Article
pubs.acs.org/biochemistry

Dynamics of Nucleosomes Assessed with Time-Lapse High-Speed
Atomic Force Microscopy
Atsushi Miyagi,† Toshio Ando,‡ and Yuri L. Lyubchenko*,††

Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska
68198-6025, United States‡
Department of Physics, Kanazawa University, Kanazawa 920-1192, JapanS Supporting Information*

ABSTRACT: A fundamental challenge of gene regulation is the accessibilityof DNA within nucleosomes. Recent studies performed by various techniques,
including single-molecule approaches, led to the realization that nucleosomes
are quite dynamic rather than static systems, as they were once considered.
Direct data are needed to characterize the dynamics of nucleosomes.
Speciﬁcally, if nucleosomes are dynamic, the following questions need to be answered. What is the range of nucleosome
dynamics? Is a non-ATP-dependent unwrapping of nucleosomes possible? What are the factors facilitating the large-scale
opening and unwrapping of nucleosomes? In previous studies using time-lapse atomic force microscopy (AFM) imaging, wewere able, for the ﬁrst time, to observe spontaneous, ATP-independent unwrapping of nucleosomes. However, low temporal
resolution did not allow visualization of various pathways of nucleosome dynamics. In the studies described here, we applied
high-speed time-lapse AFM (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale to study the
nucleosome dynamics. The mononucleosomes were assembled on a 353 bp DNA substrate containing nucleosome-speciﬁc 601
sequence. With HS-AFM, we were able to observe the dynamics of nucleosome on a subsecond time scale and visualize various
pathways of nucleosome dynamics, such as sliding and unwrapping to various extents, including complete dissociation. Thesestudies highlight an important role of electrostatic interactions in chromatin dynamics. Overall, our ﬁndings shed new light on
nucleosome dynamics and provide a novel hypothesis for the mechanisms controlling the spontaneous dynamics of chromatin.

Tsuggesting that the inherent dynamics of nucleosomes cancontribute to the chromatin unwrapping process. However,
because of the slow data acquisition rate of time-lapse AFM
techniques, many dynamic events are missing. The advent ofhigh-speed AFM (HS-AFM)27 and recent advances in thistechnique28 made it possible to directly visualize dynamic
processes, on the millisecond time scale, of site-speciﬁc DNA−
protein complexes29,30 and nucleosomes,31 allowing theidentiﬁcation of novel mechanisms of protein−DNA dynamics.
Here we applied HS-AFM to image nucleosome dynamics.
The data demonstrate that nucleosomes undergo spontaneous
unwrapping, occurring on the subsecond time scale. Althoughsite exposure is the predominant mode of nucleosome
dynamics and unwrapping, transient sliding of the nucleosome
also takes place. The sliding process is facilitated in thepresence of the zwitterion detergent, CHAPS, allowing largerange nucleosome translocation and unwrapping. The unwrapping dynamics depends on the DNA sequence facilitating thesliding pathway for sequences with low speciﬁcity for binding
histone cores. The role of intermolecular electrostaticinteractions in chromatin dynamics, dissociation, and unwrapping and the interaction with remodeling systems are discussed.

he dynamics of the nucleosome core particle (NCP), a
fundamental unit of chromatin, is a key property ofchromatin, allowing proteins involved in chromatin transcription or replication accessibility to DNA wrapped aroundthe histone core.1 Remodeling systems provide the accessibilityof DNA within nucleosomes,2 but this process can be facilitatedby the inherent dynamics of nucleosomes.3 Single-molecule
approaches of various types were instrumental in characterization of nucleosome dynamics. These methods include
ﬂuorescence resonance energy transfer,4−10 single-molecule
probing,11,12 and atomic force microscopy (AFM) imaging.13−16 Single-molecule ﬂuorescence and time-resolved
techniques revealed that nucleosomes undergo local dissociation of DNA in the absence of remodeling proteins,8,17−20 andthis process occurs on the subsecond time scale.19 Earlier, AFM
imaging was used to characterize chromatin structure at thenanoscale level.21−23 The AFM sample preparation is so gentle
that AFM allows the study of chromatin structure omitting therather traditional glutaraldehyde ﬁxation procedure of thesample.24 Note in this regard, recent studies using time-lapse
observations allowed direct observation of the dynamics andunwrapping of nucleosomes.25,26 The unwrapping of nucleosomes proceeds from the ends of the particle, resulting in
unwrapping of DNA regions as large as dozens of base pairs.
This process may lead to a complete unfolding of nucleosomes
and dissociation of the histone core from the complex.
Nucleosome dissociation occurs in the absence of ATPdependent protein systems involved in chromatin remodeling,© 2011 American Chemical Society

