ranging from 5 mg/ml to 250 mg/ml, fortwo salt concentrations Cs ¼ 15 mM and Cs ¼ 160 mM.The upper curve was equilibrated against a 12% PEGsolution at Cs ¼ 160 mM.


Discussion

Phase diagrams and kinetic effects

Despite our systematic survey of a large range ofexperimental conditions, we cannot exclude thatother phases may exist. Theoretical phasediagrams, with predictions of different structures,could have been of some help to look for otherpossible phases but, unfortunately, only simpleobjects have been considered so far: spheres, rods,platelets, spherocylinders or even cut-spheres butwith diameter to thickness ratios that do notcorrespond to NCPs.15 – 17 Moreover, discrepanciesare often observed between predictions andobservations because of the intricacies of thekinetics of the phase transitions.18 On top of that,structural details and heterogeneous chargedistributions are not considered. Such details, thatare crucial in the case of NCP, result in specificorientational ordering in the ordered phase andintroduce more complex mechanisms in thekinetics of phase formation.19

As expected, owing to the complexity of theNCP, numerous difficulties have been encounteredin the elaboration of the phase diagram. First,equilibration times may be extremely long (severalmonths). Using PEG as a stressing polymer, thesolvent was progressively extracted from the NCPsolution and this process requires times to reachthe equilibrium conditions because our samplesare macroscopic (a few mm3). The other methodthat we have been using, i.e. the evaporation ofthe solvent, may lead much faster to the formationof the dense phases, with the limitation that theconcentration of salt in the samples is notcontrolled any more. Whatever the method, a slowprocess of organization of the sample is absolutely

necessary to obtain the organization of NCP indomains large enough to be observed in opticalmicroscopy (a few mm). Anyway, we cannot certifythat samples would not remain trapped inmetastable states for years. For example, in thebiphasic intermediate salt range, we cannotascertain that the sample would not evolve furtherand that another phase could coexist with thelamellar and the hexagonal phases. Second, theformation of the dense phases was shown todepend on the initial states in the phase diagram.The initial concentration of NCP before additionof the PEG determined the formation of either a2D or a 3D (quasi) hexagonal crystal. Third, wesuspect that the stress produced by the addition ofPEG (all at once) to the NCP solution may be ofsome importance in the kinetics of phasetransitions. Indeed, it has been demonstrated howdramatically a stress can jam a system and restrictcrystallization for years.20 Other methods ofpreparation of the samples should be tried toovercome such difficulties. Fourth, the formationof oligomers of NCP piled to form columns alsointerfere with the other parameters. Finally,microphase separation phenomena were alsoobserved in the intermediate salt range. These arereminiscent of the complex structures described insolutions of virus rods in the presence of PEG.21,22

The routes by which the equilibrium phases areformed are thus complex and a lot more remainsto be understood in the organization and kineticsof phase transitions of these NCP solutions,23 butthis complex behavior may precisely offerextremely rich possibilities of adaptation ofchromatin organization in the context of the livingcell, as discussed below.

Biological relevance

To relate our present studies in vitro and theproperties of chromatin in the cell nucleus, a lotremains to be done. Of course, the extremely longorganization times reported here are not relevantfrom a biological point of view, for several reasons.First, the organization of NCP could not extendover long distances, but would be restricted tomicrodomains inside the nucleus. Second, theformation of the higher concentrated states inchromatin never starts from an isotropic solutionbut probably emerges from an already highlyorganized state in the interphase (that we do notknow) through processes that involve not onlyphysicochemical processes but also the activity ofnumerous enzymes and cofactors. These probablyhelp chromatin organize in a sophisticated way,following an unknown path in the extremelycomplex phase diagram of chromatin. We areaware that our experimental system is extremelysimplified and we do not claim that we reproduceall details of chromatin of the living cell. A lotmore remains to be done to understand thephase transition processes. We may think, forexample of a “memory” of the ordered dense

Figure 7. Tentative phase diagram of isolated NCP as afunction of monovalent salt (3 , Cs , 160 mM) andapplied osmotic pressure.


