Download Phase Diagram of Nucleosome Core Particles

doi:10.1016/j.jmb.2003.09.015
J. Mol. Biol. (2003) 333, 907–916
Phase Diagram of Nucleosome Core Particles
S. Mangenot1, A. Leforestier1, D. Durand2 and F. Livolant1*
1
Laboratoire de Physique des
Solides, CNRS UMR 8502, Bât
510, Université Paris-Sud
91405 Orsay Cedex, France
2
LURE, Université Paris-Sud
Bat. 209D, BP34, 91898 Orsay
Cedex, France
We present a phase diagram of the nucleosome core particle (NCP) as a
function of the monovalent salt concentration and applied osmotic
pressure. Above a critical pressure, NCPs stack on top of each other to
form columns that further organize into multiple columnar phases. An
isotropic (and in some cases a nematic) phase of columns is observed in
the moderate pressure range. Under higher pressure conditions, a
lamello-columnar phase and an inverse hexagonal phase form under low
salt conditions, whereas a 2D hexagonal phase or a 3D orthorhombic
phase is found at higher salt concentration. For intermediate salt concentrations, microphase separation occurs. The richness of the phase diagram
originates from the heterogeneous distribution of charges at the surface of
the NCP, which makes the particles extremely sensitive to small ionic
variations of their environment, with consequences on their interactions
and supramolecular organization. We discuss how the polymorphism of
NCP supramolecular organization may be involved in chromatin changes
in the cellular context.
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: nucleosome; phase diagram; ordered phases; supramolecular
organization; chromatin
Introduction
The eukaryotic genome is packaged into
chromatin. Recent advances have revealed that
chromatin structure is highly dynamic and subject
to reversible changes in higher-order folding and
nucleosome positioning. The structural changes
are largely mediated by enzymatic covalent modifications of DNA and of the flexible N-terminal
amino acid residues of the core histones and by
non-covalent alterations of nucleosome architecture driven by ATP-dependent chromatin
remodeling enzymes.1 – 3 Nevertheless, we seriously
lack structural data about these multiple local
changes of chromatin organization that occur
locally (at the scale of a gene or a group of genes)
inside the living cell. To overcome the difficulty of
analyzing the structural details of chromatin
organization and their changes in situ, simplified
experimental models can be used to explore the
multiple interactions and possible supramolecular
organizations that chromatin elementary units, the
nucleosome core particles (NCPs), can form over a
Abbreviations used: NCP, nucleosome core particle;
EM, electron microscopy; PEG, polyethylene glycol.
E-mail address of the corresponding author:
[email protected]
large range of experimental conditions. 3D crystals
obtained with NCP reconstituted from recombinant DNA and histones were used to determine
the atomic structure of the NCP.4 – 7 Information on
the interactions between the particles has also
been collected by the analysis of the contacts
between NCPs inside these crystals. It was shown
that these interactions depend highly on slight
changes in the charges carried by the histone
tails.8 Although interactions between NCPs inside
the crystals may differ from the interactions that
come into play inside the nucleus, they give us
information about possible relative positioning of
nucleosomes inside chromatin. However, the
dramatic limitation of these crystallographic
studies comes from the limited sets of experimental conditions that can be explored. To bypass
this limitation, some years ago we began a
systematic survey of the phases formed by the
NCP in changing conditions of ionic strength and
osmotic pressure. Multiple phases have been
observed that cover a large range of monovalent
salt and NCP concentrations. Most of them have
been characterized precisely by combining optical
and electron microscopy observations9 – 11 and
X-ray diffraction experiments.12 Our goal here is to
focus our interest on the richness of the phase
diagram under conditions of concentration and
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
908
salt that may be interesting from a biological point
of view. We set out to determine the broad conditions under which each phase forms, and where
possible to follow interconversions between them.
We show how slight changes in the ionic conditions may have tremendous effects on the interactions between particles and produce large
changes in their supramolecular organization.