Received: June 20, 2011
Revised: August 12, 2011
Published: August 17, 2011
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Article

MATERIALS AND METHODS

We used BL-AC10DS-A2 cantilevers (Olympus) modiﬁed by
the electron beam deposition method and sharpened by using aplasma etcher.27,28,39
The processing of individual frames of HS-AFM ﬁles andconventional AFM images was performed using Femtoscan
Online (Advanced Technologies Center, Moscow, Russia).

Preparation of Nucleosomal DNA. DNA used for the
nucleosome assembly is similar to that used in previous
studies.26 DNA substrate was generated by polymerase chain
reaction using plasmid pGEM3Z-601 as a template, which
encodes a high-aﬃnity nucleosome positioning sequence 601.32
The 601 sequence (147 bp) was located inside the 353 bp
DNA fragment with 127 and 79 bp arm DNA, so that themononucleosome reconstituted with the DNA substrate has
two DNA arms that diﬀer in length by 1.6-fold. A schematic for
the DNA substrate is shown in Figure S1 of the Supporting
Information.
Histone Octamer Assembly and Puriﬁcation. Histone
octamers were assembled as described in refs 26 and 33.
Octamers were separated from tetramer and dimer fractions via
size-exclusion chromatography (SEC) with Superdex 200 PC
3.2/30 columns (GE Healthcare) at 4 °C. SEC fractions wereanalyzed for purity and histone stoichiometry using sodium
dodecyl sulfate−polyacrylamide gel electrophoresis. Fractions
containing histones H2A, H2B, H3, and H4 in approximately
equal ratios were pooled and concentrated by centrifugation at
10000g.
Nucleosome Refolding. Nucleosomes were prepared as
described previously.26,34 Brieﬂy, histone octamers and DNA
containing the nucleosome positioning sequence were mixed inequimolar concentrations in 2 M NaCl and kept for 30 min atroom temperature. A series of dilutions was prepared by using
10 mM Tris-HCl to produce ﬁnal NaCl concentrations of 1,
0.67, and 0.5 M. Diluted samples were kept at 4 °C for 1 h
before being dialyzed against one change of volume of 0.2 M
NaCl overnight. Nucleosomes were concentrated using a
Microcon centrifugal ﬁlter device (molecular weight cutoﬀ of
10000) at 7000g for 10 min at 4 °C and dialyzed against one
change of 200 mL of buﬀer containing 10 mM HEPES-NaCl(pH 7.5) and 1 mM EDTA for 3 h at 4 °C.
Atomic Force Microscopy. The sample preparation
procedure for high-speed AFM was modiﬁed to accommodate
the instrument design requirements. Freshly cleaved mica was
modiﬁed with a 167 μM solution of 1-(3-aminopropyl)silatrane(APS) to make APS-mica.26,35,36 APS-mica is a positively
charged surface that binds DNA primarily electrostaticallyallowing AFM observation of segmental mobility of DNA in thefree state and in protein−DNA complexes;26,37,38 1.5 μL of an
APS solution (167 μM) was placed on the freshly cleaved mica(a disk ∼1.5 mm in diameter glued to the sample stage) for 30
min. The surface was rinsed with 3 μL of milli-Q water 10 times
and with 3 μL of “imaging” buﬀer containing 10 mM HEPES(pH 7.5) and 4 mM MgCl2 ﬁve times. A droplet of 1.5 μL of
solution containing the nucleosome sample was applied to the
APS-mica surface for 5 min and then rinsed with the imaging
buﬀer. The nucleosome sample was prepared from the stock
solution by dilution into 10 mM Tris-HCl (pH 7.5) and 4 mM
MgCl2 buﬀer.
The high-speed AFM instrument used in this work wasdeveloped by the Ando group27,28 and manufactured by RIBM
(Tsukuba, Japan). The nominal spring constant of thecantilevers was 0.1−0.2 N/m, and the resonance frequency inwater was between 400 and 1000 kHz. The scanning rate invarious experiments varied between 1 and 5.5 frames per
second.

