Volume 5 Number 12 December 1978
Nucleic Acids Research
An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size
Richard H.Simon, R.Daniel Camerini-Otero* and Gary Felsenfeld
Laboratory of Molecular Biology and *Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health,Bethesda, MD 20014, USA Received 3 October 1978
ABSTRACT Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.
INTRODUCTION There is now a large body of evidence that the arginine-rich histones, H3 and H4, play a central role in organizing the DNA of the nucleosome. Complexes of this histone pair with DNA have many of the properties of intact chromatin: digestion of the complex with nucleases gives rise to DNA fragments of discrete sizes that are a subset of those generated by digestion of chromatin (1, 2). Low angle x-ray diffraction of the complex reveals the same intensity maxima at 6.0, 3.5 and 2.8 nm that are seen in experiments with intact nucleosomes (3, 4). Furthermore, the H3/H4 pair is capable of inducing supercoiling in closed circular DNA (5, 6), and complexes of H3/H4 with circular DNA appear as beadlike structures in the electron microscope (6, 7). The arginine-rich histone pair are thus sufficient to induce many nucleosome-like characteristics in DNA. They are also necessary to this process; the other histones of the nucleosome core, H2A and H2B, do not affect DNA in this way, nor does any combination of histones from which either H3 or H4 is omitted (1, 2, 5). Although these results indicate that complexes containing the argininerich histone pair can deform DNA structure, they provide little detailed information about the stoichiometry of the DNA-histone complex or the
0 Information Retrieval Limited 1 Falconberg Court London WI V 5FG England
4805
Nucleic Acids Research extent of deformation.
To obtain this information,
we
have examined the
properties of complexes of H3/H4 with DNA of nucleosome "core" size, about 140 base pairs in length. We show that two distinct complexes are formed, one
containing
octamer.
a
tetramer of histones for each DNA molecule,
The former complex behaves hydrodynamically
ture, while the latter resembles
a
nucleosome in
as
an
the other
extended
an
struc-
compactness.
RESULTS Varying amounts of an equimolar mixture of H3 and H4 were reconstituted onto monomer DNA by gradient dialysis (see Methods). In some cases, losses of histone were observed during subsequent handling of the histone-DNA complex. For this reason, some of the reconstituted samples were fixed with
formaldehyde for comparison with the unfixed materials in the studies described below. Other precautions are described in Methods. The fixed or unfixed complexes were sedimented through a 5-20% linear sucrose gradient. Analysis of the optical absorbance at 260 nm revealed a total of three well-defined peaks; the relative amounts of material in each peak varied with the starting ratio of histones to DNA (Figs. 1 and 2).
Material from each peak was pooled. As shown in Table 1, the fastest moving peak (peak I) has a histone:DNA ratio of 1.0 gr/gr, consistent with the presence of eight histone molecules for each DNA molecule. The second peak (peak II) has a histone:DNA ratio of 0.5, while the slowest moving peak (III) has little or no histone. When the initial histone:DNA ratio is 0.5, all three components are present (Figs. la and b). At an initial ratio of 0.75, peaks I and II are observed (Fig. lc), but peak III, free DNA, is no longer present. Finally, at an initial ratio of 1.0, most of the complex sediments in peak I (Fig. 2). Although in earlier experiments (8) we experienced considerable losses of histone from the complex, recent modifications (see Methods) have resulted in much better recoveries. Thus, in the gradients shown in Fig. 2, we recover 80 to 90% of the DNA and 70 to 80% of the histone originally introduced into the reconstitution mixture. The material from peaks I and II was analyzed in the analytical ultra-
centrifuge.
As shown in Table 1, the median sedimentation coefficients
10.4S and 7.5S respectively. The fractions were relatively homoIn particular, the integral geneous in sedimentation behavior (Fig. 3). sedimentation coefficient distribution of peak I compares favorably in
were
sharpness to the distribution obtained when all four core histones (H2A, H2B, H3 and H4) are used in the reconstitution (cf. Ref. 9).
4806
Nucleic Acids Research DNA 00
8
1.0
0
0
E Ooo 0 0 ~ 000 000°° 0
X 0.5
0
00
~~~~0 ,0 o
Q
o
O.O
25 1.0
-
50
b DNA 0 0 0
~0.5
0 00 0
ii 0000 0 0
000
0
0
0
00000° 00000
00
00 O 9000,
25 1.0 -C
O
0 00
0
3 0.5
50
EHo
I
DNA
0000
0 O0
0
0 0
0
0
0
0
00000000 0000o ~~
0
~
~
~
~~on ~~~~~~0
0000000006 25 FRACTION NUMBER
Fig. 1.
50
Sucrose gradient sedimentation patterns of DNA complexes with
(See Methods.) Sedimentation is from right to left. a) Histone: DNA ratio 0.5 gr/gr. b) Same histone:DNA ratio as a), but fixed with formaldehyde before sedimentation (see Methods). c) Histone:DNA ratio 0.75 gr/gr, formaldehyde fixed. The arrow marks the position at which
H3/H4.
protein-free DNA sediments. The histone:DNA ratios given are the ratios of the starting material. All sucrose gradient sedimentation experiments were carried out as described in Methods (87,000 g for 62 hrs), except that shown in Fig. 1C, which was run at 112,000 x g for 45 hrs. The bars below each peak show the fractions pooled for subsequent study (see Figs. 3 and 4, Table 1). The histone:DNA ratios in peaks I and II are consistent with the presence respectively of a histone octamer and a tetramer bound to each 4807
Nucleic Acids Research a
DNA
I °
1.0
0
0
000 0
0
0.5
0oo O °0°0
00
00
00
25
b
0 0,0
00, 50
0
10 0
DNA
1.0 0 0
00
,0.5 0
0000 000 0 0
~~~~0 0 0 00 ~~ ~ ~ ~ °O ~~~~~~000
0
0
25
50
FRACTION NUMBER Fig. 2.
Sucrose gradient sedimentation patterns of DNA complexes with
H3/H4. a) Histone:DNA ratio 1.0 gr/gr. b) Same ratio as a), but formaldehyde fixed. DNA molecule. SDS gel electrophoresis of samples from both peaks show a 1:1 ratio of H3 to H4. To demonstrate that each complex molecule contains only one DNA molecule, i.e. that no DNA dimers or higher aggregates have formed, we measured the molecular weights of the complexes by short column equilibrium ultracentrifugation (Fig. 4, Table 1). The molecular
weights agree well with those expected for octamer and tetramer complexes with DNA. Although apparent specific volumes were estimated rather than measured, the results are not likely to be in error on this account by more than 10%, and molecules involving more than one DNA chain clearly are ruled out.
