volume 7 Number 81979
Nucleic Acids Research
Nucleosome dissociation and transfer in concentrated sah solutions
Pamela C.Stacks and Verne N.Schumaker
Department of Chemistry and Molecular Biology Institute, University of California, Los Angeles,
CA 90024, USA
Received 14 August 1979
ABSTRACT
We have examined the dissociation of nucleosomes Into histones and free,
4.5S DNA over a range of sodium chloride concentrations between 0.25 and 1 M.
We have also studied this dissociation as a function of nucleosome concentration at two salt concentrations, 0.8 M and 0.9 M. In addition, we have
measured the kinetics of transfer of histone cores from nucleosomes onto
recipient bacteriophage T7 DNA 1n 0.6, 0.7 and 0.8 M NaCl solutions. Although the mechanism of nucleosome transfer is unknown the data presented
here are consistent with either a reversible dissociation of the nucleosome
or DNA strand displacement by another DNA.
INTRODUCTION
Electrostatic
increasing
Interactions between DNA and histones are weakened with
1on1c strength.
Thus, as the Ionic strength 1s raised between
0.55 M to 0.65 M, histones HI and H5 become dissociated from chromatin1"3.
At about 0.6 M, nucleosome sliding commences^"7, although one report Indicated
sliding occurs 1n 0.15 M NaCl8.
Between 0.7 M and 1.2 M NaCl histones H2A
and H2B are eluted from chromatin using zonal techniques.
Nucleosome
histone cores are successfully transfered to exogenous DNA at an 1on1c
strength of 0.85 M NaCl and this ionic strength has been employed to Initiate
g
the reannealing of completely dissociated histones and DNA.
strengths higher than
chromatin. •
At 1on1c
1.2 H NaCl histones H3 and H4 are also eluted from
In the absence of UNA, complex equilibria are established
between various oligomeHc forms of histones which are dependent upon 1on1c
strength and p H 1 0 " 1 5 .
In the presence of nucleosomes, however, a tight
association is thought to be formed between histone core octamer and Intact
nucleosomes which stabilizes the octamer, at least at pH 8 and 0.6 M ionic
strength16.
In this communication we report the results of our studies on the
© Information Retrieval Limited 1 Falconberg Court London W1V6FG England
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extent of dissociation of nucleosomes over a range of 1on1c strengths between
0.25 and 1.0 M, and upon the equilibria obtained as a function of nucleosome
concentration. The results of studies on the kinetics of transfer of histone
core from nucleosomes to recipient DNA molecules are also presented.
MATERIALS AND METHODS
Nucleosome p r e p a r a t i o n : Chicken erythrocyte nucleosomes were prepared from 75 nil of chicken blood as described by 011ns and coworkers 17 ,
except t h a t :
(a)
The STM and STMN buffers were maintained at pH 8; (b)
Worthington micrococcal nuclease was added to a ratio of 2.86 A260 units
of nuclei/unit of nuclease.
Incubation for 90 minutes at 37° C resulted
1n a 12.8% digestion of the DNA, calculated as described 1n reference 18;
(c)
After the lysed nuclei were c l a r i f i e d by centrifugation, 2 ml of the
supernatent f l u i d was diluted 1:1 to a final concentration of 50 A26O un1ts/ml
1n 0.2 mM EDTA, pH 6.8, and then 2 ml were layered on top of each 7.5% to 25%
(w/v) linear sucrose gradient 1n a cellulose nitrate SM 27 tube (Beclunan).
The gradients also contained 2 mM EDTA, pH 6.8, 0.1 mM phenylmethylsulfonyl
fluoride (PMSF), and 0.2% Isopropanol. The gradients were centrifuged for 16
hours at 27,000 rpm and 5° C using a SW 27 r o t o r . The main peak 1n the
sucrose gradient contained 25 A26O units. I t was Isolated, dialyzed Into 0.2
mM EDTA, pH 6.8, and made 0.1 mM PMSF, 0.2% Isopropanol. The nucleosomes were
examined by analytical ultracentrifugation and by gel electrophoresis for
DNA19 and histones^O. A single sedimenting peak was observed, with a S20,w
of 11.7 S 1n lOmM T r i s , 0.25 mM EDTA pH8, containing less than 5% dimer. The
distribution of nucleosomal DNA sizes ranged from 130-180 base pairs as I n d i cated by gel electrophoresis mobility. Equimolar quantities of the core histones appeared to be present and free from proteolytic digestion. Although,
trace amounts of HI and H5 were present, no other protein bands were observed.