■RESULTS

Nucleosome Unwrapping Imaged with HS-AFM.
Previously, with the use of standard time-lapse AFM, wewere able to observe spontaneous unwrapping of nucleosomes.26,36 Here we applied the ability of HS-AFM to image
dynamics on the millisecond time scale to assess nucleosome
dynamics with substantially higher temporal resolution. The
nucleosome samples were deposited on an APS-mica surface
without a drying step (see Materials and Methods). A few
frames taken with a scanning rate of 1 frame per second areshown in Figure 1. We observed that initially, highly wrapped

Figure 1. Selected AFM images illustrating nucleosome unwrapping,obtained at the capture rate of 1 frame per second.

nucleosome particles (frame 1) start unwrapping shortly (frame3), leading to a complete unwrapping of DNA, with a histonecore remaining bound internally after 18 s (frame 18), followedby the dissociation of the histone core (frame 25). This process
is similar to our previous observations with the use of timelapse AFM with the slow data acquisition capability;26,36,40
however, the current data reveal that the dynamics takes placeon the subsecond time scale and the entire unwrapping of thenucleosome occurs in <1 min.
Transient states of the unwrapping process were detected at
the elevated scanning speed of HS-AFM. Figure 2 shows thedata obtained with a scanning rate of ∼5.5 frames per second. A
full set of the data, assembled as a movie ﬁle (Movie 1), isavailable in the Supporting Information. Images shown in set A
of Figure 2 reveal that initially a tightly overwound nucleosome(frame i) is loosening (frame ii), followed by asymmetricunwrapping in which only one arm (the bottom one) increases
in length, whereas the size of the top arm does not change
visually (frames iii and iv). This conclusion is supported by thelength measurements shown in Figure 2B; the vertical dashedlines indicate times corresponding to images in each frame in
Figure 2A. The length of the long upper arm (black squares)
remains constant, whereas the bottom short arm increases inlength, with the process occurring in a two-step fashion(indicated with arrows). The ﬁrst step appears at ∼3.5 s during
which ∼19 bp are added to the arm. At 8.5 s, the second step isobserved, with the unwrapping elongating the left arm by ∼40
bp. Note that both transitions take place on a time scale of <1 s.
The plot in Figure 2C shows the data obtained for a similar
asymmetric process for another nucleosome, in which the longarm becomes longer and the short arm remains essentiallyunchanged. See Movie 2 in the Supporting Information for theimages. The length measurements for the short arm reveal
complex nucleosome dynamics, in which the short arm
undergoes shortening and elongation processes at 7.5 and 9.5 s,
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ArticleFigure 3. Loop formation and unfolding. (A) Set of images
corresponding to 8.7 (i), 14.7 (ii), and 17.1 s (iii). In panel ii, thepositions of the DNA dissociation and unlooping events are indicatedwith a white arrow. (B) Length measurements for the looping andloop unfolding process. The lengths of the left (arm 1) and right (arm2) arms and the total DNA length are shown with black squares, reddiamonds, and green triangles, respectively. In panel B, the dashedlines correspond to the image acquisition times shown in panel A. The
scan rate is 1 frame per 301 ms.

