Originally published In Press as doi:10.1074/jbc.R300007200 on May 1, 2003
J. Biol. Chem., Vol. 278, Issue 26, 23213-23216, June 27, 2003
Minireview
Forcing Chromatin*
Jordanka Zlatanova 
From the
Departments of Chemical and Biological Sciences and Engineering,
Polytechnic University, Brooklyn, New York 11101
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INTRODUCTION
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Recent years have witnessed the emergence of single-molecule methods, a
powerful set of tools that allow the study of individual biological
macromolecules or complexes thereof, one at a time, often in real time. What
follows is a brief overview of the single-molecule approaches already applied
to investigating chromatin fiber structure and dynamics: the atomic force
microscope, optical tweezers, and magnetic tweezers. The first results are
highly encouraging, and although the field is still in its infancy, its
potential to provide insights unachievable so far cannot be overstated.
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The Chromatin Fiber and DNA Accessibility
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The DNA in the eukaryotic cell nucleus is organized as chromatin, a
nucleoprotein structure in which small basic proteins, histones, form globular
cores around which between 102 and 168 bp of DNA form left-handed superhelical
turns
(1–4).
These particles, termed nucleosomes, are spaced along the DNA at certain
distances, with the length of interconnecting, linker DNA varying according to
cell or tissue type. These nucleosomal arrays are visualized, when deposited
from low ionic strength buffers, as beads-on-a-string structures in electron
(1) and
AFM1 micrographs
(5, for some recent examples
see Refs
6–8).
Increasing the ionic strength leads to compaction of the nucleosomal arrays
until they reach dimensions compatible with the dimensions of the cell nucleus
(typically, 
1 meter of DNA is packed into the confines of a nucleus only
several micrometers in diameter). These further levels of compaction involve
additional proteins, both linker histones (so termed because of their binding
to the linker DNA, outside of the nucleosome particle) and non-histone
proteins. Understanding chromatin structure and dynamics is of paramount
importance to understanding processes requiring access to the DNA template,
such as transcription, replication, recombination, and repair.
For the DNA to be accessible to the enzymatic machineries involved in all
these processes, the compacted chromatin fiber has to undergo unraveling
(9), followed by temporary
removal of histones from the DNA in the immediate chromatin region involved.
Regulation of gene activity at the level of transcription initiation is
believed to also involve some kind of dynamic alterations to the structure
(chromatin remodeling, see Ref.
10) so as to allow binding of
sequence-specific transcription factors to their recognition sequences.
The emergence of single-molecule methods
(11,
12) has provided a powerful
set of tools to approach chromatin structure and dynamics in an unprecedented
way, allowing real-time observations on the behavior of individual chromatin
fibers and assessing the variability among individual representatives of a
fiber population. The majority of the single-molecule chromatin work has been
done using AFM, both for visualization and micromanipulation, but recently
several other single-molecule techniques have proved useful: optical tweezers,
magnetic tweezers, and flow fields. These methods allow investigation of the
mechanical properties of chromatin DNA in real time in response to application
of force, thus closely mimicking the cellular environment in which
macromolecules constantly experience forces of different magnitudes and
origin. The molecular machineries involved in DNA transactions push and pull
on the DNA, denature the double helix, introduce bends and kinks, in other
words apply forces to the double helix in the chromatin context. What is the
behavior of the chromatin fiber in such circumstances? Here we will discuss
the first meaningful results obtained upon mechanically manipulating single
chromatin fibers, comparing the different techniques in terms of their
capabilities and limitations.
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Chromatin Assembly under Applied Force
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Chromatin assembly in vivo takes place massively during DNA
replication; in addition, nucleosomes have to assemble in the wake of the
transcriptional machinery because the transcribing RNA polymerase removes
nucleosomes in its way (by itself or with the help of other factors). The
naked DNA stretches behind the polymerase have to quickly reform chromatin so
that the roles chromatin plays in compacting the DNA and regulating its
functions are restored. It is essential to realize that this reformation of
nucleosomes in the wake of RNA polymerase (probably in the wake of other
DNA-tracking enzymes as well) is a process that takes place while the DNA
molecule is still under tension as a result of the pulling exerted by the
stationary RNA polymerase (13)
on the transcribed DNA. Both RNA and DNA polymerases have been shown to be
among the strongest molecular motors, developing forces of up to 30–40
pN (14,
15). If the forces measured
in vitro are also developed in vivo, then the question
arises whether the DNA under tension can be assembled into nucleosomes and
what the force dependence of the assembly process is.
