The ATPase motor of the Chd1 chromatin remodeler stimulates DNA unwrapping from the nucleosome
Nucleic Acids Research
The ATPase motor of the Chd1 chromatin remodeler stimulates DNA unwrapping from the nucleosome
Joshua M. Tokuda 1
Ren Ren 0
Robert F. Levendosky 0
Rebecca J. Tay 0
Ming Yan 0
Lois Pollack 1
Gregory D. Bowman 0
0 T.C. Jenkins Department of Biophysics, Johns Hopkins University , Baltimore, MD 21218 USA
1 School of Applied and Engineering Physics, Cornell University , Ithaca, NY 14853 USA
Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-. Nucleosome unwrapping by Chd1, examined by bulk FRET, occurred in the presence and absence of nucleotide and did not require the Chd1 DNA-binding domain. In AMP-PNP conditions, Chd1 unwrapped one side of the Widom 601 DNA more easily than the other, consistent with previous observations of 601 asymmetry and indicating that Chd1 amplifies intrinsic sequence properties of nucleosomal DNA. Using small angle X-ray scattering (SAXS) with contrast variation, we found distinct DNA conformations depending on the nucleotide analog bound to Chd1: with AMP-PNP, DNA primarily unwrapped in-plane with the nucleosomal disk, whereas with ADP·BeF3-, a significant fraction showed distinctive out-of-plane unwrapping as well. Taken together, our findings show tight coupling between entry/exit DNA of the nucleosome and the Chd1 ATPase motor, suggesting that dynamic nucleosome unwrapping is coupled to nucleosome binding and remodeling by Chd1.
Nucleosomes are the fundamental genomic packaging unit
of eukaryotes, with approximately 146 bp of DNA tightly
wrapped around a histone octamer (
). In addition to
compacting the genome into the small space of the nucleus,
nucleosomes provide a platform for storing epigenetic
information while also limiting access to DNA. Chromatin
remodelers are an essential class of enzymes that
regulate DNA accessibility by disrupting the canonical histone–
DNA interactions that occur in the nucleosome. Typically,
remodelers alter the availability of DNA by assembling
nucleosomes from histones and naked DNA, moving existing
nucleosomes along DNA, or removing histones from DNA
). Different families of remodelers appear to be
specialized to achieve distinct biochemical outcomes, which
correlate with their roles in vivo. Here, we focus on the Chd1
remodeler, which is involved in gene transcription and is
required for stem cell pluripotency and suppression of cryptic
Chd1 has been found to act both in gene bodies and
promoters, and directly interacts with a number of key factors
involved in transcription. Chd1 contributes to nucleosome
spacing in coding regions and directly interacts with several
elongation factors such as Spt4-Spt5, the FACT complex,
and the PAF complex (
). In the absence of Chd1,
chromatin appears to be perturbed by passage of RNA
polymerase II (Pol II), which in turn leads to cryptic
). Thus, an important role of Chd1 is to help
reestablish the chromatin barrier after Pol II travels through
the coding region.
Two primary biochemical activities have previously been
demonstrated for Chd1: an ability to assemble
nucleosomes and to reposition nucleosomes along DNA (
Nucleosomes often fail to properly form when histones
are rapidly deposited on naked DNA, and instead
produce prenucleosomes, which lack the canonical
superhelical wrapping of DNA around the histone core (12). Chd1
catalyzes nucleosome assembly when histones are deposited
on DNA by chaperones (
), which coincides with the
ATP- and remodeler-dependent maturation of nucleosomes
from prenucleosomes (
). How nucleosome assembly is
achieved is not yet understood, but has been shown to be
a characteristic of ISWI-type remodelers as well.
The ability to reposition nucleosomes, also referred to as
nucleosome sliding, is common to several remodeling
families. For Chd1 and other remodelers such as SWI/SNF
and ISWI, sliding is achieved by the ATPase motor
translocating on nucleosomal DNA at an internal site called
superhelix location 2 (SHL2) (
). Due to the inherent
symmetry of the histone octamer and thus the nucleosome,
there are two such SHL2 sites where ATPase motors can
act, and two Chd1 ATPases can simultaneously bind to
these two sites (17). In addition to the ATPase motor, Chd1
possesses an N-terminal pair of chromodomains and a
Cterminal DNA-binding domain (DBD) (
chromodomains contact nucleosomal DNA at SHL1, adjacent to
the ATPase motor, whereas the DBD binds DNA exiting
the nucleosome on the opposite gyre (
negative stain and cryoEM revealed that Chd1 binding is
coupled to DNA unwrapping from the nucleosome edge
). Using single molecule FRET, DNA unwrapping
was shown to occur dynamically with Chd1 binding in the
presence and absence of nucleotide analogs (20). As seen
in a high resolution cryoEM structure (
chromodomains, ATPase motor and DBD make a tripartite unit,
with unwrapped DNA extending from the nucleosome
appearing to be held by the DBD.
Here, we use fluorescence resonance energy transfer
(FRET) and small angle X-ray scattering (SAXS) to study
how Chd1 alters the organization of nucleosomal DNA.
As shown by stopped-flow binding experiments, the
association of Chd1 with the nucleosome is strongly nucleotide
dependent, suggesting distinct conformational changes
occur in the nucleosome and/or remodeler upon binding.