us to observe sliding. Three successive frames of a nucleosome
undergoing sliding are shown in Figure 4A, and a movie ﬁle(Movie 4) is available in the Supporting Information. Initially(frame 60s), the nucleosome has a short left arm and a longright arm. In the next image (frame 62s), the left arm is longer
than the right arm, but the situation reverses to the original
position after 3 s (frame 65s). This data set shows that transient
sliding can occur. The length measurement data are assembled
in Figure 4B, demonstrating that this rather stable nucleosome
undergoes a fast transient translocation at 62 s. At this time,
one arm increases in length accompanied by a decrease inlength of another arm by the same value. The process is thenfully reversed, resulting in the arms adopting their initiallengths. Note that the size of the nucleosome does not change.
These data suggest that the nucleosome can undergo slidingthat is transient and reversible. The sliding of the histone core
along the DNA molecule occurs at a distance not exceeding 30
nm and takes 3 s.
Dynamics of the Nucleosome in the Presence of
CHAPS. We have shown recently that incubation of assembled
nucleosomes in the presence of the detergent CHAPS resulted
in the formation of nucleosomes with a histone core position
not corresponding to the location of the speciﬁc 601
sequence.40 Such nonspeciﬁc nucleosomes do not diﬀer fromthe speciﬁc ones by the size of the nucleosome particles or thelength of DNA wrapped around the histone core. We
hypothesized that the nonspeciﬁc nucleosomes were formed
from the speciﬁc ones by a sliding mechanism.40 We tested thishypothesis in this analysis by performing time-lapse imaging of
nucleosome dynamics in the presence of CHAPS.
AFM images of a set of time-lapse frames are shown in
Figure 5A−E. Panel A shows the unprocessed raw image in

Figure 2. Time-lapse HS-AFM visualization of nucleosome unwrapping in which only one end of the DNA unwraps. (A) Set of AFM
images for the nucleosome in which the bottom arm increases in size;
the initial image (i) and three frames with the same nucleosome
captured after 4.2 (ii), 7.1 (iii), and 17 s (iv) are shown. (B) Timedependence of the lengths of the upper arm (arm 1, black squares) andbottom arm (arm 2, red diamonds) along with the overall DNA length(green triangles). The dashed lines correspond to the times the images
shown in A were acquired, and the red arrows correspond to the
stepwise unwrapping process. (C) Time dependence for anothernucleosome in which only the length of arm 1 increases. See panel B
for the code. The data were obtained with a scan rate of 1 frame per
353 ms.

respectively. As in the previous graph, the unwrapping process
involving dozens of DNA base pairs occurs fast, on thesubsecond time scale.
Images in Figure 3A show details for the unwrapping process(imaging rate of 3.3 frames per second). Initially, the wrappednucleosome (frame i) unfolds generating a loop (frame ii),
followed by the loop unfolding (frame iii). The quantitative
analysis of these data is shown in Figure 3B. The vertical dashedlines in the ﬁgure indicate the times of the images shown in
Figure 3A. The long arm (squares) remains unchanged,
whereas the short arm increases in length sharply at ∼12 s,followed by a gradual increase, reaching a length close to that of
the left arm. The large set of images assembled as a movie ﬁle(Movie 3) is provided in the Supporting Information.
Reversible Octamer Sliding in Speciﬁc Nucleosomes.
Although unwrapping is the primary process by which
nucleosomes can spontaneously dissociate, HS-AFM allowed
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ArticleFigure 4. Reversible sliding of the nucleosome. (A) Selected AFM images illustrating a reversible sliding. The vertical dashed line corresponds to thecenter of nucleosomal particle at 60 s. The numerals (60s, 62s, and 65s) correspond to the times the images were captured. (B) Dependence of thearms’ lengths (arm 1, black squares, and arm 2, red diamonds) and the total arms’ lengths (green triangles) on time. The scan rate is 1 frame persecond. The scale bar in panel A is 50 nm.

