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Molecular and Cellular Biology, February 1999, p. 1470-1478, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The SWI/SNF Complex Creates Loop Domains in DNA and
Polynucleosome Arrays and Can Disrupt DNA-Histone Contacts within
These Domains
David P.
Bazett-Jones,1,*
Jacques
Côté,2
Carolyn C.
Landel,3
Craig L.
Peterson,3 and
Jerry
L.
Workman4
Department of Cell Biology and Anatomy,
University of Calgary, Calgary, Alberta, Canada T2N
4N11;
Laval University Cancer Research
Centre, Hôtel-Dieu de Québec, Québec City,
Québec, Canada G1R 2J62;
Howard
Hughes Medical Institute and Department of Biochemistry and Molecular
Biology, Pennsylvania State University, University Park,
Pennsylvania 16802-45004; and
Department
of Biochemistry and Molecular Biology, Program in Molecular
Medicine, University of Massachusetts, Worcester, Massachusetts
016053
Received 7 July 1998/Returned for modification 21 August
1998/Accepted 21 September 1998
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ABSTRACT |
To understand the mechanisms by which the chromatin-remodeling
SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and
nucleosomal arrays by using energy-filtered microscopy. By making
ATP-independent contacts with DNA at multiple sites on its
surface, SWI/SNF creates loops, bringing otherwise-distant sites into
close proximity. In the presence of ATP, SWI/SNF action leads to
the disruption of nucleosomes within domains that appear to be
topologically constrained by the complex. The data indicate that the
action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA
content from the nucleosomes. This indicates a mechanism by which
SWI/SNF unwraps part of the nucleosomal DNA.
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INTRODUCTION |
Regulatory elements of many genes
show hypersensitivity to DNase I, an indication that the disruption of
nucleosomes at these sites is required to relieve a repressive function
of chromatin. Several studies indicate that the eukaryotic cell
possesses large complexes which contribute to gene activation by
disrupting nucleosomes positioned over regulatory regions of particular
genes. Genetic studies have identified a 2-MDa multisubunit complex in
Saccharomyces cerevisiae called SWI/SNF, which can bind to
DNA, disrupt nucleosomes, and provide transcription factors with access
to nucleosomal DNA (for a review, see reference 22).
Genetic suppressors of mutant subunits of SWI/SNF in yeast have
frequently been identified as components of chromatin, such as the core
histones (11, 16, 23). Higher eukaryotes appear to have
homologs of the yeast SWI/SNF complex, which are also capable of
disrupting nucleosomes. For example, brahma (brm)
is a Drosophila melanogaster homolog of SWI2 (26)
and hbrm and BRG1 from human cells also appear to be
functional homologs of yeast SWI2 (6, 15, 19). Other complexes may perform related functions, including RSC (a large complex
like SWI/SNF), NURF, ACF, and CHRAC (a group of smaller related
complexes) (4, 14, 27, 28, 30). Similarities among subunits
of SWI/SNF, NURF, and other functional homologs indicate that the
eukaryotic cell contains a variety of related but independent
systems for activating genes by altering chromatin structure.
In biochemical studies the SWI/SNF complex has been shown
to bind naked DNA and nucleosomes with nanomolar affinity (9, 24). The SWI/SNF complex uses the energy of ATP hydrolysis to remodel nucleosome structure, which increases the affinity of transcription factors for nucleosomal DNA (7, 9, 12, 13, 17, 21,
29, 33). The remodeled conformation of the nucleosome persists
after depletion of ATP (13) and detachment of the SWI/SNF complex (9, 33). This remodeled nucleosome conformation will eventually revert to the original conformation on its own
(9) or can be converted back by further action of SWI/SNF,
indicating that SWI/SNF catalyzes the interconversion between
these two nucleosome forms (33). A little is known about the
remodeled nucleosome conformation. It retains all four core histones
(33), although the affinity of the histone octamer for DNA
is apparently diminished, as the octamer is more susceptible to
displacement by the binding of multiple GAL4-AH dimers (21).
A hallmark of the remodeled nucleosome conformation is an altered
pattern of DNase I digestion, most apparent when SWI/SNF acts on
nucleosomes in which the DNA is rotationally phased (7, 12,
17). On these nucleosomes, SWI/SNF disrupts the
sequence-specific 10-bp periodic pattern of DNase cutting, which
reflects the direction of DNA bending around the histone octamer.
However, a sequence-independent 10-bp pattern of DNase cutting is
retained over at least 70 bp of the nucleosome, indicating that, while
SWI/SNF twists or alters the path of DNA bending, part of the DNA
remains associated with the surface of the histone octamer
(9).