The group of Viovy in France has used videomicroscopy experiments in which
a single 
-DNA molecule (48.5 kbp, 16.4 µm contour length) was
attached at one end to a glass surface, the other end being free so that it
could be stretched by a flow (Fig.
1a) (16).
The assembly of chromatin on such single DNA molecules was achieved by flowing
in of Xenopus or Drosophila cell-free extracts; the
shortening of the molecules as a result of the assembly was observed by
real-time fluorescence microscopy (DNA was fluorescently labeled by
intercalation of YOYO-1). There was a clear dependence of the rate of
chromatin assembly on the shear rate, hence on the force applied to the DNA.
Assembly could proceed, albeit at a much reduced rate, up to forces of 12
pN.
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FIG. 1.
Schematic of the single-molecule chromatin assembly experiments.
a, the flow experiments of Ladoux et al.
(15). b, the optical
tweezers/flow set-up of Bennink et al.
(16). c, the magnetic
tweezers experiments of Leuba et al.
(17).
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Similar findings have been reported by using an optical tweezers/flow
set-up to follow chromatin assembly on a single -DNA molecule driven
by Xenopus egg extract
(17). In this study, a single
DNA molecule was suspended between two micron-sized beads, one held by a
pipette and the other in an optical trap
(Fig. 1b). Because the
presence of cell debris in the extract precluded the use of the optical trap
for force measurements, the optical trap was switched off during the assembly,
and the instrument was used in the laminar flow mode. Forces were estimated
using either the parameters of the laminar flow (Stokes' law) or by measuring
the Brownian motion of the freely suspended bead. The addition of the extract
led to visible shortening of the distance between the two beads, reflecting
chromatin formation. The kinetics was strongly dependent on the applied force,
with complete inhibition of assembly at forces exceeding 10 pN.
A more detailed study of single chromatin fiber assembly has been carried
out using magnetic tweezers
(18), the kind of instrument
pioneered in the laboratories of David Bensimon and Vincent Croquette in Paris
(19). In these experiments,
the -DNA molecule is suspended between a glass surface and a
micron-sized magnetic bead, and assembly is driven by the addition of a
solution of purified histone octamers and nucleosome assembly factor
(Fig. 1c). The force
on the bead can be manipulated by changing the distance between the bead and
an external magnet, and the assembly can be followed by real-time recording of
the movement of the bead on a videoscreen.
An example assembly curve is presented in
Fig. 2a. In this
experiment assembly was performed at 1.3 pN applied to the bead, i.e.
to the DNA tether. The initial fast assembly gradually slowed down, as
expected from the diminishing length of free DNA available for nucleosome
assembly. The overall dependence of the rate of assembly on the force agrees
with earlier results (16,
17). In addition, the
instrumental set-up allowed controlled adjustment of the force during a single
round of assembly by changing the distance between the external magnet and the
bead. The rheostat control of the force clearly revealed (i) that
higher forces did slow the assembly down
(Fig. 2b), and
(ii) that the response of the system to the applied tension was
instantaneous. As soon as the applied force was changed, there was a
corresponding change in the assembly rate. Finally, the data gave a clear
indication of the reversibility of the assembly process, as illustrated in the
blow-up of a portion of the assembly curve
(Fig. 2a). Note that
the general trend of DNA shortening resulting from nucleosome formation is
interrupted by occasional lengthening of the DNA tether, presumably reflecting
nucleosome disassembly. The dynamic equilibrium between an assembled and a
disassembled state could be easily shifted toward assembly at low forces and
disassembly at high forces (for further details and discussion, see Ref.
18).
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FIG. 2.
a, an example assembly curve obtained at 1.3 pN of force applied
to the magnetic bead in the magnetic tweezers experiments
(17) (see
Fig. 1c). An
enlargement of the boxed portion of the curve shows clear evidence of
occasional lengthening of the fiber during the assembly process, reflecting
the dynamic equilibrium between nucleosome assembly and disassembly.
b, assembly curves of the same DNA molecule subjected to different
forces in the course of a single assembly experiment. The distances between
the cuvette and the external magnet, and hence the corresponding forces, were
adjusted during the course of the assembly as indicated. The lines indicate
that the assembly rates change as a function of force, with ever increasing
rates at ever decreasing forces. Note the instantaneous response of the system
to the changes in force.