states that could be kept up to a certain point in thede-condensation process and help in a further re-organization of the dense states. Nonetheless, ourhypothesis is that the different organizationsobserved here may exist as well locally in thechromatin of the living cell. Indeed, as detailedbelow, the explored range of salt and NCP concen-trations conditions are biologically relevant, andsome of these phases could be kept even in thepresence of linker DNA and additional proteins.In the absence of reliable data on the local ionicconditions inside the cell nucleus, we explored alarge range of monovalent salt concentrations(3.5–160 mM), that covers the values given foreukaryotic nuclei.24 – 26 The pressures that we applyrange from 1.5 atm to 25 atm. This osmoticpressure is used here as a tool to reach the NCPconcentrations that exist inside the cell nucleus(ranging from 100 mg/ml to 500 mg/ml NCP27,28).The cell is well known to be crowded29 but, to ourknowledge, there is no experimental data availableon the pressure applied by the molecular speciesforming the eukaryotic chromatin environment.Nevertheless, for comparison, a pressure of 1 atmmaintains the bacterial nucleoıd at its initialvolume after its extraction out of the bacteria30

and DNA is maintained inside the bacteriophagecapsid under a pressure of about 30–50 atm.31,32

We also obtained the formation of condensedphases of NCPs by adding divalent or multivalentcations (Ca2þ, Mg2þ, spermidine3þ or spermine4þ

for example) to dilute NCP solutions, in theabsence of any applied pressure.33,34 In the contextof the cell, all these cations and also chargedproteins, may interact with nucleosomes in combi-nation with the macromolecular crowding effects.

The path followed by the DNA molecule insidethe chromosome is not known, and despite theactual consensus about it, the 30 nm chromatinfiber observed in vitro may not be the only possibleorganization of the chromatin fiber in vivo. Otherpossible models may be considered and liquidcrystalline states are good candidates. Numerousmodels may be proposed. As shown above, slightchanges in the ionic condition are enough toinduce phase transitions. For example, the NCPconcentration drops from 500 mg/ml to 300 mg/ml under a 5 atm pressure, when the monovalentsalt concentration, Cs, changes from 50 mM to25 mM salt. The accessibility of NCP changesaccordingly. Similar phase transitions are likely tooccur in the living cell, and may be useful tocontrol the accessibility of enzymes to the DNAmolecule. Together with the diversity of organiz-ation of the isolated NCP, we observe that thenature of the phase may depend on the initialstate and on the path followed in the phasediagram. The kinetic problems reported here maybe even more complex in chromatin, where NCPsare linked together and interact with other proteinsand multiple kinds of ions. We guess that thiscomplexity may offer extremely rich possibilitiesof adaptation of chromatin organization to

multiple local constraints in the context of theliving cell. Local phase transitions may betriggered by local changes in ionic conditions orby changes in the distribution of charges on thehistone tails, under the action of specificenzymes. Such transitions may be coupled tomacromolecular transitions (helix-coil transition)as suggested by Samulski35 and/or be involved insynchronous expression of genetic information(euchromatin to heterochromatin).

Materials and Methods

NCP

Most of the data presented here were obtained withtwo different batches of NCPs prepared from calfthymus chromatin. DNA fragments associated to thehistone core were 155(^7) bp and 165(^10) bp,respectively. A few preparations were also made withNCP with exactly 146 bp associated DNA. The integrityof the histones was checked carefully for each batch.Stock solutions were extensively dialyzed against TEbuffer (10 mM Tris–EDTA (pH 7.6)) at a concentrationof 1–3 mg/ml and concentrated by ultrafiltration up to150–200 mg/ml in the same buffer. These stock solutionswere stored at 0 8C.

Sample preparation

Aliquots of the stock NCP solutions were diluted anddialyzed against TE buffer eventually supplementedwith NaCl. The concentration of monovalent ions in thedialysis buffer (Cs) (including Trisþ and Naþ) wasadjusted to concentrations ranging from 3.5 mM to160 mM. To prepare NCP solutions of different concen-trations, while keeping constant the Cs values of theequilibration buffer, samples were prepared under con-trolled osmotic pressure. Different methods were used:for moderate pressures (0.1–4 atm), (i) samples wereplaced in dialysis bags and immersed in a large volumeof a neutral polymer solution (polyethylene glycol, Mr

