MINIREVIEW
Chromatin under mechanical stress: from single 30 nm
fibers to single nucleosomes
Jan Bednar
1,2,3
and Stefan Dimitrov
4
1 CNRS, Laboratoire de Spectrometrie Physique, St Martin d’Heres, France
2 Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, Prague, Czech Republic
3 Department of Cell Biology, Institute of Physiology, Academy of Science, Prague, Czech Republic
4 Institut Albert Bonniot, Grenoble, France
Introduction
Since the pioneering use of micromechanical and single
molecule manipulation approaches to probe biological
systems back in the late 1980s and 1990s (e.g. [1–5]),
their use has continuously expanded. In this review we
will focus mainly on the approaches using optical and
magnetic tweezers for studying the structure and con-
formational transitions of chromatin.
The basic repeating unit of chromatin, the nucleo-
some, represents the first level of the chromatin organi-
zation [6]. The major part of the nucleosome (termed
the chromatosome [7]) is composed of an octamer of
core histones (two each of H2A, H2B, H3 and H4), a
linker histone and  166 bp ( 56 nm) of DNA [6].
The histone octamer alone associates with 146 bp of
DNA ( 50 nm) wrapped round in 1.65 left-handed
superhelical turns (Fig. 1) to form the nucleosome core
particle (NCP), the structure of which has been solved
to 1.9 A
˚

maintenance of the structure of both the chromatin fiber and the individual
nucleosomes, as well as the mechanism of their unwinding under mechani-
cal stress. Experiments on the assembly of individual chromatin fibers have
illustrated the complexity of the process and the key role of certain specific
components. Nevertheless a substantial disparity exists in the data reported
from various experiments. Chromatin, unlike naked DNA, is a system
which is extremely sensitive to environmental conditions, and studies car-
ried out under even slightly different conditions are difficult to compare
directly. In this review we summarize the available data and their impact
on our knowledge of both nucleosomal structure and the dynamics of
nucleosome and chromatin fiber assembly and organization.
Abbreviations
ACF, ATP-dependent chromatin assembly and remodeling factor; HMG, high-mobility group; NAP-1, nucleosome assembly protein 1;
NCP, nucleosome core particle.
FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works 2231
of mitotic chromosomes [12,13]. The globular domain
of the linker histone is internally located in the 30 -nm
chromatin fiber [14], although how it interacts with
both the NCP and the linker DNA remains a subject
of debate [15,16].
The conformation of the 30 nm chromatin fiber is
sensitive to ionic conditions [9]. The fiber adopts a
relaxed zigzag structure at low ionic strength and
undergoes compaction with increasing salt concentra-
tion, reaching a very compact form under physiologi-
cal conditions. The linker DNA arrangement in the
most compact form of the chromatin fiber continues to
be a controversial issue [15–19].
Micromechanical approaches were used to study
three different aspects of chromatin organization: the

by 147 bp of DNA and a histone octamer, is complemented with linker histone (H1) and an additional 20 DNA bp to form the chromato-
some. Linker DNA completes and links consecutive nucleosomes which fold into the 30-nm chromatin fiber. During stretching the nucleoso-
mal array is first stretched to its contour length. Additional stretching leads to the rupture of inter-nucleosomal interactions and the array is
stretched to the beads-on-a-string configuration. Further force increase results in progressive eviction of histone octamers. The force values
are approximate (see text) (adapted from [69,80]).
Fig. 2. Optical tweezers experimental setup. The laser beam (LB)
is conducted via a dichroic mirror (DM) to the back aperture of the
objective lens (OL) which focuses the beam and creates the optical
trap (OT) at the focal point. The filament (F) (DNA, chromatin fiber
etc.) is tethered between a trapped bead (TB) and a bead (FB) held
by suction onto a micropipette (MP). The micropipette is coupled to
a high precision micro-positioning system (typically a piezoelectric
XY plate). The image of the bead is projected onto a position sen-
sor (PS). As the fiber is stretched beyond its curvilinear length, the
bead in the trap will start to displace from the center of the trap
and the force which is trying to bring the bead back is linearly pro-
portional to this displacement. Thus, the change of the fiber length
as a function of the force can be measured, resulting in a so-called
force–extension curve.
Chromatin under mechanical stress J. Bednar and S. Dimitrov
2232 FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works
dialysis, (c) nucleosome assembly protein 1 (NAP-1)
and ATP-dependent chromatin assembly and remodel-
ing factor (ACF) assembled chromatin and (d) chro-
matin assembled in nuclear extracts. These distinct
substrates have different properties and advanta-
ges ⁄ disadvantages for the experiments. Native chro-
matin fibers, isolated after light micrococcal nuclease
digestion from nuclei, exhibit heterogeneous lengths
(different numbers of nucleosomes per individual