panel E (frame 56s). Length measurements of the DNA arms
were performed, and the corresponding graphs are shown in
Figure 5F. The changes in length are detected starting at ∼15 s,
in which a gradual decrease in the length of arm 2 isaccompanied with a corresponding increase in the length ofarm 1. The ﬁrst sharp change in the lengths was observed at∼17 s. During the period of one frame, arm 1 increased inlength by ∼15 nm and arm 2 decreased in length by the samevalue. There were no substantial changes in lengths until 26 s,
when the second stepwise change of arms occurred. At thisstep, the long arm 1 increases in length by ∼20 nm (blacksquares), accompanied by a decrease in the length of the shortarm 2 by half of this value, ∼10 nm (red circles). Given the factthat one DNA turn in the nucleosome corresponds to 84 bp or∼25 nm, this stepwise translocation of the nucleosome to theend is accompanied by the nucleosome losing approximately
three-fourths of a turn due to unwrapping of long arm 1. This
unwrapping process is partially compensated by rewrapping of
the short arm, bringing back approximately one-third of thenucleosome turn. After this period, the length of the long arm 1
increases gradually, leading to a complete unwrapping of thenucleosome and the dissociation of the core at 55 s.
Figure 6 illustrates the unwrapping process of the nonspeciﬁc
nucleosome. See Movie 5 in the Supporting Information for thefull set of images. In the subset of images (Figure 6A), the ﬁrst
frame (60 s after start) corresponds to the nucleosome withapproximately two nucleosomal turns. The next image (103 s)
shows the same nucleosome after unwrapping of approximately
one turn. Note that the short arm remains unchanged;
therefore, unwrapping is associated with only elongation of
the long arm. The unwrapping is almost complete at 167 s, andthe core dissociates at 177 s. The length measurement plot isshown in Figure 6B, and a plot of time versus the number of
DNA turns calculated from the arms’ lengths is shown in Figure
6C. The vertical dashed lines in panels B and C correspond totimes at which images shown in Figure 6A were taken. A
gradual initial unwrapping process occurs during the ﬁrst 50 s,
followed by the ﬁrst step at ∼70 s during which ∼0.4
nucleosome turn is lost. The largest unwrapping step occurs at∼100 s, accompanied by unwrapping of almost one nucleosome
turn. The third step (∼140 s) leads to a complete unwrappingof the nucleosome.

Figure 5. Irreversible sliding of the nucleosome in the presence of
CHAPS. (A−E) Set of HS-AFM images. In panel A, the black dottedlines indicate the DNA arms of interest. Panels B−E show the traced
images of the molecule on a black background. (F) DNA arm length
measurements. Black squares and red diamonds correspond to thelengths of the longer and shorter arms, respectively, in the ﬁrst image.
The green triangles show the time dependence of the total DNA
length. The data were acquired with thea scan rate of 1.7 frames per
second. The dashed lines in panel F correspond to the times at which
panels B−D were acquired (from left to right, respectively).

which the selected nucleosome is indicated with black dottedlines. The nucleosome was traced and placed on a black
background to produce panel B. Panels C−E that correspondto images at later times indicated in the ﬁgures were obtained
the same way. According to the length measurements, theimage in panel B corresponds to the position of thenucleosome core at 601 sequences; we termed this a speciﬁccomplex. Shortening of the small arm is clearly seen in panel C(20 s), and the nucleosome moves to the end of the DNA
substrate in panel D (frame 32s). Note that visually, the size ofthe nucleosome remains unchanged during this repositioning
event. The size of the nucleosome decreases during further
observation, and the nucleosome dissociates completely in
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Articlethe images in Figure 3 are of high spatial resolution (the DNA
width on the images is ∼3 nm). Furthermore, they reveal thatthe dissociation of an ∼30 bp segment (12 s) led to theformation of a loop with a small curvature followed by therelaxation of the loop and repositioning of the entire arm.
Reversible Sliding. The dynamics of nucleosomes istypically described by the site exposure model,1 according towhich DNA dissociates from the histone core by theunwrapping process. Our previous time-lapse AFM data 26
were in line with this model. However, the high-temporal
resolution capability of HS-AFM showed that in addition to thesite exposure pathway, the sliding pathway is possible. The data
in Figure 4 graphically illustrate this capability. The fully
wrapped nucleosome (approximately two nucleosomal turns)
rolls quickly (∼1 s) in one direction over ∼38 bp (11 nm), after
which it returns to the original position in the same amount oftime, without any change in the number of nucleosomal turns,
the size of the nucleosome or the angle between the arms.
Thus, nucleosomes do slide, although nucleosome dynamics via
the exposure model is the predominant pathway.
Dynamics of Nonspeciﬁc Nucleosomes and Sequence
Speciﬁcity. The majority of physical, chemical, and structural
studies of nucleosomes were performed with the DNA
substrate containing 601 sequence characterized by the highest
speciﬁcity for nucleosome formation.32 Indeed, using the vast
majority of our current and previous data,26,36 the nucleosomes
are assembled at the 601 sequence leading to nucleosomal
particles with lengths of arms corresponding to the arm lengths
in Figure S1 of the Supporting Information. We have shown
recently that such high sequence speciﬁcity can be changed by
the incubation of already assembled nucleosomes in a solution
containing the zwitterionic detergent, CHAPS.40 As a result,
nucleosome particles with alternative positions relative to the
601 sequence are formed; we termed them nonspeciﬁc
nucleosomes. Such nucleosomal particles diﬀer from speciﬁcones only by the position of the nucleosome but have the samemean number of nucleosomal turns as speciﬁc particles. We
suggested previously40 that nonspeciﬁc nucleosomes areformed from speciﬁc ones by sliding, and the data in thispaper conﬁrm this hypothesis. According to Figure 5, thenucleosome slides by rolling over the short arm. The process
primarily occurs in a stepwise fashion, in which unwrapping of
the long arm is accompanied by overwrapping of the short arm.
The translocation occurs in the range of a second. In addition
to the two-step process illustrated in Figure 5, sliding can occur
in one step, demonstrated also by the set of data in Figure S2 of
the Supporting Information.
Interestingly, sliding is a one-directional process thatprimarily occurs by the elongation of the long arm andshortening of the short arm. Note that for the AFM
experiments, we used positively charged surface interactions,which are a driving force for unfolding of nucleosomes.26,36
Therefore, the long arms bind more strongly to the surface than
the short arms. Fluctuational unwrapping of the long arm
further increases the stability of its interactions with the surface,
and wrapping of the short arm compensates for the loss of thenucleosome’s free energy. As the short arm becomes smaller,
the nucleosome becomes capable of directional rolling (sliding)more eﬃciently. We did not observe this directionality for the
speciﬁc nucleosome for which the reversible roll was observed(Figure 4). We explain this observation by the eﬀect of thespeciﬁc 601 sequence that provides the most thermodynami-