Complexes of monomer-size DNA with a tetramer of H3 and H4 were also prepared directly by removing H2A and H2B from nucleosome monomers. A
4808
Nucleic Acids Research Table 1 Comparison of calculated and measured molecular weights and to DNA ratios for complexes of H3/H4 with monomer length I
Calculated Mol. Weight (daltons) Measured Mol. Weight* (daltons)
Calculated Protein/DNA (g/g) Measured Protein/DNA (g/g)
s20,w (Svedberg units)
EDTA.
protein
DNA
DNA
II
(Naked)
(Octamer)
(Tetramer)
207,000
154,000
-
162,000
-
196,000-203,000 1.04
0.52
0
1.04-0.96
0.51
-
10.4
7.5
5.3
*Molecular weights were measured in 50 mM Tris-HCl, pH 8.0, 0.1 mM Calculated molecular weights were determined assuming .DNA of mole-
cular weight 100,500. Values of apparent specific volume were calculated assuming a linear relationship with composition (0.640, 0.621 and 0.55 for peaks I and II, and for DNA; apparent specific volume = 0.74 for pure protein). Comparison of the calculated apparent specific volumes with published measurements for the nucleosome (0.661, Ref. 10) indicates that the measured molecular weights might be at most 10% too low.
concentrated solution of nucleosomes was dialyzed into 1.25 M NaCl, 10 mM Tris, pH 8, 0.1 mM Na 2EDTA. Under such conditions, H3 and H4 remain bound (This was demonstrated by to the DNA, but H2A and H2B are released. chromatography of the complex under these salt conditions on Bio-gel A-5m, followed by SDS gel electrophoresis of the fractions.) The sample was loaded directly into an analytical ultracentrifuge cell for sedimentawas tion velocity analysis. The median sedimentation coefficient of a with value found to sediment was 6.3S; no material in the boundary greater than 8.3S. s20 Samples from peaks I and II in Figs. 1 and 2 were digested with staphylococcal nuclease in approximately 10% sucrose, 30 mM Tris, pH 8, 0.1 mM CaCl 2. Only samples that had not been treated with formaldehyde were
(s20,w)
used.
The reaction
was
allowed to proceed to completion.
The limit digests,
4809
Nucleic Acids Research 1.0 r
0.SF
0.71
g 0.6
E055p -
W
0.4 03
02 -
oQF 7
8
9
10
11
12
13
SEDIMENTATION COEFFICIENT
) distribution of three Fig. 3. Integral sedimentation coefficient (s2 fractions isolated from the sucrose gradients o 'rigs. 1 and 2. o Peak II of Fig. lb (H3/H4 tetramer-DNA complex, formaldehyde fixed). A Peak I of Fig. 2a (H3/H4 octamer-DNA complex, unfixed). * Peak I of Fig. Zb (H3/H4 octamer-DNA complex, formaldehyde fixed). Arrows mark the median values of a20,w when examined in gels, displayed the regular array of double stranded fragments, predominantly about 68 base pairs in size and smaller, previously observed for limit digests of H3/H4 reconstitutes (1).
DISCUSSION Our results show that the arginine-rich histone pair, H3-H4, are capable of forming at least two discrete complexes with DNA of nucleosome core size. At low ratios of histone to DNA, the predominant species contains a tetramer of histones (two each of H3 and H4) and one DNA molecule (Fig. la, peak II). During recQnstitution the tetramer-DNA complex is in equilibrium with protein-free DNA; there is no evidence of any major intermediate species containing less than a tetramer of H3-H4. As the overall histone:DNA ratio is increased (Fig. lc) the protein-free DNA component
4810
Nucleic Acids Research
00.00 Tetramer
-1.00.
-2.001
3
4900
5000
51.00
52.00
50.00
51.00
52.00
O0tw
-1.00
3
-3.00
49.00
Fig. 4. Short column equilibrium centrifugation at 10,000 rpm of DNAH3/H4 complexes. Log (absorbance) is plotted vs. the square of the distance from the center of the rotor. a) Peak II of Fig. lb (H3/H4 tetramerDNA complex, formaldehyde fixed). b) Peak I of Fig. 2a (H3/H4 octamer-DNA complex, unfixed).
4811
Nucleic Acids Research disappears, and a new complex containing a molecule of DNA and an octamer of histones is observed. At the highest histone:DNA ratios, the octamerDNA complex is predominant (Fig. 2). It is possible that there is a minor intermediate hexamer-DNA species sedimenting between the positions of tetramer and octamer, but our methods do not have sufficient resolution to distinguish it from a mixture of tetramer- and octamer-DNA complexes. Similarly, it is possible that histone-DNA complexes containing more than an octamer or less than a tetramer of H3/H4 are present in small amounts. The composition of the sedimenting components in Fig. 1 is determined unambiguously by the measurement of their histone and DNA content, and by their molecular weight (Table 1, Fig. 4). The analysis of the sedimentation coefficient distribution (Fig. 3) suggests that each of the two components is relatively homogeneous. It is obvious that the octamer-DNA complex is a compact structure, since its median sediment coefficient, 10.4S, is quite close to that of the nucleosome core, which has a sedimentation coefficient of about llS, and contains approximately the same amount of protein and DNA as the H3/H4 octamer complex. A quantitative statement of these results is presented in Table 2A, which gives the frictional coefficients and frictional ratios (f/fo) for the octamer- and tetramer-DNA complexes. To give a more concrete representation of the expected dependence of sedimentation coefficient on shape, Table 2B shows the expected sedimentation behavior of idealized unfolded and folded tetramer and octamer species. It is again clear from the data in Table 2 that the octamer-DNA is a folded structure. The sedimentation behavior of the tetramer-DNA (Table 2) is different from that of the octamer-DNA. Its frictional ratio is greater than that of the octamer-DNA, and it sediments more slowly than would be expected for a compact nucleosome-shaped molecule possessing the mass and apparent specific volume of the tetramer complex (Table 2B). On the other hand, it does not sediment as slowly as might be expected for an idealized fully extended molecule. It should be recalled that large frictional ratios are associated either with molecular asymmetry or large amounts of hydration, so that although these data suggest that the tetramer-DNA complex is a rather extended structure, they cannot provide conclusive evidence. Recently, however, Klevan et al. (11) have used electric dichroism to measure the rotational relaxation time of this same H3/H4 tetramer-DNA complex, and find that the observed value is consistent only with an elongated molecule.
