ISWI Remodelers Slide Nucleosomes
with Coordinated Multi-Base-Pair Entry
Steps and Single-Base-Pair Exit Steps
Sebastian Deindl,1,2William L. Hwang,1,3,5Swetansu K. Hota,6Timothy R. Blosser,3Punit Prasad,6Blaine Bartholomew,6
and Xiaowei Zhuang1,2,4,*
1Howard Hughes Medical Institute
2Department of Chemistry and Chemical Biology
3Graduate Program in Biophysics
4Department of Physics
Harvard University, Cambridge, MA 02138, USA
5Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA 02115, USA
6Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
ISWI-family enzymes remodel chromatin by sliding
nucleosomes along DNA, but the nucleosome trans-
location mechanism remains unclear. Here we
use single-molecule FRET to probe nucleosome
translocation by ISWI-family remodelers. Distinct
with a similar stepping pattern maintained by the
catalytic subunit of the enzyme. Nucleosome remod-
eling begins with a 7 bp step of DNA translocation
followed by 3 bp subsequent steps toward the exit
side of nucleosomes. These multi-bp, compound
steps are comprised of 1 bp substeps. DNA move-
ment on the entry side of the nucleosome occurs
only after 7 bp of exit-side translocation, and each
entry-side step draws in a 3 bp equivalent of DNA
that allows three additional base pairs to be moved
to the exit side. Our results suggest a remodeling
mechanism with well-defined coordination at differ-
ent nucleosomal sites featuring DNA translocation
toward the exit side in 1 bp steps preceding multi-
bp steps of DNA movement on the entry side.
The packaging of genomic DNA into nucleosomes and higher-
order chromatin structures represses many essential DNA
transactions, including transcription, DNA repair, replication,
and recombination. DNA accessibility during these processes
is regulated in part by ATP-dependent chromatin-remodeling
enzymes, which utilize the energy from ATP hydrolysis to
assemble, disassemble, mobilize, or restructure nucleosomes.
These remodelers typically possess a catalytic subunit and
one or more accessory subunits. The catalytic subunits contain
a conserved ATPase domain that shares sequence homology
with superfamily 2 (SF2) helicases, as well as unique flanking
domains that give rise to four distinct remodeler families: SWI/
SNF, ISWI, CHD/Mi2, and INO80 (Clapier and Cairns, 2009;
Gangaraju and Bartholomew, 2007). The ATPase domain binds
to and translocates DNA at a site internal to the nucleosome,
which is two helical turns (or 20 bp) from the dyad and referred
to as the SHL2 site (Dang and Bartholomew, 2007; Kagalwala
et al., 2004; Lorch et al., 2005; Saha et al., 2002, 2005; Schwan-
beck et al., 2004; Whitehouse et al., 2003; Zofall et al., 2006).
Depending on the subunit composition, remodelers can display
divergent remodeling activities. For example, ISWI-family
enzymes reposition nucleosomes while maintaining their canon-
ical structure, whereas SWI/SNF-family enzymes not only can
translocate nucleosomes but also can change the nucleosome
structure, alter histone compositions, or eject histone octamers
altogether (Clapier and Cairns, 2009). Within the ISWI family,
remodelers such as human ACF and yeast ISW2 help generate
regularly spaced nucleosomal arrays (Ito et al., 1997; La ¨ngst
et al., 1999; Tsukiyama et al., 1999; Varga-Weisz et al., 1997),
whereas yeast ISW1b largely lacks nucleosome spacing activity
(Stockdale et al., 2006; Vary et al., 2003).
The mechanism by which remodeling enzymes couple ATP
hydrolysis to nucleosome translocation remains incompletely
understood. Various models have been proposed for how
remodelers reposition nucleosomes along DNA. The ‘‘twist diffu-
sion’’ model hypothesizes that remodelers generate a twist
defect in the DNA, which propagates around the histone oc-
tamer, shifting the position of the nucleosome one base pair
(bp) at a time (Flaus and Owen-Hughes, 2003; Kuli? c and Schies-
sel, 2003a; Richmond and Davey, 2003; Suto et al., 2003; van
Holde and Yager, 2003). The ‘‘loop propagation’’ model involves
a loop that propagates around the nucleosome and resolves
at the exit side (Flaus and Owen-Hughes, 2003; Kuli? c and
Schiessel, 2003b; La ¨ngst and Becker, 2004; Lorch et al., 2005;
Narlikar et al., 2002; Schwanbeck et al., 2004; Strohner et al.,
442 Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc.
2005; Widom, 2001). As a third alternative, the ‘‘octamer swivel-
ing’’ model proposes that remodelers disrupt major contacts
between the DNA and histone octamer and allow a concerted
swiveling of the DNA relative to the histone core (Bowman,
2010; Lorch et al., 2010). It remains unclear whether the true
remodeling mechanism involves one of the above models,
a combination of aspects from multiple models, or a model
distinct from any of the above. Given their distinct remodeling
outcomes, different remodeler families or different members
within the same family may also utilize distinct mechanisms to
Single-molecule experiments can provide valuable insights
into chromatin remodeling. These experiments can resolve tran-
sient intermediate states of the nucleosome during remodeling
and reveal how DNA movement at different nucleosomal sites
is coordinated, allowing various models to be tested directly.