Figure 6. Unwrapping of the nonspeciﬁc nucleosome. (A) Selected
frames corresponding to a few stages of the unwrapping process.
Times are shown in each frame. (B) Time dependence of the lengthsof the arms (squares and diamonds) and the total DNA length(triangles) measured for each frame. (C) Time dependence of thenumber of DNA turns in the nucleosome calculated from the arms’lengths. The data were obtained with a scan rate of 1.7 frames per
second. In panels B and C, the dashed lines correspond to the times ofﬁrst three images shown in A (from left to right, respectively).

■

DISCUSSION

Nucleosome Unwrapping at High Speed. Previously,
we published the ﬁnding that nucleosomes are capable ofunwrapping spontaneously, as observed by AFM.26,36 In thetime-lapse AFM experiments, nucleosomes underwent a
stepwise unwrapping process in which each step could beobserved between two consecutive frames. The time resolution
under those conditions was ∼1 min, implying that either theobserved dynamics was that slow or the kinetics on the 1 mintime scale were due to the low temporal resolution of the
instrument. High-speed AFM revealed that the unwrapping
process is fast and complex; it is accompanied by a number ofsmall fast steps spanning ≤1 s. The experiments withsubsecond temporal resolution demonstrated that unwrappingof a segment as large as 10 nm (∼30 bp) can occur in one∼300 ms step (Figure 2B). The time-resolved ﬂuorescence dataof Li et al.19 showed that the breathing dynamics ofnucleosomes occurs on the 50−250 ms time scale, but thesestudies were not able to measure the spatial range of these
ﬂuctuations. Our HS-AFM data show that DNA segments aslarge as 30 bp dissociate on the 300 ms time scale. Importantly,
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Articledistributed over the entire system, including water molecules inthe vicinity of the sample, this eﬀect is weak. Another eﬀect of
the tip is the displacement of the sample. Because of the highoscillation frequency, the time during which the tip contacts the
sample is ∼100 ns; therefore, during this period of time, thesample stage moves in the x-direction by only 0.16 nm. Thisvalue is comparable to the size of an atom, so the lateraldisplacement of the sample by the scanning tip is negligible.