strength (5 mm NaCl) and low force regime
(< 10 pN), the measured stretching curve exhibited a
rather extended plateau, which was interpreted as fiber
accordion-like extension and disruption of inter-nucle-
osomal interactions. The energy of these interactions
was estimated to be around 3.4 k
B
T per nucleosome.
The authors observed the onset of a hysteresis in
repeated stretch ⁄ relaxation cycle curves at a force of
about 20 pN. Its origin was attributed to eviction of
some histone octamers from the fiber by mechanical
stress. These experiments allowed the determination of
several physical parameters. The persistence length of
the fiber and its stretch modulus at low salt conditions
were determined to be 30 nm and 5 pN, respectively.
The chromatin fiber showed similar elastic behavior
when the experiments were performed in 40 mm NaCl.
However, the forces necessary to achieve the same
extension of the fiber were significantly higher. This
was attributed to the more compact initial conforma-
tion of the fiber. Although the compaction level of
native chromatin fibers (containing the linker histone)
is significantly higher in 150 mm NaCl than in 40 mm
[9], quite surprisingly the experiments in 150 mm did
not show significant differences in the fiber elastic
characteristics compared with those at 40 mm.
As mentioned earlier, about 166 bp of DNA is asso-
ciated with the chromatosome and 145 bp with the
NCP. The nucleosome structure can thus be consid-

and three nucleosomes, respectively.
The direct attribution of the observed released
lengths to a single nucleosomal DNA unwrapping,
however, appeared not to be straightforward. Upon
eviction of the histone octamer and histone H1 (i.e.
the disruption of the chromatosome) 166 bp of DNA
is expected to be released, i.e. 56.4 nm and not 65 nm.
J. Bednar and S. Dimitrov Chromatin under mechanical stress
FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works 2233
To explain this, it was suggested [38] that other non-
histone proteins, namely high-mobility group (HMG)
family members, abundant in the X. laevis egg extract,
were associated with the nucleosome. This would result
in reinforcing the nucleosome mechanical resistance
and in locking of additional DNA into the complex.
These suggestions were not experimentally addressed,
however.
These experiments allowed also calculation of the
assembly rate of the nucleosomes, which was found to
be about three nucleosomes per second. For the length
of k DNA (48 kbp) and the nucleosomal repeat length
(200 bp), the assembly of chromatin would thus be
complete in about 80 s under these experimental condi-
tions. This is far shorter than the chromatin assembly
time (typically a few hours) in bulk in vitro reconstitu-
tion in egg extracts [39]. When a force countering the
DNA shortening (due to nucleosome formation) was
applied, the rate of DNA shortening gradually
decreased and was finally halted at forces above 10
pN. A similar fast rate of nucleosome assembly was