When acting on arrays of nucleosomes, SWI/SNF functions
catalytically (18), increasing restriction
endonuclease sensitivity on multiple arrays. In addition, SWI/SNF
can alter the translational phasing (i.e., location) of multiple
nucleosomes within an array, although translational phasing on
nucleosome positioning sequences is regained upon removal of the
complex (21). To further investigate the interactions of
SWI/SNF with nucleosome arrays and the consequences of its action on
nucleosome structure within arrays, we have visualized SWI/SNF
complexes on naked DNA and complexes of SWI/SNF with nucleosome arrays,
using electron spectroscopic imaging (ESI) (1).
There are a number of advantages that ESI offers over conventional
electron microscopy (EM) for imaging DNA-protein complexes (2,
3). First, because electron energy loss imaging provides high
contrast, heavy-atom stains and shadows which can limit spatial resolution, are not required. Such agents also can exaggerate the
presence of or fail to contrast particular biochemical entities. Second, images recorded from particular regions of the energy loss
spectrum provide mass-sensitive information, so that molecular-mass estimates can be obtained. Finally, coupled with mass analysis, the
detection of phosphorus and the production of phosphorus maps permits
the calculation of stoichiometric relationships between the protein and
the nucleic acid and is the basis for delineating the distribution of
nucleic acid in a protein-DNA complex. The analysis presented here
complements the biochemical data obtained previously and provides new
information regarding the function of SWI/SNF. For example, multiple
sites of DNA contact on its surface result in its ability to create
loops in the DNA, bringing otherwise-distant sites into close
proximity. Furthermore, the protein-DNA stoichiometric measurements
provide insights into the nature of the conformation of
SWI/SNF-remodeled nucleosomes. The results indicate an unwrapping of
DNA from the histone octamer as the primary consequence of SWI/SNF
action. Moreover, it is apparent that a single SWI/SNF complex can
remodel a number of nucleosomes located in a region of chromatin
constrained in an intervening loop created by the complex.
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MATERIALS AND METHODS |
Imaging of SWI/SNF-DNA complexes.
Electron spectroscopic
images of SWI/SNF complexed to linear and relaxed closed-circular
plasmid DNA were obtained as follows. The circular plasmid pJH28 was
relaxed with Escherichia coli topoisomerase I as described
previously (24). The SWI/SNF protein complex was
purified as described previously (7). DNA or polynucleosome arrays (see Fig. 3 and 5) at a DNA concentration of 5 ng/µl were reacted with purified SWI/SNF at a final concentration of either 2 or 4 ng/µl in a binding buffer containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 3 mM MgCl2, 1 µM ZnCl2, 2 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 5% glycerol, and
100 ng of insulin/µl. ATP or ATP-
S was added to a final
concentration of 2 mM. The reactants were incubated for 30 min at
30°C, followed by a further fixation step with buffered EM-grade
formaldehyde (1%) and glutaraldehyde (0.5%) for 15 min at 30°C. For
ESI analysis, 1 µl of the reaction mixture was applied to a 4-µl
drop of 3 mM MgCl2-10 mM HEPES (pH 8.0) on a 1,000-mesh EM
specimen grid covered with a 3-nm-thick carbon film (3).
After 1 min, the grid was washed with pure water (prepared for tissue
culture by Gibco) and air dried.
Electron spectroscopic images were recorded on a Zeiss EM902
transmission electron microscope equipped with a prism-mirror-prism electron-imaging spectrometer. A 90-µm-diameter objective and a
400-µm-diameter condenser aperture were used. The energy-selecting slit aperture corresponded to 20 eV. Phosphorus-enhanced images were
recorded at 155 eV, and mass-sensitive reference images were recorded
at 120 eV of energy loss as described previously (references 1 and 3 and references therein).
Micrographs were recorded on electron image film (Kodak) and were
processed in full-strength D-19 developer (Kodak) for 15 min.
Phosphorus and mass analyses of nucleosomes and DNA fibers were
performed as described previously (
3). Nucleosomes were
delineated by choosing a boundary at 1 standard deviation (SD)
above
the mean of the background. The phosphorus content was calculated
by
arithmetically comparing a region in the 155-eV phosphorus-enhanced
postedge image with the same region in the 120-eV preedge
mass-sensitive
image after the two images were aligned and normalized
and background
values in the region of the particles of interest had
been subtracted.
Mass was calculated in a similar way by using only the
mass-sensitive
120-eV preedge image. Naked DNA and cospread tobacco
mosaic virus
were used as internal mass and phosphorus
standards.
Several grids of each SWI/SNF-DNA or SWI/SNF-polynucleosome
reaction were prepared to ensure that the structures were reproducible.
In addition, reproducibility between reactions was established
by
reproducing every EM experiment at least three
times.
Preparation of polynucleosomes and DNase I analysis of
SWI/SNF-induced nucleosome disruption.
Polynucleosomes were
reconstituted onto a 12-mer repeat of the 208-bp sea urchin 5S rRNA
gene nucleosome-positioning sequence by high-salt dialysis with
purified chicken erythrocyte nucleosomes (10).