Single-molecule techniques have been applied to study DNA
or nucleosome translocation by remodeling enzymes in real
time (Amitani et al., 2006; Blosser et al., 2009; Lia et al., 2006;
Prasad et al., 2007; Shundrovsky et al., 2006; Sirinakis et al.,
2011; Zhang et al., 2006). These studies have shown that
SWI/SNF enzymes can both induce DNA-loop formation on
DNA and nucleosome substrates (Lia et al., 2006; Zhang et al.,
2006) and generate canonically repositioned nucleosomes
(Shundrovsky et al., 2006). Two recent studies have revealed
that ACF, an ISWI remodeler, moves nucleosomes in ?7 or
?3 bp steps (Blosser et al., 2009), whereas RSC, a SWI/SNF
remodeler, translocates DNA substrates with a step size of
?2 bp (Sirinakis et al., 2011). Although these results place
constraints on the remodeling mechanisms, it remains unclear
how DNA is moved into the nucleosome at the entry side, prop-
agated around the octamer, and released at the exit side and
how these events are coordinated.
Moreover, the observed multi-bp translocation steps also
raised a question about their underlying mechanism. The
ATPase domains of remodeling enzymes are homologous to
those of SF2-family helicases, even though remodelers typically
do not exhibit helicase activity. An SF2 helicase has been shown
et al., 2006; Myong et al., 2007), while translocating along the
oligonucleotide backbone with 1 bp steps (Cheng et al., 2011;
Myong et al., 2007). The translocation step size of SF1 helicases
has also been reported to be 1 bp (Dillingham et al., 2000; Lee
and Yang, 2006; Park et al., 2010). Based on these results, one
may hypothesize that DNA translocation during nucleosome
remodeling also occurs in 1 bp steps. However, such 1 bp steps
have not been observed for any chromatin remodeler. Does
this discrepancy indicate that remodelers and helicases have
evolved divergent DNA translocation mechanisms, or was the
resolution of previous measurements insufficient to detect 1 bp
steps? The elementary step size of chromatin remodelers
remains an unresolved question for this family of molecular
In this study, we used single-molecule fluorescence reso-
nance energy transfer (FRET) (Ha et al., 1996; Stryer and
Haugland, 1967; Zhuang et al., 2000) to probe nucleosome
translocation by several ISWI-family remodelers. Despite their
distinct accessory subunits and remodeling outcomes, we
observed a common stepping pattern for these enzymes. DNA
translocation at the exit side of the nucleosome occurred with
an initial 7 bp step followed by 3bp subsequent steps.This step-
ping pattern was preserved even when all accessory subunits
of the enzyme were removed. The multi-bp steps were further
comprised of 1 bp elementary steps. Surprisingly, DNA move-
ment at the nucleosomal entry side appeared to occur only
after 7 bp of DNA were translocated toward the exit side and
to proceed in 3 bp increments, in accordance with the 3 bp
steps observed at the exit side after the initial 7 bp step. Our
results suggest a remodeling mechanism as follows: DNA is first
translocated toward the nucleosomal exit side by the ATPase
domain, 1 bp at a time, generating strain on the entry-side
DNA; after 7 bp of translocation, the strain becomes sufficiently
strong to trigger an enzyme action at the nucleosomal entry
side that draws DNA into the nucleosome; this action partially
releases the strain and allows three additional base pairs of
DNA to be translocated to the exit side; this 3 bp step then
repeats to generate processive DNA translocation across the
Monitoring ISWI-Induced Nucleosome-Remodeling
Dynamics at the Exit Side
To monitor the remodeling dynamics of individual nucleosomes
with histone octamers labeled with the FRET donor dye Cy3 on
histone H2A and double-stranded DNA labeled with the
acceptor dye Cy5 (Figure 1A). The 601 nucleosome positioning
sequence (Lowary and Widom, 1998) was used to place the
octamer at a well-defined position, such that the DNA wrapped
around the histone octamer in ?1.7 turns (Chua et al., 2012;
Luger et al., 1997; Makde et al., 2010; Vasudevan et al., 2010),
leaving n bp of linker DNA on the exit side and 78 bp of linker
DNA on the entry side (Table S1 available online). The nucleo-
somes were anchored onto a PEG-coated quartz surface, and
fluorescence signals from individual nucleosomes were moni-
tored with a total-internal-reflection fluorescence (TIRF) geom-
etry (Figure 1A). Although the presence of two H2A subunits
on each histone octamer led to three different donor-labeling
configurations (donor on the H2A subunit proximal to the
acceptor on the DNA, donor on the distal H2A, and donors on
both H2A subunits), their distinct FRET values allowed us to
clearly distinguish these populations at the single-nucleosome
level and to specifically select the first population for further
analyses (Figure S1A) (Blosser et al., 2009). Addition of remodel-
ers, such as yeast ISW2, and ATP to nucleosomes caused
a decrease in FRET that was not observed when the enzyme
was added without ATP (Figures 1B and S1A–S1C), consistent
with the ability of ISW2 and similar enzymes to mobilize the
histone octamer toward the center of the DNA (He et al., 2006;
Kagalwala et al., 2004; Kassabov et al., 2002; Stockdale et al.,
ously observed on the 0.01–0.05 s timescale (Li et al., 2005) was
not visible in our experiments, which have a resolution of 0.3–1 s.
To quantitatively interpret FRET changes in terms of how
many base pairs of DNA were translocated to the exit side, we
Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc. 443
generated a calibration curve of FRET versus the length of the
exit-linker DNA (Figure S1D) by measuring the FRET values for
a series of nucleosome constructs with different exit-linker
lengths (varying n, Table S1). To further validate this calibration
for determining the exit-linker length of an ISWI-induced remod-
eling product, we prepared another series of nucleosomes with
the same initial linker lengths (78 bp on the entry side and 3 bp
on the exit side) but each possessing a 2 nucleotide (nt) single-
stranded DNA (ssDNA) gap at a specified distance (m bp) from
SHL2 to stall translocation after m bp of movement (Lorch
et al., 2005; Saha et al., 2005; Schwanbeck et al., 2004; Zofall
et al., 2006). As expected, the m = 0 construct exhibited no
remodeling even upon addition of ISW2 and ATP. The FRET
value of the m > 0 constructs after remodeling by ISW2
decreased as m increased (Figure S1E). Such FRET change
was not observed when the enzyme was added alone without
ATP (Figure S1C). Quantitatively, the dependence of the post-
remodeling FRET values on m, i.e., the amount of DNA trans-
location allowed, was identical to the dependence of FRET on
the preset exit-linker length n (Figures S1D and S1E), indicating
that the observed FRET changes were due to DNA translocation
to the exit side and that the amount of DNA translocation can
be quantified based on the calibrations.