cally favorable location for the nucleosome. Moreover, in ourprevious experiment, we demonstrated that unwrapping of
nucleosomes with the 601 sequence inside the DNA substrateoccurs symmetrically, suggesting that the central part of the 601
sequence plays an important role in the sequence-speciﬁc
positioning of the nucleosome. Therefore, we hypothesize thatthe low probability of the sliding pathway observed here, as wellas in other studies with the use of the 601 sequence, is due toproperties of this sequence favoring one pathway over another.
The directionality of sliding further supports our model ofthe role of electrostatics in the stability and dynamics ofnucleosomes.26,36,40 According to this model, the strengthened
interaction of the DNA arms with a positively charged
environment (the positively charged surface in our AFM
experiments) decreases the stability of the nucleosome. We
hypothesized, therefore, that the surface of chromatin
remodeling factors accommodating the nucleosome could bepositively charged to facilitate nucleosome unfolding,26 and a
recent study from the Kornberg lab41 supports this hypothesis.
In addition to remodeling factors, chromatin interacts with thenuclear membrane that can also modulate chromatin structureand function. Characterization of these interactions isimportant for elucidating mechanisms controlling chromatinfunction. AFM as a topographic technique would be a methodof choice for such studies. Indeed, although AFM primarily uses
mica as a substrate for the imaging and the properties of mica
are diﬀerent from the properties of intracellular surfaces,
appropriate modiﬁcation of the surface may mimic the surface
features in vivo. Our studies use a positively charged APS-mica
surface, but methods for preparing surfaces with othercharacteristics are being developed.
Sequence speciﬁcity of nucleosome formation is widely
discussed in the literature (e.g., refs 42 and 43 and references
cited therein). We have shown recently40 and conﬁrmed in thispaper that the stringency for the sequence speciﬁcity of
nucleosome positioning can be decreased by adding CHAPS tothe solution. Importantly, this detergent does not dissociate thenucleosome. On the contrary, it stabilizes the nucleosomes
against dissociation at very low nucleosomal concentrations,facilitating the study of nucleosomes by single-molecule
techniques. This suggests that other compounds, in addition
to CHAPS, can modulate the sequence-speciﬁc pattern of
nucleosome positioning. Therefore, the analysis of thesequence speciﬁcity of nucleosome positioning should take
into account the possibility of changing this interaction via
environmental conditions. Although CHAPS is not aphysiological compound, its structure is rather similar to thestructure of cholesterol. We have shown that water-soluble
modiﬁcation of CHAPS stabilizes nucleosomes, although to alesser extent compared to unmodiﬁed CHAPS.40
Given the fact that AFM is a technique that directly probes
the sample, the eﬀect of the scanning tip on nucleosome
dynamics needs to be taken into consideration. The HS-AFM
instrument operates in tapping mode; therefore, we need toconsider the eﬀect of two major factors: hitting the sample withan oscillating tip and displacement of the sample during
scanning. The energy transferred by the tip to the sample is
proportional to the square of the oscillation amplitude. HSAFM operates with an amplitude of ∼1 nm, so the estimatesprovided in the Supporting Information demonstrate that theoverall energy induced by the tip is equivalent to an increase intemperature of only ∼9.6 K. Given the fact that this energy is■

ASSOCIATED CONTENT

S Supporting Information*

Potential eﬀects of the instrument characteristics on the sample
dynamics, schematics explaining the design of the DNA
substrate, graph of the time-dependent change in the lengthof the short arm during nucleosome sliding, and movies ofanimated data sets of time-lapse images. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author*Department of Pharmaceutical Sciences, University of
Nebraska Medical Center, 986025 Nebraska Medical Center,
Omaha, NE 68198-6025. Phone: (402) 559-1971. Fax: (402)559-9543. E-mail: ylyubchenko@unmc.edu.
Funding
The work is supported by grants from the U.S. Department ofEnergy (DE-FG02-08ER64579), the North Atlantic Treaty
Organization (SfP 983204), the National Science Foundation(EPS-0701892), and the Nebraska Research Initiative (NRI, allto Y.L.L.).

■ACKNOWLEDGMENTS
We thank Evgeny Menshikov, who participated in the HS-AFM
experiments, Irina Menshikova for the preparation of thenucleosome samples with CHAPS, L. Shlyakhtenko for valuable
discussions and insightful suggestions, and J. Widom and G.
Narlikar for useful comments about the results.

■

ABBREVIATIONS
AFM, atomic force microscopy; NCP, nucleosome core
particle; APS, 1-(3-aminopropyl)silatrane; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate.

■

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