Reconstitution of mononucleosomes onto a GAL4 site DNA was accomplished
by octamer transfer with H1-depleted HeLa oligonucleosomes purified and
used as described previously (8). Histone cores were
cross-linked with dimethyl suberimidate as described previously (31) during reconstitution in the DNase I nuclease
sensitivity experiments. In these experiments, the nucleosomes (25 nM)
were incubated in the presence or absence of 15 nM SWI/SNF and/or
100 nM GAL4-AH dimers and analyzed by DNase I digestion (7).
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RESULTS |
Interactions of SWI/SNF with linear and closed-circular
DNA.
Images of SWI/SNF complexes with DNA were obtained by
ESI. At a ratio of 2 ng of protein/µl to 5 ng of linear 1.4-kbp
DNA/µl, we observed that 87 of 387 (22%) DNA molecules had a single
SWI/SNF complex bound in the presence of ATP. The criteria for
identifying a SWI/SNF complex were a molecular mass estimate of 1.5 to 2.0 MDa and the appearance of subunits within the complex. In the absence of ATP, the results were similar: 45 of 292 (15%) DNA molecules had interacted with a SWI/SNF complex. With a higher input ratio of protein to DNA, 4 ng of protein/µl to 5 ng of
DNA/µl, 139 of 226 (62%) DNA molecules were complexed with a single
SWI/SNF in the presence of ATP and 61 of 142 (43%) were complexed
in the absence of ATP. Because SWI/SNF forms complexes with DNA in
the absence of ATP and because ATP produces less than a 1.5-fold
increase in the frequency of such complexes, we conclude that
SWI/SNF binds DNA by an ATP-independent mechanism.
Images of unstained and unshadowed SWI/SNF-linear DNA complexes and
SWI/SNF-relaxed closed-circular DNA complexes are shown
in Fig.
1. There are a few features that should
be noted. Though
it is difficult to represent the full dynamic range of
optical
densities contributed by the low mass density of the DNA and
the
high mass density of the SWI/SNF complex in the same
micrograph,
the subunit nature of the SWI/SNF complex is apparent
in most
of the examples shown here: it is more obvious in Fig.
1e and
f
but less obvious in Fig.
1c. Lower-contrast and magnified images
of
some complexes are shown as insets. A second feature is that
the
SWI/SNF complex is able to create a loop in the DNA, the size
of
the loop being variable even on the same DNA fragment. The
variable
size of the loop indicates that there is not a stringent
sequence
dependence for the DNA contact sites or that the specific
sequence of
the contact site occurs frequently. At an input ratio
of 4 ng of
SWI/SNF/µl to 5 ng of DNA/µl in the presence of ATP,
90 of 177 SWI/SNF-DNA complexes, or 65%, had an obvious loop created
by the
SWI/SNF complex. In the absence of ATP, 109 of 225 SWI/SNF-DNA
complexes, or 48%, displayed an obvious loop. Thus, ATP does not
significantly enhance the formation of DNA loops. The fraction
of
complexes in which a loop is formed may be greater than indicated
by
these values. Though a loop is not always seen extending from
the
SWI/SNF complex, contour length measurements (data not shown)
indicate that some complexes contain compacted DNA that could
represent
small loops superimposed on the complexes. We argue
that the loops
visualized are created by the SWI/SNF complex and
are not the
consequence of SWI/SNF binding to a loop created by
the DNA
spreading on the support film. The reason is that, in
control
experiments, a loop observed in a linear DNA fragment
of 1 kbp occurs
only in at most 2% of the molecules under the
spreading conditions
used in these experiments. With SWI/SNF,
however, the frequency of
loops is at least 25-fold higher. Furthermore,
contour length
measurements indicate that SWI/SNF causes a linear
compaction of
the DNA. Such a compaction would not be observed
if SWI/SNF
complexes were simply binding to intersections of the
DNA strands on
the support film. (In control experiments, the
SDs of the contour
lengths of 1-kbp fragments are between 1 and
2% [data not shown]).
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FIG. 1.
Electron spectroscopic images of SWI/SNF complexes
on linear plasmid DNA fragments (a to f) and relaxed closed circular
DNA (g to j). (a to c and f) A 1.0-kb fragment from pBluescript; (d and
e) 1.4-kb fragment containing promoter sequences of the human
proenkephalin gene. The insets in panels a, d, and f are magnified
twofold and reproduced at lower contrast to emphasize the subunit
structure of SWI/SNF complexes. Panels i and j are magnified
regions from panel h. Bar, 53 (a to f) and 160 (g and h) nm.
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Because of the high mass density of the SWI/SNF complex relative to
that of the DNA, it was generally not possible to form
net phosphorus
maps of the DNA-protein complexes, because the
dynamic range of the
film on which the images were collected was
insufficient. In some
cases, however, depending on (i) the exposure,
(ii) the support film
thickness, and (iii) the projection thickness
of the SWI/SNF
complex, we were able to obtain net phosphorus
images. Some examples
are shown in Fig.