Interestingly, the authors also observed a continuous
non-DNA stretching profile under a low force regime
(< 15 pN). This part of the curve was interpreted as a
continuous unwinding of nucleosomal DNA from the
histone octamer, mainly from contacts with histones
H2A ⁄ H2B where the DNA–histone interactions are
supposed to be weak [42]. The total amount of DNA
released was calculated to be 158 bp per nucleosome, a
value slightly higher than the 147 bp expected. It was
concluded that 76 bp of DNA per nucleosome is
unwound continuously in the low force regime, and
82 bp dissociates under stresses higher than 20 pN in
an all-or-none fashion. The calculated energy (using
dynamic force spectroscopy theory [43]) necessary to
dissociate the DNA from the histone octamer was 21–
22 kcalÆmol
)1
.
In the multiple stretching cycle experiments, the
reappearance of peaks was observed when the time
gap between successive cycles was sufficiently long (at
least 10 s) and the stretching force in the preceding
cycle did not exceed 50 pN. It was suggested that
forces below this value did not cause a complete evic-
tion of all histone octamers. Some of the octamers
may have remained attached to the DNA (probably at
the dyad region, where the DNA–histone interactions
are the strongest), and upon DNA relaxation nucleo-
some reassembly could occur. This phenomenon was
not observed in the case of stretching experiments

2234 FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works
and found to be 65 bp (instead of 76 bp in [41]) for
continuous release of the outer DNA turn and 72 bp
(instead of 82 bp) for disruption of the inner turn. In
all studied cases the removal or modification of histone
tails influenced the stretching profile and the effect
concerned mainly the outer DNA turn while the inner
turn was only minimally affected. A similar phenome-
non was also observed for nucleosomal arrays reconsti-
tuted with the H2A.Bbd histone variant octamer. In
this case a 2 pN drop of threshold disruption forces
(from 19 pN for conventional nucleosomes to 17 pN
for H2A.Bbd nucleosomes) was measured [45]. The
last result is in agreement with the data obtained from
other methods showing a weaker association of the
variant H2A.Bbd octamer with DNA [45,46].
Obviously, similar experiments performed on nucle-
osomal arrays prepared by salt dialysis and by assem-
bly in egg extracts (see above) gave quite divergent
results. While the threshold force values were very sim-
ilar (about 20 pN), the lengths of DNA released upon
mechanical disruption of nucleosomes were quite dif-
ferent. The values of 65 nm or 130 nm measured in
the case of egg extract assembled fibers [38] were never
observed for chromatin reconstituted by salt dialysis.
Gemmen et al. [47] performed analogous experi-
ments on nucleosomal arrays prepared in vitro by
using the histone chaperone NAP-1 and ACF which
forms nucleosomal arrays on random DNA sequences
with nucleosomal repeat of about 168 bp [48,49].

experiments with native chromatin fibers were repeated
under conditions favoring histone octamer stability
(presence of exogenous chromatin or low ionic
strength) a significant increase in the number of 50-nm
events was observed. This was interpreted as an effect
of histone octamer stabilization and the release of all
the DNA associated with a histone octamer in an all-
or-none event [50]. A similar effect was observed with
arrays containing 12 nucleosomes reconstituted on 5s
positioning sequences. Further analysis showed that
indeed under conditions typical for single molecule
experiments (where the chromatin concentration is
usually extremely low) H2A–H2B dimers as well as lin-
ker histones readily dissociated from the nucleosomes
even at moderate ionic concentrations [50]. The
remaining (H3–H4)
2
tetramers associate with only one
superhelical turn of DNA and consequently, upon
stretching, the release of only 25 nm in a single disrup-
tion event will be observed.
Why then were the peaks with 25-nm release length
not observed in experiments with egg extract reconsti-
tuted chromatin? One of the possible explanations is
the association of non-histone proteins (e.g. HMG
proteins) with chromatin, leading to an additional sta-
bilization, mainly of the outer turn. The study of Pope
and colleagues [52] showed that the situation might be
even more complex. In their work they focused mainly
on the elastic response of chromatin fibers assembled

FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works 2235
(the embryonic linker histone variant present in the
egg extract). Based on the analysis, the linker histone
contribution to nucleosomal ‘stability’ was estimated
to be rather low, about 3 k
B
T, which would reflect the
fact that no significant difference in threshold force
was observed between nucleosomal arrays with and
without linker histones [50]. In repeated stretching
experiments the number of events with high energy
barriers (28 k
B
T) rapidly decreased suggesting the per-
manent removal of B4 from the nucleosomes during
the initial stretching. No correlation between the dis-
ruption length and the energy barrier was found. The
value of the barrier was significantly lower than that
reported in [41] (16 kcalÆmol
)1
versus 20–22 kcalÆ-
mol
)1
) but again the experiments were carried out in
different ionic conditions (10 mm Tris ⁄ HCl, pH 7.5,
1mm EDTA, 150 mm NaCl, 0.05% BSA and 0.01%
NaN
3
) and the chromatin samples were assembled by
different techniques.

curve did not reach the second discontinuity. Experi-
ments conducted under a constant force regime
ranging between 2 and 3 pN revealed a bistable char-
acter of the first event with a dwell time in the
unwrapped state depending on the force value, increas-
ing with increased force. From these measurements the
free energy of the outer turn unwrapping was calcu-
lated to be  6 kcalÆmol
)1
. The unwrapping of the sec-
ond, inner turn represented by the second peak at
about 8 pN was not reversible. Its analysis with load-
ing rates in the range 2.4–11 pNÆs
)1
revealed that the
dependence of the probability of unwrapping on the
force was not linear. Therefore, the unwrapping of the
inner turn cannot be considered as a simple two-state
process but will involve some intermediate states as
well. The same experiments were also performed at
high salt concentrations (200 mm potassium acetate).
Under these conditions, the first low force transition
was transformed into a nearly continuous plateau
rather than a sharp peak and the high force transition
was shifted to lower forces.
These experiments identified at least two novel fea-
tures of the nucleosome elasticity behavior. First, the
value of the disruption force was lowered to about
half of that originally reported (9 pN versus 20 pN)
and, second, the experiments clearly showed that

the single nucleosome experiments [55], while 100 mm
Chromatin under mechanical stress J. Bednar and S. Dimitrov
2236 FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works
NaCl and 1.5 mm MgCl
2
were used for nucleosomal
array stretching [41].
An interesting approach to investigate the stability
of a single nucleosome was used by Shundrovsky et al.
[56]. Instead of pulling tethered nucleosomal templates,
they ‘unzipped’ the DNA of a reconstituted template
containing a single 601-positioned nucleosome. The
nucleosome was flanked by free DNA arms and, upon
stretching, the first 220 bp of naked DNA were
unzipped before the histone octamer was reached. The
unzipping of DNA associated with the histone octamer
was affected by histone–DNA contacts within the
nucleosome and reflected the strength of the histone–
DNA interactions. The unzipping profile of the nucleo-
some showed three distinct high force regions
(contrary to the first two, the third region was not reg-
ularly observed). Within these regions, forces up to 45
pN had to be applied in order to overcome the barrier.
The first peak was observed at about 50 bp from the
dyad upon applying an average force of 31 pN, while
the second one was observed in the vicinity of the
nucleosome dyad and at 37 pN average force. These
peaks were attributed to the disruptions of the strong
interactions between H2A–H2B dimers and H3–H4
tetramers, respectively. The attribution of the first peak

,
as well as when random DNA sequences instead of
positioning sequences were used for nucleosome recon-
stitution.
Magnetic tweezer experiments
Several experiments with magnetic tweezers have also
been reported (for the principles of magnetic tweezers
see for example [58,59]). Magnetic tweezers can mea-
sure forces about 1–2 orders smaller than optical twee-
zers and, unlike optical tweezers, they can also control
the torsion of the fiber.
Leuba et al. [60] studied NAP-1 mediated assembly
of chromatin fibers on k DNA using magnetic twee-
zers. They observed an inhibition of the fiber assembly
at forces of  10 pN, but they also registered disas-
sembly events (in an otherwise progressive assembly
process) at forces of about 5 to 7.5 pN. This suggested
that the equilibrium forces were in this range.
Experiments using a similar strategy, but in X. laevis
egg extracts, were realized by Yan et al. [61]. The
experiments were carried out either in ATP-depleted
extract or in extract containing a defined concentration
of ATP or non-hydrolyzable ATP. They found that in
ATP-depleted extract forces of only 4 pN resulted in
inhibition of nucleosome assembly. At forces below
3.5 pN, the extract was able to accomplish the assem-
bly although the number of assembled nucleosomes
was significantly lower relative to the nucleosomal
array reconstituted under optimal conditions (the mea-
sured nucleosomal repeat was only 280 bp in contrast

