volume 9 Number 201981
Nucleic Acids Research
Interaction of histone HI with superhelical DNA. Sedimentation and electron microscopical studies
at low salt concentration
Michael Bottger, Claus-Ulrich von Mickwitz, Siegfried Scherneck, Klaus Grade and Ruth Lindigkeit
Central Institute of Molecular Biology, Academy of Sciences of the GDR, 1115 Berlin-Buch, GDR
Received 3 August 1981
ABSTRACT
Complexes of histone H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na . Depending on the molar input ratio
H1/DNA three classes of sedimenting species were observed:
(1) a component sedimenting similar to superhelical DNA with
of 25 S observable up to
a sedimentation coefficient s 9
335 Mol H1/Mol DNA (w/w - 2 ) ; °'w(2) a component with s 9
120 S appearing at 135 Mol H1/Mol DNA and (3) growing
°» w
amounts of heterogeneous aggregates > 1000 S. Electron micrographs revealed the 25 S component to consist of doublefibers formed from one DNA molecule and the 120 S component
to consist of bundles of several such double-fibers. The
aggregates represent cable-like structures. The addition of
ethidium bromide to 25 S complexes induces the formation of
bundles, if H1 is present in a quantity which alone is not
sufficient to bring about this effect. This result indicates
that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.
INTRODUCTION
It has been proposed in the last few years that histone
1 2
H1 is involved in chromatin condensation in vivo • and in
vitro
and that H1 phosphorylation may be a necessary conO
dition for the modulation of the chromatin structure
C
O
»°~°.
Histone H1, on the one hand, is known to play a fundamental role in compacting the 100 8 chromatin fiber into higherorder structures ^'
» Furthermore, it stabilizes the two
full DNA turns in the nucleosome by binding at the entry and
exit points of the DNA outside of the nucleosome core
'
On the other hand, there are reports in the literature on a
cross-linking function of H1 between different oligosome
12 13
chains
'
and between high molecular weight chromatin
© IRL Press Umlted, 1 Falconberg Court, London W1V 5FG, U.K.
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fibers which are discussed to form double-fibers
1
^~'°
mechanism of H1 crosslinking activity which is also discussed
1 2
at the chromatin condensation in vivo • is still obscure.
One way to study the complex interactions between DBA
and H1 histone involved in chromatin is the use of artificially prepared complexes of H1 and DNA as simple model systems. Numerous papers on this subject have appeared so far '
17—29
28
. We refer here to a paper by von Mickwitz et al.
which demonstrates by electron microscopy the formation of
DNA double-fibers arising by DNA backfolding as a new form
of complexes between linear DNA and histone H1. A crosslinking
activity of H1 stabilizing these double-fibers can be inferred from this paper. The purpose of the present investigation
using sedimentation and electron microscopy is to show that
complexes of superhelical DNA and H1 form similar structures.
Furthermore, additional facts such as sedimentation properties, ethidium bromide probing and cooperativity of these
interesting structures are studied with the aim to get additional evidence for a possible H1 crosslinking function.
MATERIAL AND METHODS
SV40 DNA and H1 histone were prepared as described pre21
viously
. Samples of SV4O DNA used contained about 80 %
superhelical (form I) DNA and 20 % open relaxed (form II) DNA
(shown by sedimentation analysis).
H1-DNA complexes were prepared by direct mixing of H1 and
DHA in 0.1 SSC buffer corresponding to 0.02 M Na + (SSC: 0.15 H
NaCl, 0.015 M Na citrate, pH 7.0). This was performed in the
centrifuge cell into which further small volumes (5 - 10/Ul)
of concentrated H1 solution were also added after each sedimentation run by means of a Beckman micropipette. The centrifuge cells were shaken vigorously after each run to redissolve pelletted material and after H1 addition to mix the
solution. Controls with the complexes prepared in test tubes
provided the same results, for further details cf. 21. The
concentration of the DHA stock solution was 30 /Ug/ml. The
ethidium bromide titration was performed in the same way. The
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results are expressed as a function of the Mol H1/ Mol DNA
input ratio (DNA is expressed as molecules of DNA) using a
molecular weight of 3.5 • 10 6 for SV4O DNA and 21000 for H1.
Binding measurements of H1 histone to DNA were not performed.