Bacteriophage T7 DNA p r e p a r a t i o n :
After growth the bacteriophage
were concentrated with polyethylene glycol 6000 and by centrifugation 1n a
70 T1 rotor (Beckman) for 40 minutes at 40,000 rpm and 5° C21. The bacteriophage were then banded 1n a CsCl step gradient. T7 DNA was prepared
by phenol e x t r a c t i o n ^ followed by a chloroform:Isoamyl alcohol (24:1) extract i o n . The DNA was dialyzed Into 10 mM NaCl, 10 mM Tr1s, 0.1 mM EDTA, pH 8.
Nucleosome d i s s o c i a t i o n s t u d i e s :
In order to achieve the desired
salt concentrations, a stock solution of 4.4 M NaCl, 10 raM T r i s , pH 8 was
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slowly added to nucleosoraes 1n 10 mM Tr1s, pH 8.0. A 1:100 addition of 100
mH PMSF 1n Isopropanol was made t o I n h i b i t any endogeneous p r o t e o i y t i c
a c t i v i t y . The samples were Incubated at 22° C for 16 hours and subsequently
examined 1n the analytical ultracentrifuge without d i l u t i o n .
Transfer s t u d i e s :
In order t o achieve the desired s a l t concentrat i o n s a stock s o l u t i o n of 4.4 M NaCl, 10 mM T r i s was slowly added t o a
mixture of T7 DMA and Isolated chicken erythrocyte nucleosomes. The f i n a l
Incubation concentrations of T7 DNA and nucleosomes were 100 and 200 micrograms/ml, respectively.
The samples were Incubated at 22° C. To terminate
the Incubations, aliquots were diluted slowly with gentle mixing to 0.25 H
NaCl, using 10 mM T H s , pH 8.0.
A n a l y t i c a l u l t r a c e n t r i f u g a t i o n : The Beckman Model E p h o t o e l e c t r i c
scanning system at 265 run was used to follow the sedimenting boundaries.
For the more concentrated s o l u t i o n s , a wavelength between 280 to 300 nm
was selected to keep the absorbancy below 0.8.
The temperature of the
c e n t r i f u g a t i o n was monitored but not r e g u l a t e d , and was r o u t i n e l y kept
between 20 and 22° C w i t h a v a r i a t i o n of less than 0.3° C.
The obserand
ved sedimentation coefficients were corrected to S20,w>
observed concentrations were corrected to I n i t i a l concentrations using the square law of
radial d1lut1on23.
RESULTS
Nucleosome dissociation studies:
In Table 1 are l i s t e d the results
of the u l t r a c e n t r i f u g e analyses of two separate, 16 hour, 22° C Incubations of nucleosomes at a concentration of 50 ra1crograms/ml over a range of
1on1c strengths between 0.01 and 1.0 M. At the lowest 1on1c strength 1n 10 mM
T r i s , 0.25 mM EDTA, pH 8.0, the nucleosoraes sedimented as a single boundary 1n
the analytical ultracentrifuge, and as Indicated by the f l a t baseline, no free
DNA was found. In 0.25 M NaCl, a sloping base l i n e appeared In the solvent
region, which comprised less than 7% of the A265 material 1n Table 1. At
a l l higher Ionic strengths between 0.5 to 0.9 M, two sed1roent1ng boundaries
appeared with Increasing amounts of A265 material present 1n the slow boundary
with Increasing 1on1c strengths. The slower boundary had an average sedimentation coefficient of 4.5S, consistent with free nucleosomal DNA. Figure 1
shows a typical derivative plot of the two sedimenting boundaries, generated
by computer from the photoelectric scanner data.
The sedimentation coefficients
of the fast
boundaries, on the other
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TABLE I
Effect of Ionic Strength on the Sedimentation
and Dissociation Behavior of Nucleosomes
NaCl a
20 ,w
(molar)
slow boundary
S20,w
of
% raw in
of
fast boundary
slow boundary
0.0
-
11.7
-
0.25
-
9.9
-b
0.5
4.3 ± 0.05
10.0 ± 0.06
16.5 ± 0.7
0.6
4.3 ± 0.40
9.5 ± 0.01
16.5 ± 2.1
0.7
4.2 ± 0.04
9.3 ± 0.02
20.5 ± 0.07
0.8
4.5 ± 0.9
8.9 ± 0.45
29.5 ± 2.1
0.9
4.6 ± 0.32
8.5 ± 0.15
40.5 ± 6.4
1.0
4.8
8.1
51
a
10 mM Tr1s, pH 8.0, was also present.
b
A sloping baseline was observed 1n the solvent region.
hand, clearly decreased with Increasing 1on1c strength, frooi 10.OS at 0.5 M
NaCl to 8. IS at 1.0 M NaCl.