As shown by FRET, DNA unwrapping by Chd1 can
also be accomplished when the DBD is absent. Thus, the
ATPase motor of Chd1 appears to be capable of
disrupting entry/exit DNA on its own. Unexpectedly, SAXS
analysis suggested that the conformations of nucleosomes
unwrapped by Chd1 are strongly dependent on the nature
of the bound nucleotide, with in-plane unwrapping
favored by AMP–PNP and distinct out-of-plane
unwrapping stimulated by ADP·BeF3–. Additionally, both SAXS
and FRET indicated a strong asymmetry in unwrapping,
consistent with the unwrapping characteristics previously
demonstrated for the Widom 601 positioning sequence (
Taken together, our findings suggest a tight coupling
between placement of DNA exiting the nucleosome and the
conformational state of the ATPase motor of Chd1. We
propose that Chd1-dependent unwrapping represents a
previously unappreciated mechanism for altering histone–DNA
interactions and DNA accessibility that may influence
MATERIALS AND METHODS
Protein expression and purification
The Chd1 proteins used in this study were from
Saccharomyces cerevisiae and contained the chromodomains,
ATPase motor and DBD (residues 118–1274, referred to
throughout as Chd1) or only the chromodomains, ATPase
motor, and the linker prior to the DBD (residues 118–1014,
referred to as Chd1 DBD). Expression and purification
of Chd1 proteins were carried out as previously described
). Histone protein sequences were those of Xenopus
laevis, and were expressed and purified as described (23).
For fluorescence experiments using 12N80 or 80N12
nucleosomes, a single cysteine was introduced H3(V35C) and
labeled with Cy5-maleimide. Histones were prepared by
refolding H2A and H2B into dimers and H3 and H4 into
tetramers, purifying each by size exclusion chromatography.
DNA and nucleosome purification
Nucleosomal DNA was generated by large-scale PCR
using the Widom 601 sequence (
) as the template, and
purified by native acrylamide gel using a BioRad Prep Cell as
previously described (
). The DNA constructs are given
in Supplementary Figure S1. For FRET experiments, PCR
reactions used DNA oligos containing Cy3 or Cy5 at the
5 end (IDT). Nucleosomes were reconstituted by using salt
gradient dialysis to deposit purified histone octamer onto
601-containing DNA, mixed in a 1:1 ratio, as previously
The on-rates of Chd1 binding to nucleosomes were
measured using an SX20 Stopped-Flow Spectrophotometer
(Applied Photophysics). For these experiments, samples
from two syringes were rapidly mixed. One syringe
contained Chd1 at the specified concentrations, the other
contained 20 nM 12N12 nucleosome. Both samples were
suspended in 1X binding buffer (20 mM Hepes pH 7.5, 10 mM
MgCl2, 0.1 mM EDTA, 5% sucrose, 1 mM DTT, 0.02%
Nonidet P-40, 0.1 mg/ml BSA, 100 mM KCl without or
with 1 mM AMP–PNP or ADP·BeF3– [1 mM ADP, 1.2
mM BeCl2, 6 mM NaF]). The nucleosome was Cy3-labeled
on H4(A15C). The dye was excited at 510 nm and the
emission intensity collected using a 570 nm long-pass filter. For
each set of syringes, progress curves from multiple
injections were recorded at 25◦C. Typically, three to six traces
(technical replicates) were averaged together for each
experiment. Fitting was carried out in Mathematica using the
NonLinearModelFit function with the triple exponential
form, y = a1 (1 − e−k1x) + a2(1 − e−k2x) + a3(1 − e−k3x) +
c. Here an were the amplitudes (fractions of total
fluorescence range), kn were the rates (s− 1) and c was a constant.
The observed rates plotted versus Chd1 concentration
provide the apparent kon rates (M−1 ·s−1), shown in Figure 1.
FRET and fluorescence-based unwrapping assays
Fluorescently labeled nucleosomes possessed both Cy3 and
Cy5 dyes, and emission spectra were collected after
excitation at either 510 nm (for Cy3) or 645 nm (for Cy5) on a
Fluorolog 3 fluorometer (Horiba). For 16N16 nucleosomes,
experiments were performed with a 2 ml cuvette (Starna
3-Q10). Emission spectra were collected with excitation at 510
nm for 10 nM nucleosome, first in the presence of 1×
binding buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 0.1 mM
EDTA, 5% sucrose, 1 mM DTT, 0.02% Nonidet P-40, 0.1
mg/ml BSA, 100 mM KCl) and either 1 mM AMP–PNP or
1 mM ADP·BeF3–, and then again after addition of 30 nM
Two nucleosome constructs, called 12N80 and 80N12,
were also dual labeled for FRET experiments. These each
had a single Cy3 label on the 12 end, and Cy5 labels on
H3(V35C). As described in the main text, in addition to
FRET, Cy5 fluorescence was also affected by DNA
unwrapping in a Cy3-independent manner. Therefore, rather than
FRET, we describe the differences in 12N80 versus 80N12
unwrapping by plotting changes in peak Cy3 emission
(Figure 2). An advantage of this approach is that Cy3 should
report only on the 12 bp DNA end, and therefore allows us to
separately study the two sides of the Widom 601 sequence,
using 12N80 from 80N12 constructs. A disadvantage of
using Cy3 emission only is that the measurements were much
more sensitive to instrument fluctuations and differences in
nucleosome concentrations. Thus, the Cy3 data were noisier
than the FRET data calculated from the same experiments.
Experiments with 12N80 from 80N12 nucleosomes were
performed with a 100 l cuvette (Hellma 105-250-15-40).