2. Because of the high
phosphorus content in DNA relative to that of the protein, the
net
phosphorus overlay reveals the DNA path in the complex if
the
phosphorus signal-to-noise ratio is sufficient. The noise
in the
phosphorus maps is sometimes a source of confusion in tracing
the DNA
path. Contour length measurements, however, by providing
another
parameter, can be used in conjunction with the phosphorus
maps to
delineate the most likely DNA path. The contours shown
in the insets of
Fig.
2 are the most likely, based on the phosphorus
maps and the length
measurements. The contours shown measure 1,044,
988, 1,002, and 1,009 bp, respectively. In Fig.
2c, for example,
the noise along the left
side of the complex is judged not to
result from DNA because of the
slightly lower phosphorus signal
along that surface compared to the
signal elsewhere along the
DNA together with the fact that the length
of the displayed contour
is in agreement with the known length of the
DNA fragment (1 kbp).
More DNA on this left-hand surface would be
incompatible with
the contour length.
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FIG. 2.
Electron spectroscopic images of SWI/SNF-DNA
complexes. A mass-sensitive image is shown in grey levels, on which is
superimposed the net phosphorus image in magenta. The short and long
arrows indicate small and large loops, respectively, that have been
created by SWI/SNF-DNA contacts. The insets reveal the most
likely path of the DNA through or around the SWI/SNF-DNA
complex, based on the phosphorus map, the contour length, and the
topological consistency. Bar, 35 nm.
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We observe that SWI/SNF has multiple sites which make contact with
the DNA and which create small or large loops from the
intervening DNA.
From images of 23 complexes, we measured the
contour length of DNA that
appears to be in contact with the surface
of a SWI/SNF complex. The
average value was 133 ± 40 (SD) bp,
with a range from 71 to 212 bp. The measurements were made from
the point where DNA enters the
complex to where it exits at a
loop or at the final exit site. These
contact length measurements
are subject to underestimation because
the two-dimensional projection
length measured is equal to or
less than the true length in three
dimensions. The measurements,
however, can also be overestimated
because some of the DNA that
overlaps with the SWI/SNF complex
in projection may not actually be
in contact with it in solution
but deposited over it on the
two-dimensional substrate used for
EM imaging. Therefore, these values
must be regarded as only estimates
of DNA contact lengths. These
limitations notwithstanding, the
average length of DNA in contact with
SWI/SNF is in agreement
with the minimum DNA fragment length of 130 bp that is required
for a stable SWI/SNF-DNA interaction
(
24).
The data presented above are a direct visualization of the SWI/SNF
complex with DNA. Importantly, the large fraction of SWI/SNF-DNA
complexes that generate DNA loops indicates that the complex has
at
least two high-affinity binding sites which can interact with
different
DNA sequences separated by long distances. The fact
that the
SWI/SNF-DNA interactions observed here are ATP independent
is
consistent with biochemical studies showing that the binding
of
SWI/SNF to DNA (
24) and to nucleosomes (
9) is
ATP independent.
The observed simultaneous interactions of SWI/SNF
with more than
one DNA site may provide new insights into how the
complex can
perturb multiple nucleosomes within an array (
18,
21). To
determine whether the SWI/SNF complex can also
simultaneously
interact with multiple nucleosomes, we examined the
interactions
of the SWI/SNF complex with nucleosome
arrays.
Interactions of SWI/SNF with polynucleosomal DNA.
The
SWI/SNF complex has been shown to utilize the energy of ATP
hydrolysis to alter nucleosome structure, leading to an increased affinity of transcription factors (see the introduction). Very little,
however, is known about the molecular mechanisms of this function. To
obtain structural information on chromatin bound by SWI/SNF, we
imaged nucleosomes assembled onto a 12-mer repeat of the sea urchin 5S
rRNA gene sequence, a sequence with a well-characterized ability to
position a nucleosome (25). A histone-DNA ratio was chosen
so that the strands would be nearly saturated with nucleosomes. An
example of a polynucleosomal strand after reconstitution (and incubation in ATP) is shown in Fig. 3a
and b. Generally, the spacing of the nucleosomes was uniform, though on
this strand, the sixth and seventh nucleosomes from the right end are
unevenly spaced. Either the seventh nucleosome has "slid" to
contact the sixth, or it has bound in a minor positioning frame.