4812
Nucleic Acids Research Table 2 A)
Frictional Properties
Octamer (Peak I)
Tetramer (Peak
1.65 (1.54)
f (g/sec) 1.15 (1.08)
f/fo
B)
Nucleosome
DNA
2.12 (1.93)
1.61 (1.50)
2.69
1.36 (1.26)
1.14 (1.08)
1.42
II)
Comparison with Model Structures*
Sedimentation Coefficient (Svedberg units)
Octamer
Tetramer
Measured
10.4
7.5
8.7 (8.2)
Calculated, unfolded
6.9 (6.3)
Nucleosome
11.0
8.8 (8.3)
structure
10.9
Calculated, folded
8.5 (8.4>
-
structure
*The calculations are made assuming that the frictional coefficients of folded and unfolded structures are the same as that of the nucleosome and DNA respectively, but with the appropriate mass and apparent specific volume, *'. Assumed values are the same as those given in Table 1. The values in
parentheses were obtained using for the octamer the value of O' given by Olins (10) for the nucleosome (0.661) and for the tetramer the value of 0.65 used by Klevan et al. (11).
The value of the dichroism itself leads Klevan et al. to suggest that the DNA is supercoiled, but with a very large pitch, so that the overall length of the particle is not much less than that of a fully extended DNA molecule. It should be noted that the frictional ratio and protein:DNA ratio reported by Klevan et al. for the tetramer-DNA complex agree well with those reported in Table 2B and reference 8. These authors also observe more rapidly sedimenting components containing an octamer of H3/H4, corresponding to peak I in Figure 1. Similar results with respect to the octamer have been obtained by Stockley and Thomas (J. 0. Thomas, personal communi-
cation). Our
results
as
well
as
those of Klevan et al. differ from those of
Bina-Stein and Simpson (6) and Bina-Stein (12), who reported a tetramercontaining particle sedimenting at 9.8S. In none of our experiments did we observe a tetramer-DNA complex sedimenting faster than 7.5S; the faster 4813
Nucleic Acids Research moving particle sedimenting in the neighborhood of 10S always had a protein content equivalent to an octamer of histones. The solvent used in the
final stage of reconstitution and in all physical measurements was 50 mM Tris in our studies. Bina-Stein and Simpson used 0.2 M NaCl in reconstitution and 0.1 M NaCl for their measurements. We have repeated our experiments using their conditions, but we were unable to detect any 10S tetramerDNA species; under these salt conditions the majority of the nucleoprotein particles are aggregated and precipitate. We have also varied the experimental conditions in other ways in an attempt to form a tightly folded tetramer complex. Addition of Ca , Mg , or spermidine, reconstitution in the presence or absence of a-mercaptoethanol, and incubation at 37°C all have no effect on the conformation of the tetramer. It seems unlikely that the reconstitution procedure in itself is affecting the outcome of these experiments, since we find identical behavior in H3/H4 complexes that are formed by stripping the other histones from nucleosome cores. We and others have previously reported that the H3/H4 histone pair, when reconstituted with high molecular weight DNA, form complexes with many properties of the nucleosome, as discussed in the Introduction. In all of the published experiments carried out in our laboratory, such properties are elicited using 0.5 grams of histone per gram of DNA. In the experiments with 140 base pair DNA reported here (Fig. 1) and by Klevan et al. (11), the tetramer is the predominant species at this protein:DNA ratio. Our staphylococcal nuclease digestion experiments with this purified complex show that a tetramer of H3/H4 is sufficient to allow generation of many of the nuclease resistant fragments characteristic of complete nucleosomes. It may be more difficult, however, to prove that it is the tetramer, rather than the octamer, which induces supercoiling in closed circular DNA, though the model of Klevan et al. implies that some supercoiling may occur in the tetramer complex. We are thus led to the conclusion that the H3/H4 tetramer, though not capable of tightly folding the DNA into a nucleosome-like conformation, nonetheless possesses many of the other structural features of the nucleosome. Furthermore, the tetramer-DNA complex can be folded readily into the very compact structure by addition of other histones: if H2A and H2B are added, nucleosomes are formed (5, 11); if more H3 and H4 are added, the H3/H4 octamer complex is formed. As we have shown here, this octamer-DNA complex is similar in its properties to a normal nucleosome, and it is the only fully compact structure formed by H3/H4 and DNA.
4814
Nucleic Acids Research METHODS DNA monomer preparation Triton X-100 washed nuclei were prepared from duck erythrocytes as previously described (1). The nuclei were digested with staphylococcal nuclease (Worthington Biochemicals), 30 units/ml, at a DNA concentration of 0.5-1.0 mg/ml in 0.25 M sucrose, 1 mM Tris-HCl (pH 8.0), 0.3 mM CaCl2 at 370C. At the time when 15% of the DNA had become soluble in 0.8 M perchloric acid, 0.8 M NaCl (1), the digestion was stopped by adding Na2EDTA to a final concentration of 1 mM and cooling on ice. Nucleosomes were then isolated on sucrose gradients or by chromatography on Bio-gel A-5m. The DNA was isolated as previously described (1). The DNA, called "monomer" DNA, was electrophoresed on 4% polyacrylamide slab gels (1) and found to migrate as two distinct bands of 140 and 160 base pairs in length when stained with "Stains-all" (Eastman). The gels were photographed and the negative scanned with a Joyce-Loebl microdensitometer to allow calculation of the weight average molecular weight. The weight-average molecular weight for the preparation used was 150 b.p. ± 5 b.p. Preparation of histones
Histones H3/H4 and H2A/H2B were prepared by selective salt stripping of sheared duck erythrocyte chromatin that had been bound to a hydroxylapatite column (R. H. Simon and G. Felsenfeld, manuscript in preparation). The purity of the histone fractions was assessed by electrophoresis in SDS on polyacrylamide slab gels (Laemmli (13), as modified by Weintraub et al. (14)). The gels were stained with Coomassie blue, photographed, and the negative scanned on a Joyce-Loebl microdensitometer. The H2A/H2B and H3/H4 were contaminated with less than 2% of the opposite pair. To ensure that no histone was selectively lost during preparation of the various fractions, the ratios of the peaks of the individual histones in each fraction were compared and found to vary less than 10% from those present in chromatin. When H2A/H2B and H3/H4 separated in this way were mixed in equimolar proportions and reconstituted onto monomer size DNA at a ratio of 1 gram of protein to 1 gram of DNA, 60% or more of the DNA
sedimented as an llS particle. Determination of concentrations of DNA and histones DNA concentration was measured by its absorbance at 260 nm using a molar extinction of 6700 1 cm 1mol1. The protein concentrations of solutions of core histones and of H2A/H2B were determined by their absorbance at 230 nm using an extinction coefficient of 3.3 cm2mg 1 for both (15). 4815
Nucleic Acids Research To
measure
the protein content of H3/H4-containing nucleoprotein complexes,
the Lowry procedure (16)
was
used,
as
modified by Bensadoun and Weinstein
(17) to avoid interference from Tris-HCl, were
assayed using
a
The samples sucrose, and Na2EDTA. highly purified sample of H3/H4 as a primary standard.