The extent of nucleosome dissociation as monitored by the release of
4.5S ONA as a function of nucleosome concentration, 1s shown in Figure 2.
6.4
7.0
RADIUS (cm)
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Figure 1 : A derivative
plot of two sedimenting
boundaries which were
formed when nucleosomes
at a concentration of
50 micrograms/ml 1n 0.9
M NaCl pH 8 and at 22° C
were studied 1n the analyt i c a l ultracentrifuge.
The data were obtained
after 36 minutes of c e n t r i fugation at 52,000 rpra.
Nucleic Acids Research
200
400
200
QJUCLEOSOME]
400
u^/fnl
Figure 2: Effect of nucleosome concentration on the amount of
free nucleosotnal ONA present at 22° C 1n (a) 0.8 M NaCl or
(b) 0.9 M NaCl as measured by analytical ultracentHfugation.
Nucleosoroes were Incubated at 22° C for 16 hours 1n 0.8 M NaCl (F1g. 2a)
or 1n 0.9 M NaCl (F1g. 2b) over a range of nucleosorae concentrations between
25 to 500 m1crograms/ml, at pH 8.
Then, the samples were analyzed In 0.8 or
0.9 M NaCl 1n the ultracentHfuge.
The percent of DNA present 1n the slow
moving boundary 1s plotted 1n Figure 2 as a function of nucleosome concentration.
Nucleosome t r a n s f e r studies:
Chicken erythrocyte nucleosoraes and
T7 DNA of 25 m i l l i o n daltons molecular weight were used to examine the
k i n e t i c s of t r a n s f e r of hi stone cores as a function of 1on1c strength.
Since the T7 DNA has an S2o,w
of
about 30S, 1t could be separated from
the 5S DNA and the US nucleosome 1n the analytical ultracentrifuge.
The
extent of transfer was estimated both from the amount of free nucleosomal
DNA liberated and also from the enhanced sedimentation rate of the T7 DNA.
The k i n e t i c s of t r a n s f e r at 22° C are shown 1n Figure 3 f o r
strengths of 0.6, 0.7 and 0.8 M NaCl.
Ionic
At various times, aliquots were
diluted to 0.25 M NaCl, and the amount of 4.5S DNA present determined by
ultracentrifugat 1 on.
In the absence of T7 DNA, nucleosomes incubated 1n 0.6 M NaCl f o r
1 hour, and then gently diluted to 0.25 M NaCl, contained 15.2% free nucleosomal DNA.
Similar controls for the 0.7 M and 0.8 M Incubations showed
17.6% and 19% free DNA, respectively.
Controls performed with a mixture of nucleosomes and nucleosomal ONA
at concentrations of 200 and 100 ra1crograms/ral, respectively, at 0.6, 0.7
and 0.8 M NaCl demonstrated that
little
or no hi stone loss occurred upon
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F
z
Q
Figure 3: Kinetics of Mstone
core transfer onto T7 DNA at
22° C 1n (a) 0.8 M (b) 0.7 M,
or (c) 0.6 M NaCl, as measured
by the amount of free 4.5S
nucleosoraal DNA present after
d i l u t i o n to 0.25 M NaCl.
60
- 40
20
10
TIME
20
11
20
(hr)
M
- «0
20
10
TIME
20
(hr)
lowering the s a l t concentrations to 0.25 M NaCl.
DNA present
after
Thus, the amount of
4.5S
d i l u t i o n to 0.25 M NaCl was a measure of the amount of
transfer to the T7 DNA.
In t h e presence of T7 DNA, nucleosomes Incubated 1n 0.6 M NaCl did
not show any Increase 1n the amount of 4.5S
nucleosomal
ing times of Incubation, as shown 1n Figure 3C.
little
DNA with
Increas-
There appeared to be very
transfer of histones to the T 7 DNA during Incubation at 0.6 M NaCl,
10 nW Tr1s, pH 8 . 0 .
For the 0.7 mM NaCl, 10 mM Tr1s, pH 8.0 Incubations, the
zero time point was obtained by Immediately d i l u t i n g an aliquot after raising
the s a l t concentration to 0.7 M NaCl.
nucleosomes were present
as free
At t h i s zero point, 15% of the Input
nucleosomal
DNA.
Subsequent time
points
showed a gradual Increase in the amount of nucleosomal DNA which by 24 hours
had leveled off
at 51.4% of the Input nucleosome concentration, as shown 1n
Figure 3B.