Initially, we did not observe differences between the numbers of
nucleosomes in the arrays incubated in the presence of ATP and those in
the arrays incubated in the presence of ATP-S (a nonhydrolyzable
form of ATP, not usable by SWI/SNF in other assays). In a control
experiment, the number of nucleosomes per strand was 8.7 ± 2.6 (n = 129) in the presence of ATP and 9.2 ± 2.0 (n = 107) in the presence of ATP-S. From
these numbers we concluded that ATP itself does not affect the
structure or stability of the nucleosomes on this 12-mer repeat DNA
sequence. The binding of the SWI/SNF complex to the polynucleosomal
array is illustrated in Fig. 3c. Reminiscent of its interaction with
naked DNA templates (Fig. 3a and b), the SWI/SNF complex is clearly
able to make contacts with distant sites on the polynucleosome strand,
creating an intervening loop of nucleosomal DNA. The binding of the
SWI/SNF complex to polynucleosome arrays did not appear to be
significantly affected by the presence of ATP relative to its binding
in the presence of ATP-S. Strands with evenly distributed
nucleosome-like particles and with one SWI/SNF complex attached
could be found in the presence of either ATP or ATP-S.
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FIG. 3.
Electron spectroscopic images of a polynucleosome strand
in the absence of SWI/SNF (a and b) and a polynucleosome
strand with one SWI/SNF complex (c). The left and right halves of
the same strand are shown in panels a and b, respectively. The
polynucleosome arrays on a 12-mer repeat of the 208-bp sea urchin 5S
rRNA gene nucleosome-positioning sequence (25) were formed
by salt dialysis with histones purified from chicken erythrocytes
(10). The final buffer contained 25 mM HEPES. All of the
strands shown were subjected to incubation in buffer containing
ATP. The images were recorded at an energy loss of 155 eV. Bar, 24 nm.
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ESI analysis did reveal effects that SWI/SNF has on the
polynucleosome strands, effects which are dependent on a hydrolyzable
form of ATP. The effect of hydrolyzable ATP became apparent when
ESI
was used to measure the mass and the phosphorus content of
the
nucleosomes on the strands (
1,
3). Although a superficial
examination indicated that a SWI/SNF complex had bound to an array
of normal nucleosomes, the phosphorus and mass analyses of these
nucleosomes indicated otherwise (Fig.
4).
With the measurements
of total mass and total phosphorus content, the
protein and DNA
stoichiometry can be determined. The average protein
and DNA contents
of particles on strands with one SWI/SNF complex
in the presence
of ATP were 111 ± 45 kDa and 118 ± 43 bp,
respectively (
n = 59).
Whereas the average protein
content is consistent with that of
canonical nucleosomes, the DNA
content was significantly less
than that expected for nucleosomes.
Scatter plots of protein versus
DNA (Fig.
5B) show a large degree of heterogeneity
in protein
and DNA stoichiometry. The average DNA content of 118 bp is
a
value well below that expected for a canonical nucleosome. In
contrast, the average protein and DNA contents of nucleosomes
on
strands complexed with SWI/SNF in the presence of ATP-
S were
100 ± 9 kDa and 151 ± 21 bp, respectively (
n = 42). The scatter
plots show that there is far less heterogeneity
in protein and
DNA stoichiometry when ATP-
S is used in place of
hydrolyzable
ATP (Fig.
5A). In a mock reaction (minus SWI/SNF) in
the presence
of ATP, polynucleosomes also have normal stoichiometry,
containing
on average 112 ± 22 kDa of protein and 157 ± 34 bp of DNA (
n =
40) (Fig.
5C). From this stoichiometric
analysis, we conclude
that SWI/SNF has the ability to disrupt
nucleosome structure in
an ATP-dependent manner, resulting in a
reduction in and increased
heterogeneity of the extent of DNA
interacting with each histone
octamer.
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FIG. 4.
Electron spectroscopic images of
SWI/SNF-polynucleosome strands with histones not chemically
cross-linked (a) and with cross-linked histone octamers (b). The mass
and phosphorus contents of SWI/SNF-DNA and nucleosome complexes
were determined as described previously (3). (a) Protein and
DNA contents of the nucleosomes in the numbered boxes are presented in
the text. The colored insets show the net phosphorus distributions of
the indicated nucleosomes. Bar, 24 nm (insets, 12 nm). (b) Nucleosomes
indicated by arrows in the low-magnification views on the left are
shown on the right at higher magnification, with the phosphorus
represented as red. The inset in the upper-left panel is included to
clarify that a protein-free DNA writhe lies just to the right of the
nucleosome at the left of the field. Bar, 11 (left) and 5.5 (right)
nm.
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FIG. 5.
(A and B) Scatter plots of protein content versus DNA
content of particles on polynucleosome strands bound by one SWI/SNF
complex. Reactions were carried out in the presence of 2 mM ATP-S
(A) or 2 mM ATP (B). (C) Values for particles in polynucleosome strands
exposed to ATP but not SWI/SNF in a mock reaction. The mean protein
and DNA contents (± SD) are 100 ± 9 kDa and 151 ± 21 bp
(n = 42) (A), 111 ± 45 kDa and 118 ± 43 bp
(n = 59) (B), and 112 ± 22 kDa and 157 ± 34 bp (n = 40) (C). (D to F) Scatter plots of nucleosome
arrays reconstituted with cross-linked histones. Nucleosomes within a
loop created by a SWI/SNF complex (D), nucleosomes outside a loop
or on strands not bound by SWI/SNF (E), and nucleosomes on arrays
not exposed to SWI/SNF (mock-reacted with ATP) (F) were used to
obtain the values for these plots. The average amounts of protein and
DNA are 101 ± 26 kDa and 101 ± 17 bp (D), 101 ± 24 kDa and 166 ± 27 bp (E), and 102 ± 15 kDa and 155 ± 27 bp (F).