observed in the stretching profile under these condi-
tions did not correspond to an assembly of individual
nucleosomes, but rather to formation and release of
rather long ‘loops’ (200–400 nm). The fact that the
energy provided by the added ATP in the system was
not even partly used for assisted nucleosome assembly
is also surprising. However, the authors have observed
nucleosome-like disassembly steps of 50 and 100 nm
when the force was increased to over 5 pN. Impor-
tantly, no reverse (i.e. assembly) events were detected
even at low forces.
Kruithof et al. [62] carried out experiments on
strongly subsaturated oligonucleosomal arrays (one to
four nucleosomes present on 17 tandem repeats of 5s
DNA) using magnetic tweezers with sub picoNewton
resolution. This experiment is directly comparable with
the work of Mihardja et al. [55]. Although both groups
used very similar conditions, Kruithof et al. did not
observe any DNA unwrapping from the nucleosome
below forces of 6 pN, even though they used a posi-
tioning sequence with lower affinity for the histone
octamer (5s versus 601).
The data obtained by force spectroscopy of chroma-
tin are not always easy to interpret unambiguously
and to explain in terms of changes in nucleosome and
fiber structure and dynamics. While in lower salt con-
centrations (50 mm) and in the absence of bivalent
ions the inter-nucleosomal interactions can be
neglected, the situation becomes more complex when
the fiber is studied in its compact form, where the pres-

ions were depleted
from the solution, the behavior of the fibers without
linker histones changed. A disruption of the inter-nu-
cleosomal interactions at forces of about 3.5 pN and
an increasing irreversibility upon repeated stretching
cycles (in the presence of 100 mm NaCl) were
observed. Reintroduction of Mg
2+
resulted in a com-
plete recovery of the original folding pattern, suggest-
ing that, at least under these conditions, the linker
histone might not be required for proper chromatin
folding. The analysis of fiber stretching profiles, their
Hookian behavior, their length and transition to
extended beads-on-a-string structures in the third and
fourth regions of the stretching curve led the authors
to conclude that in its compact form the fiber is orga-
nized in a one-start solenoidal topology. The data
obtained on fibers with 167 bp nucleosomal repeat
were significantly different [63]. Surprisingly, their con-
tour length at 0.5 pN stretching force was longer than
for fibers with 197 bp nucleosomal repeat and their
measured stiffness was found to be 2.7-fold higher
(0.052 versus 0.019 pNÆnm
)1
). This was interpreted as
a consequence of their different topological organiza-
tion and a two-start helix topology was suggested as
best fitting the observed data.
However, the story of chromatin fiber folding is

of bivalent ions [37], using metropolis–Monte Carlo
Chromatin under mechanical stress J. Bednar and S. Dimitrov
2238 FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works
Table 1. Comparison of experimental conditions and results of selected experiments.
References Type of chromatin substrate Threshold force (pN) Disruption length
Calculated energy of
DNA–histone
octamer dissociation Ionic conditions
Cui & Bustamante [37] Native, chicken erythrocytes > 20 pN 10 m
M Tris, 2 mM EDTA pH 7.5, 5,
40 and 150 m
M NaCl, 2 mgÆmL
)1
BSA, exogenous chromatin
Bennink et al. [38] Chromatin reconstituted in
Xenopus egg extract on k DNA
> 20 pN 65, 130, 195 nm 10 m
M Tris ⁄ HCl pH 7.5, 1 mM
EDTA, 150 mM NaCl and 0.01%
(w ⁄ v) NaN
3
Brower-Toland et al. [41] Chromatin reconstituted on 17
tandem repeats of 5S DNA, no
linker histone
< 15 pN for outer turn
 20 pN for inner turn
76 bp outer turn,
continuously
unwrapped, 82 bp
for inner turn