Emission spectra were collected from 530–720 nm with
excitation at 510 nm using a 5 nm slit width. These
experiments used 20 nM nucleosomes and, when present, 1
mM nucleotide in a 140 l reaction. For AMP–PNP and
nucleotide-free (apo) conditions, nucleosomes were
equilibrated to 25◦C in binding buffer with or without AMP–
PNP. Chd1 was added, mixed, and allowed to equilibrate for
6 minutes before collecting another spectrum. For a given
experiment, successive 2 l additions of Chd1 to the
nucleosome sample resulted in Chd1 concentrations of 0, 39,
78, 153, 303, 597, 1179, 1745, 2297, 2835, 3360 nM for apo
and 0, 5, 10, 19, 38, 75, 147, 291, 574, 1134, 1680, 2212,
2732 nM for AMP–PNP. For ADP·BeF3– conditions,
reactions containing 20 nM nucleosome, 1 mM ADP·BeF3–
and varying amounts of Chd1 (0, 0.625, 1.25, 2.5, 5, 10,
20, 40, 80, 160, 320, 640 nM for Chd1 WT and 0, 10, 20,
40, 80, 160, 320, 640 and 1280 nM for Chd1 DBD) were
assembled at room temperature. Samples containing Chd1
with ADP·BeF3– were allowed a 2–4 h pre-incubation
before collecting spectra. After pre-incubation, each reaction
was placed in the cuvette and allowed to equilibrate in the
fluorometer chamber for one minute before collecting
spectra. For salt titrations, 1× binding buffer containing 4 M
NaCl was added in 5 l increments to 140 l reactions
containing 20 nM nucleosome and 1 mM AMP–PNP, yielding
final NaCl concentrations of 100, 234, 360, 477, 588, 691,
788, 880, 967, 1049, 1126 and 1200 mM. Identical titrations
were performed adding 1× binding buffer alone to assess
the impact of dilution.
For Chd1 titrations performed with ADP·BeF3–, the
responses were stoichiometric; therefore linear fits were
made to the ascending portion, and we report the point
of intersection with the saturated signal. For binding
titrations of Chd1 in apo and AMP–PNP conditions, and
Chd1 DBD in ADP·BeF3– conditions, data were fit to
binding isotherms of the form: a( x+Kxunwrap ) + c, with the
Chd1 concentration x (nM), amplitude a, apparent
unwrapping constant Kunwrap (nM) and constant c.
SAXS data collection and modeling
SAXS data were collected at G1 station at Cornell High
Energy Synchrotron Source. The X-ray energy was 11.18 keV
and sample-to-detector distance was 2.06 m (measured
using a silver-behenate standard). Scattering intensities were
collected on a Pilatus 200K detector over a q-range from
≈0.007–0.26 A˚−1. After azimuthal integration, SAXS
profiles were normalized by the intensity of the primary beam,
which was imaged directly on the detector, after attenuation
by a 200 m molybdenum beamstop. Samples were
oscillated in a 2 mm quartz capillary with 10 m thick walls.
Multiple, 10 s exposures were collected and averaged.
Profiles were carefully monitored for signs of radiation damage.
12N12 nucleosomes (5 M) were incubated with 10 M
Chd1 for greater than 60 minutes (ADP·BeF3– conditions)
or at least 10 min (all other conditions) before being
measured. Buffers contained 10 mM Tris pH 7.8, 100 mM NaCl,
2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 60% (w/v)
sucrose and ±0.5 mM AMP–PNP or ADP·BeF3– (0.5 mM
ADP, 4 mM NaF, 0.6 mM BeCl2). For each condition, three
to five separate measurements were made and analyzed
independently to confirm reproducibility.
Radius of gyration (Rg) values were calculated using
). To carry out ensemble modeling, a pool
containing 14 807 DNA structures was generated and
theoretical SAXS profiles for each structure were calculated
using CRYSOL (
) (CRYSOL parameters: number of
harmonics = 50, maximum s-value = 0.25, number of points
= 201). To select representative ensembles, we applied an
ensemble optimization method (
) which exploits a
genetic algorithm (GAJOE) to optimize the selection of
models from a pool whose summed scattering profiles best
represents the experimentally measured profile, over the q range
from 0.016–0.16 A˚ −1 (GAJOE parameters: number of
generations = 10 000; number of ensembles = 50; ensemble
size fixed = no; maximum/minimum number of curves per
ensemble = 20/5; curve repetition allowed = yes; constant
subtraction = yes; number of times genetic algorithm
repeated = 10). For the Chd1–12N12 complex measured in
the presence of ADP·BeF3– and AMP–PNP, the final best
fitting ensembles for each of the 10 iterations of the genetic
algorithm were pooled together.
The rate that Chd1 binds to nucleosomes is nucleotidedependent
Chd1 forms a high-affinity complex with the nucleosome
in the presence of both AMP–PNP and ADP·BeF3–, yet
these nucleotide analogs confer distinct properties to Chd1–
nucleosome complexes. In the presence of ADP·BeF3–,
nucleosome binding by Chd1 is less sensitive to the presence
of flanking DNA and more resistant to increased ionic
strength, with higher affinity Chd1-nucleosome complexes
observed than with AMP–PNP (
). We hypothesize that
these two nucleotide states favor distinct conformations
of Chd1 on the nucleosome. To explore potential
differences in these states, we measured the kinetics of
nucleosome binding using stopped-flow. Previous work with the
ISWI remodeler showed that nucleosomes labeled with Cy3
on the histone H4 tail (A15C) yield higher fluorescence in
the bound state (
). We discovered that for Chd1, Cy3 at
this position increased binding affinity compared with
unlabeled nucleosomes (Supplementary Figure S2), and
therefore previously reported measurements using
H4(A15CCy3) nucleosomes likely overestimated affinities (
Interestingly, reactions performed with and without the Cy3
label typically required a substantially longer time (>20
min) to equilibrate in ADP·BeF3– compared with AMP–
PNP (<5 min), suggestive of slow conformational changes.
We reasoned that the higher affinity toward H4(A15C-Cy3)
nucleosomes should increase the on-rate or decrease the
offrate, and therefore rapid kinetics of binding could still be
informative for detecting significant barriers in forming a
stable Chd1-nucleosome complex.