The nitrogen content of a solution of this material was determined by Kjeldahl analysis, and the protein concentration calculated from the published amino acid sequences (18). Bovine serum albumin (Miles Labora-
tories), used as a secondary standard, gave an optical density Lowry assay 0.825 as large as the same weight of H3/H4.
in the
Reconstitution procedure
DNA at
a
final A260 ranging from 1
to
20
was
added
to
5
M
urea,
2 M
NaCl, 10 mM Tris-HCl (pH 8.0) 0.1 mM EDTA, 10 mM 2-mercaptoethanol. Histones were then added and the mixture dialyzed for 16 hours against the The nucleoprotein was then same solution at 4°C on a rocking dialyzer.
sequentially dialyzed against 1.2 M, 1.0 M, 0.8 M, and 0.6 M NaCl for 80 minute time intervals without altering the concentration of urea, Tris-HCl, Next the urea was removed by dialysis against 0.6 M NaCl, 10 or Na2EDTA. mM Tris-HCl, 0.1 mMl Na2EDTA and finally the reconstitution was completed by dialysis into 50 mM Tris-HCl (pH 8.0), 0.1 mM Na 2EDTA. Sedimentation Analysis H3/H4-monomer DNA reconstitutes were layered over a 5-20% linear sucorse gradient in 50 mM Tris-HCl (pH 8.0), 0.1 mM Na 2EDTA and centrifuged in an SW-27 rotor at 87,000 x g for 62 hours at 4°C in a Beckman L2-65 ultracentrifuge. Fractions were collected and monitored by UV absorption at 230 and 260 nm and by Lowry protein assay.
Boundary sedimentation velocity and equilibrium experiments were performed on a Model E analytical ultracentrifuge equipped with ultraviolet optics. The base line was determined by measuring the A265 of the column of solution just outside the inner meniscus after centrifuging at 44,000 rpm for two hours and then reducing the speed to that of the original Where indicated, the apparent specific volume was calculated as the weighted average of the values for DNA and protein. The apparent specific run.
volume used for DNA
was 0.55 ml
gm
(19), and for H3/H4 was 0.74 ml gm 1
assuming that the protein behaves as an average globular protein. all cases, the solvent was 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.
In
Precautions against loss of histone We found that under certain conditions the nucleohistone complexes
4816
Nucleic Acids Research loss of H3 and H4 during either dialysis or storage following Such losses, which were time-dependent, sucrose gradient sedimentation. led to an apparent decrease in the histone:DNA ratio, and might cause a sericus error in determination of the stoichiometry of the complex. In our earlier experiments (8) and some of those reported here, we avoided such losses by fixing the complexes with formaldehyde before sucrose gradient sedimentation. This was done by dialyzing the reconstitute against 10 mM sodium phosphate buffer, pH 6.7, 0.1 mM Na2EDTA, without removing it from the reconstitution dialysis bag. One per cent formaldehyde in this buffer was then dialyzed into the bag and allowed to react for 16 hrs at 4°C. Excess formaldehyde was removed by dialysis against fresh phosphate buffer, and finally this was replaced by 50 mM Tris-HCl, pH 8.0, 0.1 mM Na2EDTA. The fixed material could be handled without loss of histone. As shown above, fixation does not affect the hydrodynamic properties
suffered
a
of the nucleoprotein fractions isolated on sucrose gradients. We have found more recently that preferential losses of H3 and H4 can be minimized, in the absence of formaldehyde fixation, by use of polyallomer (rather than cellulose nitrate) tubes for preparative centrifugation, and by the use of polycarbonate or siliconized glass tubes for the collection of fractions. With such precautions, recoveries obtained with and without fixation are virtually identical (see Figs. 1 and 2 above).
Miscellaneous Nuclei, chromatin and nucleoprotein reconstitutes were digested with staphylococcal nuclease by the procedure of Camerini-Otero et al. (1). The digests were deproteinized and electrophoresed on polyacrylamide slab gels as described previously (1).
ACKNOWLEDGMiENT We are grateful to Dr. D. Crothers and to Dr. J. 0. Thomas for communicating to us their results either in press or in preparation for publication. REFERENCES Camerini-Otero, R. D., Sollner-Webb, B. and Felsenfeld, G. (1976) Cell 1 8, 333-347. 2 Sollner-Webb, B., Camerini-Otero, R. D. and Felsenfeld, G. (1976) Cell 9, 179-193. Boseley, P. G., Bradbury, E. M., Butler-Browne, G. S., Carpenter, B. G. 3 and Stephens, R. M. (1976) Eur. J. Biochem. 62, 21-31. 4 Moss, T., Stephens, R. M., Crane-Robinson, L. and Bradbury, E. M. (1977) Nucl. Acids Res. 4, 2477-2485.
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Nucleic Acids Research 5 6 7
8 9
10 11
12 13 14 15 16 17 18 19
4818
Camerini-Otero, R. D. and Felsenfeld, G. (1977) Nucl. Acids. Res. 4, 1159-1181. Bina-Stein, M. and Simpson, R. T. (1977) Cell 11, 609-618. Oudet, P., Germond, J. P., Sures, M., Gallwitz, D., Bellard, M. and Chambon, P. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 287-300. Camerini-Otero, R. D., Sollner-Webb, B., Simon, R. H., Williamson, P., Zasloff, M. and Felsenfeld, G. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 57-75. Camerini-Otero, R. D. and Felsenfeld, G. (1977) Proc. Natl. Acad. Sci. USA 74, 5519-5523. Olins, A. L., Carlson, R. D., Wright, E. B. and Olins, D. E. (1976) Nucl. Acids Res. 3, 3271-3291. Klevan, L., Dattagupta, N., Hogan, M. and Crothers, D. M. (1978) Biochemistry 17, 4533-4540. Bina-Stein, M. (1978) J. Biol. Chem. 253, 5213-5219. Laemmli, U. K. (1970) Nature 227, 680-685. Weintraub, H., Palter, K. and Van Lente, F. (1975) Cell 6, 85-110. Hnilica, L. S. (1975) Methods in Enzymol. 40, 102-138. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241-250. Elgin, S. C. R. and Weintraub, H. (1975) Ann. Rev. Biochem. 44, 725-774. Cohen, G. and Eisenberg, H. (1968) Biopolymers 6, 1077-1100.