Moving boundary sedimentation was performed in an analytical ultracentrifuge Spinco E (Beckman) equipped with absorption optics, monochromator and photoelectric registration
as described previously
. Integral sedimentation coeffi-
cient distributions were calculated as described in 30. Calculations of the proportions (in % of the original concentration) of the sediraenting species were performed after correcting for the radial dilution effect. The relative concentrations were taken from the scanner charts. Electron microscopy was performed as described elsewhere
. The samples
were taken from the centrifuge cells and diluted to 5.0 /Ug/ml
DNA final concentration.
RESULTS
Sedimentation analysis
Depending on the molar input ratio Mol H1/Mol DNA (167
Mol H1/Mol DNA corresponds to a weight ratio w/w of 1 or
roughly complete neutralization of DNA phosphates), three
classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical form I DNA with a
sedimentation coefficient s 2 0 w of 25 S in the whole range
of H1 concentrations studied,(2) a component with about 120 S
appearing at 135 Mol H1/Mol DNA and (3) above 135 Mol H1/Mol
DNA growing amounts of heterogeneous aggregates with s 2 0 w >
1000 S. The 80
<- o, w values of the two main components of 25 S
and 120 S as a function of the Mol H1/Mol DNA ratio are shown
in Pig. 1. It is remarkable that both components show only
slowly changing s 2 o w values in a broad range of molar H1/DNA
ratios. Pig. 2 shows integral sedimentation coefficient distributions of free DNA and complexes at 300 Mol H1/Mol DNA.
Whereas form II DNA can be clearly recognized in the DNA distribution curve and at^94 Mol H1/Mol DNA (not s h o w n ) , it
sediments together with the 25 S component at > 9 4 Mol H1/Mol
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300
400
Moim/MotDNA
Pig. 1: Sedimentation coefficients B2 Q W of H1-DHA complexes
(about 80 % form I DNA) as a function of the input
Mol H1/Mol DNA ratio in 0.1 SSC, DNA concentration
approximately 30 /Ug/ml.
DNA from which it cannot be distinguished. The 120 S component sediments clearly more heterogeneously than the component with 25 S indicating the presence of H1 complexed associates as will be shown later by electron microscopy.
At the low salt concentration studied, H1 should be bound well
to DNA. Therefore, the three components observed should represent H1-DNA complexes, although there is no information on the
23
distribution of H1 molecules between them
. The 25 S component sediments only little faster than free DHA (23 S and
18 S for forms I and II at 0.1 SSC, respectively! This component is present also at high H1/DNA ratios at which DNA is not
expected to be free of H1 and should be therefore a H1-DNA
complex. This can also be derived from the electron microscopy
of this material shown in the next section. Furthermore, SgQ w
remains constant with growing H1 input. The increasing tnole5256
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1
'
1.0
|
0.8
0.6
0A
0.2
-
I
I
(1)
I
- if
-
-
0
i
i
50
100
i
150s20.w(S)
Pig. 2i Integral s 2 o w distributions of H1-free SV4O DNA (1)
and H1-S740 DNA complexes at 300 Mol Hi/Mol DNA (2) at
three different sedimentation times, DNA concentration
30 yug/ml, 0.1 SSC.
cular weight of the 25 S complexes, therefore, has to be compensated by increasing frictional ratios for holding s 2 w
values approximately constant. This indicates an asymmetrical
shape of the complexes (cf. electron microscopic results).
In Pig. 3 the percentages of the 25 S and 120 S components are given as a function of the molar H1/DNA ratio. Up
to /v 135 Mol H1/Mol DNA only the 25 S component is present
(including the 20 % of form II DNA-H1 complex which is not
shown for clarity). At about 135 Mol H1/Mol DNA the 120 S
component appears and increases continuously in quantity at
the expense of the 25 S component which diminishes. It should
be mentioned that there is a certain variation in the H1 concentration at which the 120 S component can be safely detected on the centrifuge scans. Because above 135 Mol H1/Mol
DNA both components are present simultaneously and show unchanged s 2 o w values, the 120 S component has to be formed
in a cooperative way. The amount of aggregated material with
8
2o w v a l u e 8 > 1000 S i8 somewhat uncertain at high H1/DNA
ratios. A part of this material is irreversibly pelletted at
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Pig. 3:
Proportions in
% of the 25 S
(•) and 120 S
(°) components
and of the aggregated material (•) from
Pig. 1 as a
function of the
input Mol
H1/Mol DNA
ratio, data obtained by moving
boundary sedimentation.
300
400
Mol H1/Mol DNA
the bottom of the centrifuge cell leading to somewhat shifted
proportions.