Results
Dense phases of NCP formed under low
salt conditions
Lamello-columnar phase of NCP
The lamello-columnar phase is found for
monovalent salt concentrations ranging from
Cs ¼ 3.5 mM to Cs ¼ 25 mM and for pressures
ranging from about 3 atm to 25 atm. Within this
range of experimental conditions, multiple textures
are observed in optical microscopy (Figure 1d –f).
The evolution of these textures was followed by
increasing the applied osmotic pressures at a salt
concentration Cs ¼ 15 mM. Isolated tubes form
first, under pressures ranging from 3 atm to 5 atm
(Figure 1d). They progressively shorten and new
connected tubes form at their extremities
(5 – 10 atm) leading to the formation of dense
spherulites (15 – 25 atm) (Figure 1e and f). The
walls of the tubes are formed by coiling of a series
of stacked layers, as seen in freeze-fracture EM
when the fracture plane is normal to the axis of
the tube (Figure 1c). These various textures are
also observed independently of the presence of a
dialysis membrane separating the NCP from the
PEG. Whatever the observed textures, the phase is
lamello-columnar. A first description of this phase
was given using cryo-sections of vitrified material
observed in cryoEM.10 It was further confirmed by
X-ray diffraction analysis.12 The structure of this
phase is sketched in Figure 1, in a perspective
view (Figure 1a0 ) and in a section plane, normal to
the plane of the layers (Figure 1a00 ). Bilayers of
NCP columns (L) alternate with layers of solvent
(stars in Figure 1b). NCPs are stacked on top of
each other in the columns and oriented with their
dyad axis more or less normal to the plane of the
bilayer. The front sides of the NCP (F, with the
DNA ends) are facing the solvent, while the NCP
back sides (B) are oriented inwards the bilayer. As
seen on the cryo-section of Figure 1b, the circular
(or slightly elliptical) shape of the NCP is observed
when the column is perfectly seen in top view. The
fact that NCP cannot be recognized everywhere
along a given bilayer reveals slight deviations of
the orientation of the columns. The orientation of
the columns may also be different in adjacent
bilayers. We have discussed how these bilayers
are most probably stabilized by attractive interactions mediated by the amino terminal tail of histone H2B.10 We suspect that the formation of these
tubes and of the more complex textures presented
Phase Diagram of Nucleosome Core Particles
here originate from the chiral and electrostatic
interactions that come into play between NCP
columns in the bilayers and between bilayers
themselves (unpublished results).
The lamellar phase was studied by X-ray for
monovalent salt concentrations Cs ranging from
15 mM to 25 mM and under pressures ranging
from 5 atm to 25 atm. The scattering profiles
(Figure 4, below) provide three types of
information. (i) In the small q-range three
diffraction peaks are characteristic of the lamellar
organization with an inter-lamellar distance
decreasing from 376 Å to 358 Å when the osmotic
pressure is increased. (ii) Other diffraction peaks
observed at higher q-values sign the existence of a
bi-dimensional monoclinic ordering within each
layer of a lamella. (iii) Three broad scattering
maxima superimposed to the narrow diffraction
peaks indicate that the NCPs are more disordered
in some parts of the sample. From X-ray data the
NCP concentration can be estimated to 280 –
320 mg/ml in the salt and PEG concentration
ranges mentioned above.
Inverse columnar hexagonal phase
For higher pressures (above 25 atm), NCPs selforganize in a different way. Freeze-fracture
electron microscopy does not reveal any lamellar
structure any more. Instead, hexagonal patterns
can be seen over large domains (Figure 2b0 ). In
other regions of the same replicas, parallel columns
of stacked NCP can be seen (Figure 2a0 ). Aligned
side-by-side, these columns also form bilayers
(Figure 2a0 , in the circle) but instead of extending
laterally over long distances as they did in the
lamellar phase, these bilayers form a honeycomb
hexagonal network of parameter a (sketched in
Figure 2a and b). The solvent is located in parallel
channels separated from each other by the bilayers.