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The protein-DNA stoichiometric disruption was often reflected in a
disruption in morphology, which was particularly apparent
in the
phosphorus distribution maps. In Fig.
4a, for example,
particles 1 (80 kDa; 152 bp) and 2 (120 kDa; 138 bp) have near-normal
levels of protein
and DNA. Particle 1 has the normal en face morphology
commonly seen in
ESI images of nucleosomes, where the phosphorus
(inset) is concentrated
on the particle's periphery. In contrast,
particle 3 (104 kDa; 85 bp)
has a higher-than-average protein/DNA
ratio, probably reflecting the
loss of a turn of DNA. The inset
for particle 3, showing the phosphorus
(or DNA) distribution,
also indicates that this nucleosome has a
disrupted morphology.
The protein content of particle 4 (80 kDa; 170 bp), located outside
the loop created by the SWI/SNF complex, is
close to the canonical
value, and there has been no loss of DNA in the
particle. A value
greater than 146 bp of DNA may indicate that linker
DNA is superimposed
on the particle in the
projection.
The altered morphology of SWI/SNF-remodeled nucleosomes is further
illustrated in Fig.
4b. The DNA distribution, based on
phosphorus
imaging of the nucleosome in the SWI/SNF-induced loop
(upper-left
panel), does not have the characteristic "doughnut"
profile typical
of ESI images of nucleosomes, such as the nucleosome
outside of the
loop (see the high-magnification phosphorus maps
of both nucleosomes in
the right-hand panels). (Because of the
low contrast of the DNA in the
image reproduction, the inset was
used for clarification, showing that
a protein-free writhe in
the DNA is present just to the upper right of
the nucleosome.)
This nucleosome in the SWI/SNF loop has a protein
mass of 107
kDa and a DNA content of 107 bp, with a protein/DNA mass
ratio
of 1.53. The nucleosome in the SWI/SNF-induced loop in the
lower-left
panel appears less disrupted than its counterpart in the
upper-left
panel. It does, however, show a larger amount of phosphorus
on
its left than on its right side. Its protein content is normal
(114 kDa), but it has a reduced amount of DNA (100 bp) compared
to that of a
canonical nucleosome. The nucleosome outside the
loop is located at the
end of the polynucleosome strand. Its phosphorus
profile is similar to
that of its counterpart in the panel above,
typical of en face views of
intact nucleosomes in arrays visualized
by ESI (
3,
20).
The analytical microscope emphasizes important features of nucleosome
disruption by SWI/SNF. First, structures within loops
created by
SWI/SNF may appear to be nucleosomes, based on a superficial
examination. Comparisons of mass- and phosphorus-sensitive images
indicate, however, that only some structures have the characteristics
of canonical nucleosomes while other structures have abnormally
low
amounts of phosphorus compared to those expected for canonical
nucleosomes. Earlier in this paper we reported that polynucleosome
arrays complexed with one SWI/SNF were observed with both ATP-
S
and ATP. Subsequent comparisons of mass and phosphorus contents,
however, indicate that not all of the structures initially scored
as
nucleosomes were actually canonical nucleosomes. SWI/SNF together
with ATP, but not ATP-
S, does indeed affect the number of normal
or
intact nucleosomes on an array. Second, only some nucleosomes
within
loops are disrupted (e.g., Fig.
4a, particle 3) while some
appear intact (Fig.
4a, particles 1 and 2). Based on the mean
values of
protein and DNA content (Fig.
5B), we conclude that
SWI/SNF is able
to remove DNA from the histone octamer. The heterogeneity
in the
protein content, however, indicates that protein loss can
also occur,
perhaps as a consequence of reduced DNA binding. An
H2A-H2B dimer, for
example, may be lost along with the disruption
of DNA from the histone
core on some particles. Particles with
higher-than-average protein
content may arise from atypical associations
with additional histones
liberated from other disrupted
nucleosomes.
SWI/SNF disruption involves loss of histone-DNA contacts but
does not require loss of histones.
The images and stoichiometric
analysis by ESI (Fig. 4 and 5) indicate that ATP-dependent
disruption of multiple nucleosomes in an array by SWI/SNF favors a
loss of DNA over a loss of histones from the particles. Thus,
disruption of nucleosomes by SWI/SNF might only require alteration
of histone-DNA contacts, resulting in loss of the DNA, perhaps by
unwinding, without the loss of histones from the nucleosome core. To
test the possibility that SWI/SNF disrupts nucleosomes without
requiring the loss of histones from the octamer core, we analyzed
SWI/SNF disruption of nucleosomes biochemically, using nucleosome
cores which contained covalently cross-linked histone octamers.
