From the Program in Molecular Biology, Microbiology,
and Molecular Biology and Department of Biochemistry and Molecular
Biology, Southern Illinois University School of Medicine, Carbondale,
Illinois 62901-4413, the § Program in Molecular Medicine
and Department of Biochemistry and Molecular Biology, University of
Massachusetts Medical School Worcester, Massachusetts 01605, and the
Huntsman Cancer Institute and Department of Oncological
Sciences, University of Utah School of Medicine, Salt Lake City, Utah
84112
Received for publication, November 20, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Interactions of the yeast chromatin-remodeling
complexes SWI/SNF and RSC with nucleosomes were probed using
site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with
photoreactive nucleotides incorporated at different sites in DNA to
scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The
Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently
cross-linked at several positions in the nucleosome, whereas only Snf6
was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA
photoaffinity labeling of RSC showed that the Rsc4 subunit is in close
proximity to nucleosomal DNA and not when RSC is bound to free DNA.
After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer
detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.
An important element of eukaryotic gene regulation is the dynamic
nature of chromatin because of the ability of various protein complexes
to alter the structure of chromatin. Chromatin is altered by chemical
modification of the core histone proteins through acetylation,
phosphorylation, methylation, and ubiquitination. Acetylation of the
N-terminal tails has been shown to play a key role in the regulation of
gene expression (1-4). Another approach to alter chromatin structure
uses the mechanical energy provided by hydrolysis of the The first of these complexes to be extensively purified and
characterized was the SWI/SNF complex from Saccharomyces
cerevisiae (13, 16). The yeast SWI/SNF complex has 11 different
subunits and a total molecular mass of ~2 MDa. The largest subunit of
this complex, Swi2/Snf2, is a 194-kDa protein containing seven
highly conserved subdomains corresponding to the
DNA-dependent ATPase domain and a bromodomain located
toward the C terminus. Single amino acid changes in the
DNA-dependent ATPase domain eliminate both the ATPase
activity of Swi2/Snf2 and the chromatin-remodeling activity of
SWI/SNF (13, 17). The next largest subunit of SWI/SNF, Swi1 (148 kDa),
had been shown to interact with DNA by UV cross-linking of SWI/SNF
bound to naked DNA and contains an AT-rich interactive domain
DNA-binding domain that may be involved in SWI/SNF binding of DNA
(18). The RSC complex is another ATP-dependent chromatin-remodeling complex found in yeast that is closely related to
SWI/SNF. The Sth1 subunit of RSC is 72% identical over 661 amino acid
residues of Swi2/Snf2 (11). Three subunits of SWI/SNF, Swi3,
Snf5, and Swp73, are also paralogs of RSC components; similar subunits
are also found in the human and Drosophila SWI/SNF complexes (14). Two subunits of SWI/SNF are shared with the RSC complex and are
the actin-related proteins Arp7 and Arp9 (19, 20). The ability of Arp7
and Arp9 to bind or hydrolyze ATP appears not to be important for their
role in chromatin remodeling (20).
Direct correlation of the ATPase activity of the Swi2/Snf2
subunit with the chromatin-remodeling activity of SWI/SNF through mutational analysis indicates that the Swi2/Snf2 subunit is the catalytic core of SWI/SNF. These observations are also reflected in the
mutational analysis of Swi2/Snf2 homologs in other SWI/SNF-like complexes. Purified recombinant Swi2-like proteins, human BRG1 and
hBRM, and the Drosophila ISWI proteins possess some or
essentially complete chromatin-remodeling activity characteristic of
their respective intact multi-subunit complexes (9, 21-23). Although the mechanical energy for chromatin remodeling is provided through the
ATP hydrolysis of Swi2/Snf2 or its homologs, there is no data showing the direct interaction of the Swi2/Snf2 subunit with the nucleosome or DNA. The ATPase activity of SWI/SNF and RSC is equally stimulated by naked DNA or nucleosomes. UV cross-linking of SWI/SNF bound to naked DNA did not show any Swi2/Snf2 cross-linking but instead cross-linked a protein with an electrophoretic mobility corresponding to the Swi1 subunit with lesser amounts of two proteins, p68 and p78 (18). Under these conditions Swi2/Snf2 does not appear to make extensive contact with DNA as evident by its inability to be cross-linked to DNA. Indirect evidence suggests that the Swi2/Snf2 subunit may be able to interact with the N-terminal tails of histones H3 and H4. A bromodomain, similar to that found in
the C terminus of Swi2/Snf2, has been shown in vitro
to bind selectively to the tails of H3 and H4 but not to the tails of H2A and H2B (24).
Using a photochemical approach with site-specific modified
photoreactive DNAs, we have investigated the interactions of yeast SWI/SNF with the nucleosome and present evidence for the direct interaction of Swi2/Snf2 in the SWI/SNF complex and Rsc4 in the RSC complex with DNA in a nucleosome-dependent manner. The
5 S rDNA template was chosen for these studies, because it has been well characterized to help phase the nucleosome on DNA and to cause
tight rotational positioning of the nucleosome (25, 26). Although
rotationally positioned, the 5 S rDNA nucleosome has more than one
translational position (25, 27, 28). We have taken advantage of the
several translational positions of the 5 S rDNA to scan for the
interactions of SWI/SNF with nucleosomal DNA and to compare that with
its interactions with naked DNA. Another important outcome of our
results is the data showing significant changes in the interface
between the nucleosome and the chromatin-remodeling complex after
hydrolysis of ATP, which is indicative of key conformational changes of
SWI/SNF or changes in the sites of DNA bound in the nucleosome.