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Disassembly of Chromatin Fibers under Force
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The application of mechanical stretching to chromatin DNA is expected to
lead to chromatin disassembly, i.e. dissociation of histone octamers
from the DNA. The force required for this dissociation reflects nucleosomal
strength and depends on the number, kinds, and strength of interactions that
need to be broken during dissociation. Chromatin fiber disassembly under
applied force has been studied successfully so far only with optical
tweezers.
Earlier attempts to use the AFM for mechanically stretching chromatin
fibers have run into a rather unexpected artifact. Long native chromatin
fibers isolated from chicken erythrocytes or fibers assembled in
vitro from purified histones and relatively short, tandemly repeated DNA
sequences (capable of accommodating 12 regularly spaced nucleosomes; compare
with the 240 nucleosomes assembled on -DNA) were deposited on
mica or glass surfaces and pulled with the AFM tip
(20,
21). In such stretching
experiments the scanning of the sample in the x and y
direction needed for imaging was disabled, and the cantilever-mounted tip was
allowed to move only in the z direction, i.e. upwards and
downwards, away and toward the surface.
When the AFM tip is pushed into the sample, it may attach to a fiber by
nonspecific adsorption; upon retraction, it stretches the fiber and
force-extension curves are recorded (Fig.
3, a and b). These curves exhibited a sawtooth
pattern, similar to the patterns obtained upon stretching of multidomain
proteins like titin (22) or
tenascin (23)
(Fig. 3c). Each of the
peaks in these patterns arises because the initial entropic stretching of the
polymer chain, accompanied by a gradual build-up of tension in the molecule,
is followed by enthalpic changes in the internal organization (unraveling) of
a certain individually folded domain. As a result of the unraveling of a
domain the chain elongates in a jump, which in turn leads to an abrupt fall in
the tension of the molecule (Fig.
3d). Successive unraveling events of individual domains
in a multidomain polypeptide chain lead to the appearance of multiple peaks in
the force-extension curve, hence the sawtooth pattern.
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FIG. 3.
Stretching chromatin fibers and multidomain proteins with the AFM.
a, schematic of the AFM chromatin pulling experiments. b,
example force-extension curves on isolated chicken erythrocyte chromatin
fibers (19). c,
schematic of a typical force-extension curve obtained on pulling a single
titin molecule, as in the experiments of Rief et al.
(21). d, explanation
of the sawtooth pattern of the force-extension curves by successive unfolding
of individual protein domains.
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The force-extension curves of both chromatin fibers extracted from cells
and fibers reconstituted in vitro exhibited the multipeak appearance
(Fig. 3b), but a
closer look at the distances between individual peaks made it clear that the
sought after unraveling of individual nucleosomes as a result of mechanical
stretching did not occur, despite the relatively high forces applied, in the
range of 300–600 pN. Control experiments with glutaraldehyde-fixed
fibers (in which unraveling is precluded because of the fixation) and analysis
of AFM images of the fibers being stretched suggested that the jumps in the
force curves corresponded to removal of successive intact nucleosomes from the
glass surface, followed by stretching of the naked DNA between the nucleosomes
attached to the tip and the surface. Earlier work on stretching pieces of
naked double-helical DNA with optical tweezers
(24), optical fibers
(25), or AFM
(26,
27) has demonstrated a
structural transition from the B-form DNA to the so-called S (stretched)-form,
in which DNA was extended to 2-fold over its original contour length. The
force causing the B-to-S transition was measured to be 70 pN in both the
optical tweezers and optical fibers experiments and 120 pN in the AFM
work. Despite the apparent inconsistency in these numbers and the lack of
agreement about the nature of the structural changes occurring during the
"overstretching transition" (for further discussion, see Refs.
28–30),
it is clear that the forces applied in the chromatin stretching experiments
(20,
21) were by far exceeding
those needed for the B-to-S transition; thus, the scenario explaining the
observed sawtooth pattern by a succession of nucleosomes popping off from the
surface followed by stretching of linker DNA seems highly likely.
Three papers have reported results on stretching individual chromatin
fibers with optical tweezers, consistently measuring nucleosomal strength in
the range of 20–40 pN. Cui and Bustamante
(31) stretched isolated
chicken erythrocyte fibers, Bennink et al.