To monitor Chd1 binding, we designed a nucleosome
with 12 bp of DNA on either side of the Widom 601
strong nucleosome positioning sequence (12N12)
containing H4(A15C-Cy3). To determine on-rates, rapid mixing
experiments with 10 nM nucleosomes were carried out with
increasing concentrations of Chd1 (Figure 1A). Binding
reactions for both AMP–PNP and ADP·BeF3– proceeded in
several stages and were best fit with triple exponential
equations. Plotting the observed rates with respect to
concentration yielded apparent on-rates for both nucleotide
conditions (Figure 1B). For ADP·BeF3–, all three rates were
concentration dependent, whereas for AMP–PNP, only the
fastest rate showed a clear concentration dependence.
Interestingly, despite a higher apparent affinity for ADP·BeF3–,
the fastest on-rate (2.7 ± 0.3 × 106 M–1 s–1) was ∼5-fold
slower than that of AMP–PNP (1.3 ± 0.1 × 107 M–1 s–1).
These different binding characteristics suggest that Chd1
adopts distinct conformations in each nucleotide state,
possibly leading to specific, nucleotide-dependent
rearrangements of the nucleosome.
FRET reveals asymmetric unwrapping by Chd1
Reorganization of histone–DNA contacts within the
nucleosome is believed to be principally driven by DNA
translocation of the Chd1 ATPase motor, which requires turnover
of ATP. However, recent work suggests that Chd1
binding unwraps DNA from the edge of the nucleosome in the
presence of the non-hydrolyzable ATP mimics,
independently from active ATP hydrolysis (
hypothesized that the nucleotide dependent binding dynamics we
observed may correlate with altered conformations of
nucleosomal DNA. To investigate this idea, we designed a
FRET-labeled nucleosome having 16 bp of flanking DNA
on each side of the strong Widom 601 positioning sequence
(16N16), with Cy3 and Cy5 dyes at the DNA ends
(Figure 2A). This labeling scheme yielded average FRET
efficiencies of 0.45 ± 0.02 for the nucleosome alone, consistent
with trajectories of entry/exit DNA of nucleosome
crystal structures and indicative of a fully wrapped state.
Addition of Chd1 in the presence of AMP–PNP or ADP·BeF3–
markedly reduced FRET efficiencies to 0.088 ± 0.005 and
0.065 ± 0.015, respectively, consistent with DNA
unwrapping by Chd1 (Figure 2B). A decrease in FRET was also
observed with Chd1 in the absence of nucleotide (apo state),
but required the addition of considerably more Chd1 (1 M
for apo versus 30 nM for nucleotide analogs) to achieve a
smaller change in FRET (from 0.45 to 0.32)
(Supplementary Figure S3). This suggests that Chd1 is capable of
unwrapping DNA in the apo state, but does so more readily in
the presence of nucleotide, possibly due to more stable
binding or by adopting conformations favorable for unwrapping
when bound to ATP-mimicking analogs.
One disadvantage of monitoring DNA unwrapping with
the 16N16 construct is its sensitivity to small DNA
movements. By labeling both flanking DNA segments, FRET
changes can be amplified by the 2-fold symmetry of the
nucleosome, as simultaneous unwrapping on both sides would
double the change in dye separation. Additionally, due to
the superhelical geometry of the nucleosome, the DNA ends
experience the greatest changes in position upon
unwrapping, resulting in high FRET sensitivity for small changes
in the angle of entry/exit DNA. Another drawback of this
construct is the inability to distinguish between
unwrapping from either side of the nucleosome. To resolve these
issues, we designed different nucleosomes with Cy3/Cy5
FRET pairs that would only respond to unwrapping on
one side and would require a greater degree of unwrapping
to separate the dye pair. Following the original FRET
design by Widom (
), Cy5 maleimide was attached to histone
H3(V35C) adjacent to DNA entering/exiting the
nucleosome, and Cy3 was attached to the DNA end, 12 bp
outside the nucleosome on 12N80 and 80N12 constructs
(Figure 2C). Using these two constructs enabled the comparison
of unwrapping from either side of the Widom 601, which is
asymmetric with respect to the number of phased TpA
dinucleotides (TA steps) located where the minor groove faces
the octamer. These TA steps are thought to facilitate the
bending of DNA around the histone octamer, and previous
force spectroscopy studies have shown that higher force is
required to disrupt histone-DNA contacts from the TA-rich
side of the Widom 601 (
To monitor FRET, we collected emission spectra while
exciting Cy3 at 510 nm. Titrations showed that increasing
amounts of Chd1 yielded progressively more intense Cy3
emission peaks and less intense Cy5 emission peaks (Figure
2C). While the dependence of Cy5 fluorescence on Cy3
excitation indicates FRET, some of the decrease in Cy5
intensity might have resulted from Chd1 binding rather than
separation from the Cy3 donor. To investigate this possibility,
emission spectra were also collected with direct excitation
of Cy5 (645 nm). As shown in Supplementary Figure S4A,
the Cy5 emission peak did in fact decrease with addition of
Chd1, indicating that, in addition to FRET, environmental
changes surrounding Cy5 also decreased fluorescence
intensity. To determine whether the change in Cy5 intensity was
due to Chd1 binding directly, or was an indirect effect due
to unwrapping, we monitored fluorescence with alternating
Cy3 and Cy5 excitation during salt-induced disassembly of
the nucleosome (Supplementary Figure S5). The salt
titrations revealed dramatic drops in Cy5 fluorescence during
excitation of either Cy3 or Cy5, indicating that the
Cy3independent changes in Cy5 fluorescence likely arose from
In comparing Chd1 titrations for 12N80 and 80N12
nucleosomes in AMP–PNP conditions, emission peaks from
Cy5 direct excitation were generally quite similar, whereas
the profiles of Cy3 emission peaks differed between these
two nucleosomes (Supplementary Figure S4B). This
discrepancy may be explained by the labeling scheme: Cy3 was
uniquely on one DNA end for each nucleosome, whereas
Cy5 was in two symmetrically related positions on the H3
tail. We therefore focused on changes in Cy3 emission as a
more appropriate reporter for how Chd1 titrations affected
unwrapping of the two sides of the nucleosome. Each
titration was fit to a binding isotherm to calculate Kunwrap,
corresponding to the Chd1 concentration required to achieve
half of the maximum signal change. In AMP–PNP
conditions, Kunwrap was dramatically different for these two
nucleosome constructs, with a much larger value of 700 ± 200
nM for 12N80 compared with 13 ± 2 nM for 80N12
(Figure 2D, green symbols). Interpreting these differences with
respect to the Widom 601 sequence, it appears that the TA
poor side unwraps more readily by Chd1 than the TA rich
side, consistent with previously published work (
Chd1-dependent changes in Cy3 and Cy5 fluorescence
were also observed in apo and ADP·BeF3– conditions.