The Translocation Step Sizes Are Conserved among
Different ISWI-Family Members
We studied nucleosome remodeling by three representative
yeast ISWI enzymes with different accessory proteins: (1)
ISW2, which is comprised of a catalytic subunit, Isw2
(130 kDa), and three accessory subunits, Itc1 (146 kDa), Dpb4
(22 kDa), and Dls1 (18 kDa) (McConnell et al., 2004); (2) ISW1b,
which is comprised of the catalytic subunit Isw1 (131 kDa),
homologous to Isw2, and two different accessory subunits,
Ioc2 (93 kDa) and Ioc4 (55 kDa) (Vary et al., 2003); and (3) the
catalytic subunit of ISW2 alone, which will be referred to as
Isw2p for clarity. Isw1 and Isw2 each contain a single ATP-
We first added ISW2 and ATP to nucleosomes with 3 bp of
linker DNA on the exit side (n = 3 bp; Table S1). We observed
a stepwise decrease in FRET with pauses at FRET values of
0.46 ± 0.03 and 0.26 ± 0.04 (Figures 2A and S2A). These pauses
correspond to a translocation of 6.9 ± 0.6 bp of DNA prior to the
A similar stepping behavior was also observed for nucleosomes
with a different initial exit-linker length (n = ?3 bp, i.e., 3 bp
omitted from the 601 sequence) (Table S1), except that the
higher initial FRET value allowed us to observe two additional
pauses that occurred after further translocation by 3.2 ± 0.5 bp
and 3.6 ± 0.8 bp, respectively (Figures 2A and S2A). These
data indicate that nucleosome translocation by ISW2 involves
a unique first translocation step of approximately 7 bp in size
and subsequent steps that are approximately 3 bp each. These
step sizes are quantitatively similar to the ones previously
observed for the human ISWI remodeler ACF (Blosser et al.,
2009), the catalytic subunit of which shares sequence homology
with that of ISW2 (Hota and Bartholomew, 2011).
To test whether this stepping pattern was also shared by
other ISWI enzymes with similar catalytic subunits, we probed
nucleosome-remodeling dynamics by ISW1b, which possesses
a catalytic subunit homologous to that of ISW2. Notably, nucle-
osome remodeling by ISW1b exhibited translocation step sizes
virtually identical to the ones observed for ISW2 (Figures 2B
and S2B), despite their different compositions of accessory
subunits and nucleosome-spacing activities (McConnell et al.,
2004; Stockdale et al., 2006; Tsukiyama et al., 1999; Vary
et al., 2003).
Next, we purified the catalytic subunit of ISW2, Isw2p, without
any accessory subunits. Due to its low nucleosome-binding
affinity and processivity (Hota and Bartholomew, 2011), nucleo-
somes exhibited only a limited amount of DNA translocation
before enzyme dissociation. Therefore, typically only two trans-
location pauses were detected, independent of the initial exit-
linker length (n = 3 or ?3 bp). Nonetheless, the first pause
again occurred after ?7 bp of DNA translocation and the second
pause after an additional ?3 bp (Figures 2C and S2C).
For ISW1b and Isw2p, a fraction of the remodeling traces
(?36% and ?31%, respectively) displayed reversals of the
translocation direction, as reflected by back-and-forth FRET
changes, within the dynamic range of our measurement. Such
direction reversal was rarely observed for intact ISW2 com-
plexes. Analysis of the FRET values at direction-reversal points
suggests that direction reversal by Isw2p preferably occurred
at the 7 bp pause (Figure S2D). In contrast, the direction-reversal
positions of the ISW1b enzymes seemed to coincide with the
Figure 1. Probing DNA Translocation on the
Exit Side of the Nucleosome by Single-
(A) Schematic of FRET detection for DNA trans-
location on the exit side of the nucleosome. The
nucleosomes are labeled with the FRET donor,
Cy3 (green star), and acceptor, Cy5 (red star). The
histone octamerand DNAare depictedas ayellow
cylinder and a blue line, respectively.
(B) Representative Cy3 (green) and Cy5 (red)
fluorescence and FRET (blue) time traces showing
translocation of asingle nucleosome after addition
of the enzyme and ATP at time zero.
See also Table S1 and Figure S1.
444 Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc.
5 bp and 10 bp periodicities of the nucleosome (Figure S2E) but
did not correlate with the translocation step sizes determined
from traces that did not exhibit direction reversal.
The Multi-bp DNA Translocation Steps at the
Nucleosomal Exit Side Are Comprised of 1 bp
In order to explore whether the observed multi-bp steps are
further comprised of hidden translocation events with a smaller
step size, we measured the dwell times (t1and t2) of the first
two translocation phases during ISW2-induced remodeling
for the n = 3 bp nucleosomes (Figure 3A). In a simple model
where each transition consists of a series of irreversible elemen-
tary steps with an identical rate constant k, the corresponding
transition time t should follow a G-distribution, AtN?1exp(-kt)
(Dumont et al., 2006; Myong et al., 2007). Depending on whether
the stepping transient itself or the wait time between steps is
rate-limiting, the total number of elementary steps within the
transition would be either N or N + 1, respectively. Notably, the
dwell time (t1and t2) distributions were both well described by
a G-distribution, with N = 6.5 ± 0.4 and 3.4 ± 0.4 for the first
and second translocation phases, respectively (Figure 3B).
