J. Mol. Biol. (1977)
116, 769-781
Characterization
of the Octamer of Histones Free in Solution
JEAN
0.
THOMAS
AND
P. J. G. BUTLER
Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge, CB2 l&W, England and Medical
Research Council Laboratory of Molecular Biology Hills Road, Cambridge, CBZ 2&H, England
(Received 6 April
1977, and in revised form, 27 July 1977)
The nucleosome “core protein” isolated from chromatin in high-salt solutions (2 M-NaCl) has been characterized in detail. The preparation described yields material which is stable for prolonged periods at either 4°C or 37°C. It has an apparent partial specific volume of 0.767 ml/g and a sedimentation coefficient (si,,) of 4.77 (hO.04) S. The molecular weight, determined by sedimentation eqiilibrium, is 107,500 (5 7700), which is compatible with an octameric structure of the form (HSA),(H2B),(H3),(H4),, as previously proposed. The sedimentation coefficient and molecular weight are very similar to those of cross-linked core protein, which is known to be octameric.
1. Introduction It is now well established that the fundamental unit of organization in chromatin is a nucleoprotein particle cont,aining an octameric core of histone surrounded by about 200 base-pairs of DNA, as first proposed by Kornberg (1974) (for references set Kornberg, 1977). A chromatin fibre consists of a contiguous array of such particles, t>ermed nucleosomes (Oudet et al., 1975). The products of chemical cross-linking of chromatin or of isolated nucleosomes with dimethyl suberimidate are consistent with the proposed composition of the octamer, (HZA),(H2B),(H3),(H4), (Thomas & Kornberg, 1975a). Hydrodyna’mic measurements on the 140-base-pair “core part’iclc”. which contains no Hl (Shaw et aE., 1976; No11 8.1Kornberg. 1977), and studies of its protein and DNA components by low-angle neutron scattering (Pardon et al., 1975: Hjelm et aE., 1976) are also consistent with an octameric core. We suggested earlier (Thomas & Kornberg, 1975a) that when DNA is displaced from the histone core in rat liver chromatin by 2 M-NaCI at pH 9, the integrity of t,he octamer is maintained (presumably the high salt concentration screens positive charges previously neutralized by DNA phosphates) ; the evidence for this came from an analysis of the subunit structure by cross-linking with dimethyl suberimidatc. The composition of the octamer, and of the hexamer and dimer into which it dissociates upon dilution, were determined by the combined use of cleavable cross-links and two-dimensional gel analysis (Thomas & Kornberg, 19756), and were consistent. with the structure (HZA),(HSB),(H3),(H4),. On the other hand, Weintraub et al. (1975) concluded from their data that removal 769
770
J.
0.
THOMAS
AND
P.
J.
G.
BUTLER
of DNA from the octameric core of the chicken erythrocyte nucleosome in 2 M-NaCI at pH 7 caused the release of two “heterotypic tetramers”, H2A.H2BSH3.H4. Further, Campbell & Cotter (1976) h ave recently presented laser-light-scattering results in support of a tetrameric rather than an octameric “core protein” in 2 ?vr-NaCl for both calf thymus and chicken erythrocytes. We now present evidence in support of our previous claim (Thomas & Kornberg, 1975a,b) that the “core protein” isolated in 2 M-NaCl is an octamer. We describe the preparation of pure octamer from rat liver, and studies of its behaviour by hydrodynamic and chemical cross-linking methods.
2. Materials and Methods (a) Preparation
of core protein
long (average 40 nucleosomes) was prepared from rat liver (Hewish & Burgoyne, 1973) as described previously (No11 et al., 1975) by digesting with 15 units micrococcal nuclease/ml (Worthington or P-L BioNative
chromatin
about
15 to
150 nucleosomes
chemicals) for 30 s at 37°C. In a typical preparation 40 ml of a suspension of nuclei (A2s0 in 1.0 M-NaOH of approx. 45) were converted into 30 ml chromatin at an A,,, of 15 to 20. The chromatin solution in 0.2 mn-NaEDTA (pH 7) was made 5 mM in Tris*HCl (pH 7), 0.25 mM in phenylmethylsulphonyl fluoride (from a 50 mM stock in propan-2-ol), and 0.6 M in NaCl (addition of 5 M stock), and about 5 ml layered on to 10% sucrose in 5 mM-Tris*HCl (pH 7), 0.25 mM-phenylmethylsulphonyl fluoride, 0.55 M-NaCl in each of six SW27 tubes (1 in x 34 in). The pellets obtained after centrifugation at 27,000 revs/min for 16 h were swollen by shaking at 0°C with 4.8 ml 0.2 mM-NaEDTA, 0.5 mM-phenylmethylsulphonyl fluoride for about 3 h, and 5 ml 4 M-NaCl, 100 m&r-Tris.HCl (pH 7), or 5 ml 3.95 M-Nacl, 137 m&f-Na borate (pH 9), were then added. After shaking at 0°C for a further hour the solution was gently pipetted with a wide-tip pipette until homogeneous, and centrifuged in two B-ml polypropylene tubes at 50,000 revs/min in an SW50.1 rotor for 18 h. The supernatant (10 ml) was concentrated about S-fold at 4°C either against Sephadex G200 or in a “Minicon” concentration cell. The concentrated protein solution was dialysed either against 2 M-NaCl, 50 mna-TrisaHCl (pH 7), or against 1.975 M-NaCl, 68.5 mM-Na borate (pH 9), and its absorption at 230, 260 and 280 nm read in water against the dialysis buffer. The absorbances for a typical preparation (about 1.3 ml) were A,,, (12.6), A,,, (0.98) and 4s0 (1.64). Protein concentrations were determined accurately by amino acid analysis after total acid hydrolysis and the absorption coefficient (A:% = 4.2), determined experimentally for the core protein prepared as described, was used only as a rough guide to concentration to avoid possible errors arising from adventitious contamination with traces of nucleotides. In order to check the quality of the preparation by analysis in SDSt/gels, a small sample was precipitated with an equal volume of 50% trichloroacetic acid, and washed twice with acetone. of total hi&one (b) Preparation A solution of native chromatin prepared as described in O-2 mM-NaEDTA but at a of approx. 30) was diluted with an equal volume of buffer at higher concentration (A,,, an ionic strength of about 4 as described above, phenylmethylsulphonyl fluoride was added to a final concentration of 0.5 mM, and the solution centrifuged at 50,000 revs/min as described. The supernatant was “total histone” and its purity was checked in SDS/gels. (c) Cross-linking
&u&es
Cross-linking of core protein at pH 9, I = 2, was carried out at 22°C at a protein concentration of 1.1 to 2.0 mg/ml and a reagent concentration of 1 mg/ml by a procedure otherwise identical with that described previously (Thomas & Kornberg, 1975a,1977). Final cross-linkedproductswereroutinelyexaminedinan SDS/18~opolyacrylamideslabgel. t Abbreviation
used: SDS, sodium dodecyl sulphate.