In another series of experiments we added the intercalating dye ethidium bromide (EB) to the H1-D2JA complexes at
20 mM N a + . The complexes should be sensitive to EB which unwinds DNA double helical and superhelical turns if properties
of the secondary and tertiary structure of DNA are involved in
the complex formation. In Pig. 4 82 w values of the observed
sedimenting components are given as a function of the Mol H1/
Mol DNA ratio and subsequently of the Mol iSB/ Mol nucleotide
ratio. As is shown in Pig. 4b, the formation of a rapidly sedimenting component with 60 - 100 S is favoured by £B if H1 is
present in a quantity which alone is not sufficient to induce
this component. Too low an H1 content does not result in rapidly sedimenting complexes after EB addition (Pig. 4 a ) . The
120 S component already present associates further in the
presence of iSB (Pig. 4c). There is another interesting aspect
regarding the 25 S component. As can be seen from Pig. 4b the
8
2o w v a l u e s o f this component decrease to approximately 15 S
in that range of &B/ nucleotide ratios in which the 60 - 100 S
component is formed. This effect was not observed at low and
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50 " a
25
0
20
WO
b
"20.W
IS)
50
50 100
200
100
°50 150 200 0
Mol H1/Mol DNA
0.1
0.2
0.3
MolEB/MoiNucl.
Pig. 4: Sedimentation coefficients S2o.w of H1-SV4O DNA complexes
after addition of ethidium bromide (SB) as a function of the inDUt Mol H1/Mol DHA and the input Mol EB/Mol nucleotide ratios.
DNA concentration 30 jug/ml, 0.1 S5C. i£B addition was started at
58 Mol H1/Hol DHA a),H6 Mol H1/MolDWA b)'and 234 Mol H1/
Mol DNA c)
high H1/DKA ratios (Pig. 4a, c). These findings might be indicative for a SB induced redistribution of H1 molecules. At
Mol EB/Mol nucleotide > 0.2 the rapidly Bedimenting component
breaks down, possibly as a result of beginning H1 release.
Pig. 5 shows the quantitative proportions of both complex
components given in Pig. 4b as a function of the Mol H1/Mol
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'50
100
150
Mol HI/MolDNA
0.1
0.2
0.3
0.4
Mol EB/Mol Nucl.
Pig. 5: Proportions in % of the 25 S (•) and (o) 60 - 100 S
components from Pig. 4b as a function, of the Mol H1/Mol DNA
and the Mol EB/Mol nucleotide ratios.
DNA and Mol EB/Mol nucleotide ratios. An interesting feature
of this plot is the appearance of a reversion point at 0.15
Mol EB/Mol nucleotide. Up to this point the amount of rapidly
sedimenting material increases, afterwards it decreases. The
slow component behaves inversely. This could be a result of
the suggested redistribution of H1 molecules induced by EB.
Prom the coexistence of the both different complexes it can
be concluded that the 60 - 100 S component is cooperatively
formed.
Electron microscopy
Electron microscopy was performed in parallel with sedimentation; the samples were taken from the centrifuge cell after thorough shaking it at different H1 and EB concentrations.
Pig. 6a shows SV40 DNA at 20 mM N a + as a control. In Pig. 6b
and c complexes of H1 and SV4O DNA at 146 and 234 Mol H1/DNA,
respectively, are given. The complexes at 146 Mol H1/Mol DNA
can clearly be distinguished from the superhelical DNA. The
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superhelix turns are less well visible and uniform threads
with double-fibrillar character of roughly half the DNA length
are formed. The double-fibrillar structure of these threads
can often be recognized only at few sites where open loops are
present. Besides these double-fibers aingle-fibrillar material
can be observed (Pig. 6 b ) . At 234 Mol H1/Mol DNA twisted associates of several of such double-fibers as a new characteristic structure can be seen (Pig. 6c). It seems that not more
than 4 - 7 double-fibers are packed side-by-side to a bundle
which appears either loosely or densely twisted. Besides bundles of roughly half the DNA length (as has to
be expected
for double-fibers), sometimes longer cable-lilce structures are
present which contain the DNA molecules connected in a headto-teil arrangement. The diameter of the densely twisted cables is markedly constant and amounts to about 13«0 nm in
shadowed specimens. The cables arise by a stronger twisting
or spinning of the loose bundles. Again, besides the bundles
and cables single- and double-fibrillar material is present.
The simultaneous presence of all these structures supports the
idea that the formation of double-fibers, bundles and cables
is cooperative.
Fig.
6d - f shows the complexes in the presence of EB.