Histone-histone cross-linking was performed with dimethyl
suberimidate (31). This converts the histone octamer into a
single 100-kDa complex (Fig. 6A), which retains the ability to reconstitute DNA into nucleosome cores (Fig. 6B)
(reference 31 and references therein). Note that, although the same amount of protein was loaded, the cross-linked octamer is poorly contrasted because it takes up less silver than the
control histones. The individual core histones are resolved in the
control octamer lane but are not present in the cross-linked octamer
lane. Though the silver staining suggests that the core histones are
not present in equal stoichiometric amounts, the Coomassie stain (not
shown) confirmed that they are present at equal levels.
View larger version (88K):
[in this window]
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|
FIG. 6.
Histone octamer cross-linking does not affect
nucleosomal DNA perturbation and enhanced GAL4-AH binding by
SWI/SNF. Histone cross-linking conditions with dimethyl
suberimidate and nucleosome reconstitution by transfer were described
previously (31). (A) Sodium dodecyl sulfate-18%
polyacrylamide gel showing that cross-linking conditions with dimethyl
suberimidate create a protein band at around 100 kDa (arrowhead shows
migration of 97.5-kDa marker) with no detectable free histone or
partially cross-linked product (50 ng of histone loaded; 172-bp 5S DNA
probe [21] used in the reconstitution). Note that the
cross-linked histones take up less stain than control histones. The
band that migrates more slowly than the core histones corresponds to
bovine serum albumin, used to maintain stability. (B) Mobility shift
gel showing that the cross-linked octamers do not significantly change
the migration of the 5S mononucleosome. (C) Nucleosomes (25 nM) from
the preparation used for panel B were incubated with 1 mM ATP in the
presence (+) or absence ( ) of purified SWI/SNF (2.5 nM) and
analyzed by DNase I digestion. The bullets show where the intensity of
a band is modified by SWI/SNF action. (D) A single-GAL4-site DNA
probe (7) was reconstituted in nucleosome cores by using
cross-linked (lanes 2 to 6) or non-cross-linked (lanes 7 to 11) histone
octamers. The nucleosomes (25 nM) were incubated in the presence (+) or
absence () of 15 nM SWI/SNF and/or 100 nM GAL4-AH dimers as
indicated and were analyzed by DNase I digestion. Mg-ATP (1 mM) is also
present in all of the lanes. The bracket indicates the GAL4-AH binding
site. XLed and X-linked, cross-linked; unXLed and unX-linked, not
cross-linked.
|
|
Treatment of the cross-linked nucleosomes with the SWI/SNF complex
in the presence of ATP led to a disruption of the nucleosomal
DNase I
digestion pattern that is indistinguishable from the disruption
observed with the control nucleosomes, which were not cross-linked
(Fig.
6C, compare lane 1 to lane 3, and Fig.
6D, compare lanes
2 and 4 to lanes 7 and 9). The effect of SWI/SNF on the mononucleosome
is
less in Fig.
6C than in 6D, because the molar ratio of SWI/SNF
to
mononucleosomes was 10-fold higher in the reaction represented
in 6D.
Furthermore, we observe that the SWI/SNF complex is also
able to
stimulate the binding of GAL4-AH to cross-linked nucleosomes
with the
same efficiency as to control nucleosomes (Fig.
6D, compare
lanes 3 and
5, and 8 and 10). Thus, the consequences of SWI/SNF
action on the
nucleosomes composed of cross-linked histones and
on the control
nucleosome cores were indistinguishable by the
DNase I protection
assay. This assay of mononucleosomes fully
supports the conclusions
from the ESI data on nucleosomal arrays
that showed that SWI/SNF
disrupts nucleosomes by targeting histone-DNA
contacts without
requiring the eviction of histones from the core
particle.
To provide structural information to help interpret the DNase digestion
studies of cross-linked histone nucleosomes, we used
ESI to examine
arrays of nucleosomes reconstituted with cross-linked
octamers.
Nucleosomes reconstituted into arrays with cross-linked
octamers were
morphologically indistinguishable from control nucleosomes
(images not
shown; compare Fig.
5F). Quantitative analysis revealed
that
nucleosomes within loops created by SWI/SNF had a protein
content
equivalent to that of canonical nucleosomes (101 ± 26
[SD] kDa)
but had lost a significant amount of DNA (101 ± 17 bp);
the
protein/DNA mass ratio was 1.61 ± 0.36 (Fig.