OCTAMER
OF
HISTONES
IN
SOJLUTION
771
In samples to be used for hydrodynamic measurements, cross-linking was terminated at the appropriate time (established by gel analysis) by addition of 0.1 vol. ammonium bicarbonate (0.1 M) which was allowed to react for 10 min at 22”C, after which time tjhe sa.mple was chilled. (d) SDSjpolyacrylamide geb SDS/l 8% polyacrylamide slab gels were run as described earlier (Thomas & Kornberg, 1975a, 1977). All slabs were about 15 cm x 15 cm and 0.15 cm thick, and were run vertically. (e) Amino
acid analysis
Samples of core protein (dried down in vucuo from 2 M-Salt solution) were hydrolysed with 0.2 ml 6 ~-HC1-0.1% phenol (Sanger & Thompson, 1963) in sealed tubes at 105°C for 24 11.The hydrolysate was dried down in 2rucuo over NaOH pellets, dissolved in analyser column buffer containing norleucine as internal standard and analysed on a Rank Hilger Chromaspek single-column analyser coupled with a Digico micro 16 V computer. (f)
Apparent
partial
spec$c
&ztme
The apparent partial specific volume, +‘t, for the core protein in 2 M-NaCl was calculated from the density increment, with all other components at constant chemical potential, (5p/&,),, measured for the protein at dialysis equilibrium (Casassa & Eisenberg, 1964) using the equation : (1 - $‘PO) = (aP/w,. (1) Core protein was dialysed against the final buffer for between 48 and 72 h and the density of the protein solution and the buffer measured with a Digital Precision Density meter DMA 02D (Anton Paar, Graz, Austria) (Kratky et al., 1973), calibrated with air and water. Buffer densities were measured for both the external buffer and also a sample of buffer dialysed in the same flask as the protein solution, to check for any possible effect of the dialysis membrane in the high-salt solution. (No such effect was observed.) Protein samples were checked on SDS/polyacrylamide gels after measurement of the sohltjion density, to ensure that no degradation had occurred during the prolonged dialysis. Protein concentrations were determined from amino acid analyses of 6 to 10 replicate samples for each concentration. Weight concentrations were determined from the yields of Asp, Glu, Pro, Gly, Ala, Leu, Tyr, Phe and Arg and the amino acid compositions of the histones (H2A, H2B, H3 and H4) which were assumed to be present in equimolar amounts (Kornberg, 1974; Olins et al., 1976; Joffe et al., 1977). (Compositions were taken to b(, those of calf thymus histones; sequences given in Elgin & Weintraub, 1975.) Any value for an individual amino acid within each sample, or for a sample compared with the others, falling outside I.5 standard deviations was discarded, and t,he estimated concentration (and standard deviation of the estimate) calculated from the remaining r,atues. Us@ this mettlod of detorminat,ion, 4’ will be that of the salt-free prot,ein and calculation of ttlc molrcular weight using this 4’ will give the correct value for t,tie core protein alone (Casussn R Eisezlberg, 1964). (g) Sedimentation measztrements Analytical ultracentrifugation was performed with an MSE analytical ultracentrifugc~ MkII, fitted with an ultraviolet scanner. All samples were at, dialysis equilibrium, as described above, thus permitting t,he correct use of the measured value of 4’ (Casassa 8: Eisenberg, 1964). Sedimentation coefficients were corrected to give values of ,Q~,~ (Svedberg & Pederson, 1940) using viscosities and densities from ttle International Critical Tables for both water and NaCl solutions, and the measured value of 4’. Values of .$&were obtained by extrapolation from the values of .szo w measured at several protein cl>ncentrations, using the equation: 1/920.w =
I/&,
+ kCZ.
(2,
t 4’. apparent partial specific volume; ca, protein concentration; pO, buffer density; (ap/a~)~, density increment with respect to component 2 (i.e. protein), all other components at constant chemical potentials; (an/&),,, refractive increment with respect to component 2.
772
J.
0.
THOMAS
AND
P.
J-. G.
BUTLER
Sedimentation equilibrium experiments were performed by the low-speed method. Establishment of equilibrium was confirmed by comparison of scans taken 24 h apart. When no difference could be seen between successive traces, the later trace was taken to show the equilibrium distribution. Baseline absorbances were measured by overspeeding to sediment the protein, followed by reduction to the previous equilibrium speed before scanning again. Detailed handling of the data is discussed below.
3. Results (a) Isolation
and cross-linking
of the core protein
Core protein isolated at pH 7 or at pH 9 contained the four main histones H3, H4, H2A and H2B in the same relative amounts as in chromatin, as estimated by densitometry (not shown) of a stained poIyacrylamide gel such as that shown in Figure I. Figure 1 shows the quality of the core protein which is essentially free of any higher molecular degradation product. The weight contaminants and entirely free of any proteolytic material shows an identical gel pattern after storage for at least two weeks at 073, and even for several days (at least) at 37°C ; this strengthens our belief that it contains no proteolytic activity. Treatment of the core protein with dimethyl suberimidate at I = 2, pH 9, gave a major cross-linked product after 45 minutes (Fig. 1). This product is completely soluble under the cross-linking conditions : the solution is neither opalescent nor turbid. Further modification of the protein does however begin to cause aggregation and turbidity, but this may be prevented by termination of the reaction (see Materials and Methods). When the progress of the cross-linking reaction is examined at intermediate times (Thomas, 1977) bands from l-mer, 2-mer . . . up to 8-mer are present in an SDS/ polyacrylamide gel. The 8mer corresponds in electrophoretic mobility with the final product of cross-linking shown in Figure 1, and this final product is thereby identified unambiguously as a cross-linked octamer. Since there is a steady transition from low to high molecular weight cross-linked products, as shown in the gel (Thomas, 1977), it is unlikely that a cross-linked octamer arises as an artefact by the association of two Weintraub et al., 1975); in such a situation half-octamers (“heterotypic tetramers”, a build-up of cross-linked tetramer during the course of the cross-linking reaction might be expected but is not observed. (b) Density
increment
In our best measurements (at the highest concentration then available), the density of the protein solution was measured as 1.08604 (*l x 10d5) g/ml, while that of the buffer was 1.08574 (&l x 10e5) g/ml, g iving a density difference of 0.30 (56%) mg/ml. The protein concentration was 1.796 (&O-4%) mg/ml, giving a density incremen.t of 0.167 ( +65o/o). Together with the measured solvent density, this corresponds to 4’ = 0.767 (+l-8%) ml/g (eqn (1)). In practice the value of the molecular weight is a linear function of the density increment (i.e. the usual equation includes the term (1 - 4’~~)) and so the measured molecular weight will have a standard error of 65% due to the 4’ determination. Measurements were also made using total histones extracted directly from chromatin with 2 M-NaC1 without prior removal of Hl (Fig. 1). In this case the density difference between the protein and buffer solutions was 0.58 (+3-5%) mg/ml and the protein concentration was 3.456 (&O-4%) mg/ml. Th ese lead to a density increment of
OCTAMER
OF
HISTONES
IN
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773
H4
(a)
(b)
(cl
1. Histonos extracted from rat liver chromatin at ionic strength 2.0. (a) Core protein, pH 9; (b) total histones, pH 7; (c) cross-linked core protein, pH 9 (the positiona of the cross-linked octtlmrr and of small amounts of hexamw and dimrr are marked by ELITOWS on the right). FIG.
0.167 (&4x) and 4’ of 0.769 (-&1*5x) ml/g. Th ese values confirm bhose measured for the core protein alone which were used in all calculations. (c) Sedimentation
velocity measurewuxts
Core protein extracted at pH 7 or at pH 9 sedimented with a well-defined boundary (Fig. 2(a)). The dependence of the value of .szO,Won concentration is shown in Figure
774
J.
0.
THOMAS
AND
P.
J.
G.
BUTLER ib)
(0)
Proieln cm
img/mi)
Fm. 2. Sedimentation of core protein in 2 M-N&I, pH 9. (a) and (b) Show profiles of untreated and cross-linked samples, respectively, sedimented at 59,600 revs/min, &O”C, scanned at 280 nm after approximately 60 min (sedimentation is from right to left). (-- 0 -- 0 --) and cross-linked (c) Shows the dependence of 8a0,W on ca for untreated ( . . 0 . . 0 .) core protein. The lines show regression fits of eqn (2) to the data.
2(c) and this leads to an siO,, of 4.77 (hO.04) S. Cross-linked core protein (Fig. 2(b) and (c)) has a sedimentation coefficient (sg,,,) of 5.24 (,tO.OS) S, but this value may vary slightly with different extents of cross-linking. There is no indication of any dissociation of core protein and we ascribe the increase in sedimentation coefficient of the structure plus some contribution from the on cross-linking to a “tightening” increased molecular weight due to the cross-links. We think it unlikely to be due to formation of a cross-linked octamer from two tetramers; if both proteins were spherical, the value would be expected to rise by about 60% (i.e. to 22/3 of its former value) and for any less symmetrical tetramers forming a compact octamer, the predicted rise would be still greater. (d) Sedimentation
equilibrium
Sedimentation equilibrium experiments were performed on the core protein at both pH 7 and pH 9, and also after cross-linking. The optical density was measured (at 280 or 285 nm) at between 40 and 60 points along each scanner trace, and the apparent weight average molecular weight at each point determined from the slope (fitted by regression) of the line through sets of 11 data points on a plot of In(c,) against radius squared; the concentration was determined from the optical density of the midpoint
OCl’AMER
8
OF
HISTONES
IN
SOLUTION
775
:/ --cd1 - -- ---VW_--_
75.000~
2 1-s
[ 50,000
I-
25,000
/
Protein cone (mg/ml) (b)
(a) FIG.