The volume of these channels varies, as also the
hexagonal parameter a to follow any change in the
NCP concentration. As a matter of fact, the
diameter of the NCP (11 nm), which is large compared to the dimensions of the hexagonal network,
imposes discrete steps of variation of a. Multiple
values of a have been measured: a < 38 nm,
a < 45 nm and a < 54 nm (^ 2 nm). Two networks
may coexist under equilibrium conditions. From
these different values of a, and from the corresponding NCP organization (not shown), the NCP
concentration was calculated to vary from about
320 mg/ml to 420 mg/ml in this phase. X-ray
diffraction analyses will be necessary to get more
information on the fine details of the structure. We
named this phase “inverse hexagonal phase” by
analogy with amphiphilic systems in which polar
molecules also form bilayer structures.
The textures of this phase, observed in polarizing microscopy, are consistent with the structure
described above. Rigid rods, either isolated or
organized into 3D spiky spheres (Figure 2c), or
more classical hexagonal textures (not shown) can
Phase Diagram of Nucleosome Core Particles
909
Figure 1. Lamello-columnar phase of NCP formed under low salt conditions (Cs , 25 mM). a– a00 , Sketch of the
lamello-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 (in
perspective view) or circles (in top view) with a black dot that identifies their front side (F) with the two free ends of
DNA. Nucleosomes are stacked on top of each other to form columns and these columns are aligned in parallel to
form bilayers (L). The front sides of the NCP, with the two free ends of the DNA strand, face the solvent layer separating the bilayers, and the back sides (B) are oriented inwards to the bilayers. Bilayers themselves form a lamellar
structure with a period dL. b, Cryo EM section normal to the plane of the bilayers (as drawn in a00 ). Bilayers (L) are
separated by solvent layers (p ). NCPs can be seen in top views in regions where the axis of the columns is perfectly
normal 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 almost
along its axis on a EM freeze-fracture replica. d – f, Textures of the lamello-columnar phase formed under increasing
osmotic pressure conditions (PEG 19 – 35%): isolated tubes in side view (d), tubes budding new tubes at their
extremities (d and e), and spherulites (f). d, e and f, Interferential Nomarski contrast.
be seen. Between crossed polars, the brightness of
each rod is intense and its extinction occurs at
once for given orientations of the rod, which
reveals a unidirectional orientation of the NCP
columns in these monodomains.
Dense phases of NCP formed under high
salt conditions
For salt concentrations ranging from 50 mM to
160 mM, and under applied pressures varying
from 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 closely
packed to form a columnar quasi-hexagonal
structure, as sketched in Figure 3a – a00 . In a first
approximation the crystalline cell is orthorhombic,
like that observed in crystals obtained with
reconstituted NCP in the presence of divalent
ions.4,13,14 Some additional Bragg peaks could result
from a superstructure along the column axis or
from a slight distortion of the orthorhombic unit
cell. Increasing the osmotic pressure from 4.7 atm
to 13 atm produces similar reductions of all three
910
Phase Diagram of Nucleosome Core Particles
Figure 3. Dense phases of NCP prepared under high
salt conditions (Cs . 50 mM NaCl). a, Columns align in
parallel in a close-packing arrangement, either hexagonal
(a00 ) or orthorhombic (quasi-hexagonal) (a0 ). b and c
textures observed in polarizing microscopy: typical
hexagonal germs with their six branches (noted 1 –6) (b)
or hexagonal textures (c).
Figure 2. Inverse hexagonal phase, found under high
pressure, and under low salt conditions (Cs , 25 mM). a
and b, Schematic views of the hexagonal structure of
parameter a with the corresponding patterns observed
in freeze-fracture EM in planes either parallel (a0 ) or
normal (b0 ) to the orientation of the columns. a0 , One
can recognize the stacking of NCP in the columns
(arrow), and the association of two series of columns to
form one bilayer (in the circle). The structure of each
bilayer is similar to that described in Figure 1a and b,
but it does not extend laterally over large distances. The
bilayers separate the solvent channels. In b0 the solvent
channels (red dots) form an hexagonal network, that
extends over large distances. The bilayer structure of
the walls that separate these channels (drawn in b) is
not resolved here. This inverse hexagonal phase may
appear in the form of rigid birefringent rods in polarizing microscopy (c).