Purification of SWI/SNF Complex--
The SWI/SNF complex was
purified from S. cerevisiae strain CY396 containing
HA1-tagged and
His6-tagged Swi2. The purification involved
nickel-nitrilotriacetic acid agarose, DNA cellulose, MonoQ column
chromatography, and glycerol gradient ultracentrifugation as described
previously (29). The concentration of the SWI/SNF was determined by
quantitative Western blot analysis using a polyclonal anti-Snf5 or
anti-Swp73 antibody.
DNA Probe Synthesis--
A 214-bp
EcoRI-DdeI fragment of DNA derived from plasmid
pXP-10 (30), which includes the Xenopus borealis somatic 5 S
rRNA gene, was used to construct DNA photoaffinity probes. Each probe had photoreactive nucleotide analogs AB-dUMP
(5-[N-(4-azidobenzoyl)-3-aminoallyl]dUMP) and/or AB-dCMP
(4-[N-(p-azidobenzoyl)-2-aminoethyl]dCMP)
incorporated adjacent to radiolabeled nucleotides at specific sites in
the DNA (31). Four DNA probes, designated as 47/55, 1/5, Reconstitution of Nucleosomes--
Nucleosomes were
reconstituted by transferring histone octamers from HeLa
oligonucleosomes onto probe DNA by the octamer transfer method as
described previously (26). H1-depleted oligonucleosomes from HeLa cells
were prepared as described previously. Mock nucleosomal/naked DNA was
prepared by carrying out octamer transfer in the absence of probe DNA.
Probe DNA was added after the final step of octamer transfer.
Photoaffinity Labeling--
A binding reaction was set up
containing ~30-40 nM SWI/SNF and 30 nM
nucleosomes in buffer A (20 mM Na-HEPES, pH 7.8, 100 mM NaCl, 3 mM MgCl2, 0.1% Nonidet
P-40, 5% glycerol, 2 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 0.01% Tween 20, and 0.2 mg/ml bovine serum albumin). The reaction was incubated at
30 °C for 30 min. ATP, ATP
For photoaffinity labeling with RSC, the binding reactions contained 20 nM RSC and were in buffer B (20 mM K-HEPES, pH
7.8, 4 mM MgCl2, 30 mM potassium
acetate, and 75 µg/ml bovine serum albumin). ATP was included
optionally in the binding reaction at a final concentration of 500 µM. The reaction was incubated at 30 °C for 30 min as before.
After the incubation, 4 µl of the binding reaction was loaded on a
4% native polyacrylamide gel in 0.5× TBE at 4 °C
(acrylamide:bisacrylamide of 38.9:1.1). Alternatively, a 5 or 6%
native polyacrylamide gel (acrylamide: bisacrylamide of 60:1) in 0.2×
TBE was used to resolve the different translational positions of the
nucleosome. Of the RSC binding reaction, 4 µl was loaded on a 3.2%
native polyacrylamide gel in TE buffer (10 mM Tris-HCl, pH
7.5, and 1 mM EDTA).
The remainder of the sample was irradiated with ultraviolet light as
described previously (31). The samples were treated with DNase I and S1
nuclease and analyzed on a 4-20% SDS-polyacrylamide gel. The dried
gel was analyzed using a Cyclone phosphorimaging device from
Packard Instruments.
Western Blot Analysis--
SWI/SNF was separated on a 4-20%
SDS-polyacrylamide gel. After electroblotting onto a nitrocellulose
membrane and the blocking of membrane, several antibodies were used to
probe the membrane (32). Antibodies to Swi3, Swp82, Swp73, Arp7, Arp9,
Snf6, and Tfg3/Taf30 were provided by Bradley Cairns, and antibodies to Swi1 and Snf5 were provided by Craig Peterson. High affinity anti-HA antibody (Roche Molecular Biochemicals) was used for the detection of
the Swi2/Snf2 subunit.
Restriction Endonuclease Cleavage Assay--
A 50 µl-binding
reaction, as described for photoaffinity labeling, was incubated at
30 °C for 30 min. Eighty units of EcoRV were added to
each reaction followed by a 5-min incubation at 37 °C. The cleavage
reaction was stopped by the addition of 10 mM EDTA. The
aqueous layer was extracted first with a phenol/chloroform mixture
(1:1) and then with chloroform. The DNA was precipitated by the
addition of 0.1 volume of 10 M LiCl and 2.5 volumes of 100% ethanol. 10 µg of plasmid DNA was added as a carrier for the
precipitation. The DNA was resuspended in 10 µl of TE containing 0.05% Tween 20 and analyzed on a 9% native polyacrylamide gel.
Immunoprecipitation--
A 67-µl photoaffinity labeling
reaction was incubated at 30 °C for 30 min. Four microliters of the
sample was loaded on a 4% native polyacrylamide gel in 0.5× TBE. The
remainder of the sample was irradiated with ultraviolet light. After
irradiation, one-third of the reaction mixture was treated with DNase I
and S1 nuclease as before. The remaining two-thirds of the sample were
immunoprecipitated essentially as described previously (16). Protein
A-Sepharose 4B resin (40 µl, Sigma) was equilibrated with buffer C
(20 mM K-Hepes, pH 7.8, 10% glycerol, 12.5 mM
MgCl2, 0.1 mM EDTA, 0.2% Tween 20, 0.1 mM dithiothreitol, and 0.1 M potassium acetate). Anti-Swp73 antibody (7 µl) was bound to 20 µl of
equilibrated resin for 30 min at 4 °C with mixing at 1300 rpm every
2 min. Unbound antibody was removed by washing the resin with 500 µl of buffer C.