3. Dependence
of the apparent
weight
average
molecular
weight, of core protein
on con-
(a) Untreated and (b) cross-linked samples sedimented to equilibrium at 9000 revs/min, 5.O”C. (---) Data for individual cells. (-----) The averaged value for the untreated core protein, allowing for the non-ideality (see text); this same line is shown in (b) for comparison. ((b) Shows t,he sensitivity to a small proportion of non-equilibrating lower or higher molecular weight material--see text.)
of each set of 11. Plots of apparent weight average molecular weight against concentration are shown in Figure 3(a). These include data from experiments at both pH 7 and pH 9; as no difference could be observed between the results at these pH values. The data were fitted by regression to the equation : w
w.app = WKo
+ %
(3)
where R,,.,,, and xW, 0 are the apparent and true weight average molecular weights at, finite and zero protein concentrations, respectively, B is the second osmotic virial coefficient and c2 is the protein concentration (Casassa & Eisenberg, 1974). The curve generated from this equation, showing the effect of non-ideality on the apparent molecular weight, is shown as a heavy broken line in Figure 3. There is no evidence for either aggregation or dissociation over the concentration range studied (between 0.5 and 2-O mg/mI) and the weight average molecular weight from these data, allowing for the effect of non-ideality, is 107,500, while the second virial coefficient (B) is 0.2 x IV6 ml mg-l dalton-‘. This estimate of the molecular weight has an error of 3,930 dalt,ons due to errors in the sedimentation data and, more significant, an error of +9”/; due to the need to use (?p/ZcJlr for its determination (see eqn (4)). This method of handling the data is capable of detecting both scatter in the data points and also any variation in the apparent molecular weight with concentration, whether due to non-ideality or to aggregation or dissociation. Fitting the regression curve averages out the random errors (which anyway fluctuate only between 90.000 and 110,000 daltons) without losing the sensitivity required to detect non-ideality. A direct plot of ln(c,) against radius squared for two arbitrarily chosen cells from Figure 3(a) shows the much lower sensitivity of this frequently used method (Fig. 4). The lines underneath each set of data indicate the slope expected for the molecular weight corresponding to the mid-concentration in the cell, showing the close agreement.
r
46.0
47.0
48.0 12 (cm?)
FIQ. 4. Plots of In(c,) against radius squared for core protein. Samples (not cross-linked) sedimented to equilibrium at 9000 revs/min, 6.0°C and pH 7 (lower curve) or pH 9 (upper curve). Lines show the slope expected (from Fig. 3(a)) with the protein at the mid-concentration in each cell.
As an additional cheek of the apparent partial specific volume used to calculate the molecular weight of the core protein, the molecular weight was also determined, using this same value, for the cross-linked protein which had been shown to be octameric in SDS/gels. While this material is not as homogeneous as the native core protein, yet throughout most of the length of each cell the apparent weight average molecular weight is in good agreement with that determined for the core protein (Fig. 3(b)); some aggregation may be visible near the base of each cell and some dissociation near the meniscus (see Discussion), although this could be due to errors in the data, which are particularly likely to occur near the meniscus. These effects, if real, are not concentration-dependent since the cross-linking has rendered them irreversible and therefore a chemical equilibrium is not established together with the sedimentation equilibrium.
4. Discussion (a) Conclusions from cross-linlcing and sedimentation results The product of cross-linking of salt-extracted core protein at pH 9 is defined unambiguously as an octamer by gel analysis of the final reaction product (Fig. 1) and of the kinetic intermediates (2-mer, 3-mer . . . 7-mer, 8-mer) in its formation (Thomas, 1977). The cross-linked octamer has a molecular weight, measured by sedimentation equilibrium, indistinguishable within experimental error from that of the unmodified core protein extracted at either pH 7 or pH 9 (Figs 3 and 4), so the core protein itself is also identified as an octamer. Both the velocity run (Fig. 2(b)) and the equilibrium run (Fig. 3(b)) on cross-linked
OCTAMER
OF
HISTONES
IN
SOLUTION
77-l
core protein show evidence of some aggregated material in addition to octamer (in the equilibrium run this accounts for the sharp rise in the molecular weight towards tht: bottom of each cell whereas material distributed throughout most of the cell has the molecular weight of an octamer) and this is also seen as a weak band near the top of t’he gel in Figure 1, There is, in addition, possible evidence of slight dissociation, more prominent in the cross-linked than in the uncross-linked material, which may be due to some dissociat’ion of the octamer into hexamer and dimer (Thomas & Kornberg, 1975a,h) at the particular concentration used for cross-linking. The small amounts of aggregated and of dissociated forms do not affect the conclusion that the crosxlinked core protein has the molecular weight of a histone octamer. (As already noted (see Results), a plot of ln(c,) against radius squared would obscure much of the fluct.uat~ion in molecular weight highlighted by a plot of the concentration dependenct! of molecular weight such as that in Fig. 3(b), and is in fact essentially linear for crosslinked protein (not shown).) The apparent partial specific volume of 0.767 ml/g measured for the core protein in the high-salt conditions (essentially 2 M-NaCl) is significantly higher than the value frequently taken for a protein in low-salt conditions (i.e. 0.725 to 0.73 ml/g). However. a high value would be expected for histones since the value calculat.ed for the partial specific volume from the amino acid composition of the core protein, using the data of Cohn & Edsall (1943), is 0.746 ml/g. In addition, an elevated value of the apparent partial specific volume is observed for other proteins in high-salt conditions and some change is indeed predictable if any preferential interaction occurs between the protein and &her water or bhe salt (Casassa & Eisenberg, 1964). Probably the hest-characteriztxd system is that of haemoglobin in NaCl solutions (Kellett, 1971), where the value: of 4’ is 0.03 ml/g higher in 2-O M than in 0.09 M-NaCl (0.779 as compared with 0.749 ml/g). Lt should be noted that while such a rise in +’ can be described as preferential ti>-dration, with relative exclusion of the salt ions from the bound water layer, it does not rule out, the specific binding of either anions or cations of the salt, (or bot)h), but, merely represents the algebraic sum of these effects (Tanford, 1969). Using the measured value of $‘, the weight average molecular weight of t,he con‘ protein in 2 M-x&l is 107,500, with a standard error in this estimate: of 7700. Such a value is compatible only with an octameric structure for the core protein under thestk conditions, the molecular weight of the octamer (HZA),(HZB),(H3),(H4), being 109.000. This is still true with the ca,lculated partial specific volume, which leads to it mo1ccula.r weight. of 94,600. The reliability of the estimated molecular weight wa< confirmed by the closely similar value found for cross-linked material which had bee11 shown to be octamer bv SDS/polyacrylamide gel electrophoresid. From the measured molecular weight, sedimentation coefficient and apparent’ partial specific volume, the frictional ratio (f/j,,) is 1.24. This leads to a calculated value for tbc axial ratio of the octamer (assuming a hydration of 0.5 g water/g protein) ot approxima,telp 2. This is consistent with the protein core of a disk-shaped nucleosomt~ of 110 -\ Y, 110 Lk 2. 55 A (Finch t Klug, 1977). .1\11of these measurements therefore lead to the conclusion that on extraction from cbromatin with 2 .w-NaCI under the conditions described, whether at pH 7 or pH 9, t,tlc* (or(’ protein can be obtained as an octameric complex. consistent with t,lia composition (HZA),(H2B),(H3),(H4), (Thomas & Kornberg. 1975a,b). It is, of course, possible tha.t octamers dissociate, perhaps irreversibly, to heterotypic tetramers under certain conditions, and this may explain the conflicting conclusions (Thomas &
778
J.
0.
THOMAS
AND
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G.