Xenopus egg extract
 20 pN 30, 59 and 117 nm 14.5 and
16 kcalÆmol
)1
10 mM Tris ⁄ HCl pH 7.5, 1 mM
EDTA, 150 mM NaCl, 0.05% BSA
and 0.01% NaN
3
Mihardja et al. [55] Single nucleosome reconstituted
on 601 sequence
3 pN outer turn
8–9 pN inner turn
21 nm outer turn
22 nm inner turn
6 kcalÆmol
)1
for
outer turn
10 mM Tris-acetate, 50 mM
potassium acetate, 10 mM
magnesium acetate, 1 mM
dithiothreitol, 0.1 mgÆml
)1
BSA
Kruithof et al. [62] Strongly subsaturated nucleosomal
arrays reconstituted on 17 tandem
repeats of 5S DNA, no linker
histone
> 6 pN (no DNA
unwrapping observed

FEBS Journal 278 (2011) 2231–2243 Journal compilation ª 2011 FEBS. No claim to original French government works 2239
simulation [69], proposed the zigzag organization of
the fiber as the best fitting to measured elastic profiles.
Therefore, the organization of the chromatin fiber in
its compact state remains an open issue and it is very
likely that variable topologies can be adopted depend-
ing on the given conditions [18].
Chromatin arrays under twist
The group of Viovy used magnetic tweezers to study
the behavior of a 36 nucleosome long array reconsti-
tuted on the tandem repeat of 5s DNA under torsional
stress [70]. The acquired data allowed the determina-
tion of several elastic parameters of the fiber, namely
the persistence length (28 nm) and the stretch modulus
(8 pN), which are quite close to the values obtained
for native chromatin fibers (30 nm persistence length
and 8 pN stretch modulus) determined in [37]. How-
ever, the determined torsional persistence length
(5 nm) differed markedly from the value of 35 nm
obtained by WLC (worm-like chain) modeling of simi-
lar arrays, using canonical nucleosomes [71]. A new
model of the fiber was therefore proposed, where the
nucleosomes could exist in three different configura-
tions according to the crossing of the entry ⁄ exit DNA
segments: negatively crossed, open and positively
crossed. Transitions between the different configura-
tions are possible and energies of 0.4 kcalÆmol
)1
and
1.2 kcalÆmol

as documented in Table 1 where data obtained in
selected studies are compared. As can be seen, in some
cases the data from very similar experiments are quite
divergent. This reflects the high sensitivity of the stud-
ied chromatin samples to a number of parameters.
Obviously, the traction parameters, i.e. the loading
rate, turns out to be particularly important. It is there-
fore not surprising that data from early experiments,
using in general quite high loads, are quite similar (e.g.
a disruption force around 20 pN), but very different
from the latest data (3–11 pN). The ionic conditions
and the buffer composition are also very important
factors, as they can influence the octamer stability or
the DNA–octamer association strength. It is also clear
that the choice of DNA substrate has an impact on
the results [57]. The question of the effect of the linker
histone association still remains an open issue as most
of the array stretching experiments were carried out in
the absence of linker histone. Although substantial
progress has been made in the micromanipulation of
chromatin substrates, many additional experiments will
certainly be needed in order to evaluate the effects of
individual factors that potentially influence the
mechanical properties of chromatin substrates.
Acknowledgement
This work was supported by grants from INSERM
and CNRS. S.D. acknowledges ANR-09-BLAN-
NT09-485720 ‘CHROREMBER’. J.B. acknowledges
the support of the Ministry of Education, Youth and
Sports (MSM0021620806 and LC535) and the Acad-

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