Given the 6.9 ± 0.6 bp and 3.4 ± 0.6 bp of DNA translocation
observed during the two phases (Figure 2A), these N values
correspond to a mean elementary step size close to 1 bp
(1.1 ± 0.2 bp or 0.9 ± 0.1 bp for the t1phase and 1.0 ± 0.3 bp
of steps equals N or N + 1, respectively). These results suggest
that the multi-bp steps are likely compound steps consisting of
1 bp elementary steps.
Considering that the assumptions underlying the G-distribu-
tion may not be fully satisfied for the translocation phases, we
set out to detect the elementary 1 bp steps directly. To this
end, we reduced the stepping rate in two alternative ways. First,
we used low ATP concentrations in combination with high
concentrations of the nucleotide analog ATP-g-S, which hydro-
lyzes at a dramatically reduced rate. Using this approach, we
directly observed 1 bp translocation steps in single-molecule
FRET traces (Figure 3C). In addition to the 1 bp steps, the traces
also showed larger step sizes of 2 and 3 bp. Both 1 bp and larger
Figure 2. Identical Step Sizes of DNA Translocation on the Exit Side of the Nucleosome Induced by Different ISWI-Family Enzymes
(A) Remodeling of nucleosomes with initial exit-linker DNA length of n = 3 bp or n = ?3 bp by ISW2. Left: FRET time trace showing ISW2-induced translocation of
a single n = 3 bp nucleosome. 6.2 nM ISW2 and 2 mM ATP were added at time zero. Middle: Histogram of the FRET values at translocation pauses constructed
from n = 3 bp nucleosomes. Right: FRET histograms of the translocation pauses constructed from n = ?3 bp nucleosomes.
(B) Remodeling of the n = 3 bp and n = ?3 bp nucleosomes by ISW1b. FRET histograms of the pauses for n = 3 bp (left) and n = ?3 bp (right) nucleosomes in the
presence of 8.8 nM ISW1b and 10–150 mM ATP.
presence of 69 nM Isw2p and 1 mM ATP.
The nucleosome schemes display the footprint of the histone octamer (yellow oval) on the DNA (blue line). Numbers above double-headed arrows shown in the
histograms represent mean step sizes.
See also Figure S2.
Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc. 445
steps occurred randomly at varying positions in different traces,
consistent with a uniform step size of 1 bp where some pauses
were too short to be resolved. Indeed, a simulation based on
the experimentally determined stepping rate constant and
FRET signal-to-noise ratio suggests that we would miss ?50%
of the 1 bp steps. Although we cannot rule out a translocation
mechanism that involves heterogeneous steps of varying sizes,
we consider such a mechanism less likely given that the dura-
tions of the multi-bp steps follow a G-distribution with the
number of elementary steps matching the number of base pairs
translocated (Figures 3A and 3B).
For automated step identification, we utilized a hidden Markov
modeling (HMM) algorithm (McKinney et al., 2006) to determine
the distinct FRET states (plateaus) present in the FRET traces
(Figure 3C). We separately analyzed two FRET regions, 0.32 %
FRET % 0.62 and 0.59 % FRET % 1, and allowed 10 initial states
in each region. The HMM analysis of the FRET traces converged
to ?4–5 states in each region, suggesting that the state identifi-
cation was unlikely influenced by the initial parameter setting.
Moreover, we obtained nearly identical fits with an alternative
step-finding algorithm (Kerssemakers et al., 2006) (Figure S3A).
Remarkably, the histograms of the FRET plateau values exhibit
well-defined peaks each separated by 1.0 ± 0.2 bp (Figures 3D
Alternatively, to reduce the stepping rate without using
ATP-g-S, we monitored remodeling at a lower temperature of
15?C, instead of the 30?C used for the above experiments.
Again, 1 bp steps were observed (Figure S3B). A histogram of
the FRET plateau values shows peaks separated by 1.0 ±
0.1 bp (Figure S3B). However, because of the reduced
enzyme-binding affinity and slower translocation kinetics at
this lower temperature, photobleaching restricted analysis to
only the first four 1 bp translocation steps (Figure S3B). Taken
together, our data suggest that the multi-bp translocation steps
comprised of 1 bp elementary steps.
Roles of ATP Binding and Hydrolysis during Nucleosome
To dissect the roles of ATP binding and hydrolysis during nucle-
osome translocation, we examined the dwell times associated
with individual 1 bp steps at various concentrations of ATP
and ATP-g-S at 30?C. Photobleaching limited our analysis to
the first nine steps. At low ATP-g-S concentrations, the pause
duration after the seventh 1 bp step, tp,7, was noticeably longer
than the dwell times associated with all other 1 bp steps (Fig-
ure S4A). As the concentration of ATP-g-S increased, the pause
duration after the seventh step (tp,7) decreased (Figures 4A and
Figure 3. The Multi-bp Translocation Steps on the Exit Side of the Nucleosome Are Comprised of 1 bp Elementary Steps
(A) FRET time trace of a nucleosome indicating the dwell times of the first two translocation phases (t1and t2).
(B) Histogram of t1and t2values constructed from many nucleosomes. Fits to the G-distribution AtN?1exp(-kt) (black lines) yield N = 6.5 ± 0.4 and k = 0.52 ±
0.04 s?1for the t1phase (left) and N = 3.4 ± 0.4 and k = 0.67 ± 0.11 s?1for the t2phase (right).