BUTLER
Kornberg, 1975a; Weintraub et al., 1975; Campbell & Cotter, 1976) about the nature of the “core protein” in 2 M-NaCl (see below). The evidence currently available on octamer dissociation, however, is that on dilution in 2 ivr-Nacl, the octamer dissociates first to hexamer by loss of a lysine-rich dimer, and eventually to tetramers and dimers (Thomas & Kornberg, 1975aJ); further, lowering the ionic strength of a 2 M-salt extract of chromatin (van der Westhuyzen & von Holt, 1971) gives argininerich tetramers and lysine-rich dimers or polymers (Kornberg & Thomas, 1974). Nonetheless there might well be conditions in which heterotypic tetramers are formed. In this context it should be noted that the proposed octameric structure (HZA),(H2B),(H3),(H4), for the core protein is entirely consistent with (although of course it does not prove) the possible existence of a dyad symmetry axis (either strict or else quasi or local) for the core protein, either as the free octamer or in a “core particle” combined with 140 base-pairs of DNA. There is certainly no contradiction with the possibility that in combination with DNA and in appropriate conditions the octamer might split symmetrically into two heterotypic tetramer domains ; in the extreme this could account for the observation of “semi-nucleosomes” in the electron microscope (Oudet et al., 1977). (b) Comparison
with
other measurements
Various other workers have also examined the core protein in 2 M-NaCl (Weintraub et al., 1975; Campbell & Cotter, 1976) and have concluded that it is a tetramer. A possible cause of a genuinely low molecular weight of the “core protein” is species difference (rat octamer versus chicken erythrocyte and calf thymus tet.ramer). More probably, damage to the octamer during preparation, for example by proteolysis or even deamidation, might lead to dissociation even though it was too slight to be readily detected otherwise (for example in SDS/polyacrylamide gels). The experiments which are claimed to establish the existence of a tetramer are, however, open to certain criticisms because of either the assumption of parameters or insufficiently careful analysis of possible errors. The most unreliable parameter in any estimation of the molecular weight of the core protein is the protein concentration, particularly if this is determined from absorbance. In this case it will be liable to both systematic error in the extinction coefficient employed and also to an error, systematic for any given sample, due to possible nucleotide contamination, as well as the usual random errors inherent in any measurement. In sedimentation equilibrium experiments (Weintraub et al., 1975; this paper), the absolute concentration is required only for the measurement of (i3p/i3~~)~, which appears linearly in the molecular weight (giving rise in our experiments to an error of 49%). Systematic errors in c2 for a particular sample will not affect the value of d ln(c,)/dG, and hence not the apparent molecular weight, since the form of the equation is : iI? w.app = 2RT(d ln(c2)/drz)/(w2(ap/ac2)~), (4) where R is the gas constant, T is the temperature, o is the angular velocity and r is the distance from the axis. Such systematic error in c2 will alter the estimate of B (eqn 3). The error in x, due to random errors in the data can be estimated from the regression analysis of l/M upon c2, amounting in our experiments to &O*9°/0. In the original work suggesting a heterotypic tetramer (Weintraub et al., 1975), the molecular weight was estimated using a partial specific volume in 2 M-salt which was
OCTAMER
OF
HISTONES
IN
SOLUTION
i5!)
tZaken to be 0.725 ml/g (compared with our measured value of 0.767 ml/g). Moreover the plot of ln(c,) against radius squared was not linear, as it should be for a homogeneous species ; using the partial specific volume assumed by these workers, this plot gives average molecular weights varying between 35,000 near the meniscus (M-her the contaminating histones Hl and H5 will be apparent) and 100,000 near the bottom of t,hr cell. (Taking our measured 4’. these values should then be increased by about %O?/,.) Tn light-scattering experiments the absolute concentration is required for t,he estimation both of the refractive increment (an/a& and of the apparent molecular weight. In the analysis to derive molecular weight, the concentration and refractive increment8 occur as the product (%/ac,)~c, (since the refractive coefficient: H, involves t,he term (2rb/&,)z). Thus it is still true that the molecular weight is affected linearl? Iby errors in cZ1 as in the case of sedimentat.ion equilibrium. provided that t,he SU’Y)I.P sample is used in both the light-scattering experiment and for the determination of (&A/&,),. However, if the same sample is not used for both measurements then the errors for the different samples (whether in actual measurement of concentrations OI systematic within any sample) will multiply to the third power, i.e. they can have a markedly increased effect upon the apparent molecular weight. Clearly any estimate of the dependence of molecular weight upon concentration will be still further affected I))- error in the estimated concentration. From the studies of core proteins from both chicken erythrocytes and calf thymus by laser-light scattering (Campbell C Cotter. 1976). it was concluded that these materials are very simila,r: with molecular weigh& of about 56,500 and 60,000, respectively. Surprisingly, however, t.he extrapolations t,o zero concentration suggest that the molecular weight in each ease decreases E&?~,mrb~f with protein concentration (rather than by an inverse relationship as is usual, see tqn (3)) and, furt’hermore, the dependence upon concentration is about 26,000 daltons mljmp for the chicken eryt,hrocyte protein and 1500 daltons ml/mg for the calf thymus probein (compared with 2100 daltons for 1 mg/ml given by our data for rat liver protein). This difference of over an order of magnitude in t,he second virial coefficient between the calf and chicken proteins is somewhat surprising when it, occurs between two samples of essentially similar material. Since we cannot. reliabl) estimate the errors in these experiments (as discussed above), a full evaluation of the results is not possible. The apparent characterization of a “heterotypic tetramer” may be due to underestimation of the molecular weight of the octamer. Such underestimation of the moIecular weight of :L protein in high-salt. solution has been a well-known phenomenon in various systems previously. Perhaps the best-characterized has been that of haemoglobin, where it gave rise to the so-called “salt paradox”, from which it, was suggested that u/3 dimers showed co-operativity in 2 M-S& identical with that found for the a$2 tet’ramer in 0.09 M-salt. This paradox was found to be due ent,irely to a gross underestimation of the molecular weight in the 2 M-salt, with a consequent overestimation of the extent of dissociation to dimer. When sufficiently careful work \vas performed, whether by sedimentation (Kellett, 1971; Edelstein & Gibson, 1971) or by light scattering (Noren et al., 1971), the haemoglobin was found to be still largely tetrameric at the high-salt concentration. Another possible cause of the difference in the reported molecular weights of t,he core protein might be that material prepared by different workers is in different stat,es of aggregation. However, while it would appear unlikely that a tetramer of the .io
786
J. 0. THOMAS
AND
P. J. G. BUTLER
histones could, by mishandling, be induced to associate into a highly specific octamer apparently identical to the one found in intact chromatin (Thomas & Kornberg, 1975a,b), the possibility of the accidental breakdown of such material is rather great. We would therefore conclude that, as previously proposed, the core protein of the nucleosome exists as an octamer in its most native state in high-salt solutions, although this structure may be relatively labile and readily damaged. We are grateful to Valerie Furber for expert technical assistance, to Graham Parr for the amino acid analyses and to the Science Research Council for a grant (B/RG/3508.3) to one of us (J. 0. T.). We thank Dr A. Klug for helpful comments on the manuscript.
REFERENCES Campbell, A. M. & Cotter, R. I. (1976). FEBS Letters, 70, 2099211. Casassa, E. F. & Eisenberg, H. (1964). Advan. Protein Chem. 19, 287-395. Cohn, E. J. & Edsall, J. ‘IL’. (1943). Proteins, Amino Acids and Peptides, Reinhold Publishing Corp., New York. Edelstein, S. J. & Gibson, Q. H. (1971). In Probes of Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T. & Mildvan, A. S., eds), vol. 2, pp. 417-429, Academic Press, New York. Elgin, S. C. R. & Weintraub, H. (1975). Annu. Rev. Biochem. 44, 725-774. Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. & Klug, A. (1977). Nature (London), 269, 29-36. Hewish, D. R. & Burgoyne, L. A. (1973). Biochem. Biophys. Res. Commun. 52, 504510. Hjelm, R. P., Kneale, G. G., Suau, P., Baldwin, J. P., Bradbury, E. M. & Ibel, K. (1977). CeEZ,10, 139-151. International Critical Tables (1933). Natn. Acad. Sci., U.S.A. McGraw-Hill, New York. Joffe, J., Keene, M. & Weintraub, H. (1977). Biochemistry, 16, 1236-1238. Kellett, G. L. (1971). J. Mol. Biol. 59, 401-424. Kornberg, R. D. (1974). Science, 184, 868-871. Kornberg, R. D. (1977). Annu. Rev. Biochem. 46, 931-954. Kornberg, R. D. & Thomas, J. 0. (1974). Science, 184, 865868. Kratky, O., Leopold, H. & Stabinger, H. (1973). Methods Enzymol. 27 (D), 98-110. Noll, M. & Kornberg, R. D. (1977).J. Mol. BioZ. 109, 393-404. Noll, M., Thomas, J. 0. & Kornberg, R. D. (1975). Science, 187, 1203-1206. Noren, I. B. E., Ho, C. & Casassa, E. F. (1971). Biochemistry, 10, 3222-3229. Olins, A. L., Carlson, R. D., Wright, E. B. $ Olins, D. E. (1976). NucZ. Acida Res. 3, 3271-3291. Oudet, P., Gross-Bellard, M. & Chambon, P. (1975). Cell, 4, 281-300. Oudet, P., Germond, J. E., Bellard, M. & Chambon, P. (1977). Phil. Trans. Roy. SOC. London, in the press. Pardon, J. F., Worcester, D. L., Wooley, J. C., Tatchell, K., Van Holde, K. E. & Richards, B. M. (1975). NucZ. Acids Res. 2, 2163-2176. Sanger, F. & Thompson, E. 0. P. (1963). Biochim. Biophys. A&, 71,468-471. Shaw, B. R., Herman, T. M., Kovacic, R. T., Beaudreau, G. S. & Van Holde, K. E. (1976). Proc. Nat. Acad.Sci., U.S.A. 73, 505-509. Svedberg, T. & Pederson, K. 0. (1940). The Ultracentrijuge, Oxford University Press, London. Tanford, C. (1969). J. Mol. BioZ. 39, 539-544. Thomas, J. 0. (1977). In Molecular BioZogy of the Mammalian Genetic Apparatus (Ts’o, P. 0. P., ed.), vol. 1, pp. 1999209, Elsevier/North-Holland Biomedical Press, Amsterdam. Thomas, J. 0. & Kornberg, R. D. (1975a). Proc. Nat. Acad. Sk., U.S.A. 72, 2626-2630. Thomas, J. 0. & Kornberg, R. D. (1975b). FEBS Letters, 58, 353-358. Thomas, J. 0. t Kornberg, R. D. (1977). Methods Cell BioZ. 18,429-440.