orthorhombic parameters: a, b and c decrease from
115.4 Å, 203.6 Å and 119.0 Å to 110.1 Å, 194.0 Å
and 112.7 Å, respectively. A 2D hexagonal mesophase may also form under identical pressure and
salt conditions: we found that the initial NCP concentration before addition of the PEG solution
determines the formation of the 2D or 3D columnar
phase (see Mangenot et al.12 for details). The lateral
packing of the NCP columns is then perfectly
hexagonal (Figure 3a and a00 ), and there is no
correlation between the longitudinal order of NCP
along the column and the lateral organization of
columns. Whatever the 2D or 3D ordering of NCP
in these phases, the final NCP concentrations are
quite the same, close to 500 mg/ml, under a
pressure of 4.7 atm. This concentration may rise
up to about 610 mg/ml under higher pressures
(23.5 atm) at the expense of a good ordering. The
important point we raise here is that different
final states can be obtained depending on the
story of the sample preparation, while all final
concentrations are identical.
We reported previously the formation of a
columnar phase under high salt conditions, based
upon optical and electron microscopy observations.9,11 Spectacular macroscopic hexagonal
domains form, that further divide into six branches
(Figure 3b), as a consequence of the chiral structure
of the NCP.11 We suspect that these germs are 3D
quasi-hexagonal crystals because their textures
and defects are not characteristic of 2D mesophases. They also lack any fluidity and never fuse
together. More rarely, more classical hexagonal
textures are also observed (Figure 3c). We assume
that these latter textures correspond to the 2D
mesophase. Micro-focus diffraction experiments
on selected regions of both samples would be
necessary to assign a structure to each texture
unambiguously.
911
Phase Diagram of Nucleosome Core Particles
Phases formed at intermediate
salt concentrations
The organization of NCP is more complex in the
intermediate
range
of
salt
concentration
From
X-ray
diffraction
(25 , Cs , 50 mM).
experiments (Figure 4), we observed that a dense
phase of columns forms progressively without any
long-range ordering between columns and with
NCPs irregularly piled along a column. Further
on, de-mixing progressively takes place and
several months later the lamello-columnar phase
and the 2D hexagonal (or 3D quasi-hexagonal)
phases, described above under low salt and high
salt conditions, respectively, are found to coexist.12
Samples prepared under similar conditions and
observed in the polarizing microscope reveal
unexpected textures due to microphase separation
effects. After a complex process of organization
(unpublished results), a superimposed series of
layers forms parallel to the observation plane.
They are made of platelets with a puzzle-piece
shape (Figure 5b and b0 ). It is remarkable that the
limits of the domains do not superimpose but are
quite systematically out of phase in the successive
layers. At a higher magnification, thin tubes (the
same as described in the low salt lamello-columnar
phase) can be seen on both surfaces of the layers.
Figure 5. Microphase separation effect observed in the
intermediate salt range (Cs ¼ 37.5 mM) in flat capillaries
(0.2– 0.4 mm thick) and observed with interferential
Nomarski contrast as sketched in a. b, b0 , The same
region of the preparation observed at two different foci,
showing puzzle-pieces of the hexagonal phase separated
from one another by a layer of solvent. An enlarged view
of the surface of these plates reveals a network of tubes
of the lamello-columnar phase (c).
Their density may be rather low, or quite high as
shown in Figure 5c. The tubes do correspond
unambiguously to the lamello-columnar structure
and we suppose from the X-ray data12 that the
layers correspond to the (quasi) hexagonal phase,
although they are not characteristic of hexagonal
textures. This organization of these two phases
into micro domains is sketched in Figure 5a.
Isotropic to ordered phases transition
Figure 4. Profiles of the integrated intensities IðqÞ for
different salt concentrations: 25 mM (lamello-columnar
phase), 160 mM (3D orthorhombic crystal), 50 mM (2D
hexagonal phase) and 37 mM (biphasic sample before
and after phase separation).