(32) pulled on fibers directly
reconstituted in the flow cell from -DNA and purified histones with
the help of Xenopus extracts (Fig.
4a), and Brower-Toland et al.
(33) used preassembled
nucleosomal arrays containing 17 nucleosomes as their stretching substrate
(Fig. 5a). Up to 20
pN, the fibers underwent reversible stretching, but applying stretching forces
above 20 pN led to irreversible alterations, interpreted in terms of removal
of histone octamers from the fibers with recovery of the mechanical properties
of naked DNA.
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FIG. 5.
a, schematic of the experimental approach of Brower-Toland et
al. (32). Preassembled,
fully saturated nucleosomal arrays containing 17 positioned nucleosomes were
mechanically stretched by moving the coverslip at a constant velocity relative
to the bead; the position of the bead was kept constant by modulating the
light intensity of the trapping laser (velocity clamp mode of operation).
b, a representative force curve: the low force range and high force
range are designated, with discrete opening events seen only in the high force
range (redrawn or modified from Ref.
32).
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The high speed of data acquisition in the Bennink et al.
(32) experiments allowed
recording of force curves in which discrete, sudden drops in force could be
observed upon fiber stretching, reflecting discrete opening events in fiber
structure (Fig. 4b).
These opening events were quantized at increments in fiber lengths of 65
nm and were attributed to unwrapping of individual nucleosomal particles. The
high resolution data allowed the first direct measurements of the forces
needed to unspool individual nucleosomes: these forces ranged between 20 and
40 pN. As Fig. 5b
shows, the forces to unravel the 17 individual nucleosomes in the preassembled
nucleosomal array (33) were
exactly in the same range.
One important difference in the results reported by Bennink et al.
(32) and Brower-Toland et
al. (33) lies in the way
the nucleosome yielded to the applied tension. The step size with which the
chromatin fiber elongated at each opening event was 65 nm in Ref.
32; this was interpreted to
mean that the entire nucleosomal DNA unwrapped from around the histone octamer
in a single dissociation event. Contrary to that, the step size in the
Brower-Toland et al.
(33) work was only about
25 nm. A model was proposed in which the dissociation process occurred in
steps, determined by points of stronger contact between the histone octamer
and the DNA that served as roadblocks to unraveling. It was suggested that the
application of low forces led initially to unwrapping of the ends of the
nucleosomal DNA from the octamer until positions +4 and –4 of the DNA
superhelix were reached and that the application of higher forces
(Fig. 5b) caused
disruption of the +4 and –4 DNA/protein contacts, which led to
unraveling of the remaining DNA is a jump. The peaks in the force curves
corresponded only to this second step in the unraveling of individual
particles, whereas the first step had no recognizable signature related to
nucleosomes. The fiber lengthened in a continuous manner in the low force
regime of stretching. The way nucleosomes unraveled in the two types of
experiments can be, at least in part, explained by the much different rates of
load application (for further discussion on this and some related points, see
Refs. 29 and
34).
It may be important to mention here that the mechanical forces needed to
unravel individual nucleosomes are in the same range as the stall forces
developed by RNA and DNA polymerases
(14,
15), the enzymes that
encounter nucleosomes while reading the information in the DNA. This may mean
that the polymerases are by themselves capable of removing the nucleosomes in
their way.
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Conclusions
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This brief overview of the single-molecule approaches to understanding
chromatin fiber structure and dynamics convincingly shows the potential these
methods have for providing insights unachievable until now. The application of
these methods to chromatin studies is still in its infancy but would
undoubtedly constitute the research focus of more and more laboratories in the
future.
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FOOTNOTES
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* This minireview will be reprinted in the 2003 Minireview Compendium, which
will be available in January, 2004. 
To whom correspondence should be addressed: Dept. of Chemistry and Chemical
Engineering, Polytechnic University, 6 MetroTech Center, Brooklyn, NY 11201.
Tel.: 718-260-3176; Fax: 786-524-5899; E-mail:
jzlatano{at}duke.poly.edu.
1 The abbreviation used is: AFM, atomic force microscope.
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ACKNOWLEDGMENTS
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I thank Dr. Ken van Holde for critical reading of the manuscript and Drs.
Sanford Leuba and Mikhail Karymov for providing unpublished data and help with
figures.
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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INTRODUCTION
The Chromatin Fiber and...
xn. The values for