(C) FRET time trace, before (gray) and after (blue) 5-point averaging, of a single n = 3 bp nucleosome in the presence of 6.2 nM ISW2, 2 mM ATP, and 2 mM
ATP-g-S. A HMM fit is shown by the red line. The horizontal orange dotted lines indicate 1 bp intervals (derived from the calibration in Figure S1D).
See also Figure S3.
446 Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc.
S4A), whereas the dwell times of the other steps (tp) increased
(Figures 4B and S4A). As a result, all steps became equal in
duration at saturating ATP-g-S concentrations (Figure S4A). At
saturating ATP-g-S concentrations, the duration of these 1 bp
steps (tp) decreased with increasing ATP concentrations (Fig-
ures 4C and S4B). Given that ATP-g-S hydrolyzes much more
slowly than ATP and competes with ATP for binding to the
enzyme, the above results indicate that the individual 1 bp
translocation steps require ATP hydrolysis, whereas the pausing
observed after 7 bp of translocation involves an additional
ATP-binding event of the enzyme. Binding of ATP-g-S can thus
facilitate this event.
Monitoring ISWI-Induced Nucleosome-Remodeling
Dynamics at the Entry Side
osome, wemoved the FRETacceptor dye Cy5fromthe exitDNA
linker to the entry DNA linker, 10 bp away from the nucleosomal
edge, but kept the entry- and exit-linker lengths at 78 bp and
3 bp, respectively (Figure 5A). As is the case for the exit-side
labeling scheme, we were able to distinguish the different
donor-labeling configurations at the single-nucleosome level
and select for further analysis only those with a single Cy3 dye
on the proximal H2A (Figure S5A).
Uponaddition ofISW2andATP andafterawaiting periodtwait,
the FRET time traces displayed an increase in FRET as the Cy5
dye on the entry DNA linker was moved closer to the octamer
(Figure 5B). As DNA continues to move into the nucleosome,
the Cy5 dye is expected to eventually pass the Cy3 dye, causing
a FRET decrease. Indeed, such a nonmonotonic FRET change
was observed (Figure 5B). No FRET change was observed
when ISW2 was added without ATP (Figure S5B).
Coordination of the DNA Movement on the Entry
and Exit Sides of the Nucleosome
To investigate the coordination of entry-side and exit-side re-
modeling activity, we first compared the waiting times before
the onset of any FRET change, twait, at both entry and exit sides
(Figures 1B and 5B). Surprisingly, the average twait value
measured at the entry side was substantially longer than that
at the exit side (Figure 5C), suggesting that exit-side DNA trans-
location likely occurred prior to any DNA movement at the entry
side. The delay between entry-side and exit-side movements
decreased as the ATP concentration increased (Figure 5C).
Because DNA translocation at the exit side occurred in 1 bp
steps, we reason that this movement was caused by the SF2-
homologous ATPase domain bound at the SHL2 site of the
nucleosome. Our observations may thus be interpreted as
DNA translocation at SHL2 by the ATPase occurring prior to
any DNA movement into the nucleosomal entry side.
Because twaitwas measured at the entry and exit sides with
differently labeled constructs, the observed time difference
provides only an indirect measure of the order of these events.
To further test whether DNA translocation at SHL2 by the
ATPase domain is indeed required for DNA movement at the
entry side, we generated entry-side-labeled nucleosomes with
a 2 nt ssDNA gap positioned at the SHL2 site (m = 0 bp, Fig-
ure 6A), which prevented any DNA translocation to the exit
side (Figure S1E). Interestingly, addition of ISW2 and ATP to
these nucleosomes did not cause any FRET change at the entry
side (Figures 6B and 6C), indicating that DNA movement at the
entry side was also inhibited.
Next, we generated a series of entry-side-labeled nucleosome
constructs with ssDNA gaps at varying distances (m bp) from
the SHL2 site (Figure 6A), which allow only m bp of DNA to
be translocated to the exit side as shown in Figure S1E. Remark-
ably, after addition of ISW2 enzyme and 2 mM ATP, no entry-side
FRET change was observed for nucleosomes with m % 7 bp,
whereas long-lasting FRET increases were observed for
nucleosomes with m > 7 bp (Figures 6B and 6C). At higher
ATP concentrations (R200 mM), the m < 7 bp nucleosomes
still showed no change in FRET. A minor fraction (?40%) of
the m = 7 bp nucleosomes showed an increase in FRET but
mostly with only transient excursions to higher FRET, whereas
Figure 4. Dependence of the Stepping Kinetics on the Concentrations of ATP and ATP-g-S
(A) The pause duration after the first, 7 bp compound step (tp,7) at various ATP-g-S concentrations and 2 mM ATP.
(B) The pause duration between each 1 bp elementary step (tp) at various ATP-g-S concentrations and 2 mM ATP. All pauses except for the ones after 6, 7, and
8bpof translocationwerepooled todetermine tp.Asshownin Figure S4,inadditiontothe 7thpause, the6thand 8thpauses also appearlongerthantheremaining
ones, likely due to errors in pause identification. The value of tpat 0 mM ATP-g-S was derived from 1/k value obtained from the G-distribution in Figure 3B.
equal durations and were pooled to determine tp.
All data are shown as the mean ± standard error of the mean (SEM) (N = 15–100 events). See also Figure S4.
Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc. 447
only <20% of the nucleosomes showed a stable increase in
FRET. In contrast, the vast majority (?75%–90%) of m > 7 bp
nucleosomes exhibited an increase in FRET, among which
most (80%–90%) showed stable FRET changes. These obser-
vations are consistent with DNA movement at the entry side
occurring only after the ATPase has translocated 7 bp of DNA
toward the exit side, likely because a certain amount of strain
on the entry-side DNA is required to trigger any movement into
the nucleosome. ssDNA gaps not only limit the amount of DNA
translocation by the ATPase domain but can also inhibit the
generation or propagation of DNA torsion. Among these two
factors, entry-side DNA movement was more likely inhibited by
the limited amount of ATPase translocation because the inhibi-
tion was only observed for the m % 7 nucleosomes, whereas
ssDNA gaps at m > 7 should also relax torsional strain on the
entry-side DNA. Further supporting this interpretation, the twait
values observed for the m > 7 nucleosomes were quantitatively
similar to those observed for intact nucleosomes without any
gap (Figure S6A), suggesting that the gaps did not perturb the
strain required to trigger entry-side movement. These obser-
vations are also consistent with the long pause observed
after the first 7 bp of DNA translocation to the exit side for intact
nucleosomes without gaps (Figure 2A). We reason that the
accumulated strain after 7 bp of DNA translocation stalls exit-
side translocation momentarily, and that an entry-side move-
ment needs to be triggered to partially relax the strain and allow
additional DNA to be pumped to the exit side, giving rise to the
Notably, the post-remodeling FRET values at the entry side
were identical within error for the nucleosomes with gaps at
m = 8, 9, or 10 bp but distinct from those observed for the
m = 11, 12, and 13 bp nucleosomes, which were also identical
to each other (Figures 6C). These observations are consistent
with a 3 bp equivalent of DNA being moved into the nucleosome
per step on the entry side, which in turn allows an additional
3 bp of DNA to be translocated to the exit side. The rationale is
as follows. Since 7 bp of DNA can be translocated to the exit
Figure 5. DNA Movement on the Exit Side
of the Nucleosome Precedes that on the
(A) Schematic of the nucleosome construct used
for measuring DNA movement at the entry side.
(B) Donor signal (green), acceptor signal (red),
and FRET (blue) time traces showing ISW2-
induced remodeling after adding 12 nM ISW2
and 2 mM ATP at time zero. The dashed line
indicates the onset of FRET change after an initial
wait time, twait.
(C) Comparison of twaiton the entry (yellow bars)
and exit sides (purple bars) of the nucleosome
under identical enzyme and ATP concentrations.
Data are shown as the mean ± SEM (N = 80–220
See also Figure S5.
side without any entry-side DNA move-
ment,if each step of the entry-side move-
ment allows an additional 3 bp of DNA
to be pumped to the exit side, the cases of translocating 8, 9,
and 10 bp to the exit side would all require only one entry step,
leading to the same post-remodeling entry-side FRET values
for the m = 8, 9, and 10 bp nucleosomes. The cases of translo-
cating 11, 12, and 13 bp to the exit side would all require two
entry steps instead, and thus the post-remodeling entry-side
FRET values for the m = 11, 12, and 13 bp nucleosomes would
be identical to each other but different from those of the m = 8,
9, and 10 bp nucleosomes. Our observations agree with these
predictions (Figure 6C), demonstrating that the first entry-side
step moved a 3 bp equivalent of DNA into the nucleosome,
allowing 3 bp to be translocated to the exit side. Our data are
also consistent with a second 3 bp entry-side step, though it is
formally possible that the second step size is greater than 3 bp
because the FRET values have not been measured for the
m > 13 bp nucleosomes. However, given that after the initial
7 bp translocation step, the subsequent exit-side translocation
steps are all ?3 bp in size, DNA movement on the entry side
likely also occurs in increments of ?3 bp, giving rise to
the ?3 bp compound steps observed at the exit side.
Interestingly, even though up to 7 bp of DNA can be translo-
cated to the exit side without any observable action at the
entry side, the exit-side translocation of the m < 7 nucleosomes
appeared less stable: the majority of the m = 5 nucleosomes
exhibited direction reversals (Figure S6B). In contrast, such
direction reversal was rarely observed for m > 7 nucleosomes
(Figure S6B), suggesting that action on the entry side helps
prevent direction reversal in DNA translocation.
Chromatin remodelers utilize the energy from ATP hydrolysis
to disrupt DNA-histone contacts and mobilize nucleosomes
along DNA. In this work, we used single-molecule FRET to
study nucleosome translocation by ISWI-family remodelers.
We observed DNA movement at different sites of the nucleo-
some and determined how movements at these sites were
448 Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc.
for nucleosome translocation by ISWI remodelers.
We showed that several representative ISWI remodelers
from yeast, despite their highly distinct accessory subunits and
remodeling outcomes, all translocated nucleosomes with a
common stepping pattern (Figure 2). Exit-side translocation
occurred with an initial 7 bp step followed by 3 bp subsequent
steps. This stepping behavior was preserved even upon removal
of all accessory subunits, leaving only the catalytic subunit for
remodeling. These step sizes were also identical to the ones
previously observed for the human ISWI remodeler ACF, and
previous evidence on ACF suggests that the step sizes are
possibly independent of the DNA sequence used (Blosser
et al., 2009). Taken together, these results suggest a common
remodeling mechanism for ISWI remodelers that is enabled by
the catalytic subunit and conserved from yeast to humans.
Notably, the step sizes observed here are not identical to the
5 bp or 10 bp periodicity of nucleosomal DNA-histone contacts
(Hall et al., 2009; Luger et al., 1997) and thus are likely influenced
by the remodeling enzymes, though we do not preclude the
possibility that the step sizes are determined by a combination
of enzyme and nucleosome properties. Moreover, the energy
landscape of the nucleosomal substrates could affect the trans-
location kinetics quantitatively by modulating the dwell times
between steps, which may explain why the remodeling products
observed in ensemble biochemical analyses tend to exhibit
?10 bp intervals for DNA translocation (Schwanbeck et al.,
2004; Zofall et al., 2006).