Nucleic Acids Research
An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size
Richard H.Simon, R.Daniel Camerini-Otero* and Gary Felsenfeld
Laboratory of Molecular Biology and *Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health,Bethesda, MD 20014, USA Received 3 October 1978
ABSTRACT Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.
INTRODUCTION There is now a large body of evidence that the arginine-rich histones, H3 and H4, play a central role in organizing the DNA of the nucleosome. Complexes of this histone pair with DNA have many of the properties of intact chromatin: digestion of the complex with nucleases gives rise to DNA fragments of discrete sizes that are a subset of those generated by digestion of chromatin (1, 2). Low angle x-ray diffraction of the complex reveals the same intensity maxima at 6.0, 3.5 and 2.8 nm that are seen in experiments with intact nucleosomes (3, 4). Furthermore, the H3/H4 pair is capable of inducing supercoiling in closed circular DNA (5, 6), and complexes of H3/H4 with circular DNA appear as beadlike structures in the electron microscope (6, 7). The arginine-rich histone pair are thus sufficient to induce many nucleosome-like characteristics in DNA. They are also necessary to this process; the other histones of the nucleosome core, H2A and H2B, do not affect DNA in this way, nor does any combination of histones from which either H3 or H4 is omitted (1, 2, 5). Although these results indicate that complexes containing the argininerich histone pair can deform DNA structure, they provide little detailed information about the stoichiometry of the DNA-histone complex or the
0 Information Retrieval Limited 1 Falconberg Court London WI V 5FG England
4805
Nucleic Acids Research extent of deformation.
To obtain this information,
we
have examined the
properties of complexes of H3/H4 with DNA of nucleosome "core" size, about 140 base pairs in length. We show that two distinct complexes are formed, one
containing
octamer.
a
tetramer of histones for each DNA molecule,
The former complex behaves hydrodynamically
ture, while the latter resembles
a
nucleosome in
as
an
the other
extended
an
struc-
compactness.
RESULTS Varying amounts of an equimolar mixture of H3 and H4 were reconstituted onto monomer DNA by gradient dialysis (see Methods). In some cases, losses of histone were observed during subsequent handling of the histone-DNA complex. For this reason, some of the reconstituted samples were fixed with
formaldehyde for comparison with the unfixed materials in the studies described below. Other precautions are described in Methods. The fixed or unfixed complexes were sedimented through a 5-20% linear sucrose gradient. Analysis of the optical absorbance at 260 nm revealed a total of three well-defined peaks; the relative amounts of material in each peak varied with the starting ratio of histones to DNA (Figs. 1 and 2).
Material from each peak was pooled. As shown in Table 1, the fastest moving peak (peak I) has a histone:DNA ratio of 1.0 gr/gr, consistent with the presence of eight histone molecules for each DNA molecule. The second peak (peak II) has a histone:DNA ratio of 0.5, while the slowest moving peak (III) has little or no histone. When the initial histone:DNA ratio is 0.5, all three components are present (Figs. la and b). At an initial ratio of 0.75, peaks I and II are observed (Fig. lc), but peak III, free DNA, is no longer present. Finally, at an initial ratio of 1.0, most of the complex sediments in peak I (Fig. 2). Although in earlier experiments (8) we experienced considerable losses of histone from the complex, recent modifications (see Methods) have resulted in much better recoveries. Thus, in the gradients shown in Fig. 2, we recover 80 to 90% of the DNA and 70 to 80% of the histone originally introduced into the reconstitution mixture. The material from peaks I and II was analyzed in the analytical ultra-
centrifuge.
As shown in Table 1, the median sedimentation coefficients
10.4S and 7.5S respectively. The fractions were relatively homoIn particular, the integral geneous in sedimentation behavior (Fig. 3). sedimentation coefficient distribution of peak I compares favorably in
were
sharpness to the distribution obtained when all four core histones (H2A, H2B, H3 and H4) are used in the reconstitution (cf. Ref. 9).
4806
Nucleic Acids Research DNA 00
8
1.0
0
0
E Ooo 0 0 ~ 000 000°° 0
X 0.5
0
00
~~~~0 ,0 o
Q
o
O.O
25 1.0
-
50
b DNA 0 0 0
~0.5
0 00 0
ii 0000 0 0
000
0
0
0
00000° 00000
00
00 O 9000,
25 1.0 -C
O
0 00
0
3 0.5
50
EHo
I
DNA
0000
0 O0
0
0 0
0
0
0
0
00000000 0000o ~~
0
~
~
~
~~on ~~~~~~0
0000000006 25 FRACTION NUMBER
Fig. 1.
50
Sucrose gradient sedimentation patterns of DNA complexes with
(See Methods.) Sedimentation is from right to left. a) Histone: DNA ratio 0.5 gr/gr. b) Same histone:DNA ratio as a), but fixed with formaldehyde before sedimentation (see Methods). c) Histone:DNA ratio 0.75 gr/gr, formaldehyde fixed. The arrow marks the position at which
H3/H4.
protein-free DNA sediments. The histone:DNA ratios given are the ratios of the starting material. All sucrose gradient sedimentation experiments were carried out as described in Methods (87,000 g for 62 hrs), except that shown in Fig. 1C, which was run at 112,000 x g for 45 hrs. The bars below each peak show the fractions pooled for subsequent study (see Figs. 3 and 4, Table 1). The histone:DNA ratios in peaks I and II are consistent with the presence respectively of a histone octamer and a tetramer bound to each 4807
Nucleic Acids Research a
DNA
I °
1.0
0
0
000 0
0
0.5
0oo O °0°0
00
00
00
25
b
0 0,0
00, 50
0
10 0
DNA
1.0 0 0
00
,0.5 0
0000 000 0 0
~~~~0 0 0 00 ~~ ~ ~ ~ °O ~~~~~~000
0
0
25
50
FRACTION NUMBER Fig. 2.
Sucrose gradient sedimentation patterns of DNA complexes with
H3/H4. a) Histone:DNA ratio 1.0 gr/gr. b) Same ratio as a), but formaldehyde fixed. DNA molecule. SDS gel electrophoresis of samples from both peaks show a 1:1 ratio of H3 to H4. To demonstrate that each complex molecule contains only one DNA molecule, i.e. that no DNA dimers or higher aggregates have formed, we measured the molecular weights of the complexes by short column equilibrium ultracentrifugation (Fig. 4, Table 1). The molecular
weights agree well with those expected for octamer and tetramer complexes with DNA. Although apparent specific volumes were estimated rather than measured, the results are not likely to be in error on this account by more than 10%, and molecules involving more than one DNA chain clearly are ruled out.