5D). Cross-linked
nucleosomes outside a SWI/SNF-induced loop or on strands not
occupied
by a SWI/SNF complex had near-normal levels of protein and
DNA
(101 ± 24 kDa of protein and 166 ± 27 bp of DNA;
protein/DNA mass
ratio, 0.95 ± 0.26) (Fig.
5E). Similarly,
nucleosomes on arrays
not exposed to SWI/SNF but mock-reacted in
the presence of ATP
were canonical on the basis of their protein and
DNA contents
(102 ± 15 kDa of protein and 155 ± 27 bp of
DNA; protein/DNA mass
ratio, 1.02 ± 0.24) (Fig.
5F). In contrast
to nucleosomes that
were not cross-linked, the cross-linked nucleosomes
were more
homogeneous in protein content in the presence of SWI/SNF
and
ATP (compare Fig.
5B and D). Thus, the cross-linked nucleosomes
underwent only loss of DNA without substantial protein redistribution,
yet they displayed the biochemical hallmarks of the
SWI/SNF-remodeled
nucleosomes (i.e., altered DNase digestion
patterns and increased
factor binding [Fig.
6]). This indicates that
protein redistribution
is a secondary consequence of nucleosome
disruption which is not
required for enhancement of transcription
factor binding. Protein
redistribution, however, may be a requirement
in other aspects
of transcriptional activation or
elongation.
| |
DISCUSSION |
The structural studies presented here extend the previous
biochemical analysis in two important respects. (i) While the
SWI/SNF complex has been shown to perturb the DNase I digestion
pattern of nucleosomal DNA (7, 12, 17), this effect could
easily result from either the loss of histone proteins or the unwinding of nucleosomal DNA. Our stoichiometric measurements and DNase I
protection analysis of cross-linked histone octamers both demonstrate that it is DNA that is generally lost from the nucleosome,
supporting a mechanism where SWI/SNF peels DNA off the histone
octamer surface. (ii) While previous biochemical studies have shown
that the SWI/SNF complex can perturb multiple nucleosomes within an
array (18, 21), it was unclear whether this would occur
sequentially, perhaps requiring multiple SWI/SNF complexes per
polynucleosome array. The structural analysis presented here
illustrates clearly that one SWI/SNF complex can lead to the
disruption of multiple nucleosomes in a polynucleosome array. This
multinucleosome disruption is also in agreement with our previous
report, in which we showed that nucleosome arrays can be disrupted
efficiently at very low SWI/SNF ratios (18).
These data argue that the action of the SWI/SNF complex on an array
of nucleosomes can lead to a region of nucleosome disruption. We think
that the domains in which nucleosomes appear disrupted result from an
interaction with only the SWI/SNF complex that is visualized and
not with the visualized complex and another complex that had previously
interacted and then moved on. One reason for this interpretation is
that the input ratio of SWI/SNF to DNA is quite low, so that only
about one-third of the nucleosome strands have one SWI/SNF complex
associated and strands with more than one SWI/SNF complex are
extremely rare (
1%). Secondly, cross-linked nucleosomes
outside of SWI/SNF loops and on strands that are not occupied by SWI/SNF are not, on average, disrupted, based on mass and phosphorus analyses. Nevertheless, though it is unlikely, we cannot
definitively rule out the possibility that disruption of nucleosomes
around a bound SWI/SNF could have involved another SWI/SNF
molecule in a "hit-and-run" mechanism. However, we favor a
mechanism whereby a loop domain is created by SWI/SNF, followed by
a sequential disruption of nucleosomes within that domain, probably by
direct contacts with SWI/SNF by linear diffusion, while the domain
is maintained in a topologically constrained state. Evidence for a
topologically constrained region is frequently observed in
SWI/SNF-nucleosome complexes formed in the presence of ATP, where
the DNA in the loops is interwound with itself. Such interwound DNA is
rarely seen when ATP-S is used instead (data not shown). Indeed, the
fact that the SWI/SNF complex can interact with multiple DNA sites,
generating a loop between them, raises the intriguing possibility that
the SWI/SNF complex might be able to simultaneously disrupt
nucleosomes at distal regulatory elements, i.e., enhancers and promoters.
| |
ACKNOWLEDGMENTS |
This work was supported by research grants from the Natural
Sciences and Engineering Research Council of Canada to D.P.B.-J., from
the Medical Research Council of Canada to J.C., and from the NIH-NIGMS
to C.L.P. and J.L.W. J.C. was a Centennial Fellow of the Medical
Research Council of Canada, and C.L.P. is a Scholar of the Leukemia
Society of America. J.L.W. is an Associate Investigator of the Howard
Hughes Medical Institute.
We thank Michael J. Hendzel for comments on the manuscript and Manfred
Herfort for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology and Anatomy, University of Calgary, Calgary, AB, Canada T2N 4N1. Phone: (403) 220-3025. Fax: (403) 270-0737. E-mail:
bazett{at}acs.ucalgary.ca.
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Molecular and Cellular Biology, February 1999, p. 1470-1478, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