OCTAMER
OF
HISTONES
IN
SOLUTION
iS1
Thomas, J. 0. & Thompson, R. D. (1977). Cell, 10, 633-640. van der Westhuyzen, D. R. & von Holt, C. (1971). FEBS Letters, 14, 333~-342. Weintraub, H., Palter, K. & Van Lente, F. (1975). Cell, 5, 85--l 10.
Note added in proof. Pardon et al. (1977) consider the core protein in 2 M-NaCl to be H. heterotypic tetramer and the cross-linked core protein to be an octamer. However, they find that uncross-linked and cross-linked core protein have very similar radii of gyrat,ion as measured by neutron scattering. They explain this apparent paradox by suggestsing t,hat, the heterotypic tetramer is a flat disk and that in the cross-linked octamer two such disks are st.acked directly on top of each other. From our results both the untreated and t.hr (aross-linked core protein would be expected to have essentially the samr radius of gyration, since both are octamers. REFERENCE Pardon, J. F., Worcester, D. L., Wooley, J. C., Cotter, R. M. (1977). NucZ. Acida Res. 4, 3199-3214.
R. I., Lilley,
D. M. J. & Richards,
116, 769-781
Characterization
of the Octamer of Histones Free in Solution
JEAN
0.
THOMAS
AND
P. J. G. BUTLER
Department of Biochemistry, University of Cambridge Tennis Court Road, Cambridge, CB2 l&W, England and Medical
Research Council Laboratory of Molecular Biology Hills Road, Cambridge, CBZ 2&H, England
(Received 6 April
1977, and in revised form, 27 July 1977)
The nucleosome “core protein” isolated from chromatin in high-salt solutions (2 M-NaCl) has been characterized in detail. The preparation described yields material which is stable for prolonged periods at either 4°C or 37°C. It has an apparent partial specific volume of 0.767 ml/g and a sedimentation coefficient (si,,) of 4.77 (hO.04) S. The molecular weight, determined by sedimentation eqiilibrium, is 107,500 (5 7700), which is compatible with an octameric structure of the form (HSA),(H2B),(H3),(H4),, as previously proposed. The sedimentation coefficient and molecular weight are very similar to those of cross-linked core protein, which is known to be octameric.
1. Introduction It is now well established that the fundamental unit of organization in chromatin is a nucleoprotein particle cont,aining an octameric core of histone surrounded by about 200 base-pairs of DNA, as first proposed by Kornberg (1974) (for references set Kornberg, 1977). A chromatin fibre consists of a contiguous array of such particles, t>ermed nucleosomes (Oudet et al., 1975). The products of chemical cross-linking of chromatin or of isolated nucleosomes with dimethyl suberimidate are consistent with the proposed composition of the octamer, (HZA),(H2B),(H3),(H4), (Thomas & Kornberg, 1975a). Hydrodyna’mic measurements on the 140-base-pair “core part’iclc”. which contains no Hl (Shaw et aE., 1976; No11 8.1Kornberg. 1977), and studies of its protein and DNA components by low-angle neutron scattering (Pardon et al., 1975: Hjelm et aE., 1976) are also consistent with an octameric core. We suggested earlier (Thomas & Kornberg, 1975a) that when DNA is displaced from the histone core in rat liver chromatin by 2 M-NaCI at pH 9, the integrity of t,he octamer is maintained (presumably the high salt concentration screens positive charges previously neutralized by DNA phosphates) ; the evidence for this came from an analysis of the subunit structure by cross-linking with dimethyl suberimidatc. The composition of the octamer, and of the hexamer and dimer into which it dissociates upon dilution, were determined by the combined use of cleavable cross-links and two-dimensional gel analysis (Thomas & Kornberg, 19756), and were consistent. with the structure (HZA),(HSB),(H3),(H4),. On the other hand, Weintraub et al. (1975) concluded from their data that removal 769
770
J.
0.
THOMAS
AND
P.
J.
G.
BUTLER
of DNA from the octameric core of the chicken erythrocyte nucleosome in 2 M-NaCI at pH 7 caused the release of two “heterotypic tetramers”, H2A.H2BSH3.H4. Further, Campbell & Cotter (1976) h ave recently presented laser-light-scattering results in support of a tetrameric rather than an octameric “core protein” in 2 ?vr-NaCl for both calf thymus and chicken erythrocytes. We now present evidence in support of our previous claim (Thomas & Kornberg, 1975a,b) that the “core protein” isolated in 2 M-NaCl is an octamer. We describe the preparation of pure octamer from rat liver, and studies of its behaviour by hydrodynamic and chemical cross-linking methods.
2. Materials and Methods (a) Preparation
of core protein
long (average 40 nucleosomes) was prepared from rat liver (Hewish & Burgoyne, 1973) as described previously (No11 et al., 1975) by digesting with 15 units micrococcal nuclease/ml (Worthington or P-L BioNative
chromatin
about
15 to
150 nucleosomes
chemicals) for 30 s at 37°C. In a typical preparation 40 ml of a suspension of nuclei (A2s0 in 1.0 M-NaOH of approx. 45) were converted into 30 ml chromatin at an A,,, of 15 to 20. The chromatin solution in 0.2 mn-NaEDTA (pH 7) was made 5 mM in Tris*HCl (pH 7), 0.25 mM in phenylmethylsulphonyl fluoride (from a 50 mM stock in propan-2-ol), and 0.6 M in NaCl (addition of 5 M stock), and about 5 ml layered on to 10% sucrose in 5 mM-Tris*HCl (pH 7), 0.25 mM-phenylmethylsulphonyl fluoride, 0.55 M-NaCl in each of six SW27 tubes (1 in x 34 in). The pellets obtained after centrifugation at 27,000 revs/min for 16 h were swollen by shaking at 0°C with 4.8 ml 0.2 mM-NaEDTA, 0.5 mM-phenylmethylsulphonyl fluoride for about 3 h, and 5 ml 4 M-NaCl, 100 m&r-Tris.HCl (pH 7), or 5 ml 3.95 M-Nacl, 137 m&f-Na borate (pH 9), were then added. After shaking at 0°C for a further hour the solution was gently pipetted with a wide-tip pipette until homogeneous, and centrifuged in two B-ml polypropylene tubes at 50,000 revs/min in an SW50.1 rotor for 18 h. The supernatant (10 ml) was concentrated about S-fold at 4°C either against Sephadex G200 or in a “Minicon” concentration cell. The concentrated protein solution was dialysed either against 2 M-NaCl, 50 mna-TrisaHCl (pH 7), or against 1.975 M-NaCl, 68.5 mM-Na borate (pH 9), and its absorption at 230, 260 and 280 nm read in water against the dialysis buffer. The absorbances for a typical preparation (about 1.3 ml) were A,,, (12.6), A,,, (0.98) and 4s0 (1.64). Protein concentrations were determined accurately by amino acid analysis after total acid hydrolysis and the absorption coefficient (A:% = 4.2), determined experimentally for the core protein prepared as described, was used only as a rough guide to concentration to avoid possible errors arising from adventitious contamination with traces of nucleotides. In order to check the quality of the preparation by analysis in SDSt/gels, a small sample was precipitated with an equal volume of 50% trichloroacetic acid, and washed twice with acetone. of total hi&one (b) Preparation A solution of native chromatin prepared as described in O-2 mM-NaEDTA but at a of approx. 30) was diluted with an equal volume of buffer at higher concentration (A,,, an ionic strength of about 4 as described above, phenylmethylsulphonyl fluoride was added to a final concentration of 0.5 mM, and the solution centrifuged at 50,000 revs/min as described. The supernatant was “total histone” and its purity was checked in SDS/gels. (c) Cross-linking
&u&es
Cross-linking of core protein at pH 9, I = 2, was carried out at 22°C at a protein concentration of 1.1 to 2.0 mg/ml and a reagent concentration of 1 mg/ml by a procedure otherwise identical with that described previously (Thomas & Kornberg, 1975a,1977). Final cross-linkedproductswereroutinelyexaminedinan SDS/18~opolyacrylamideslabgel. t Abbreviation
used: SDS, sodium dodecyl sulphate.