In a previous work10 we described, by cryoEM of
thin films, how NCPs stack to form isolated
columns which themselves form an isotropic
solution, under low salt conditions (, 25 mM) at a
concentration just below the transition to the
lamello-columnar phase. NCP solutions prepared
for Cs ¼ 15 mM and for NCP concentrations
ranging from 5 mg/ml to 250 mg/ml (in the
absence of PEG) give IðqÞ profiles slightly different
for q , 0:05 Å21 that reflect the evolution of the
structure factor with NCP concentration. For
CNCP ¼ 250 mg/ml the curve is also slightly
different around q ¼ 0.11 Å21 (Figure 6), which
912
Phase Diagram of Nucleosome Core Particles
Phase diagram
Figure 6. Scattered intensity IðqÞ of NCP solutions of
concentration ranging from 5 mg/ml to 250 mg/ml, for
two salt concentrations Cs ¼ 15 mM and Cs ¼ 160 mM.
The upper curve was equilibrated against a 12% PEG
solution at Cs ¼ 160 mM.
suggest that a fraction of NCP self-assembled into
short columns. At CNCP < 270 mg/ml, the lamellocolumnar phase is already formed. Therefore,
isolated columns may exist under low salt conditions over a very narrow concentration range
(between 250 mg/ml and 270 mg/ml).
Under high salt conditions (Cs ¼ 160 mM), the
formation of the columns was not seen for
CNCP # 250 mg/ml (Figure 6). They are clearly visible in solutions to which a pressure of 1.6 atm
(12% (w/v) PEG) was applied. The X-ray profile
exhibits three broad maxima that are characteristic
of a disordered phase of columns, with some disorder along the columns. The NCP concentration
evaluated from the position of the first maximum
is higher than 450 mg/ml (Figure 6). In the
absence of samples prepared at intermediate NCP
concentrations, we cannot predict the range of
concentrations where the columns form. From the
spectra analysis, we cannot predict whether the
columns form an isotropic or a nematic phase.
Optical microscopy observations show that both
can exist. The isotropic phase turns birefringent
under flow, which is further evidence of the
formation of the columns, and the nematic
phase is observed for slightly higher NCP
concentrations, in a narrow domain of the phase
diagram.
The combination of experimental data coming
from X-ray diffraction, optical and electron
microscopy lead us to present the tentative NCP
phase diagram given in Figure 7. More than 30
different experimental conditions (PEG and salt)
have been analyzed. A larger range of salt
conditions has been explored more qualitatively in
the absence of applied pressure. The same
structures were found using both methods. A
remarkable self-assembling property of NCP is to
form isolated columns, by stacking on top of each
other, in the absence of any divalent cation and of
DNA linking the NCPs together. These columns
form before any lateral organization is set between
NCPs and whatever the ionic conditions are. They
are found in a narrow concentration range
(250 , CNCP , 270 mg/ml) at low salt. We suspect
this range to be larger under high salt conditions,
although NCP concentrations were not determined
precisely. These columns further align to form a
columnar nematic phase. Under an osmotic
pressure fixed to 4.7 atm, a series of ordered 2D or
3D-ordered phases are found: a lamello-columnar
phase below 25 mM monovalent salt, a 3D
orthorhombic crystal or a 2D columnar hexagonal
phase above 50 mM. The lamello-columnar
phase and the high salt phase (2D or 3D) coexists
in the intermediate salt range, and a microphase
separation occurs. An inverse hexagonal phase is
found in the low salt range for pressures above
23 atm.
We observed the formation of 3D crystals using
NCP carrying DNA fragments with some polydispersity and longer than 146 bp. The average
distance between columns is just slightly larger
than in 3D crystals diffracting at high
resolution.4,5,13 Nevertheless, the 3D packing is lost
when DNA fragments become too long (170 bp) or
too polydisperse. On the contrary, the lamellocolumnar and the inverse hexagonal phases afford
significant variations of the DNA length carried
by the NCP (at least 146– 170 bp). This is not
surprising, since the free ends of the DNA may
extend on both external faces of the bilayers
without any geometrical constraint. We observe
that the boundaries between these different phases,
determined by ionic and pressures conditions,
move with the DNA fragment length. This is due
to the increase in the negative phosphate charges
that changes the net charge of the NCP. For the
same reasons, displacements of the phase
boundaries are also observed when the histone
tails of the NCP are partly removed. As a
consequence, in samples containing NCP with a
well-defined DNA length, half intact and half
deleted of their H3 and H2B tails, a segregation
occurs under low salt conditions: intact NCP form
microdomains of the lamello-columnar phase
while the other NCPs pack into 3D hexagonal
crystals.