We further showed that the multi-bp steps observed on the
nucleosomal exit side were compound steps comprised of
1bp elementary steps(Figure 3). Given thatthe ATPasedomains
of the ISWI remodelers share sequence homology with SF2
helicases, which translocate DNA with 1 bp elementary steps
(Cheng et al., 2011; Myong et al., 2007), the 1 bp steps of the
remodelers most likely reflect an intrinsic translocation property
of their ATPase domains. Given that the ATPase domain binds
to the SHL2 site of the nucleosome 20 bp from the dyad (Dang
and Bartholomew, 2007), our results suggest that the ATPase
domain translocates DNA at SHL2 1 bp at a time, which then
propagates to the exit side, resulting in the observation of 1 bp
steps at the exit side. Although such translocation likely tracks
a DNA strand, the ATPase domain may partially disengage
from the SHL2 site from time to time, and hence there may not
be substantial accumulation of DNA rotation during remodeling
(Bowman, 2010; Cairns, 2007). It has been shown previously
that ssDNA gaps placed between SHL2 and the exit site do
notinterfere with nucleosome slidingbyISWIenzymes(Schwan-
beck et al., 2004; Zofall et al., 2006). It is thus possible that the
propagation of DNA from the SHL2 site to the exit side also
does not require torsional strain.
What is the mechanism underlying the multi-bp, compound
steps for ISWI remodelers? Because DNA translocation at
SHL2 toward the exit side occurs in 1 bp steps, it is reasonable
to hypothesize that the multi-bp steps are a result of actions at
the entry side. Surprisingly, we found that DNA movement at
the entry side appeared to only occur after 7 bp of DNA were
translocated toward the exit side (Figures 5 and 6). A net trans-
location to the exit side without any DNA being moved into the
nucleosome will cause strain on the entry-side DNA. We thus
hypothesize that a certain amount of strain needs to accumulate
on the DNA before entry-side movement is triggered. Such
strain may take the form of DNA stretching or transient con-
formational changes of the octamer or both. DNA stretching
under force (Smith et al., 1996) or in nucleosome structures
(Ong et al., 2007), as well as conformational changes of the
octamer (Bo ¨hm et al., 2011), have been previously observed.
According to this picture, as the ATP concentration increases,
the strain on the entry-side DNA created by translocation at
SHL2 should accumulate faster, and thus the time lag between
DNA movements at the SHL2 and entry sites should decrease.
Indeed, the time difference observed between entry- and exit-
side movements decreased as the ATP concentration was
increased (Figure 5).
Interestingly, once entry-side movement was triggered, it
appeared to proceed in 3 bp increments that allowed 3 addi-
tional base pairs to be moved to the exit side (Figure 6), which
Figure 6. Entry-Side DNA Movement Occurs after 7 bp of DNA Translocation toward the Exit Side and Proceeds in 3 bp Steps
(A) Schematic of the nucleosome constructs used to monitor DNA movement at the entry side when exit-side translocation is restricted by a 2 nt ssDNA gap. The
gap is located m bp away from the SHL2 site (shown as a purple line) such that m bp of DNA can be translocated to the exit side.
(C)FRET valuesbefore (red bar) andafter(blue bars)remodelingby ISW2as afunction ofthedistance mtotheSHL2site. Because theDNApathonthe entryside
may involve bending and/or twisting due to the direct interaction with the remodeling enzyme, we do not expect a similar linear dependence of FRET on the linker
DNA length as on the exit side where the linker DNA is largely free of enzyme-induced distortion. Data are shown as the mean ± SEM (N = 80–150 nucleosomes).
See also Figure S6.
Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc. 449
provides a simple explanation for why exit-side DNA trans-
location occurred with an initial 7 bp step followed by 3 bp
subsequent steps. Given that the pauses preceding the 3 bp
steps on the exit side can be shortened by addition of ATP-g-S
(Figure 4), indicating that an ATP-binding event is needed
for exiting the pause, one may hypothesize that this event is
related to the entry-side action. The entry-side step potentially
involves an enzyme action, for instance, a conformational
change of a linker-DNA-binding domain that draws in a 3 bp
equivalent of DNA. The HAND-SANT-SLIDE module, which
binds to the linker DNA on the entry side (Dang and Bartholo-
mew, 2007; Yamada et al., 2011), may play a role in this
process. Supporting this notion, mutations in the SLIDE domain
of ISW2 inhibited DNA movement on the entry side yet still
allowed a substantial amount of DNA translocation to the exit
side (Hota et al., 2013). It has been reported recently that the
DNA-translocation activity of the ISWI ATPase is inhibited by
a neighboring NegC region, and that binding of the HAND-
SANT-SLIDE module to linker DNA relieves this inhibition
(Clapier and Cairns, 2012). Thus, the HAND-SANT-SLIDE
module potentially plays two roles in nucleosome remodeling,
helping both DNA translocation to the exit side and DNA move-
ment on the entry side.
Based on the above results, we propose the following model
for nucleosome remodeling by ISWI-family enzymes (Figure 7).
ISWI-induced nucleosome remodeling starts with the ATPase
domain translocating DNA at the SHL2 site. This translocase
activity pumps DNA toward the exit side, 1 bp at a time, utilizing
energy from ATP hydrolysis. Translocation at SHL2 induces
strain on the entry-side DNA, which initially remains immobile.