Consistent with the 16N16 experiments, higher amounts of
Chd1 were required in apo conditions to elicit changes in
Cy3, corresponding to apparent Kunwrap values of 800± 400
nM for 12N80 and 700 ± 200 nM for 80N12 (gray
symbols). In sharp contrast, ADP·BeF3– conditions promoted
maximal changes in Cy3 fluorescence with stoichiometric
amounts of Chd1 (magenta symbols). The saturation point
was 51 ± 5 nM, close to 2:1, for the 12N80 and 22 ± 2 nM,
or roughly 1:1, for the 80N12, again consistent with the
TApoor edge of the Widom 601 more easily unwrapping at low
concentrations of Chd1. Thus, DNA unwrapping by Chd1
is strongly affected by both the sequence of the DNA and
the nucleotide state of the remodeler.
Recent cross-linking and EM data suggest that all
three domains of Chd1 work in concert to span both
gyres of DNA and unwrap DNA from the nucleosome
). We suspected that a specific,
nucleotidedependent organization of Chd1 domains held the DBD in
a position that favored an unwrapped trajectory of DNA
exiting the nucleosome. To test this idea, we examined the
extent that increases in Cy3 intensity could be stimulated
by a Chd1 construct lacking the DBD. Binding was
performed in the presence of ADP·BeF3–, since this nucleotide
state appeared to yield the tightest Chd1-nucleosome
complex. Titrating Chd1 DBD in the presence of ADP·BeF3–
gave a strong change in Cy3 fluorescence, indicative of
unwrapping (Figure 2E). The amplitude of the fluorescence
changes were comparable to titrations with Chd1, yet the
Chd1 DBD titrations showed that a much higher protein
concentration was required to approach saturation
(Supplementary Figure S6). While these data show the same trends,
individual experiments unfortunately were noisier
(Supplementary Figure S6), resulting in greater variation in Kunwrap
values, which were 500 ± 300 nM for the 12N80 and 300 ±
200 nM for the 80N12. Despite the poorer fits, these data
indicate that the chromo-ATPase portion of Chd1 is sufficient
for unwrapping DNA from the nucleosome. While
unwrapping can be achieved by binding of a transcription factor to
the outer DNA gyre of the nucleosome (
), it seems
unlikely that binding to the outer DNA gyre could explain the
unwrapping observed with Chd1 DBD. In previous work,
we found that a chromo-ATPase construct was unable to
stably associate with naked DNA (
). Thus, even the
M concentrations of Chd1 DBD used here would likely
be insufficient for significant unwrapping by mass action.
These results are consistent with the idea that the DBD is
required for high affinity binding, yet not necessary for
stimulating changes in DNA trajectory. Given the direct contact
that the ATPase motor makes with the opposite gyre DNA
as seen by cryoEM (
), as well as the sensitivity of
unwrapping to bound nucleotide (Figure 2D), we favor the notion
that the ATPase motor can disrupt wrapping at the edge of
the nucleosome when bound at the internal SHL2 site.
The structural distributions of unwrapped nucleosome states are sensitive to the nucleotide analog bound by Chd1
While the sensitivity of fluorescent probes provides a
powerful tool to detect perturbations of nucleosomal DNA,
we sought to obtain additional insight into what an
altered structure of the nucleosome might look like. We
therefore turned to SAXS, which can provide insight into the
global structures of individual macromolecules in solution.
For large complexes containing multiple components with
different electron densities (e.g. proteins and DNA),
however, interpretation of SAXS data is limited due to the
difficulty in resolving each component’s contribution to the
measured SAXS profile (
). Therefore, we applied
contrast variation small angle x-ray scattering (CV-SAXS) to
resolve the nucleotide-dependent conformation(s) of
nucleosomal DNA changes upon Chd1 binding. In CV-SAXS,
sucrose is added to the bulk solvent until its electron
density equals that of the lower electron density component,
in this case the proteins. Under this matched condition, the
protein components no longer contribute to the signal and
details of the DNA conformation can be resolved. We
previously used CV-SAXS to study DNA unwrapping during
the salt-induced disassembly of nucleosomes (
we applied this technique to determine whether Chd1
altered the canonically wrapped conformation of
nucleosomal DNA when bound in different nucleotide states.