OCTAMER
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SOJLUTION
771
In samples to be used for hydrodynamic measurements, cross-linking was terminated at the appropriate time (established by gel analysis) by addition of 0.1 vol. ammonium bicarbonate (0.1 M) which was allowed to react for 10 min at 22”C, after which time tjhe sa.mple was chilled. (d) SDSjpolyacrylamide geb SDS/l 8% polyacrylamide slab gels were run as described earlier (Thomas & Kornberg, 1975a, 1977). All slabs were about 15 cm x 15 cm and 0.15 cm thick, and were run vertically. (e) Amino
acid analysis
Samples of core protein (dried down in vucuo from 2 M-Salt solution) were hydrolysed with 0.2 ml 6 ~-HC1-0.1% phenol (Sanger & Thompson, 1963) in sealed tubes at 105°C for 24 11.The hydrolysate was dried down in 2rucuo over NaOH pellets, dissolved in analyser column buffer containing norleucine as internal standard and analysed on a Rank Hilger Chromaspek single-column analyser coupled with a Digico micro 16 V computer. (f)
Apparent
partial
spec$c
&ztme
The apparent partial specific volume, +‘t, for the core protein in 2 M-NaCl was calculated from the density increment, with all other components at constant chemical potential, (5p/&,),, measured for the protein at dialysis equilibrium (Casassa & Eisenberg, 1964) using the equation : (1 - $‘PO) = (aP/w,. (1) Core protein was dialysed against the final buffer for between 48 and 72 h and the density of the protein solution and the buffer measured with a Digital Precision Density meter DMA 02D (Anton Paar, Graz, Austria) (Kratky et al., 1973), calibrated with air and water. Buffer densities were measured for both the external buffer and also a sample of buffer dialysed in the same flask as the protein solution, to check for any possible effect of the dialysis membrane in the high-salt solution. (No such effect was observed.) Protein samples were checked on SDS/polyacrylamide gels after measurement of the sohltjion density, to ensure that no degradation had occurred during the prolonged dialysis. Protein concentrations were determined from amino acid analyses of 6 to 10 replicate samples for each concentration. Weight concentrations were determined from the yields of Asp, Glu, Pro, Gly, Ala, Leu, Tyr, Phe and Arg and the amino acid compositions of the histones (H2A, H2B, H3 and H4) which were assumed to be present in equimolar amounts (Kornberg, 1974; Olins et al., 1976; Joffe et al., 1977). (Compositions were taken to b(, those of calf thymus histones; sequences given in Elgin & Weintraub, 1975.) Any value for an individual amino acid within each sample, or for a sample compared with the others, falling outside I.5 standard deviations was discarded, and t,he estimated concentration (and standard deviation of the estimate) calculated from the remaining r,atues. Us@ this mettlod of detorminat,ion, 4’ will be that of the salt-free prot,ein and calculation of ttlc molrcular weight using this 4’ will give the correct value for t,tie core protein alone (Casussn R Eisezlberg, 1964). (g) Sedimentation measztrements Analytical ultracentrifugation was performed with an MSE analytical ultracentrifugc~ MkII, fitted with an ultraviolet scanner. All samples were at, dialysis equilibrium, as described above, thus permitting t,he correct use of the measured value of 4’ (Casassa 8: Eisenberg, 1964). Sedimentation coefficients were corrected to give values of ,Q~,~ (Svedberg & Pederson, 1940) using viscosities and densities from ttle International Critical Tables for both water and NaCl solutions, and the measured value of 4’. Values of .$&were obtained by extrapolation from the values of .szo w measured at several protein cl>ncentrations, using the equation: 1/920.w =
I/&,
+ kCZ.
(2,
t 4’. apparent partial specific volume; ca, protein concentration; pO, buffer density; (ap/a~)~, density increment with respect to component 2 (i.e. protein), all other components at constant chemical potentials; (an/&),,, refractive increment with respect to component 2.
772
J.
0.
THOMAS
AND
P.
J-. G.
BUTLER
Sedimentation equilibrium experiments were performed by the low-speed method. Establishment of equilibrium was confirmed by comparison of scans taken 24 h apart. When no difference could be seen between successive traces, the later trace was taken to show the equilibrium distribution. Baseline absorbances were measured by overspeeding to sediment the protein, followed by reduction to the previous equilibrium speed before scanning again. Detailed handling of the data is discussed below.
3. Results (a) Isolation
and cross-linking
of the core protein
Core protein isolated at pH 7 or at pH 9 contained the four main histones H3, H4, H2A and H2B in the same relative amounts as in chromatin, as estimated by densitometry (not shown) of a stained poIyacrylamide gel such as that shown in Figure I. Figure 1 shows the quality of the core protein which is essentially free of any higher molecular degradation product. The weight contaminants and entirely free of any proteolytic material shows an identical gel pattern after storage for at least two weeks at 073, and even for several days (at least) at 37°C ; this strengthens our belief that it contains no proteolytic activity. Treatment of the core protein with dimethyl suberimidate at I = 2, pH 9, gave a major cross-linked product after 45 minutes (Fig. 1). This product is completely soluble under the cross-linking conditions : the solution is neither opalescent nor turbid. Further modification of the protein does however begin to cause aggregation and turbidity, but this may be prevented by termination of the reaction (see Materials and Methods). When the progress of the cross-linking reaction is examined at intermediate times (Thomas, 1977) bands from l-mer, 2-mer . . . up to 8-mer are present in an SDS/ polyacrylamide gel. The 8mer corresponds in electrophoretic mobility with the final product of cross-linking shown in Figure 1, and this final product is thereby identified unambiguously as a cross-linked octamer. Since there is a steady transition from low to high molecular weight cross-linked products, as shown in the gel (Thomas, 1977), it is unlikely that a cross-linked octamer arises as an artefact by the association of two Weintraub et al., 1975); in such a situation half-octamers (“heterotypic tetramers”, a build-up of cross-linked tetramer during the course of the cross-linking reaction might be expected but is not observed. (b) Density
increment
In our best measurements (at the highest concentration then available), the density of the protein solution was measured as 1.08604 (*l x 10d5) g/ml, while that of the buffer was 1.08574 (&l x 10e5) g/ml, g iving a density difference of 0.30 (56%) mg/ml. The protein concentration was 1.796 (&O-4%) mg/ml, giving a density incremen.t of 0.167 ( +65o/o). Together with the measured solvent density, this corresponds to 4’ = 0.767 (+l-8%) ml/g (eqn (1)). In practice the value of the molecular weight is a linear function of the density increment (i.e. the usual equation includes the term (1 - 4’~~)) and so the measured molecular weight will have a standard error of 65% due to the 4’ determination. Measurements were also made using total histones extracted directly from chromatin with 2 M-NaC1 without prior removal of Hl (Fig. 1). In this case the density difference between the protein and buffer solutions was 0.58 (+3-5%) mg/ml and the protein concentration was 3.456 (&O-4%) mg/ml. Th ese lead to a density increment of
OCTAMER
OF
HISTONES
IN
SOLUTION
773
H4
(a)
(b)
(cl
1. Histonos extracted from rat liver chromatin at ionic strength 2.0. (a) Core protein, pH 9; (b) total histones, pH 7; (c) cross-linked core protein, pH 9 (the positiona of the cross-linked octtlmrr and of small amounts of hexamw and dimrr are marked by ELITOWS on the right). FIG.