After 7 bp of DNA are translocated, the strain becomes
sufficiently strong to trigger action on the entry side. This
entry-side action potentially involves a conformational change
between the linker-DNA-binding domain (possibly the SLIDE
domain) and the ATPase domain, which pushes a 3 bp equiva-
lent of DNA into the nucleosome, allowing an additional 3 bp
of DNA to be pumped to the exit side. After these 3 bp are
translocated to the exit side, the strain on the entry-side DNA
becomes sufficiently strong again to trigger another action on
the entry side, allowing another 3 bp to be translocated to the
exit side. This cycle repeats to allow processive nucleosome
Preparation of Dye-Labeled Mononucleosomes
Double-stranded dye-labeled DNA constructs with varying flanking linker
lengths on the two sides of the 601 nucleosome positioning sequence and/
or a 2 nt ssDNA gap at specific locations were generated using a standard
PCR or annealing approach. Mononucleosomes were reconstituted from
Cy5-labeled DNA and histone octamers, labeled with Cy3 on histone H2A,
by salt dialysis and purified by gradient ultracentrifugation (Luger et al.,
1997) (see Extended Experimental Procedures).
Preparation of ISW2, Isw2p, and ISW1b
ISW2 and ISW1b were affinity-purified from Saccharomyces cerevisiae strains
BY4742 and YTT449, respectively, as previously described (Gangaraju and
Bartholomew, 2007; Tsukiyama et al., 1999) (see Extended Experimental
Procedures). For the isolation of the catalytic subunit Isw2p, we deleted
a portion of the ITC1 gene to disrupt the ITC1-Isw2p interaction and immuno-
purified the isolated catalytic subunit Isw2p.
Biotinylated and dye-labeled mononucleosomes were surface-anchored
on poly(ethylene glycol)-coated quartz microscope slides through biotin-
streptavidin linkage, which did not inhibit the remodeling activity (Blosser
et al., 2009). Immobilized nucleosomes were excited with a 532 nm Nd:YAG
laser (CrystaLaser), and fluorescence emissions from Cy3 and Cy5
were detected using a prism-type TIRF microscope, filtered with a 550 nm
Figure 7. A Model for Nucleosome Translo-
cation by ISWI-Family Remodelers
black/gray/red, yellow, and blue/green, respec-
as solid black and dashed gray lines, respectively.
Each base pair of DNA translocated to the exit
side is shown by a red dot. A cartoon representa-
tion of the remodeler is shown as a semi-
transparent light blue or light green shape, and the
locations of the ATPase and linker-DNA-binding
domains as blue and green spheres, respectively.
ISWI-induced remodeling starts with the ATPase
domain translocating DNA from the SHL2 site
toward the exit side, 1 bp at a time. The trans-
location by the ATPase domain generates strain
on the entry-side DNA (depicted by magenta/
purple coloring of the DNA), which initially remains
immobile. After 7 bp of DNA translocation, the
accumulated strain is sufficiently strong to trigger
an entry-side action, possibly a conformational
change of the enzyme, which pushes a 3 bp
partially relaxes the strain and allows three addi-
tional base pairs of DNA to be translocated
to the exit side. This cycle then repeats to allow
processive nucleosome translocation.
450 Cell 152, 442–452, January 31, 2013 ª2013 Elsevier Inc.
long-pass filter (Chroma Technology), spectrally separated by a 630 nm
dichroic mirror (Chroma Technology), and imaged onto the two halves of
a CCD camera (Andor iXonEM+ 888 1024 3 1024).
In order to obtain nucleosomes labeled with a single donor (Cy3) dye and
a single acceptor dye (Cy5), we reconstituted nucleosomes with a mixture of
Cy3-labeled and unlabeled H2A. The presence of two H2A subunits in each
histone octamer gives rise to a heterogeneous population of nucleosomes
with three different labeling configurations, which can be separated in FRET
measurements at the single-molecule level (Figures S1 and S5) (Blosser
et al., 2009). In this work, we selected nucleosomes containing a single
donor on the H2A subunit proximal to the acceptor dye on DNA for further
analysis. The imaging buffer contained 12 mM HEPES, 40 mM Tris (pH 7.5),
60 mM KCl, 0.32 mM EDTA, 3 mM MgCl2, 10% glycerol, 0.02% Igepal (Sigma
Aldrich), an oxygen scavenging system (10% glucose, 800 mg ml?1glucose
oxidase, 40 mg ml?1catalase) to reduce photobleaching, 2 mM Trolox (Sigma)
to reduce photoblinking of the dyes (Rasnik et al., 2006), and 0.1 mg ml?1BSA
(Promega). Imaging was performed at 30?C, unless otherwise mentioned.
Remodeling was induced by infusing the sample chamber with the imaging
buffer supplemented with remodeling enzyme, ATP or ATP + ATP-g-S, and
additional MgCl2equimolar to the total amount of added nucleotide using
a syringe pump (KD Scientific).
Automated Step-Identification Analyses of FRET Time Traces
Nucleosome translocationsteps wereidentified by fitting the FRET time traces
before photobleaching with a staircase function using a HMM algorithm
(McKinney et al., 2006) (http://bio.physics.illinois.edu/HaMMy.html) or an
alternative step-finding algorithm (Kerssemakers et al., 2006) (see Extended
To assign a number k that identifies the position of each pause for analyses
shown in Figure S4, the FRET value of the corresponding plateau was con-
verted into the exit DNA-linker length (in bp) and then rounded to the nearest
Supplemental Information includes Extended Experimental Procedures, six
figures, and one table and can be found with this article online at http://dx.
We thank J. Widom for providing the plasmid containing the 601 positioning
sequence and G. Narlikar for providing histone protein expression plasmids.
This work is supported in part by the Howard Hughes Medical Institute (to
X.Z.) and the National Institutes of Health GM 70864 (to B.B.). X.Z. is a Howard
Hughes Medical Institute investigator. S.D. is a Merck fellow of the Jane Coffin
Childs Memorial Fund for Medical Research. W.L.H. is supported by the NIH
Molecular Biophysics Training Grant and Medical Scientist Training Program.
Received: September 8, 2012
Revised: October 16, 2012
Accepted: December 17, 2012
Published: January 31, 2013
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