Complexes of monomer-size DNA with a tetramer of H3 and H4 were also prepared directly by removing H2A and H2B from nucleosome monomers. A
4808
Nucleic Acids Research Table 1 Comparison of calculated and measured molecular weights and to DNA ratios for complexes of H3/H4 with monomer length I
Calculated Mol. Weight (daltons) Measured Mol. Weight* (daltons)
Calculated Protein/DNA (g/g) Measured Protein/DNA (g/g)
s20,w (Svedberg units)
EDTA.
protein
DNA
DNA
II
(Naked)
(Octamer)
(Tetramer)
207,000
154,000
-
162,000
-
196,000-203,000 1.04
0.52
0
1.04-0.96
0.51
-
10.4
7.5
5.3
*Molecular weights were measured in 50 mM Tris-HCl, pH 8.0, 0.1 mM Calculated molecular weights were determined assuming .DNA of mole-
cular weight 100,500. Values of apparent specific volume were calculated assuming a linear relationship with composition (0.640, 0.621 and 0.55 for peaks I and II, and for DNA; apparent specific volume = 0.74 for pure protein). Comparison of the calculated apparent specific volumes with published measurements for the nucleosome (0.661, Ref. 10) indicates that the measured molecular weights might be at most 10% too low.
concentrated solution of nucleosomes was dialyzed into 1.25 M NaCl, 10 mM Tris, pH 8, 0.1 mM Na 2EDTA. Under such conditions, H3 and H4 remain bound (This was demonstrated by to the DNA, but H2A and H2B are released. chromatography of the complex under these salt conditions on Bio-gel A-5m, followed by SDS gel electrophoresis of the fractions.) The sample was loaded directly into an analytical ultracentrifuge cell for sedimentawas tion velocity analysis. The median sedimentation coefficient of a with value found to sediment was 6.3S; no material in the boundary greater than 8.3S. s20 Samples from peaks I and II in Figs. 1 and 2 were digested with staphylococcal nuclease in approximately 10% sucrose, 30 mM Tris, pH 8, 0.1 mM CaCl 2. Only samples that had not been treated with formaldehyde were
(s20,w)
used.
The reaction
was
allowed to proceed to completion.
The limit digests,
4809
Nucleic Acids Research 1.0 r
0.SF
0.71
g 0.6
E055p -
W
0.4 03
02 -
oQF 7
8
9
10
11
12
13
SEDIMENTATION COEFFICIENT
) distribution of three Fig. 3. Integral sedimentation coefficient (s2 fractions isolated from the sucrose gradients o 'rigs. 1 and 2. o Peak II of Fig. lb (H3/H4 tetramer-DNA complex, formaldehyde fixed). A Peak I of Fig. 2a (H3/H4 octamer-DNA complex, unfixed). * Peak I of Fig. Zb (H3/H4 octamer-DNA complex, formaldehyde fixed). Arrows mark the median values of a20,w when examined in gels, displayed the regular array of double stranded fragments, predominantly about 68 base pairs in size and smaller, previously observed for limit digests of H3/H4 reconstitutes (1).
DISCUSSION Our results show that the arginine-rich histone pair, H3-H4, are capable of forming at least two discrete complexes with DNA of nucleosome core size. At low ratios of histone to DNA, the predominant species contains a tetramer of histones (two each of H3 and H4) and one DNA molecule (Fig. la, peak II). During recQnstitution the tetramer-DNA complex is in equilibrium with protein-free DNA; there is no evidence of any major intermediate species containing less than a tetramer of H3-H4. As the overall histone:DNA ratio is increased (Fig. lc) the protein-free DNA component
4810
Nucleic Acids Research
00.00 Tetramer
-1.00.
-2.001
3
4900
5000
51.00
52.00
50.00
51.00
52.00
O0tw
-1.00
3
-3.00
49.00
Fig. 4. Short column equilibrium centrifugation at 10,000 rpm of DNAH3/H4 complexes. Log (absorbance) is plotted vs. the square of the distance from the center of the rotor. a) Peak II of Fig. lb (H3/H4 tetramerDNA complex, formaldehyde fixed). b) Peak I of Fig. 2a (H3/H4 octamer-DNA complex, unfixed).
4811
Nucleic Acids Research disappears, and a new complex containing a molecule of DNA and an octamer of histones is observed. At the highest histone:DNA ratios, the octamerDNA complex is predominant (Fig. 2). It is possible that there is a minor intermediate hexamer-DNA species sedimenting between the positions of tetramer and octamer, but our methods do not have sufficient resolution to distinguish it from a mixture of tetramer- and octamer-DNA complexes. Similarly, it is possible that histone-DNA complexes containing more than an octamer or less than a tetramer of H3/H4 are present in small amounts. The composition of the sedimenting components in Fig. 1 is determined unambiguously by the measurement of their histone and DNA content, and by their molecular weight (Table 1, Fig. 4). The analysis of the sedimentation coefficient distribution (Fig. 3) suggests that each of the two components is relatively homogeneous. It is obvious that the octamer-DNA complex is a compact structure, since its median sediment coefficient, 10.4S, is quite close to that of the nucleosome core, which has a sedimentation coefficient of about llS, and contains approximately the same amount of protein and DNA as the H3/H4 octamer complex. A quantitative statement of these results is presented in Table 2A, which gives the frictional coefficients and frictional ratios (f/fo) for the octamer- and tetramer-DNA complexes. To give a more concrete representation of the expected dependence of sedimentation coefficient on shape, Table 2B shows the expected sedimentation behavior of idealized unfolded and folded tetramer and octamer species. It is again clear from the data in Table 2 that the octamer-DNA is a folded structure. The sedimentation behavior of the tetramer-DNA (Table 2) is different from that of the octamer-DNA. Its frictional ratio is greater than that of the octamer-DNA, and it sediments more slowly than would be expected for a compact nucleosome-shaped molecule possessing the mass and apparent specific volume of the tetramer complex (Table 2B). On the other hand, it does not sediment as slowly as might be expected for an idealized fully extended molecule. It should be recalled that large frictional ratios are associated either with molecular asymmetry or large amounts of hydration, so that although these data suggest that the tetramer-DNA complex is a rather extended structure, they cannot provide conclusive evidence. Recently, however, Klevan et al. (11) have used electric dichroism to measure the rotational relaxation time of this same H3/H4 tetramer-DNA complex, and find that the observed value is consistent only with an elongated molecule.