The sample was pre-cleared by adding it to 20 µl of equilibrated
resin. The supernatant was added to the antibody-bound protein A-Sepharose resin and incubated for 3 h at 4 °C with mixing as described earlier. The supernatant was removed, and the resin was
washed three times with 500 µl of buffer D (20 mM
K-Hepes, pH 7.8, 10% glycerol, 12.5 mM MgCl2,
0.1 mM EDTA, 0.2% Tween 20, 0.1 mM
dithiothreitol, and 0.6 M potassium acetate) and once with buffer A. The resin was resuspended in 20 µl of buffer A, and the
digestion of the DNA with DNase I and S1 nuclease was carried out as
described earlier. The samples were analyzed on a 4-20% SDS-polyacrylamide gel, and the dried gel was subjected to phosphorimaging.
The Swi2/Snf2 and Snf6 Subunits of Yeast SWI/SNF Are in
Close Proximity to DNA in the SWI/SNF-Nucleosome
Complex--
Site-specific DNA photoaffinity labeling was used to
understand how SWI/SNF interacts with the 5 S rDNA nucleosome before and after remodeling. Nucleosomes were reconstituted using a 214-bp fragment of DNA that included the X. borealis somatic 5 S
rRNA gene. Nucleosomes reconstituted with this DNA are highly
positioned rotationally and occupy several well defined translational
positions (25, 27, 28). Four major dyad axes have been mapped on this DNA to base pairs
To ascertain the effect of this kind of DNA modification on the ability
of SWI/SNF to remodel the modified nucleosome, a restriction endonuclease accessibility assay was used with the most modified DNA
probe, probe 47/55. Unmodified DNA was synthesized with a single
radioactive label incorporated at bp
A similar effect was seen with probe 47/55 (Fig. 1C). The
efficiency of nucleosome formation with probe 47/55 was better (95%) than that of the unmodified DNA (data not shown); therefore, only 6%
of the DNA was accessible to the enzyme in the absence of SWI/SNF (lane 2). At lower amounts of SWI/SNF, the modified DNA was
cleaved to a slightly higher extent (Fig. 1, B and
C, compare lanes 4, 6, and 8) than the
unmodified nucleosome. As with unmodified DNA, the extent of cleavage
increased as the amount of SWI/SNF added was increased until it reached
a molar ratio of 1:3, when almost 90% of the DNA was cleaved (Fig.
1C, lanes 9 and 10). These
results show that DNA modification does not interfere with the
interaction of SWI/SNF with the 5 S nucleosome.
The formation of discrete SWI/SNF·nucleosome complexes was detected
by gel shift assay with all four modified nucleosomes (Fig.
2A, lanes 3, 6, 9, and 12). The efficiency of nucleosome formation with all
four probe DNAs was comparable with between 87 and 93% of the DNA
being assembled as assayed by gel shift analysis. The amount of SWI/SNF
required for quantitative binding was empirically determined by
titrating SWI/SNF with a fixed amount of nucleosomal probe and assaying
by gel shift (data not shown). The addition of ATP to the
SWI/SNF·nucleosome complex caused a further reduction in the
electrophoretic mobility of the complex, resulting in the complex not
migrating into the gel (lanes 4, 7, 10, and 13).
The binding of SWI/SNF to free DNA also formed a complex that does not
migrate into the gel (lanes 5, 8, 11, and 14).
The electrophoretic properties of the SWI/SNF:DNA complex suggest that
several SWI/SNF molecules may be bound per DNA. The SWI/SNF·nucleosome complexes were photocross-linked at the four different regions in the nucleosome by irradiating the complexes and
then digesting away most of the cross-linked DNA with DNase I and S1
nuclease. The short labeled DNA fragment left covalently attached to
either histones or SWI/SNF serves as a radioactive tag to identify the
components of the complex cross-linked at the various sites on the 5 S
rDNA. DNA probe 47/55 most efficiently cross-linked histones and a
~205-kDa protein (Fig. 2B, lane 1). The
electrophoretic mobility of the 205-kDa protein was shown to be similar
to that of the Swi2/Snf2 protein by Western blotting analysis
with anti-HA antibody detection of the
Swi2/Snf2-HA-His6 (data not shown). Minor
cross-linking of several other proteins was also observed. These
polypeptides with apparent molecular masses of 127, 92, 76, 63, and 50 kDa were found by Western blot analysis to have electrophoretic
mobilities similar to that of Swi3/Snf5, Swp82, Swp73, Arp7/Arp9, and
Snf6, respectively.
Cross-linking of the 205-kDa polypeptide was dramatically reduced by
the addition of ATP (Fig. 2B, lane 2). These
results indicate that the Swi2/Snf2 subunit is located close to
nucleosomal DNA upon binding to the nucleosome but after chromatin
remodeling is displaced from the nucleosome so that it cannot be
cross-linked to DNA. In the experiment with cross-linking of SWI/SNF
bound to naked DNA, the Swi2/Snf2 subunit is not cross-linked at base pairs 47/55 in the 5 S rDNA (lane 3). Thus, these data
indicate that the association of Swi2/Snf2 near DNA is
nucleosome-dependent. Proteins with relative mobilities
similar to that of Swi1, Arp7 or Arp9, and Snf6 are most readily
cross-linked in the SWI/SNF bound to naked DNA in the absence of ATP.
Similarly, these same proteins are cross-linked when SWI/SNF is
associated with the nucleosome in the presence of ATP, with the
exception of the ~60- and ~63-kDa protein (Arp7 and Arp9). Other
positions on the 5 S rDNA, some near the ends of the DNA, were also
probed but did not efficiently cross-link SWI/SNF (data not shown).