913
Phase Diagram of Nucleosome Core Particles
Figure 7. Tentative phase diagram of isolated NCP as a
function of monovalent salt (3 , Cs , 160 mM) and
applied osmotic pressure.
Discussion
Phase diagrams and kinetic effects
Despite our systematic survey of a large range of
experimental conditions, we cannot exclude that
other phases may exist. Theoretical phase
diagrams, with predictions of different structures,
could have been of some help to look for other
possible phases but, unfortunately, only simple
objects have been considered so far: spheres, rods,
platelets, spherocylinders or even cut-spheres but
with diameter to thickness ratios that do not
correspond to NCPs.15 – 17 Moreover, discrepancies
are often observed between predictions and
observations because of the intricacies of the
kinetics of the phase transitions.18 On top of that,
structural details and heterogeneous charge
distributions are not considered. Such details, that
are crucial in the case of NCP, result in specific
orientational ordering in the ordered phase and
introduce more complex mechanisms in the
kinetics of phase formation.19
As expected, owing to the complexity of the
NCP, numerous difficulties have been encountered
in the elaboration of the phase diagram. First,
equilibration times may be extremely long (several
months). Using PEG as a stressing polymer, the
solvent was progressively extracted from the NCP
solution and this process requires times to reach
the equilibrium conditions because our samples
are macroscopic (a few mm3). The other method
that we have been using, i.e. the evaporation of
the solvent, may lead much faster to the formation
of the dense phases, with the limitation that the
concentration of salt in the samples is not
controlled any more. Whatever the method, a slow
process of organization of the sample is absolutely
necessary to obtain the organization of NCP in
domains large enough to be observed in optical
microscopy (a few mm). Anyway, we cannot certify
that samples would not remain trapped in
metastable states for years. For example, in the
biphasic intermediate salt range, we cannot
ascertain that the sample would not evolve further
and that another phase could coexist with the
lamellar and the hexagonal phases. Second, the
formation of the dense phases was shown to
depend on the initial states in the phase diagram.
The initial concentration of NCP before addition
of the PEG determined the formation of either a
2D or a 3D (quasi) hexagonal crystal. Third, we
suspect that the stress produced by the addition of
PEG (all at once) to the NCP solution may be of
some importance in the kinetics of phase
transitions. Indeed, it has been demonstrated how
dramatically a stress can jam a system and restrict
crystallization for years.20 Other methods of
preparation of the samples should be tried to
overcome such difficulties. Fourth, the formation
of oligomers of NCP piled to form columns also
interfere with the other parameters. Finally,
microphase separation phenomena were also
observed in the intermediate salt range. These are
reminiscent of the complex structures described in
solutions of virus rods in the presence of PEG.21,22
The routes by which the equilibrium phases are
formed are thus complex and a lot more remains
to be understood in the organization and kinetics
of phase transitions of these NCP solutions,23 but
this complex behavior may precisely offer
extremely rich possibilities of adaptation of
chromatin organization in the context of the living
cell, as discussed below.
Biological relevance
To relate our present studies in vitro and the
properties of chromatin in the cell nucleus, a lot
remains to be done. Of course, the extremely long
organization times reported here are not relevant
from a biological point of view, for several reasons.
First, the organization of NCP could not extend
over long distances, but would be restricted to
microdomains inside the nucleus. Second, the
formation of the higher concentrated states in
chromatin never starts from an isotropic solution
but probably emerges from an already highly
organized state in the interphase (that we do not
know) through processes that involve not only
physicochemical processes but also the activity of
numerous enzymes and cofactors. These probably
help chromatin organize in a sophisticated way,
following an unknown path in the extremely
complex phase diagram of chromatin. We are
aware that our experimental system is extremely
simplified and we do not claim that we reproduce
all details of chromatin of the living cell. A lot
more remains to be done to understand the
phase transition processes. We may think, for
example of a “memory” of the ordered dense
914
states that could be kept up to a certain point in the
de-condensation process and help in a further reorganization of the dense states. Nonetheless, our
hypothesis is that the different organizations
observed here may exist as well locally in the
chromatin of the living cell. Indeed, as detailed
below, the explored range of salt and NCP concentrations conditions are biologically relevant, and
some of these phases could be kept even in the
presence of linker DNA and additional proteins.