The concentration of sucrose required to mask the
protein signals was experimentally determined. Initially, SAXS
profiles of the DNA (5 M), Chd1 (15 M), and histones
(5 M) were separately measured in solutions containing
varying percentages of sucrose (Figure 3A). In 60% sucrose,
the signals from protein components (Chd1 and histones)
were effectively eliminated, but, due to its higher electron
density, sufficient signal remained from the DNA to
measure its conformation in the presence (or absence) of protein
Figure 3B shows SAXS profiles measured in 60% sucrose
for 12N12 nucleosomes (5 M), with and without Chd1
(10 M). All measured SAXS profiles are qualitatively
similar, displaying the characteristic supercoiled shape of DNA
around the histone octamer, suggesting that most of the
DNA remains wrapped in nucleosomal structures. As a
control, we measured SAXS profiles for nucleosomes alone in
the presence of different nucleotides, but without the
remodeler. These profiles are identical (Supplementary
Figure S7A and B). Small but significant differences were
detected when Chd1 was present, dependent on the presence
of ADP·BeF3– or AMP–PNP, or the absence of nucleotide
(apo) (Supplementary Figure S7C and D). The radius of
gyration (Rg) for the 12N12 nucleosome alone was 49.4 ± 0.3
A˚, consistent with expectations for a fully wrapped
structure. In the presence of Chd1, the Rg increased to 56.6 ±
0.7 A˚ and 55.3 ± 0.5 A˚ with the addition of ADP·BeF3–
and AMP–PNP, respectively, consistent with the partial
unwrapping of the DNA ends observed using FRET (Figure
2D). The complex (Chd1–12N12) in the apo state had an
Rg of 50.4 ± 0.2 A˚. This decreased response is consistent
with the weaker fluorescence changes and a requirement for
higher Chd1 concentrations in the apo state (Figure 2D and
Supplementary Figure S3).
To gain structural insights that go beyond the average,
measured Rg values, we applied an ensemble
optimization method (EOM) to identify potential DNA structures
present in the various complexes. Starting from a large pool
of possible structures, EOM finds the subset of structures
(‘ensemble’) that best recapitulates the SAXS data.
Following the strategies in ref (
), we manually unwrapped
DNA to generate a pool of structures. These structures
were based on the nucleosome crystal structure 1KX5 (37)
with varying amounts of DNA removed and replaced with
straight segments (linear B-form DNA). This procedure
produced 9182 structures with the DNA unwrapped along
the natural trajectory of the nucleosomal DNA. To further
diversify the DNA pool, we generated 5625 variations of
unwrapped nucleosomal DNAs that contained kinks directing
DNA out-of-plane with the nucleosome disk
(Supplementary Figure S8). These kinks were generated by
introducing various combinations of bends into 12N12 nucleosomal
DNA using 3D-DART (
). From this pool of 14 807
total structures, a genetic algorithm (
ensembles of DNA structures using the SAXS data collected for
12N12 nucleosomes with and without Chd1 in 60% sucrose.
The resulting fits to the data are shown in Figure 4A–D.
To characterize the ensembles, we calculated Rg
distributions for the structures selected by EOM (Figure 4E and
F). Although the pool contained structures that ranged in
size from 45–165 A˚ (Figure 4E), the structures within the
selected ensembles cluster in size between 45–70 A˚
(Figure 4F), consistent with DNA in 12N12 nucleosomes
being mostly wrapped in the presence of Chd1 regardless of
the nucleotide state. The nucleosomes alone are represented
by the peak centered around 49 A˚. For Chd1–12N12
complexes in the presence of AMP–PNP and ADP·BeF3–, the
Rg distributions showed a prominent peak centered around
55 A˚, reflecting partially unwrapped nucleosome structures.
For Chd1–12N12 in the apo state, the Rg distribution
suggests a mixture between mostly wrapped nucleosomes and
a small population of partially unwrapped structures. The
EOM pools therefore suggest subtle but measurable
differences in DNA conformation in response to
nucleotidedependent conformations of Chd1.
Ensembles for each condition are shown and compared
in Figure 4G–J. The structures in each ensemble were
assigned colors according to shared characteristic features.
Under all conditions, at least some DNA remained mostly
wrapped (green in Figure 4G–J). These wrapped states are
most prevalent for the nucleosomes without Chd1 (100%)
and Chd1–12N12 in the apo state (64%). Between a
quarter and a third of the structures remain mostly wrapped for
Chd1–12N12 in AMP–PNP (27%) and ADP·BeF3– (31%).
This distribution is consistent with dynamic unwrapping, as
has been illustrated for Chd1-nucleosome complexes in apo
and AMP–PNP states (
The partially unwrapped structures observed in the
presence of Chd1 showed distinct trajectories (blue, yellow, and
red in Figure 4G-J) that were present with varying weights
depending on the nucleotide state. In the unwrapped
structures, the DNA end typically peeled away from the histone
at a common internal site, up to 25 bp from the
nucleosome edge, which is consistent with the cryoEM structure of
Chd1 partially unwrapping one side of a nucleosome (
While most structures showed some degree of unwrapping
on both sides, unwrapping was typically more prominent on
one side than the other. To visualize the extent of
asymmetry, we calculated the extent that each half of each model
deviated from an idealized, symmetrically wrapped model
(Figure 4K). Models that were mostly wrapped, which in
this analysis showed deviations up to but not beyond ∼10
A˚ , showed the least asymmetry. Most of the AMP–PNP
and ADP·BeF3– models with greater deviations were
asymmetric, with more unwrapping on one side than the other.
An exception was one of the ADP·BeF3– models, which
showed a similar out-of-plane unwrapping on both sides.
Although a 2-fold molar equivalent of Chd1 was present
relative to nucleosomes, some asymmetry may have arisen
from incomplete saturation of nucleosomes, resulting in 1:1
(Chd1:nucleosome) complexes. Since SAXS is not
sensitive to chirality, this analysis cannot distinguish
unwrapping from a particular side of the nucleosome, though we
would expect the unwrapping of the TA-poor side to be
favored, based on the FRET experiments with 12N80 and
80N12 nucleosomes in AMP–PNP conditions (Figure 2D).