The photoaffinity-labeled SWI/SNF complex was purified away from
labeled histones and other proteins by immunoprecipitation using an
anti-Swp73 antibody. Swp73 is a highly conserved subunit of SWI/SNF
that is tightly associated with the complex, thus making it possible to
immunoaffinity purify complete SWI/SNF complexes (33). SWI/SNF is
dissociated from the nucleosome under the salt concentrations used in
the immunoprecipitation. After extensive washing of the beads with
SWI/SNF still bound to the protein A-Sepharose, the sample is digested
with DNase I and S1 nuclease, and the protein is eluted by adding a
sample loading buffer. Six of the proteins cross-linked with probe
47/55 were immunoprecipitated and correspond by relative molecular mass
to the Swi2/Snf2, Swi3, Swp82, Swp73, Arp7/Arp9, and Snf6
subunits of the SWI/SNF complex (Fig. 2B, lane
4). Swi2/Snf2 was the most intensely cross-linked subunit, suggesting again that it makes close contact with nucleosomal DNA. The
161-, 103-, and 35-kDa polypeptides (compare lane 1 with lane 4, *) did not immunoprecipitate and are probably
contaminants present in SWI/SNF. The histone proteins were also removed
by immunoprecipitation and facilitated in identifying whether the Snf11
subunit of SWI/SNF was cross-linked.
Immunoprecipitation of the SWI/SNF·nucleosome complex after
remodeling showed that the interactions of SWI/SNF with the nucleosome were greatly altered (lane 5). The Swi2/Snf2 subunit
was no longer cross-linked efficiently, suggesting that it does not
make close contact with DNA at this position in the remodeled
nucleosome. The most efficiently cross-linked subunit of SWI/SNF after
remodeling is Snf6. Although much less efficiently cross-linked in the
initial complex, the contacts of the Swp82 and Arp7 or Arp9 subunits
were also lost upon the addition of ATP. On the other hand, the
contacts of Swp73 and Snf6 were increased, and in addition, Swi1, Snf5, and Tfg3/Taf30 were cross-linked to DNA, although weakly only after the
addition of ATP. These altered contacts of SWI/SNF with the nucleosome
after remodeling bear great similarity to the interaction of SWI/SNF
with naked DNA (compare lane 5 with lane 6).
To examine SWI/SNF contacts at other positions across the nucleosome,
several other probes were synthesized. Three probes, 1/5, Hydrolysis and Not Just Binding of ATP Is Required for Changes in
the Interface between SWI/SNF and the Nucleosome--
To ascertain if
the binding of ATP alone or hydrolysis of the Altered Conformation of the Nucleosome after Remodeling Is Detected
by Gel Shift Assay--
Because the cross-linking of SWI/SNF to the
nucleosome after the addition of ATP resembles that of SWI/SNF bound to
DNA alone, it was important to determine whether the probe DNA was
still bound to the nucleosome after the remodeling reaction. After
remodeling, a change in the nucleosome complexes was observed by
high-resolution gel shift assay after competing SWI/SNF away with an
excess of competitor nucleosomes (Fig.
4A, compare lanes 2 and 6). The initial 5 S rDNA nucleosome complex has three
distinct forms that can be resolved by gel electrophoresis (lane
2). Previously, a centrally located nucleosome had been observed
to have a lower electrophoretic mobility than one located closer to the
ends of DNA (34, 35). After remodeling there was still some of the N1
complex, but there was a distinct loss of the more acentrically
positioned nucleosomes (N2 and N3) (lane 6). An increased
smear in the lane and the generation of additional free DNA were
observed after remodeling. The total amount of free DNA generated after
remodeling was only approximately 10% after taking into account the
original amount of free DNA present in the nucleosome assembly. Thus,
most of the DNA in the starting nucleosome core particle is still bound
but not in a discrete nucleosome complex. The binding of SWI/SNF to the
nucleosome alone was not sufficient to alter the nucleosome complex
(lane 4). After remodeling, the band migrating higher than
the original N1 complex migrates similarly to the reported dinucleosome
reported for yeast RSC and human SWI/SNF (36, 37). This complex was not
consistently observed in our experiments.
The loss of discrete nucleosome complexes was assayed at different
ratios of SWI/SNF to nucleosome to determine whether this same change
could be detected at substoichiometric amounts of SWI/SNF (Fig.
4B). At less than stoichiometric amounts of SWI/SNF and
without competitor, a loss of discrete nucleosome complexes and the
release of some DNA from the nucleosome were detected upon remodeling
(compare lanes 3, 5, 7, and 9 with lanes 4, 6, 8, and 10). These results show that less than
stoichiometric amounts of SWI/SNF are sufficient to cause a loss of
discrete nucleosome complex and enhance the release of DNA from the nucleosome.
The Interactions of RSC with a 5 S Nucleosome Differ from
Those of SWI/SNF--
RSC formed a discrete complex with nucleosomal
and naked DNA (Fig. 5A, lanes
2 and 5). In the presence of ATP, the electrophoretic mobility of the RSC·nucleosome complex was unchanged under those gel
conditions (lane 3). Photoaffinity labeling with probe 47/55 revealed that four subunits of the RSC complex were cross-linked to the
nucleosomal DNA (Fig. 5B, lane 2), and these
included Sth1 the (Swi2/Snf2 homolog), Rsc3, and Rsc4, with
minor cross-linking of Rsc2. Unlike the Swi2/Snf2 subunit of the
SWI/SNF complex, Sth1 did not appear to have a specificity for
nucleosomal DNA and was cross-linked equally efficiently to naked DNA
(compare lane 2 with lane 5). The only subunit
that was cross-linked in a nucleosome-specific manner was Rsc4.
As with the SWI/SNF complex, the cross-linking of RSC after the
addition of ATP strongly resembled that obtained with naked DNA. In the
presence of ATP, the cross-linking of Sth1 and Rsc3 was unchanged;
however, the cross-linking of the Rsc2 subunit was increased, whereas
that of Rsc4 was eliminated (lane 3). Another subunit of RSC
was also cross-linked to the remodeled nucleosome complex or the RSC
bound to DNA alone, and it is one of the Rsc5-10 or Sfh1 subunits, but
it could not be uniquely identified because of the inadequate
separation of these subunits.