In the absence of reliable data on the local ionic
conditions inside the cell nucleus, we explored a
large range of monovalent salt concentrations
(3.5 – 160 mM), that covers the values given for
eukaryotic nuclei.24 – 26 The pressures that we apply
range from 1.5 atm to 25 atm. This osmotic
pressure is used here as a tool to reach the NCP
concentrations 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 our
knowledge, there is no experimental data available
on the pressure applied by the molecular species
forming the eukaryotic chromatin environment.
Nevertheless, for comparison, a pressure of 1 atm
maintains the bacterial nucleoı̈d at its initial
volume after its extraction out of the bacteria30
and DNA is maintained inside the bacteriophage
capsid under a pressure of about 30 – 50 atm.31,32
We also obtained the formation of condensed
phases of NCPs by adding divalent or multivalent
cations (Ca2þ, Mg2þ, spermidine3þ or spermine4þ
for example) to dilute NCP solutions, in the
absence of any applied pressure.33,34 In the context
of the cell, all these cations and also charged
proteins, may interact with nucleosomes in combination with the macromolecular crowding effects.
The path followed by the DNA molecule inside
the chromosome is not known, and despite the
actual consensus about it, the 30 nm chromatin
fiber observed in vitro may not be the only possible
organization of the chromatin fiber in vivo. Other
possible models may be considered and liquid
crystalline states are good candidates. Numerous
models may be proposed. As shown above, slight
changes in the ionic condition are enough to
induce phase transitions. For example, the NCP
concentration drops from 500 mg/ml to 300 mg/
ml under a 5 atm pressure, when the monovalent
salt concentration, Cs, changes from 50 mM to
25 mM salt. The accessibility of NCP changes
accordingly. Similar phase transitions are likely to
occur in the living cell, and may be useful to
control the accessibility of enzymes to the DNA
molecule. Together with the diversity of organization of the isolated NCP, we observe that the
nature of the phase may depend on the initial
state and on the path followed in the phase
diagram. The kinetic problems reported here may
be even more complex in chromatin, where NCPs
are linked together and interact with other proteins
and multiple kinds of ions. We guess that this
complexity may offer extremely rich possibilities
of adaptation of chromatin organization to
Phase Diagram of Nucleosome Core Particles
multiple local constraints in the context of the
living cell. Local phase transitions may be
triggered by local changes in ionic conditions or
by changes in the distribution of charges on the
histone tails, under the action of specific
enzymes. Such transitions may be coupled to
macromolecular transitions (helix-coil transition)
as suggested by Samulski35 and/or be involved in
synchronous expression of genetic information
(euchromatin to heterochromatin).
Materials and Methods
NCP
Most of the data presented here were obtained with
two different batches of NCPs prepared from calf
thymus chromatin. DNA fragments associated to the
histone core were 155(^7) bp and 165(^ 10) bp,
respectively. A few preparations were also made with
NCP with exactly 146 bp associated DNA. The integrity
of the histones was checked carefully for each batch.
Stock solutions were extensively dialyzed against TE
buffer (10 mM Tris –EDTA (pH 7.6)) at a concentration
of 1 – 3 mg/ml and concentrated by ultrafiltration up to
150– 200 mg/ml in the same buffer. These stock solutions
were stored at 0 8C.