With ADP·BeF3–, FRET showed that both sides of the
nucleosome were highly sensitive to Chd1, with apparent
saturation at 2:1. It was therefore unexpected to observe
significant asymmetry for most EOM models for ADP·BeF3–
Comparison of the EOM-selected models to the
Chd1nucleosome complex solved by cryoEM (
interesting nucleotide-specific trends. In the cryoEM structure,
which was solved in an ADP·BeF3– state, the nucleosomal
DNA adjacent to where Chd1 is bound is unwrapped by
∼2 helical turns and is slightly out-of-plane (
Alignment to this structure shows that the SAXS-derived, AMP–
PNP models have a wider range of unwrapping, yet always
remain more in-plane than DNA in the cryoEM complex
(Figure 5A). In contrast, the SAXS-derived, ADP·BeF3–
models show approximately the same magnitude of in-plane
unwrapping as the cryoEM structure, yet more significant
unwrapping in the out-of-plane direction (Figure 5B). To
evaluate how critical these unique features were for fitting
the SAXS data, an additional round of EOM selections was
performed where the starting pool was limited to the
selected models of the other nucleotide state. In both cases,
the fits between the available models and data were worse:
0A.9D7P(·cBoemFp3–adreadtatofit w2it=h A0.M89Pf–oPrNAPDmP·oBdeeFls3–yimeloddedelas,
F2ig=ure 4D), and AMP–PNP data fit with ADP·BeF3– models
yielded a 2 = 1.28 (compared to 2 = 0.87 for AMP–PNP
models, Figure 4C). The EOM-based interpretation of the
SAXS data therefore supports the conclusion that the
distributions of DNA shapes are distinct. These differences in
unwrapping geometry imply that the nucleotide bound by
Chd1 significantly alters the dynamics or conformational
state of the ATPase motor on the nucleosome, which in turn
influences both the extent and direction of unwrapping.
This work explores the ability of the Chd1 chromatin
remodeler to unwrap DNA from the edge of the
nucleosome. Recent studies have revealed that the ATPase
tor and DBD of Chd1 interact across the gyres of the
nucleosome, with the ATPase motor bound to the internal
SHL2 site and the DBD engaged with DNA exiting the
). While both of these domains appear
to stabilize an unwrapped state of the nucleosome in a
recent cryoEM structure (19), we show here that the DBD is
not required for unwrapping (Figure 2E). We therefore
propose that the Chd1 ATPase motor is sufficient for
destabilizing histone–DNA contacts at the nucleosome edge.
Consistent with this idea, we observed that unwrapping was
strongly correlated to the nature of the bound nucleotide,
with significant differences for ADP·BeF3– compared to
AMP–PNP. FRET experiments showed that ADP·BeF3–
promoted Chd1-dependent unwrapping of the 601
nucleosome more readily than AMP–PNP (Figure 2D). More
striking, however, was the finding that these two
nucleotidebound states favored distinct conformations of unwrapped
Chd1-nucleosome complexes. As determined by EOM
analysis of CV-SAXS data, Chd1 loaded with AMP–PNP
unwrapped DNA at a wider angle but essentially in-plane with
the nucleosome disk, whereas a significant fraction of DNA
unwrapped more distinctly out-of-plane with ADP·BeF3–
(Figures 4 and 5).
As for all ATPase motors, Chd1 is expected to be
structurally sensitive to its nucleotide-bound state.
Accordingly, we observed distinct kinetics of nucleosome
binding in AMP–PNP versus ADP·BeF3– conditions (Figure 1),
which we interpret as reflecting conformational changes in
Chd1 domains and/or the nucleosome upon binding.
Together with our findings that these nucleotide analogs
produce structurally unique distributions of unwrapped
nucleosomes, these results indicate that DNA unwrapping
is tightly coupled to the dynamics and/or conformational
states of the ATPase motor. Although it is unclear whether
each cycle of ATP hydrolysis produces a specific in-plane
to out-of-plane pattern of unwrapping, this tight coupling
suggests that the position of exit DNA on the opposite
gyre could influence ATPase activity. We speculate that
particular conformations of flanking DNA––whether biased
or stabilized via the Chd1 DBD, DNA sequence, or other
factors––would affect the conformational landscape of the
ATPase motor during the ATP binding and hydrolysis cycle,
and thus could provide an external means of regulation.
Nucleosome repositioning and assembly are two defining
characteristics of Chd1 (
), but it seems unlikely that
the DNA unwrapping described here is required for either
of these processes. When bound at SHL2, the ATPase
motor of Chd1 directly contacts the DNA being unwrapped
from the opposite gyre (19). It has been suggested that
contact between the ATPase motor and opposite gyre DNA
may be a common characteristic of chromatin remodelers: a
cryoEM structure of the SWI/SNF ATPase bound to a
nucleosome identified conserved ATPase residues in contact
with opposite gyre DNA, and mutation of these residues
reduced sliding activity (
). The SWI/SNF-nucleosome
complex, however, showed no unwrapping, suggesting that
those ATPase contacts are insufficient for the unpeeling
of DNA observed in the Chd1–nucleosome complex.