We have focused on examining the interface between the
chromatin-remodeling complexes SWI/SNF or RSC, and the nucleosome by 1)
identifying which subunits of SWI/SNF or RSC are near the nucleosomal DNA, 2) comparing or contrasting these interactions with how SWI/SNF and RSC bind naked DNA, and 3) determining how contacts with the nucleosome change after the remodeling reaction. Our approach has been
to attach photoreactive side chains to specific sites on a DNA, which
has intrinsic nucleosome-positioning properties. Different X. borealis 5 S rDNA probes were synthesized and assembled into
nucleosomes to investigate regions facing both in toward the octamer
and away from the octamer. Each of these DNA probes has several
positions on the octamer, because each is bound to the nucleosome in
one of several translational positions.
Site-specific DNA-protein cross-linking has shown that the
Swi2/Snf2 and Snf6 subunits of the yeast SWI/SNF complex are in close proximity to nucleosomal DNA in the SWI/SNF·nucleosome complex. SWI/SNF was shown to bind to the nucleosome and form a discrete complex
by gel shift or electrophoretic mobility shift assay under conditions
that were also optimal for cross-linking the 205- and 50-kDa proteins.
The photoaffinity-labeled 205- and 50-kDa polypeptides were shown to be
the Swi2/Snf2 and Snf6 subunits of SWI/SNF by comparing the
electrophoretic mobility of the labeled proteins with that of the
SWI/SNF subunits detected by immunoblotting with subunit-specific
polyclonal antibodies. These labeled proteins were further shown to be
part of the SWI/SNF complex by immunoprecipitation with anti-Swp73
antibodies in a manner similar to that originally used by others to
purify SWI/SNF (16, 33). The Swi2/Snf2 subunit is consistently
the most or one of the most readily cross-linked subunits regardless of
where the nucleosome is probed. The Snf6 subunit is also one of the
most efficiently cross-linked subunits of SWI/SNF with three of the
four different DNA probes used in this study. Several other SWI/SNF
subunits were cross-linked with a low efficiency, suggesting that they
are not as closely associated with nucleosomal DNA as are Swi2/Snf2 and Snf6.
The Swi2/Snf2 and Snf6 subunits, thus presumably, play an
important role in the chromatin-remodeling reaction because of their position in the interface between the chromatin-remodeling complex and
the nucleosome. Although this is the first evidence of direct interaction between the Swi2/Snf2 subunit and nucleosomal DNA, other lines of evidence have shown that Swi2/Snf2 or its homolog is the catalytic subunit of ATP-dependent
chromatin-remodeling complexes and as such might be expected to
interact directly with the nucleosome. Mutational analysis of
Swi2/Snf2 has shown that the elimination of the ATPase activity
of Swi2/Snf2 inactivates the remodeling activity of SWI/SNF (13,
17). Recombinant human Swi2/Snf2 homologs, hBrm and Brg1, have
been shown alone to have minimal remodeling activity that is stimulated
by the presence of three other subunits of the human SWI/SNF complex
related to the Swi3 and Snf5 subunits of yeast SWI/SNF (23). Also, the recombinant Drosophila protein ISWI, a Swi2/Snf2
homolog, has been shown alone to have comparable levels of remodeling
activity with that of the NURF, CHRAC, and ACF complexes that
contain ISWI (9). These results indicate that the catalytic center of
the ATP-dependent chromatin-remodeling complexes resides in
the Swi2/Snf2-like subunit, and now we present evidence that the
catalytic center containing the subunit contacts the nucleosome.
Less is known about the Snf6 subunit, an essential subunit of SWI/SNF,
than the Swi2/Snf2 subunit and there is no known homolog in
other chromatin-remodeling complexes (38). The C terminus, containing
an acidic region and a glutamine-rich region, is not absolutely
required in vivo for SWI/SNF activity, and the extreme N
terminus contains a highly basic region (39). The association of Snf6,
unlike Swi2/Snf2, with DNA is not dependent on the packaging of
DNA into a nucleosome core particle. Snf6 is cross-linked
efficiently by all four DNA probes when SWI/SNF is bound to DNA alone.
Therefore, Snf6 presumably has a general affinity for DNA as assembled
in the SWI/SNF complex and may help promote SWI/SNF binding to free DNA
or to nucleosomal DNA.
It is significant that Swi2/Snf2 only cross-links well to
nucleosomal DNA and not to free DNA. The cross-linking data indicate that the histone octamer helps recruit the Swi2/Snf2 subunit to the surface of the DNA or stabilizes its interaction with DNA in the
SWI/SNF·nucleosome complex. Sequence analysis of SWI2/SNF2 reveals a
bromodomain located at the C terminus, and a similar bromodomain in
GCN5 and TAFII250 has been indicated to bind to the
N-terminal tails of histones H3 and H4 (24, 40). An interaction between
the bromodomain of Swi2/Snf2 and histones H3 and H4 would be
consistent with the nucleosome-specific cross-linking of
Swi2/Snf2 observed with all four DNA photoaffinity probes.
Although the bromodomain may facilitate in the interaction of SWI/SNF
with the nucleosome, it is not essential in vivo for its
chromatin-remodeling activity as shown by Laurent and Carlson (38).
SWI/SNF and RSC can also remodel trypsinized histone octamers,
suggesting that tail interactions are not essential for remodeling (41,
42).