4812
Nucleic Acids Research Table 2 A)
Frictional Properties
Octamer (Peak I)
Tetramer (Peak
1.65 (1.54)
f (g/sec) 1.15 (1.08)
f/fo
B)
Nucleosome
DNA
2.12 (1.93)
1.61 (1.50)
2.69
1.36 (1.26)
1.14 (1.08)
1.42
II)
Comparison with Model Structures*
Sedimentation Coefficient (Svedberg units)
Octamer
Tetramer
Measured
10.4
7.5
8.7 (8.2)
Calculated, unfolded
6.9 (6.3)
Nucleosome
11.0
8.8 (8.3)
structure
10.9
Calculated, folded
8.5 (8.4>
-
structure
*The calculations are made assuming that the frictional coefficients of folded and unfolded structures are the same as that of the nucleosome and DNA respectively, but with the appropriate mass and apparent specific volume, *'. Assumed values are the same as those given in Table 1. The values in
parentheses were obtained using for the octamer the value of O' given by Olins (10) for the nucleosome (0.661) and for the tetramer the value of 0.65 used by Klevan et al. (11).
The value of the dichroism itself leads Klevan et al. to suggest that the DNA is supercoiled, but with a very large pitch, so that the overall length of the particle is not much less than that of a fully extended DNA molecule. It should be noted that the frictional ratio and protein:DNA ratio reported by Klevan et al. for the tetramer-DNA complex agree well with those reported in Table 2B and reference 8. These authors also observe more rapidly sedimenting components containing an octamer of H3/H4, corresponding to peak I in Figure 1. Similar results with respect to the octamer have been obtained by Stockley and Thomas (J. 0. Thomas, personal communi-
cation). Our
results
as
well
as
those of Klevan et al. differ from those of
Bina-Stein and Simpson (6) and Bina-Stein (12), who reported a tetramercontaining particle sedimenting at 9.8S. In none of our experiments did we observe a tetramer-DNA complex sedimenting faster than 7.5S; the faster 4813
Nucleic Acids Research moving particle sedimenting in the neighborhood of 10S always had a protein content equivalent to an octamer of histones. The solvent used in the
final stage of reconstitution and in all physical measurements was 50 mM Tris in our studies. Bina-Stein and Simpson used 0.2 M NaCl in reconstitution and 0.1 M NaCl for their measurements. We have repeated our experiments using their conditions, but we were unable to detect any 10S tetramerDNA species; under these salt conditions the majority of the nucleoprotein particles are aggregated and precipitate. We have also varied the experimental conditions in other ways in an attempt to form a tightly folded tetramer complex. Addition of Ca , Mg , or spermidine, reconstitution in the presence or absence of a-mercaptoethanol, and incubation at 37°C all have no effect on the conformation of the tetramer. It seems unlikely that the reconstitution procedure in itself is affecting the outcome of these experiments, since we find identical behavior in H3/H4 complexes that are formed by stripping the other histones from nucleosome cores. We and others have previously reported that the H3/H4 histone pair, when reconstituted with high molecular weight DNA, form complexes with many properties of the nucleosome, as discussed in the Introduction. In all of the published experiments carried out in our laboratory, such properties are elicited using 0.5 grams of histone per gram of DNA. In the experiments with 140 base pair DNA reported here (Fig. 1) and by Klevan et al. (11), the tetramer is the predominant species at this protein:DNA ratio. Our staphylococcal nuclease digestion experiments with this purified complex show that a tetramer of H3/H4 is sufficient to allow generation of many of the nuclease resistant fragments characteristic of complete nucleosomes. It may be more difficult, however, to prove that it is the tetramer, rather than the octamer, which induces supercoiling in closed circular DNA, though the model of Klevan et al. implies that some supercoiling may occur in the tetramer complex. We are thus led to the conclusion that the H3/H4 tetramer, though not capable of tightly folding the DNA into a nucleosome-like conformation, nonetheless possesses many of the other structural features of the nucleosome. Furthermore, the tetramer-DNA complex can be folded readily into the very compact structure by addition of other histones: if H2A and H2B are added, nucleosomes are formed (5, 11); if more H3 and H4 are added, the H3/H4 octamer complex is formed. As we have shown here, this octamer-DNA complex is similar in its properties to a normal nucleosome, and it is the only fully compact structure formed by H3/H4 and DNA.
4814
Nucleic Acids Research METHODS DNA monomer preparation Triton X-100 washed nuclei were prepared from duck erythrocytes as previously described (1). The nuclei were digested with staphylococcal nuclease (Worthington Biochemicals), 30 units/ml, at a DNA concentration of 0.5-1.0 mg/ml in 0.25 M sucrose, 1 mM Tris-HCl (pH 8.0), 0.3 mM CaCl2 at 370C. At the time when 15% of the DNA had become soluble in 0.8 M perchloric acid, 0.8 M NaCl (1), the digestion was stopped by adding Na2EDTA to a final concentration of 1 mM and cooling on ice. Nucleosomes were then isolated on sucrose gradients or by chromatography on Bio-gel A-5m. The DNA was isolated as previously described (1). The DNA, called "monomer" DNA, was electrophoresed on 4% polyacrylamide slab gels (1) and found to migrate as two distinct bands of 140 and 160 base pairs in length when stained with "Stains-all" (Eastman). The gels were photographed and the negative scanned with a Joyce-Loebl microdensitometer to allow calculation of the weight average molecular weight. The weight-average molecular weight for the preparation used was 150 b.p. ± 5 b.p. Preparation of histones
Histones H3/H4 and H2A/H2B were prepared by selective salt stripping of sheared duck erythrocyte chromatin that had been bound to a hydroxylapatite column (R. H. Simon and G. Felsenfeld, manuscript in preparation). The purity of the histone fractions was assessed by electrophoresis in SDS on polyacrylamide slab gels (Laemmli (13), as modified by Weintraub et al. (14)). The gels were stained with Coomassie blue, photographed, and the negative scanned on a Joyce-Loebl microdensitometer. The H2A/H2B and H3/H4 were contaminated with less than 2% of the opposite pair. To ensure that no histone was selectively lost during preparation of the various fractions, the ratios of the peaks of the individual histones in each fraction were compared and found to vary less than 10% from those present in chromatin. When H2A/H2B and H3/H4 separated in this way were mixed in equimolar proportions and reconstituted onto monomer size DNA at a ratio of 1 gram of protein to 1 gram of DNA, 60% or more of the DNA
sedimented as an llS particle. Determination of concentrations of DNA and histones DNA concentration was measured by its absorbance at 260 nm using a molar extinction of 6700 1 cm 1mol1. The protein concentrations of solutions of core histones and of H2A/H2B were determined by their absorbance at 230 nm using an extinction coefficient of 3.3 cm2mg 1 for both (15). 4815
Nucleic Acids Research To
measure
the protein content of H3/H4-containing nucleoprotein complexes,
the Lowry procedure (16)
was
used,
as
modified by Bensadoun and Weinstein
(17) to avoid interference from Tris-HCl, were
assayed using
a
The samples sucrose, and Na2EDTA. highly purified sample of H3/H4 as a primary standard.