In contrast with the gradual
release at 0.7 M, the 0.8 M NaCl samples
showed a moderately rapid r e l e a s e of f r e e
tine
Incubation mixture contained
48.5% of
the
Input
nucleosomal
23% free
nucleosomal
nucleosoraal
DNA was present
DNA.
DNA.
The "zero"
By one hour,
as free 4.5S DNA, and the
amount q u i c k l y l e v e l e d off a t 58.5% of the m a t e r i a l , as shown 1n Figure
3A.
The sedimentation
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behavior
of
the
T7 DNA a f t e r
the
transfer
reaction
Nucleic Acids Research
was a function of the number of nucleosomes acquired. Formation of T7 DNAnucleosome complexes resulted in an Increase 1n molecular weight, a decrease
1n particle density, and a decrease 1n the f r i c t i o n a l coefficient of the T7
DNA due to a seven-fold compaction of each section of DNA associated with a
nucleosome2^.
The appropriate equation allowing a conversion between the
sedimentation coefficient of the T7 DNA-nucleosome complex and the number of
bound nucleosomes was derived 1n reference 25, and i t 1s plotted 1n Figure 4.
I t has been reported that at least hi stones H3 and H4 are required for the
observed Increased sedimentation of reconstituted DNA26"29.
In Table 2 are l i s t e d the observed sedimentation coefficients of the
T7 DNA-nucleosome complexes after 16 hours of Incubation and d i l u t i o n to
0.25 H NaCl. The number of Mstone cores t r a n s f e r r e d to the T7 DNA was
estimated from the release of 4.5S DNA (Table 2, under the heading of "% 4.5S
DNA". The number of histone cores transferred was also estimated from the
values of Sgo.w measured for the T7 DNA-nucleosorae complexes and the conversion curve of Figure 4. These values also are l i s t e d 1n Table 2. There 1s
good agreement between the two sets of estimates for the amount of histone
core transfer.
That the transferred hi stones were present on the T7 DNA 1n the form
of Intact nucleosomes was strongly suggested by the results of digestion
experiments with DNase I .
An autoradiograra of a gel of DNA resulting from
digestion of uniformly labelled 32p_y7 DNA-nucleosome complexes displayed
a ladder pattern with DNA fragments, separated by 10 base pairs, ranging i n
size from 20 to 130 nucleotides (not shown). This was Indicative of nucleosomal structure present on the T7 DNAlf*.
A control mixture of 32P-T7 DNA
and unlabelled nucleosomes which had been maintained at low 1on1c strength
resulted 1n a smear of radioactive DNA of no specific length after DNase I
digestion.
Figure 4:
The calculated
s
20,w of T7 DNA-nucleosome' complexes 1s plotted
as a function of the number
of nucleosoraes associated
with the T7 DNA.
NUCLEOSOMES/T7 DNA
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TABLE 2
Number of Nucleosoraes per T7 DNA Predicted from the Sedimentation
Rates of the Complexes or the Release of 4.5S DNA
NaCl
(molar)
s°20,wa
0.6
35.0
16
15
35
0.7
61.5
104
50
116
0.8
68.6
119
58
134
n/T7 DNAb
% 4.5S DNAC
n/T7 DNAd
a
The T7 DNA concentration during the centrifugation was
25 ug/ml.
A correction of +13% was used to correct the
observed sedimentation rates to values of s°20,w
D
Values obtained by use of Fig. 4 and the values of s°20,w
l i s t e d 1n the preceding column.
c
Values of % 4.5S DNA released obtained from F1g. 3.
d
Values predicted from the percent release of 4.5S DNA, as
l i s t e d in the preceding column.
DISCUSSION
In the presence of high concentrations of sodium chloride, 0.6 to 1.0M,
the l i g h t absorbing material was observed to sediment as two d i s t i n c t boundaries (Table I ) .
Since the histones absorb very l i t t l e l i g h t at 265nm, t h i s
light absorbing material represents two d i f f e r e n t , DNA-containing particles.
The sedimentation rate of the slow boundary was the same as that of phenol extracted, protein-free, nucleosomal DNA. This was an unexpected observation
for 1t has been reported that while histones H2A and H2B are released at lower
ionic strengths, H3 and H4 remain associated with the DNA until about 1.3M28.