The cross-linking data suggest that the Swi2/Snf2 subunit
is displaced out of the interface between the nucleosome and SWI/SNF after remodeling and is therefore not able to be cross-linked to DNA
after the addition of ATP. The conformational change observed by both
changes in DNA photoaffinity labeling and gel shift analysis required
the hydrolysis of the We have compared the interactions of the RSC complex with the 5 S
nucleosome to those of the SWI/SNF complex. The Sth1 protein has a 72%
identity over 661 amino acids to the Swi2/Snf2 protein and could
be expected to interact with the nucleosome in a manner similar to that
of Swi2/Snf2 (45, 46). However, unlike Swi2/Snf2, Sth1 is
cross-linked to DNA both in the DNA alone bound to RSC and in the RSC
bound to the nucleosome. The Sth1 subunit also differs from
Swi2/Snf2, because it is not displaced from the nucleosomal DNA
after remodeling. Both Sth1 and Swi2/Snf2 proteins contain bromodomains at the C terminus and potentially could interact directly
with the nucleosome. The cross-linking demonstrates that Rsc4 is
specifically recruited by the nucleosome to be near the DNA, as is
Swi2/Snf2 in the SWI/SNF complex. Rsc4 has been characterized as
a protein that may interact with histone
tails,3 consistent with the
observed nucleosome-specific cross-linking. Rsc4 and Swi2/Snf2
behave similarly in that both are displaced from the surface of DNA
after remodeling. The key difference between the mode of action for
SWI/SNF and that of RSC is that only in SWI/SNF is the catalytic center
apparently displaced from DNA. This potential difference in their mode
of action is also suggested in genetic studies showing that SWI/SNF has
different interactions with chromatin than RSC. Mutations in histone
and nonhistone proteins that suppress swi/snf defects were
found to enhance the defects of temperature-sensitive mutants of
sth1 (47).
Next, the Rsc2 subunit apparently takes the place of Rsc4 near DNA
after remodeling, because Rsc2 is only cross-linked to DNA in the
RSC·nucleosome complex after the addition of ATP. When RSC
binds to naked DNA, the Rsc1 and Rsc2 subunits are also efficiently cross-linked. Rsc1 and Rsc2 contain an AT hook domain that has been associated with DNA binding activity and could account for why
these two subunits make close contact with DNA when RSC is bound to DNA
alone (48).
The interface between the chromatin-remodeling complexes (SWI/SNF and
RSC) and the nucleosome core particle has been probed using
site-specific DNA photoaffinity labeling. Not only were some of the
subunits of these complexes shown to be located at this interface, but
also these interactions changed upon hydrolysis of ATP. In the future,
it will be important to use this approach to examine changes in the
nucleosome core particle after remodeling to better understand the
structure of the "remodeled" nucleosome.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate
of ATP without covalent modification of the nucleosome.
ATP-dependent chromatin-remodeling complexes have been
purified from yeast, fly, frog, and human and can range in subunit
complexity from 2 to 15 different subunits/complex (5-13). These
chromatin-remodeling machines have been shown in vitro to 1)
enhance accessibility of nucleosomal DNA without the loss of the
nucleosome, 2) alter the path of DNA in the nucleosome, 3) cause
sliding of the nucleosome along DNA, and 4) promote octamer transfer to
another DNA (14, 15). As of now, little is known about how these
enzymes interact with the nucleosome and how their contacts with the
nucleosome change upon the binding and hydrolysis of ATP.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13/
14, and
27/
30, were synthesized as described previously (26). The numbers
indicate the base pair position or range within which a modified
nucleotide is incorporated with +1 being the start site of
transcription. In addition, an unmodified DNA probe containing only a
single radiolabeled nucleotide at bp
70 and no photoreactive nucleotides was synthesized. Probes 47/55, 1/5, and
27/
30
were on the transcribed strand, whereas probes
13/
14 and
70 were on the nontranscribed strand.
S, ADP, AMP, GTP, CTP, and UTP were
included optionally in the binding reaction at a final concentration of 100 µM. For competitor experiments, 1.8 µg (20-fold
excess) of cold oligonucleosomes was added to the reaction after
the binding reaction and incubated for an additional 30 min at
30 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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44,
24,
3 and +7 with +1 being the start site
of transcription for the 5 S rRNA gene. Four photoreactive DNA probes
were found that efficiently cross-linked SWI/SNF and that are
distributed at varied positions in the 5 S rDNA (Fig. 1A). One of the modified
regions was located in the nontranscribed strand from bp
13 to
14
(referred to as
13/
14), and the other three DNA probes were in the
transcribed strand from bp
27 to
30 (
27/
30), bp +1 to +5 (1/5),
and bp +47 to +55 (47/55).
View larger version (25K):
[in a new window]
Fig. 1.
A, schematic diagram of 5 S rDNA probes.
Each probe contains one or more modified nucleotides, AB-dUMP and/or
AB-dCMP, as indicated by ^. [ -32P]Deoxynucleotide
was incorporated adjacent to the photoreactive nucleotide as indicated
by *. The face of the DNA pointing in toward the nucleosome is
shaded on the DNA sequence as previously determined by
hydroxyl radical footprinting (27, 49). The four previously mapped dyad
axes are indicated by arrows above the sequence and are
labeled A, B, C, and D. Restriction enzyme
cleavage of nucleosomes reconstituted with unmodified (B)
and modified (C) DNA. Increasing amounts of purified SWI/SNF
were added to a constant amount of nucleosomal DNA (30 nM),
and the final concentration of SWI/SNF is indicated above each
lane (lanes 3-10). The modified DNA was probe
47/55 and contained a total of 7 modified deoxynucleotides, 4 AB-dUMP
and 3 AB-dCMP. After incubation with SWI/SNF and with or without ATP
added, the nucleosomes were cleaved with 80 units of EcoRV
for 5 min. After phenol-chloroform extraction and DNA precipitation,
the samples were analyzed on a 9% native polyacrylamide gel.