0.167 (&4x) and 4’ of 0.769 (-&1*5x) ml/g. Th ese values confirm bhose measured for the core protein alone which were used in all calculations. (c) Sedimentation
velocity measurewuxts
Core protein extracted at pH 7 or at pH 9 sedimented with a well-defined boundary (Fig. 2(a)). The dependence of the value of .szO,Won concentration is shown in Figure
774
J.
0.
THOMAS
AND
P.
J.
G.
BUTLER ib)
(0)
Proieln cm
img/mi)
Fm. 2. Sedimentation of core protein in 2 M-N&I, pH 9. (a) and (b) Show profiles of untreated and cross-linked samples, respectively, sedimented at 59,600 revs/min, &O”C, scanned at 280 nm after approximately 60 min (sedimentation is from right to left). (-- 0 -- 0 --) and cross-linked (c) Shows the dependence of 8a0,W on ca for untreated ( . . 0 . . 0 .) core protein. The lines show regression fits of eqn (2) to the data.
2(c) and this leads to an siO,, of 4.77 (hO.04) S. Cross-linked core protein (Fig. 2(b) and (c)) has a sedimentation coefficient (sg,,,) of 5.24 (,tO.OS) S, but this value may vary slightly with different extents of cross-linking. There is no indication of any dissociation of core protein and we ascribe the increase in sedimentation coefficient of the structure plus some contribution from the on cross-linking to a “tightening” increased molecular weight due to the cross-links. We think it unlikely to be due to formation of a cross-linked octamer from two tetramers; if both proteins were spherical, the value would be expected to rise by about 60% (i.e. to 22/3 of its former value) and for any less symmetrical tetramers forming a compact octamer, the predicted rise would be still greater. (d) Sedimentation
equilibrium
Sedimentation equilibrium experiments were performed on the core protein at both pH 7 and pH 9, and also after cross-linking. The optical density was measured (at 280 or 285 nm) at between 40 and 60 points along each scanner trace, and the apparent weight average molecular weight at each point determined from the slope (fitted by regression) of the line through sets of 11 data points on a plot of In(c,) against radius squared; the concentration was determined from the optical density of the midpoint
OCl’AMER
8
OF
HISTONES
IN
SOLUTION
775
:/ --cd1 - -- ---VW_--_
75.000~
2 1-s
[ 50,000
I-
25,000
/
Protein cone (mg/ml) (b)
(a) FIG.
3. Dependence
of the apparent
weight
average
molecular
weight, of core protein
on con-
(a) Untreated and (b) cross-linked samples sedimented to equilibrium at 9000 revs/min, 5.O”C. (---) Data for individual cells. (-----) The averaged value for the untreated core protein, allowing for the non-ideality (see text); this same line is shown in (b) for comparison. ((b) Shows t,he sensitivity to a small proportion of non-equilibrating lower or higher molecular weight material--see text.)
of each set of 11. Plots of apparent weight average molecular weight against concentration are shown in Figure 3(a). These include data from experiments at both pH 7 and pH 9; as no difference could be observed between the results at these pH values. The data were fitted by regression to the equation : w
w.app = WKo
+ %
(3)
where R,,.,,, and xW, 0 are the apparent and true weight average molecular weights at, finite and zero protein concentrations, respectively, B is the second osmotic virial coefficient and c2 is the protein concentration (Casassa & Eisenberg, 1974). The curve generated from this equation, showing the effect of non-ideality on the apparent molecular weight, is shown as a heavy broken line in Figure 3. There is no evidence for either aggregation or dissociation over the concentration range studied (between 0.5 and 2-O mg/mI) and the weight average molecular weight from these data, allowing for the effect of non-ideality, is 107,500, while the second virial coefficient (B) is 0.2 x IV6 ml mg-l dalton-‘. This estimate of the molecular weight has an error of 3,930 dalt,ons due to errors in the sedimentation data and, more significant, an error of +9”/; due to the need to use (?p/ZcJlr for its determination (see eqn (4)). This method of handling the data is capable of detecting both scatter in the data points and also any variation in the apparent molecular weight with concentration, whether due to non-ideality or to aggregation or dissociation. Fitting the regression curve averages out the random errors (which anyway fluctuate only between 90.000 and 110,000 daltons) without losing the sensitivity required to detect non-ideality. A direct plot of ln(c,) against radius squared for two arbitrarily chosen cells from Figure 3(a) shows the much lower sensitivity of this frequently used method (Fig. 4). The lines underneath each set of data indicate the slope expected for the molecular weight corresponding to the mid-concentration in the cell, showing the close agreement.
r
46.0
47.0
48.0 12 (cm?)
FIQ. 4. Plots of In(c,) against radius squared for core protein. Samples (not cross-linked) sedimented to equilibrium at 9000 revs/min, 6.0°C and pH 7 (lower curve) or pH 9 (upper curve). Lines show the slope expected (from Fig. 3(a)) with the protein at the mid-concentration in each cell.
As an additional cheek of the apparent partial specific volume used to calculate the molecular weight of the core protein, the molecular weight was also determined, using this same value, for the cross-linked protein which had been shown to be octameric in SDS/gels. While this material is not as homogeneous as the native core protein, yet throughout most of the length of each cell the apparent weight average molecular weight is in good agreement with that determined for the core protein (Fig. 3(b)); some aggregation may be visible near the base of each cell and some dissociation near the meniscus (see Discussion), although this could be due to errors in the data, which are particularly likely to occur near the meniscus. These effects, if real, are not concentration-dependent since the cross-linking has rendered them irreversible and therefore a chemical equilibrium is not established together with the sedimentation equilibrium.