Sample preparation
Aliquots of the stock NCP solutions were diluted and
dialyzed against TE buffer eventually supplemented
with NaCl. The concentration of monovalent ions in the
dialysis buffer (Cs) (including Trisþ and Naþ) was
adjusted to concentrations ranging from 3.5 mM to
160 mM. To prepare NCP solutions of different concentrations, while keeping constant the Cs values of the
equilibration buffer, samples were prepared under controlled osmotic pressure. Different methods were used:
for moderate pressures (0.1– 4 atm), (i) samples were
placed in dialysis bags and immersed in a large volume
of a neutral polymer solution (polyethylene glycol, Mr
20,000 from Sigma), prepared in the same buffer, with
the same Cs concentration; or (ii) samples were
equilibrated by ultrafiltration through a nitrocellulose
membrane under nitrogen pressure. To reach higher
pressures (4.7– 23 atm), the NCP solution was first concentrated by one of the two methods described above
up to a concentration of about C ¼ 225– 260 mg/ml.
Equilibrated samples for X-ray and microscopy experiments are prepared as detailed below. The osmotic
pressures are related to the PEG concentration
(calculated with the help of the empirical expression in
Ref. 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 10
atm; 28% PEG ¼ 13 atm; 35% PEG ¼ 23 atm. For
microscopy and X-ray experiments, the following
protocols were used.
Microscopy
A 20 ml sample of the NCP solution was introduced
into flat capillaries (Vitro Dynamics). The PEG solution
(with the same salt concentration, Cs) was then introduced in the capillary, pushing the NCP solution and
creating a PEG gradient from one extremity of the
915
Phase Diagram of Nucleosome Core Particles
capillary to the other. The two extremities of the capillary
were sealed with wax and the specimen left to stabilize.
We did not observe any change in the specimen after a
few weeks. To explore rapidly the phase diagram,
another method was used in some cases: drops of NCP
solutions of defined salt and NCP concentration were
deposited between slide and coverslip and left to concentrate progressively by slow dehydration. Salt and
NCP concentrations corresponding to the observed
textures were calculated a posteriori from the volume
variation between the initial and final steps.
Observations were made with a Nikon polarizing
microscope equipped for interferential Nomarski
contrast.
For electron microscopy, drops of the phases prepared
in flat capillaries or in dialysis bags were deposited onto
gold discs and stabilized in a humid atmosphere. Drops
of the same samples deposited onto glass slides were
used as controls to check the presence of the expected
textures in the polarizing microscope. Samples were
frozen by projection onto a copper surface cooled down
to 10 K with liquid helium (Cryovacublock Reichert).
Freeze fracture was realized in a Balzers BAF 400T
apparatus and replicas observed in a Philips CM12 TEM
at 80 kV. Cryo-sections (40– 80 nm thick) were realized
at 113 K under nitrogen atmosphere in a cryo-ultramicrotome (Leica) and observed at 100 K in a Philips
CM12 cryo-TEM at 80 kV. The vitreous state of water
was checked by electron diffraction, and images
recorded in low-dose mode at a direct magnification of
45,000 £ and 600– 700 nm defocus.
X-ray diffraction
About 15– 20 ml of nucleosome solution and 200 ml of
polymer solution were successively added into quartz
capillaries ,1.5 mm in diameter and left to equilibrate
for more than four weeks at room temperature. In some
cases, in particular for the dense phases obtained at
high salt, the sample-containing capillaries were placed
in a magnetic field higher than 7 T immediately after
addition of the PEG solution to the NCP solution and
kept during the whole formation of the dense phase. It
produced an alignment of the NCP columns that was
kept after removal of the magnetic field and helped in
the understanding of diffraction patterns. X-ray experiments were carried out on the D24 instrument installed
on the storage ring LURE-DCI (Orsay, France) and on
beam line ID2 at the European Synchrotron Radiation
Facility (ESRF, Grenoble, France). On D24 (ID2) the
wavelength was 1.488 Å (0.989 Å) and the sample-todetector distance was 2500 mm (4730 mm), respectively.
These setups gave access to scattering vectors q (where
q ¼ 4p sin u=l; 2u is the scattering angle) ranging from
0.01 Å21 to 0.25 Å21 (0.01 – 0.35 Å21) and to an instrumental resolution Dq (full width at half maximum
FWHM value) of 0.00145 Å21 (0.0008 Å21).
Acknowledgements
This work has been supported by The National
Center for Scientific Research (CNRS), by the
French Research Department and the Research
Association Research against Cancer (ARC) who
provided one of us (S.M.) with a three year and a
three 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)