Phosphorimaging analysis was used to determine the percentage of the
cleaved product relative to the total amount of DNA. The adjusted
percent cleavage is with the amount of free DNA cleaved subtracted out
(compare lanes 3, 5, 7, and 9 with lane
2) before determining the amount of DNA cleaved by
EcoRV as a result of the addition of SWI/SNF and ATP.
70. Nucleosomes reconstituted with the unmodified or modified DNA probes were cleaved with
EcoRV in the presence or absence of SWI/SNF. The cleavage
site for EcoRV is ~105 bp from either end of the DNA and
is well occluded within the nucleosome. In the absence of SWI/SNF, 24%
of the unmodified nucleosomal DNA was accessible to the enzyme (Fig.
1B, lane 2). This finding is consistent with the efficiency
of nucleosome formation being ~80% as assayed by gel shift (data not
shown). The extent to which the nucleosomal probe is cleaved increases
as the amount of SWI/SNF added is increased. At a molar ratio of 1:3 of
SWI/SNF to nucleosome, almost 90% of the nucleosomes are accessible to EcoRV cleavage (Fig. 1B, lanes 9 and
10).
View larger version (34K):
[in a new window]
Fig. 2.
The Swi2/Snf2 and Swi6 subunits of
SWI/SNF are positioned near nucleosomal DNA in the SWI/SNF·nucleosome
complex. A, gel shift analysis. Probe DNAs were
reconstituted into nucleosomes by the octamer transfer method and
analyzed by gel shift assay on a 4% native polyacrylamide gel in 0.5×
TBE. Mock nucleosome reconstitutions were performed also using the
octamer transfer method, except that the probe DNA was added last after
doing the final dilution of the donor nucleosomes to 100 mM
NaCl (lanes 5, 8, 11, and 14). These samples are
referred to as free DNA because no octamer transfer occurs under these
conditions. The amount of SWI/SNF added in lanes 3-14 was
stoichiometric and was shown empirically by titrating with different
amounts of SWI/SNF in a gel shift binding assay with a fixed amount of
nucleosomes. Remodeling reactions in lanes 4, 7, 10, and
13 contained 100 µM ATP. B, DNA
photoaffinity labeling. A large portion of the same samples from
A were irradiated and digested with DNase I and S1 nuclease
leaving a radiolabeled DNA tag cross-linked to protein. The samples
were analyzed by 4-20% SDS-polyacrylamide gel electrophoresis and
phosphorimaging analysis. The electrophoretic mobilities of the SWI/SNF
subunits were determined by Western blotting using subunit-specific
polyclonal antibodies and are indicated on the left. Samples
in lanes 4-15 were immunoprecipitated using an anti-SWP73
antibody before degradation of the DNA probe and subsequent
analysis.
13/
14,
and
27/
30, cross-linked the Swi2/Snf2 subunit of SWI/SNF in
a nucleosome-specific manner. Immunoprecipitation of the SWI/SNF
subunits cross-linked to nucleosomal probes 1/5,
13/
14, and
27/
30 showed that Swi2/Snf2 and Snf6 were efficiently cross-linked to DNA (Fig. 2B, lanes 7, 10, and
13). The cross-linking of Swi2/Snf2 was the most
dramatically decreased in the presence of ATP as with probe 47/55
(compare lanes 7, 10, and 13 with lanes 8, 11, and 14). Minor cross-linking of other subunits,
including Swi1, Swi3/Snf5, Swp82, Swp73, Arp9 and Tfg3/Taf30, was also
obtained. The two weak bands immediately below the Swi2/Snf2
subunit are probably degradation products of Swi2/Snf2 and
varied in intensity in different preparations of SWI/SNF. Snf6 appears
to interact closely with free DNA, because it is cross-linked
efficiently when SWI/SNF is bound to DNA alone (lanes 9, 12, and 15). At these three positions, the efficiency of Snf6
cross-linking does decrease with the addition of ATP. Swp73 also
interacts with nucleosomal and naked DNA but to a lesser extent as
compared with Snf6.
-phosphate of ATP was
required for the loss of photoaffinity labeling of the Swi2/Snf2
subunit or changes in the SWI/SNF·nucleosome complex, several ATP
analogs as well as other nucleotide triphosphates were used. As before,
the addition of ATP resulted in a change in the gel-shifted complex,
which no longer entered into the gel (Fig.
3A, lanes 4 and 5).
ATP
S and AMP-PNP did not change the electrophoretic mobility of the
initial SWI/SNF·nucleosome complex (lanes 6 and
7). Similarly, ADP, AMP, and other nucleotide triphosphates did not change the mobility of the complex (data not shown). Consistent with the observation that a hydrolyzable form of ATP was required for
significant changes in the electrophoretic mobility of the SWI/SNF·nucleosome complex, a loss in DNA photoaffinity labeling of
the Swi2/Snf2 protein with probe 47/55 was also observed to require hydrolysis of the
-phosphate of ATP (Fig. 3B). In
the presence of ATP, there was a decrease in histone-DNA contacts and a
loss of Swi2/Snf2 cross-linking as well as an increase in cross-linking Swp73 and Snf6 subunits of SWI/SNF (Fig. 3B,
lanes 2 and 3). However, there was no change in
the efficient cross-linking of Swi2/Snf2 with ATP
S, ADP, AMP,
GTP, CTP, or UTP (lanes 4-9). The requirement for the
hydrolysis of the
-phosphate of ATP for loss of Swi2/Snf2
cross-linking was also observed with probes 1/5,
13/
14, and
27/
30 (data not shown).