Although it is not yet known whether ISWI remodelers also
stimulate nucleosome unwrapping, it is interesting that both
Chd1 and ISWI remodelers slide and assemble nucleosomes
yet are differently affected by the presence of linker histones,
which stabilize a wrapped form of nucleosomes (
cannot incorporate histone H1 during nucleosome
), and the presence of H1 and H5 interfere with
nucleosome sliding by Chd1 (
). In contrast, ISWI
remodelers can both assemble and slide nucleosomes in the presence
of linker histones (
Notably, in the Chd1–nucleosome complex visualized
by cryoEM, a loop protruding from the ATPase motor
packs against the phosphate backbone where DNA
unwraps from the histone core (
) (Supplementary Figure
S9A). Sequence alignment of different remodeler families
reveals that this loop has conserved basic character and
is longer in Chd1 orthologs than SWI/SNF or ISWI
remodelers (Supplementary Figure S9B). Based on its
location in the cryoEM structure and positively charged
character, we propose that this family-specific loop would assist in
stimulating and/or stabilizing DNA unwrapping. This loop
is longer in S. cerevisiae than human Chd1, and therefore
the propensity to unwrap may also differ among Chd1
orthologs. It is also interesting to note that the two other CHD
subfamilies also have a pronounced loop in this region, with
a significantly longer and highly charged segment for the
CHD3/CHD4/CHD5 group. Future work will be needed
to determine the extent that this ATPase loop is involved in
Nucleosomal DNA can unwrap spontaneously and
transiently, which is important for allowing access to
transcription factor binding sites at the nucleosome edge (
DNA sequence determines the intrinsic stability of histone–
DNA contacts, and the most favored sequences, such as the
widely used Widom 601, contain phased motifs (such as TA
steps) that bias DNA bending in one direction (
Despite its high affinity, however, the strength of histone–DNA
contacts are not symmetrically distributed for the Widom
), and under tension, 601 nucleosomes preferentially
unwrap on the less flexible side, which has fewer TA steps
). Asymmetric unwrapping was prevalent in our SAXS
experiments (Figure 4), and FRET indicated that with
AMP–PNP, Chd1 more easily unwrapped nucleosomes on
the TA poor side (Figure 2). These findings indicate that
Chd1 amplifies the natural unwrapping tendencies of the
underlying sequence. Such amplification of nucleosome
dynamics should likewise increase the sequence-dependent
accessibility for chromatin-associated factors. In addition to
transcription factors, histone modifying enzymes are also
likely to be affected. As observed in the first and many
subsequent nucleosome crystal structures, residues 39–42
of histone H3 pass between the DNA gyres of the
nucleosome at the DNA entry/exit site (
). DNA unwrapping by
Chd1 exposes this N-terminal segment of histone H3 (
which is consistent with our observation of
unwrappingcoupled environmental changes of Cy5-labeled H3(V35C)
(Supplementary Figure S5). Unwrapping would therefore
increase accessibility for post-translational modifications
such as H3R42 methylation and H3Y41 and H3T45
). In addition, methylation and
acetylation of the nearby H3K36 is strongly coupled to
), and unwrapping may also favor deposition or
recognition of these marks.
In addition to increasing DNA and histone
accessibility, nucleosome unwrapping by Chd1 may potentially
influence transcriptional elongation. Nucleosomes present a
barrier to elongating polymerases such as RNA polymerase
II (Pol II), causing transcriptional pausing (
Studitsky and coworkers have shown that DNA unwrapping
from the upstream edge of the nucleosome facilitates
transcription of Pol II through the outermost turn of
nucleosomal DNA, whereas unwrapping from the downstream
edge of the nucleosome greatly diminishes the major Pol
II pause site at the upstream SHL2 (57). Engagement of
Chd1 with the downstream SHL2 would unwrap the
upstream edge of the nucleosome and therefore potentially
help Pol II entry. However, the presence of Pol II at the
upstream SHL2, a major site of pausing, would sterically
block Chd1 from unwrapping the downstream edge.
Interestingly, Pol II has more difficulty transcribing through the
upstream SHL2 site of 601 nucleosomes if the upstream
DNA immediately behind the polymerase does not rewrap
). DNA unwrapping, if maintained by Chd1 on the
entry side, therefore could be antagonistic to passage of Pol
II through nucleosomes. Pol II transcription through
nucleosomes is assisted by several elongation factors, and the
FACT complex both reduces polymerase pausing and
stabilizes histone–DNA contacts during transcription (
Although its mechanism of action is not presently clear, FACT
appears to act in opposition to Chd1, with FACT mutant
phenotypes in budding yeast rescued by Chd1 defects (
It has been shown that two polymerases traveling together
evict nucleosomes (
), and therefore while speculative,
controlled pausing may help keep Pol II complexes separated
from each other. Determining whether DNA unwrapping
by Chd1 contributes to transcription-related processes is an
exciting new direction for future studies.
SAXS data and the EOM-selected models described here
have been deposited in SASBDB (https://www.sasbdb.org/)
with the following accession numbers: SASDCT6 (12N12
nucleosome alone in 60% sucrose, ADP·BeF3– buffer
conditions); SASDCU6 (Chd1–12N12 complex in 60%
sucrose, nucleotide-free buffer); SASDCV6 (Chd1–12N12
complex in 60% sucrose, ADP·BeF3– buffer); and
SASDCW6 (Chd1–12N12 complex in 60% sucrose, AMP–PNP
Supplementary Data are available at NAR Online.
We thank Sarah Woodson for use of her stopped-flow and
steady state fluorometers. We thank Arthur Woll for
technical assistance at the CHESS G1 beamline.
National Institutes of Health [R35-GM122514 to L.P.,
R01-GM084192 and R01-GM113240 to G.D.B.,
T32GM007231 to R.J.T.]; National Science Foundation (NSF)
& National Institutes of Health/National Institute of
General Medical Sciences (NIH/NIGMS) via NSF award
[DMR-0936384 to CHESS]. Funding for open access
charge: National Institutes of Health [R01-GM113240].
Conflict of interest statement. None declared.
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