The nitrogen content of a solution of this material was determined by Kjeldahl analysis, and the protein concentration calculated from the published amino acid sequences (18). Bovine serum albumin (Miles Labora-
tories), used as a secondary standard, gave an optical density Lowry assay 0.825 as large as the same weight of H3/H4.
in the
Reconstitution procedure
DNA at
a
final A260 ranging from 1
to
20
was
added
to
5
M
urea,
2 M
NaCl, 10 mM Tris-HCl (pH 8.0) 0.1 mM EDTA, 10 mM 2-mercaptoethanol. Histones were then added and the mixture dialyzed for 16 hours against the The nucleoprotein was then same solution at 4°C on a rocking dialyzer.
sequentially dialyzed against 1.2 M, 1.0 M, 0.8 M, and 0.6 M NaCl for 80 minute time intervals without altering the concentration of urea, Tris-HCl, Next the urea was removed by dialysis against 0.6 M NaCl, 10 or Na2EDTA. mM Tris-HCl, 0.1 mMl Na2EDTA and finally the reconstitution was completed by dialysis into 50 mM Tris-HCl (pH 8.0), 0.1 mM Na 2EDTA. Sedimentation Analysis H3/H4-monomer DNA reconstitutes were layered over a 5-20% linear sucorse gradient in 50 mM Tris-HCl (pH 8.0), 0.1 mM Na 2EDTA and centrifuged in an SW-27 rotor at 87,000 x g for 62 hours at 4°C in a Beckman L2-65 ultracentrifuge. Fractions were collected and monitored by UV absorption at 230 and 260 nm and by Lowry protein assay.
Boundary sedimentation velocity and equilibrium experiments were performed on a Model E analytical ultracentrifuge equipped with ultraviolet optics. The base line was determined by measuring the A265 of the column of solution just outside the inner meniscus after centrifuging at 44,000 rpm for two hours and then reducing the speed to that of the original Where indicated, the apparent specific volume was calculated as the weighted average of the values for DNA and protein. The apparent specific run.
volume used for DNA
was 0.55 ml
gm
(19), and for H3/H4 was 0.74 ml gm 1
assuming that the protein behaves as an average globular protein. all cases, the solvent was 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.
In
Precautions against loss of histone We found that under certain conditions the nucleohistone complexes
4816
Nucleic Acids Research loss of H3 and H4 during either dialysis or storage following Such losses, which were time-dependent, sucrose gradient sedimentation. led to an apparent decrease in the histone:DNA ratio, and might cause a sericus error in determination of the stoichiometry of the complex. In our earlier experiments (8) and some of those reported here, we avoided such losses by fixing the complexes with formaldehyde before sucrose gradient sedimentation. This was done by dialyzing the reconstitute against 10 mM sodium phosphate buffer, pH 6.7, 0.1 mM Na2EDTA, without removing it from the reconstitution dialysis bag. One per cent formaldehyde in this buffer was then dialyzed into the bag and allowed to react for 16 hrs at 4°C. Excess formaldehyde was removed by dialysis against fresh phosphate buffer, and finally this was replaced by 50 mM Tris-HCl, pH 8.0, 0.1 mM Na2EDTA. The fixed material could be handled without loss of histone. As shown above, fixation does not affect the hydrodynamic properties
suffered
a
of the nucleoprotein fractions isolated on sucrose gradients. We have found more recently that preferential losses of H3 and H4 can be minimized, in the absence of formaldehyde fixation, by use of polyallomer (rather than cellulose nitrate) tubes for preparative centrifugation, and by the use of polycarbonate or siliconized glass tubes for the collection of fractions. With such precautions, recoveries obtained with and without fixation are virtually identical (see Figs. 1 and 2 above).
Miscellaneous Nuclei, chromatin and nucleoprotein reconstitutes were digested with staphylococcal nuclease by the procedure of Camerini-Otero et al. (1). The digests were deproteinized and electrophoresed on polyacrylamide slab gels as described previously (1).
ACKNOWLEDGMiENT We are grateful to Dr. D. Crothers and to Dr. J. 0. Thomas for communicating to us their results either in press or in preparation for publication. REFERENCES Camerini-Otero, R. D., Sollner-Webb, B. and Felsenfeld, G. (1976) Cell 1 8, 333-347. 2 Sollner-Webb, B., Camerini-Otero, R. D. and Felsenfeld, G. (1976) Cell 9, 179-193. Boseley, P. G., Bradbury, E. M., Butler-Browne, G. S., Carpenter, B. G. 3 and Stephens, R. M. (1976) Eur. J. Biochem. 62, 21-31. 4 Moss, T., Stephens, R. M., Crane-Robinson, L. and Bradbury, E. M. (1977) Nucl. Acids Res. 4, 2477-2485.
4817
Nucleic Acids Research 5 6 7
8 9
10 11
12 13 14 15 16 17 18 19
4818
Camerini-Otero, R. D. and Felsenfeld, G. (1977) Nucl. Acids. Res. 4, 1159-1181. Bina-Stein, M. and Simpson, R. T. (1977) Cell 11, 609-618. Oudet, P., Germond, J. P., Sures, M., Gallwitz, D., Bellard, M. and Chambon, P. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 287-300. Camerini-Otero, R. D., Sollner-Webb, B., Simon, R. H., Williamson, P., Zasloff, M. and Felsenfeld, G. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 57-75. Camerini-Otero, R. D. and Felsenfeld, G. (1977) Proc. Natl. Acad. Sci. USA 74, 5519-5523. Olins, A. L., Carlson, R. D., Wright, E. B. and Olins, D. E. (1976) Nucl. Acids Res. 3, 3271-3291. Klevan, L., Dattagupta, N., Hogan, M. and Crothers, D. M. (1978) Biochemistry 17, 4533-4540. Bina-Stein, M. (1978) J. Biol. Chem. 253, 5213-5219. Laemmli, U. K. (1970) Nature 227, 680-685. Weintraub, H., Palter, K. and Van Lente, F. (1975) Cell 6, 85-110. Hnilica, L. S. (1975) Methods in Enzymol. 40, 102-138. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241-250. Elgin, S. C. R. and Weintraub, H. (1975) Ann. Rev. Biochem. 44, 725-774. Cohen, G. and Eisenberg, H. (1968) Biopolymers 6, 1077-1100.