I t does not appear possible that substantial quantities of H3 and H4
could be attached to the 5S DNA since 1t has been reported that the complex
composed of a single H3-H4 tetramer and a single piece of nucleosomal DNA
sediments at 9.8S29. Moreover, i f a single H3-H4 diraer were to be associated
with a 5S DNA, the sedimentation rate of the 5S DNA may be calculated to
Increase by 25% due to the increase in buoyant molecular weight even i f
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the frictional coefficient remained that of 5S DNA. Therefore, we conclude
that the 5S boundary represents essentially protein-free, nucleosomal DNA
released by salt concentrations in the range of 0.6 to 1.0M.
The appearance of a large amount of free, 5S DNA at the high 1on1c
strengths could be due to a reversible dissociation of the nucleosomes with
free DNA forming one of the products. In this case, it would be reasonable
to expect that the equilibrium constant would be a function of 1on1c strength,
with increasing dissociation occurring as the 1on1c strength were Increased.
Moreover, a reversible dissociation 1n which several products were formed
from a single nucleosome would be expected to be a function of nucleosome
concentration. This has been tested, as shown 1n Figures 2A and 2B, and,
Indeed, a strong concentration dependence has been found to exist. To us,
this 1s convincing evidence for the existence of a reversible reaction 1n
which the nucleosome dissociates to form a number of products including free,
5S DNA. However, the curves of Figure 2 appear to approach limiting values of
about 16%, which would Indicate the existence of a population of nucleosomes
not participating 1n the equilibrium reaction. Therefore, we feel that the
best explanation for the release of the free 5S DNA involves both a reversible
dissociation of the nucleosomes and nucleosome heterogeneity. More than 80%
of the nucleosomes appear to undergo a reversible dissociation over the range
between 0.6 to 1.0 M. In addition, there appears to be a subpopulation of
about 16% which 1s more susceptible to dissociation.
We would expect the reversible dissociation to be a slow reaction because
the two sedimenting boundaries were well resolved. Since the hi stones are not
directly detected during centrifugation, the only product of the dissociation
that was detected was the 5S DNA. Because not all of the products of the
dissociation are known, 1t 1s not possible to make a boundary analysis along
the lines suggested by Cann and Kegeles (27) to support our assumption that
the dissociation 1s slow. More direct evidence comes from a study of the
half-Hves of the nucleosome dissociations as measured by the transfer experiments.
The kinetics of nucleosomal transfer at high ionic strengths have been
measured by monitoring the appearance of free nucleosomal DNA (Figure 3). In
contrast to the experiments just discussed above, 1n which dissociation was
studied during centrifugation at high 1on1c strengths, 1n the transfer experiments the centrifuge runs were performed after the 1on1c strength had been
readjusted to 0.25M. The rate of transfer was gradual with time and was
dependent upon the Ionic strength with half-Hves of 6.8 hours In 0.7M NaCl
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and 45 minutes 1n 0.8M NaCl. Since an equal weight ratio of donor and recipient DNA was used 1n these experiments and since the reaction appeared to
reach completion at between 50 and 60% free nucleosomal DNA, we conclude
there 1s no preference for longer over shorter pieces of DNA during nucleosome
formation.
The sedimentation rate of the recipient T7 DNA Increased 1n a
manner consistant with the appearance of free nucleosomal DNA (Table 2).
Since the presence of histones H3 and H4 are required for Increased sedimentation (26,29) and DNase I digestion Indicated the presence of nucleosomal
structures on the T7 DNA after transfer (see t e x t ) , we have concluded that the
transfer of nucleosomes onto the T7 DNA had occurred 1n these experiments.
The mechanism of nucleosome transfer 1s not known.
I t could Involve the
complete dissociation of the histones from the DNA. On the other hand,
transfer might be a displacement process, with one piece of DNA gradually
displacing a second from the histone core, keeping most of the histone DNA
bonds Intact at all times.
I t seems evident from the data presented here that most of the nucleosomes neither dissociate nor transfer until the 1on1c strength 1s raised above
0.6M.
Between 0.7 to 0.9M, the particles appear to reversibly dissociate.
These transitions are not sudden, but rather depend upon the salt concentration and the nucleosome concentrations 1n a fashion consistent with mass law
e q u i l i b r i a . The overall rates of both transfer and of dissociation and
reassodation appear to be slow and dependent upon salt concentration.
Careful study of this system as a function of temperature, 1on1c strength and
concentration should yield valuable thermodynanvic and kinetic Information
leading to a better understanding of the mechanism of binding of DNA by the
core histones.
ACKNOWLEDGMENTS
Our thanks t o Anita Wadel for her e f f o r t s I n the typing of t h i s manus c r i p t . This research was supported by National I n s t i t u t e s of Health Grant to
V.N. Schumaker (GM13914).
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