The H1 concentration was taken to 146 Mol H1/Mol DNA, a concentration at which bundle-like structures are seldom observable. At an input ratio Mol BB/llol nucleotide of 0.048 the
formation of associates but not of typical bundles as described previously was seen (Pig. 6 d ) . Typical bundles and
especially cable-like structures were observed at 0.072 Mol
SB/Mol nucleotide (Pig. 6 e ) . These structures can hardly be
distinguished from those obtained in the presence of H1 alone.
Furthermore, single- and double-fibrillar material was present.
Pig. 6f shows complexes at 0.373 Mol SB/Mol nucleotide. According to the sedimentation results the associates break down
again at this high EB concentration. There is mainly present
double- and single-fibrillar material.
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DISCUSSION
The mode of interaction of histone H1 with DNA is complex
and depends on the ionic strength 4,20,23,24,26,27^ t h g s t r u c _
ture of DNA, linear (open circular) or superhelical
' ,
and probably the way to produce the complexes
. Here we
have studied the structure of complexes of histone H1 with
superhelical DNA prepared at 20 mM Na + by direct mixing. We
varied the H1 concentration in the H1-DNA mixtures by direct
titration of H1 into the centrifuge cell in order to obtain
information on effects on DNA by H1 itself. Variation of the
salt concentration at constant H1/DNA ratio as performed by
other authors was omitted.
A novel interesting result is the conversion of the
superhelical DNA molecule into a regular double-fibrillar
structure in the presence of H1 at molar input ratios < 135
Mol H1/Mol DNA ( < H 6 Mol H1/Mol DNA by electron microscopy)
and the formation of bundles of such double-fibrillar structures at > 135 Mol H1/Mol DNA (Pig. 6b, c ) . The formation
of the H1 mediated double-fibers seems not to result in pronounced changes of the sedimentation behaviour compared with
superhelical DNA. Double-fibers and superhelical DNA sediment
with 25 S and 23 S, respectively. Bundles of 4 - 7 such
double-fibers sediment with 120 S and increase in quantity
with increasing H1 concentration in a cooperative way. We
can only speculate about the mode of operation of H1 in such
double-fibrillar particles. It is necessary, however, first
to consider some relevant data from the literature.
28
Von Mickwitz et al.
were the first to demonstrate by elec-
tron microscopy similar double-chains in complexes of H1
and linear DNA at 20 mM N a + . Prom this study it can be inPig. 6: Electron micrographs of histone H1-SV40 DNA complexes.
SV40 DNA (about 80 % form I ) , control a ) , H1-SV40 DNA
complexes with input ratios of 146 Mol HI/Mol DNA b)
and 234 Mol H1/Mol DNA c ) . H1-SV40 DNA complexes with
input ratios of 146 Mol H1/Mol DNA after addition of
ethidium bromide (EB) in an input ratio of 0.048 Hoi
iili/liol nucleotide d ) , 0.072 Mol SB/Mol nucleotide e)
and 0.373 Mol SB/tool nucleotide f ) . All samples in
0.1 SSC buffer. The bars indicate 100 nm.
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ferred that these structures are produced by hairpin-like
intramolecular backfolding of the DNA on itself because of
the presence of loops at one end. These double-fibers form
cable-like structures at higher H1 content (£
1.05 w/w)
in analogy with the results described here. Sedimentation
measurements of Renz and Day
at similar H1 and salt con-
centration resulted in the appearance of two components with
sedimentation coefficients of 26 S and 80 S. The 80 S component was discussed to contain one DNA molecule and to be
formed by a. cooperative conformational transition from the
26 3 component. Two similar components were observed by
27
D'Anna et al.
and in this study after interaction of H1
with superhelical DNA. The parallel electron microscopical
representation enables us now to identify these components
as double-fibers (25 S) and bundleB of double-fibers (120 S)
or cables (heterogeneouely sedimenting material), respective28
ly, independently of the structure of the DNA as linear
or superhelical (present results). This is in contradiction
to Renz and Day
who discussed the 26 S component as H1-DNA
complex of DNA-like structure with the H1 molecules bound
randomly.
It seems surprising that the double-fibers sediment
similar to DNA. A big difference was found, however, in the
intrinsic viscosity C i ] of complexes of linear DNA and H1
between 0.3 mil and 15 ml/i N a + by Prisman et al. 1 7 » ^ 2 . [i?] decreases cooperatively by one-half, already at a histone/DNA
weight input ratio of 0.12 and does not change up to 0.35.