View larger version (24K):
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Fig. 3.
Loss of Swi2/Snf2 cross-linking and
change of the electrophoretic mobility of the SWI/SNF·nucleosome
requires hydrolysis of the -phosphate of
ATP. A, gel shift analysis of the SWI/SNF·nucleosome
complex with DNA probe 47/55 in the presence of ATP (lane
5), ATP
S (lane 6), and AMP-PNP (lane
7). Lane 1 is the DNA alone, and lanes 2 and
3 are the 5 S nucleosome alone with and without ATP.
B, DNA photoaffinity labeling. Nucleosomes reconstituted
with probe 47/55 were bound to SWI/SNF in the presence of ATP
(lane 3), ATP
S (lane 4), ADP (lane
5), AMP (lane 6) or other nucleoside triphosphates
(lanes 7-9). After cross-linking, the samples were analyzed
by 4-20% SDS-polyacrylamide gel electrophoresis. The band
marked with an asterisk (*) in lane 7 was not reproducible
and was probably caused by incomplete digestion of DNA.
View larger version (24K):
[in a new window]
Fig. 4.
Altered conformation of the nucleosome after
remodeling as detected by gel shift assay. A, a
stoichiometric amount of SWI/SNF was used in lanes 3-6.
SWI/SNF was competed away from the radiolabeled 5 S nucleosome by the
addition of a 20-fold excess of cold oligonucleosomes before and after
remodeling (lanes 4 and 6, respectively). The
samples were analyzed by native 5% polyacrylamide gel electrophoresis
(0.2× TBE) at 4 °C. The nucleosome occupies at least three
different translational positions with N1 and N3 being the most centric
and acentric translational positions, respectively. B, the
amount of SWI/SNF was varied while maintaining a constant amount of
nucleosome to determine the amount of SWI/SNF required to see changes
in the 5 S nucleosome with no competitor added. The concentrations of
SWI/SNF are indicated above each lane, and lanes 4, 6, 8, 10, and 12 all contained 100 µM
ATP.
View larger version (18K):
[in a new window]
Fig. 5.
The interactions of the RSC complex with the
5 S rDNA nucleosome differ from those of SWI/SNF. A,
Gel shift analysis. 5 S rDNA nucleosomes assembled with probe 47/55
(lanes 1-3) or free DNA (lanes 4-5) were bound
to RSC either in the presence (lane 3) and/or absence of 500 µM ATP. The complexes were analyzed on a 3.2% native
polyacrylamide gel in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA. Free DNA samples were prepared the same as
described in Fig. 2. B, DNA photoaffinity labeling. A
portion of each sample from A was irradiated, enzymatically
digested, and analyzed by 4-20% SDS-polyacrylamide gel
electrophoresis and phosphorimaging analysis. The pre-stained molecular
mass standards are as indicated on the right.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate of ATP and did not occur by binding
ATP alone or with other ribonucleotide triphosphates. There is a
significant similarity in both the gel shift and DNA cross-linking of
the SWI/SNF·nucleosome complex after remodeling with that of SWI/SNF
bound to free DNA. These similarities are consistent with the
nucleosome structure being altered so significantly that SWI/SNF can
bind to the complex essentially as it does to free DNA. It is clear
from the gel shift data that although some of the octamer have been
displaced, most had not been displaced from the DNA probe. The
competition of SWI/SNF away from the remodeled nucleosomes demonstrates
that most of the DNA are still bound to the octamer and that the
nucleosome after remodeling is changed sufficiently to alter its
electrophoretic mobility. Changes in the electrophoretic mobility of
the nucleosome could be caused by deformation of the nucleosome or by
sliding of the octamer on DNA to many different translational
positions. It is not possible to differentiate between these two
possibilities. Other data have suggested that SWI/SNF can slide
the nucleosome along DNA similar to that of other chromatin remodeling
complexes, such as NURF and CHRAC (21, 22, 43, 44). Sliding mediated by
ISWI complexes of mononucleosome substrates has shown sliding to a
preferred translational position. Similarly, the yeast ISW2 complex
preferentially slides the nucleosome on the same 5 S rDNA probes used
in this report to a unique more centric translational
position.2 These sliding
results are noticeably different from that observed for SWI/SNF in that
there is no preferred translational position but rather a diffuse smear
of many potentially alternative translational positions. Although
nucleosome sliding could be the cause of the reduced Swi2/Snf2
cross-linking, it would be expected that at some positions on DNA,
there should be enhanced cross-linking of Swi2/Snf2 after
remodeling. The data however indicate that the loss of Swi2/Snf2 most
probably is not attributed primarily to nucleosome sliding, because at
no position probed is there an increase in the cross-linking of
Swi2/Snf2. There is no increase in Swi2/Snf2
cross-linking even near the ends of the DNA (data not shown).
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ACKNOWLEDGEMENTS |
---|
We thank Stefan Kassabov and Martin Zofall for helpful discussions and comments regarding this work.
![]() |
FOOTNOTES |
---|
* This work was supported by the Public Health Service Grant GM48413 from National Institutes of Health (NIGMS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Nijmegen University, Moleculaire Biologie, Toernooiveld 1, 6525-EDNijmegen, The Netherlands.
** To whom correspondence should be addressed. Tel.: 618-453-6437; Fax: 618-453-6440; E-mail: bbartholomew@siumed.edu.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M10470200
2 N. Henry and B. Bartholomew, personal communication.
3 H. Szerlong and B. Cairns, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
HA, hemagglutinin;
TBE, 90 mM Tris borate, 2 mM EDTA;
bp, base pair(s);
ATPS, adenosine
5'-3-O-(thio)triphosphate;
AMP-PNP, adenosine
5'-(
,
-imino)triphosphate.
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