20,000 from Sigma), prepared in the same buffer, withthe same Cs concentration; or (ii) samples wereequilibrated by ultrafiltration through a nitrocellulosemembrane under nitrogen pressure. To reach higherpressures (4.7–23 atm), the NCP solution was first con-centrated by one of the two methods described aboveup to a concentration of about C ¼ 225–260 mg/ml.Equilibrated samples for X-ray and microscopy experi-ments are prepared as detailed below. The osmoticpressures are related to the PEG concentration(calculated with the help of the empirical expression inRef. 36: 12% PEG ¼ 1.6 atm; 16% PEG ¼ 3.1 atm; 19%PEG ¼ 4.7 atm; 23% PEG ¼ 7.6 atm; 25.5% PEG ¼ 10atm; 28% PEG ¼ 13 atm; 35% PEG ¼ 23 atm. Formicroscopy and X-ray experiments, the followingprotocols were used.

Microscopy

A 20 ml sample of the NCP solution was introducedinto flat capillaries (Vitro Dynamics). The PEG solution(with the same salt concentration, Cs) was then intro-duced in the capillary, pushing the NCP solution andcreating a PEG gradient from one extremity of the


capillary to the other. The two extremities of the capillarywere sealed with wax and the specimen left to stabilize.We did not observe any change in the specimen after afew weeks. To explore rapidly the phase diagram,another method was used in some cases: drops of NCPsolutions of defined salt and NCP concentration weredeposited between slide and coverslip and left to con-centrate progressively by slow dehydration. Salt andNCP concentrations corresponding to the observedtextures were calculated a posteriori from the volumevariation between the initial and final steps.Observations were made with a Nikon polarizingmicroscope equipped for interferential Nomarskicontrast.

For electron microscopy, drops of the phases preparedin flat capillaries or in dialysis bags were deposited ontogold discs and stabilized in a humid atmosphere. Dropsof the same samples deposited onto glass slides wereused as controls to check the presence of the expectedtextures in the polarizing microscope. Samples werefrozen by projection onto a copper surface cooled downto 10 K with liquid helium (Cryovacublock Reichert).Freeze fracture was realized in a Balzers BAF 400Tapparatus and replicas observed in a Philips CM12 TEMat 80 kV. Cryo-sections (40–80 nm thick) were realizedat 113 K under nitrogen atmosphere in a cryo-ultra-microtome (Leica) and observed at 100 K in a PhilipsCM12 cryo-TEM at 80 kV. The vitreous state of waterwas checked by electron diffraction, and imagesrecorded in low-dose mode at a direct magnification of45,000 £ and 600–700 nm defocus.

X-ray diffraction

About 15–20 ml of nucleosome solution and 200 ml ofpolymer solution were successively added into quartzcapillaries ,1.5 mm in diameter and left to equilibratefor more than four weeks at room temperature. In somecases, in particular for the dense phases obtained athigh salt, the sample-containing capillaries were placedin a magnetic field higher than 7 T immediately afteraddition of the PEG solution to the NCP solution andkept during the whole formation of the dense phase. Itproduced an alignment of the NCP columns that waskept after removal of the magnetic field and helped inthe understanding of diffraction patterns. X-ray experi-ments were carried out on the D24 instrument installedon the storage ring LURE-DCI (Orsay, France) and onbeam line ID2 at the European Synchrotron RadiationFacility (ESRF, Grenoble, France). On D24 (ID2) thewavelength was 1.488 A (0.989 A) and the sample-to-detector distance was 2500 mm (4730 mm), respectively.These setups gave access to scattering vectors q (whereq ¼ 4p sin u=l; 2u is the scattering angle) ranging from0.01 A21 to 0.25 A21 (0.01–0.35 A21) and to an instru-mental resolution Dq (full width at half maximumFWHM value) of 0.00145 A21 (0.0008 A21).

Acknowledgements

This work has been supported by The NationalCenter for Scientific Research (CNRS), by theFrench Research Department and the ResearchAssociation Research against Cancer (ARC) who

provided one of us (S.M.) with a three year and athree month fellowship, respectively.

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Edited by J. O. Thomas

(Received 28 May 2003; received in revised form 3 September 2003; accepted 6 September 2003)


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