Whereas, evidently, the present sedimentation data do not
allow to identify a particular structure, this result might
be consistent with a strong shortening of the DNA contour
length as has to be expected for double-fibers. In this connection it should be noted that, analogously to Prisman et
32
al.
, double-fibers from calf thymus DNA can be demonstrated
electron microscopically down to 1 mM N a + (C.-U. v. Kickwitz,
G. Burckhardt, unpublished).
26
Knippers et al.
found that complexes of H1 and superhelical
DNA are cooperatively trapped on nitrocellulose filters at
20 mil N a + . A similar conclusion on cooperative complex forraa-
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tion can also be drawn from the data of Singer and Singer
23
although these authors do not discuss this problem. Linear
DNA complexed with H1 is bound to filters cooperatively above
We argue here that the formation of doub20 - 40 mM N a + .
le-fibers is a cooperative process although there is no direct
evidence from our experiments with superhelical DNA, compare
1 7 3 2
Manning
suggested a territorial binding mode of H1 to
DNA. Accordingly, the H1 molecule or especially the lysinerich C-terminal segment (residues 117 - 212) is bound in a
region of 7 8 in thickness which contains the condensed counterions and surrounds the DNA. H1 can move free of energy
along the DNA axis. This binding mode allows to explain such
known phenomena as selectivity of H1 for AT rich and high
molecular weight DNA
• . On the other hand, H1 is discussed to be preferentially bound to cross-over points of
opposite DNA segments of superhelical DNA arising from the
22
superhelix turns
. The apolar globular H1 region extending
from residues 39 - 116 is able to recognize specifically
these cross-overs.
We propose that cross-overs of opposite DNA segments are
cramped together by a crosslinlcing action of H1 which is
conceivable as polycationic bridge formed by different segments of the H1 molecule. Further crosslinking could be facilitated by the free mobility of H1 along the DNA axis
leading to a clustered, linear array of H1 molecules which
should be necessary to induce such regular structures as
double-fibers. This could be the basis for a cooperative
complex formation as suggested above. Polycationic H1 bridges
resulting in big DNA aggregates have already been postulated
9A
by other authors
.
The formation of bundles of double-fibers P I S O seems to
be a cooperative process as our data have revealed. It begins
formally at complete charge neutralization on a 1 : 1 input
basis of H1 lysines and DNA phosphates. The mechanism of
bundle formation is unclear as yet. One possibility would be
that it is a property of neutralized DNA molecules themselves
12
which tend to aggregate
. However, in this case, the known
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Nucleic Acids Research
donut-like structures are observed
12
. More attractive to us
seems a mechanism in which H1 is directly involved, e. g.
also in form of polycationic bridges. Bundles of doublefibers and cables at still higher H1 input might then be the
next higher level in H1 induced DNA packing.
Obviously, the process of bundle formation can also be
initiated at lower H1 input in the presence of ethidiutn bromide (Pig. 4 b ) . We have suggested that E 3 induces a redistribution of H1 molecules which leads to the formation of bundles and cables. If the H1 concentration is too low to form
bundles in the presence of E£ (Pig. 4a) and if bundles ore
already present (Pig. 4 c ) , no noticeable EB-induced redistribution of H1 is observed (the S 2 0w values of the 25 S component remain constant then, see ref. 34). The EB induced release
of a H1 ssgment which can react with another double-fiber
would already be sufficient to bring about the bundles.
V/hether an unwinding of the DHA double helix by EB intercalation or the introduction of a fixed positive charge by
ethidium leads to this H1 redistribution is not clear. It
should be mentioned, however, that 3B is able to split off
H1 histone from chromatin in the presence of salt (ionic
strength 0.3 H) ^' . At very high 3B concentration (> 0.15
Mol EB/Mol nucleotide) the bundles break down possibly as a
result of the beginning release of H1 histone.
The existence of double-fibrillar structures in this system
possibly maintained by a crosslinking function of H1 might
indicate that nucleosome double-fibers, as described repeatedly
'-* might be real structures. Here H1 histone
25
which r e p r e s e n t s a m u l t i f u n c t i o n a l p r o t e i n
possibly f u l f i l s a new i n t e r e s t i n g f u n c t i o n . Furthermore, the c r o s s l i n k i n g mechanism of H1 a t c r o s s - o v e r s of opposite DNA s e g ments as suggested here i s s i m i l a r to the binding mode of H1
at the entry and e x i t p o i n t of DHA a t the nucleosome as suggested by Allan e t e l .
RgPEREHCES
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