4. Discussion (a) Conclusions from cross-linlcing and sedimentation results The product of cross-linking of salt-extracted core protein at pH 9 is defined unambiguously as an octamer by gel analysis of the final reaction product (Fig. 1) and of the kinetic intermediates (2-mer, 3-mer . . . 7-mer, 8-mer) in its formation (Thomas, 1977). The cross-linked octamer has a molecular weight, measured by sedimentation equilibrium, indistinguishable within experimental error from that of the unmodified core protein extracted at either pH 7 or pH 9 (Figs 3 and 4), so the core protein itself is also identified as an octamer. Both the velocity run (Fig. 2(b)) and the equilibrium run (Fig. 3(b)) on cross-linked
OCTAMER
OF
HISTONES
IN
SOLUTION
77-l
core protein show evidence of some aggregated material in addition to octamer (in the equilibrium run this accounts for the sharp rise in the molecular weight towards tht: bottom of each cell whereas material distributed throughout most of the cell has the molecular weight of an octamer) and this is also seen as a weak band near the top of t’he gel in Figure 1, There is, in addition, possible evidence of slight dissociation, more prominent in the cross-linked than in the uncross-linked material, which may be due to some dissociat’ion of the octamer into hexamer and dimer (Thomas & Kornberg, 1975a,h) at the particular concentration used for cross-linking. The small amounts of aggregated and of dissociated forms do not affect the conclusion that the crosxlinked core protein has the molecular weight of a histone octamer. (As already noted (see Results), a plot of ln(c,) against radius squared would obscure much of the fluct.uat~ion in molecular weight highlighted by a plot of the concentration dependenct! of molecular weight such as that in Fig. 3(b), and is in fact essentially linear for crosslinked protein (not shown).) The apparent partial specific volume of 0.767 ml/g measured for the core protein in the high-salt conditions (essentially 2 M-NaCl) is significantly higher than the value frequently taken for a protein in low-salt conditions (i.e. 0.725 to 0.73 ml/g). However. a high value would be expected for histones since the value calculat.ed for the partial specific volume from the amino acid composition of the core protein, using the data of Cohn & Edsall (1943), is 0.746 ml/g. In addition, an elevated value of the apparent partial specific volume is observed for other proteins in high-salt conditions and some change is indeed predictable if any preferential interaction occurs between the protein and &her water or bhe salt (Casassa & Eisenberg, 1964). Probably the hest-characteriztxd system is that of haemoglobin in NaCl solutions (Kellett, 1971), where the value: of 4’ is 0.03 ml/g higher in 2-O M than in 0.09 M-NaCl (0.779 as compared with 0.749 ml/g). Lt should be noted that while such a rise in +’ can be described as preferential ti>-dration, with relative exclusion of the salt ions from the bound water layer, it does not rule out, the specific binding of either anions or cations of the salt, (or bot)h), but, merely represents the algebraic sum of these effects (Tanford, 1969). Using the measured value of $‘, the weight average molecular weight of t,he con‘ protein in 2 M-x&l is 107,500, with a standard error in this estimate: of 7700. Such a value is compatible only with an octameric structure for the core protein under thestk conditions, the molecular weight of the octamer (HZA),(HZB),(H3),(H4), being 109.000. This is still true with the ca,lculated partial specific volume, which leads to it mo1ccula.r weight. of 94,600. The reliability of the estimated molecular weight wa< confirmed by the closely similar value found for cross-linked material which had bee11 shown to be octamer bv SDS/polyacrylamide gel electrophoresid. From the measured molecular weight, sedimentation coefficient and apparent’ partial specific volume, the frictional ratio (f/j,,) is 1.24. This leads to a calculated value for tbc axial ratio of the octamer (assuming a hydration of 0.5 g water/g protein) ot approxima,telp 2. This is consistent with the protein core of a disk-shaped nucleosomt~ of 110 -\ Y, 110 Lk 2. 55 A (Finch t Klug, 1977). .1\11of these measurements therefore lead to the conclusion that on extraction from cbromatin with 2 .w-NaCI under the conditions described, whether at pH 7 or pH 9, t,tlc* (or(’ protein can be obtained as an octameric complex. consistent with t,lia composition (HZA),(H2B),(H3),(H4), (Thomas & Kornberg. 1975a,b). It is, of course, possible tha.t octamers dissociate, perhaps irreversibly, to heterotypic tetramers under certain conditions, and this may explain the conflicting conclusions (Thomas &
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THOMAS
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Kornberg, 1975a; Weintraub et al., 1975; Campbell & Cotter, 1976) about the nature of the “core protein” in 2 M-NaCl (see below). The evidence currently available on octamer dissociation, however, is that on dilution in 2 ivr-Nacl, the octamer dissociates first to hexamer by loss of a lysine-rich dimer, and eventually to tetramers and dimers (Thomas & Kornberg, 1975aJ); further, lowering the ionic strength of a 2 M-salt extract of chromatin (van der Westhuyzen & von Holt, 1971) gives argininerich tetramers and lysine-rich dimers or polymers (Kornberg & Thomas, 1974). Nonetheless there might well be conditions in which heterotypic tetramers are formed. In this context it should be noted that the proposed octameric structure (HZA),(H2B),(H3),(H4), for the core protein is entirely consistent with (although of course it does not prove) the possible existence of a dyad symmetry axis (either strict or else quasi or local) for the core protein, either as the free octamer or in a “core particle” combined with 140 base-pairs of DNA. There is certainly no contradiction with the possibility that in combination with DNA and in appropriate conditions the octamer might split symmetrically into two heterotypic tetramer domains ; in the extreme this could account for the observation of “semi-nucleosomes” in the electron microscope (Oudet et al., 1977). (b) Comparison
with
other measurements
Various other workers have also examined the core protein in 2 M-NaCl (Weintraub et al., 1975; Campbell & Cotter, 1976) and have concluded that it is a tetramer. A possible cause of a genuinely low molecular weight of the “core protein” is species difference (rat octamer versus chicken erythrocyte and calf thymus tet.ramer). More probably, damage to the octamer during preparation, for example by proteolysis or even deamidation, might lead to dissociation even though it was too slight to be readily detected otherwise (for example in SDS/polyacrylamide gels). The experiments which are claimed to establish the existence of a tetramer are, however, open to certain criticisms because of either the assumption of parameters or insufficiently careful analysis of possible errors. The most unreliable parameter in any estimation of the molecular weight of the core protein is the protein concentration, particularly if this is determined from absorbance. In this case it will be liable to both systematic error in the extinction coefficient employed and also to an error, systematic for any given sample, due to possible nucleotide contamination, as well as the usual random errors inherent in any measurement. In sedimentation equilibrium experiments (Weintraub et al., 1975; this paper), the absolute concentration is required only for the measurement of (i3p/i3~~)~, which appears linearly in the molecular weight (giving rise in our experiments to an error of 49%). Systematic errors in c2 for a particular sample will not affect the value of d ln(c,)/dG, and hence not the apparent molecular weight, since the form of the equation is : iI? w.app = 2RT(d ln(c2)/drz)/(w2(ap/ac2)~), (4) where R is the gas constant, T is the temperature, o is the angular velocity and r is the distance from the axis. Such systematic error in c2 will alter the estimate of B (eqn 3). The error in x, due to random errors in the data can be estimated from the regression analysis of l/M upon c2, amounting in our experiments to &O*9°/0. In the original work suggesting a heterotypic tetramer (Weintraub et al., 1975), the molecular weight was estimated using a partial specific volume in 2 M-salt which was
OCTAMER
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tZaken to be 0.725 ml/g (compared with our measured value of 0.767 ml/g). Moreover the plot of ln(c,) against radius squared was not linear, as it should be for a homogeneous species ; using the partial specific volume assumed by these workers, this plot gives average molecular weights varying between 35,000 near the meniscus (M-her the contaminating histones Hl and H5 will be apparent) and 100,000 near the bottom of t,hr cell. (Taking our measured 4’. these values should then be increased by about %O?/,.) Tn light-scattering experiments the absolute concentration is required for t,he estimation both of the refractive increment (an/a& and of the apparent molecular weight. In the analysis to derive molecular weight, the concentration and refractive increment8 occur as the product (%/ac,)~c, (since the refractive coefficient: H, involves t,he term (2rb/&,)z). Thus it is still true that the molecular weight is affected linearl? Iby errors in cZ1 as in the case of sedimentat.ion equilibrium. provided that t,he SU’Y)I.P sample is used in both the light-scattering experiment and for the determination of (&A/&,),. However, if the same sample is not used for both measurements then the errors for the different samples (whether in actual measurement of concentrations OI systematic within any sample) will multiply to the third power, i.e. they can have a markedly increased effect upon the apparent molecular weight. Clearly any estimate of the dependence of molecular weight upon concentration will be still further affected I))- error in the estimated concentration. From the studies of core proteins from both chicken erythrocytes and calf thymus by laser-light scattering (Campbell C Cotter. 1976). it was concluded that these materials are very simila,r: with molecular weigh& of about 56,500 and 60,000, respectively. Surprisingly, however, t.he extrapolations t,o zero concentration suggest that the molecular weight in each ease decreases E&?~,mrb~f with protein concentration (rather than by an inverse relationship as is usual, see tqn (3)) and, furt’hermore, the dependence upon concentration is about 26,000 daltons mljmp for the chicken eryt,hrocyte protein and 1500 daltons ml/mg for the calf thymus probein (compared with 2100 daltons for 1 mg/ml given by our data for rat liver protein). This difference of over an order of magnitude in t,he second virial coefficient between the calf and chicken proteins is somewhat surprising when it, occurs between two samples of essentially similar material. Since we cannot. reliabl) estimate the errors in these experiments (as discussed above), a full evaluation of the results is not possible. The apparent characterization of a “heterotypic tetramer” may be due to underestimation of the molecular weight of the octamer. Such underestimation of the moIecular weight of :L protein in high-salt. solution has been a well-known phenomenon in various systems previously. Perhaps the best-characterized has been that of haemoglobin, where it gave rise to the so-called “salt paradox”, from which it, was suggested that u/3 dimers showed co-operativity in 2 M-S& identical with that found for the a$2 tet’ramer in 0.09 M-salt. This paradox was found to be due ent,irely to a gross underestimation of the molecular weight in the 2 M-salt, with a consequent overestimation of the extent of dissociation to dimer. When sufficiently careful work \vas performed, whether by sedimentation (Kellett, 1971; Edelstein & Gibson, 1971) or by light scattering (Noren et al., 1971), the haemoglobin was found to be still largely tetrameric at the high-salt concentration. Another possible cause of the difference in the reported molecular weights of t,he core protein might be that material prepared by different workers is in different stat,es of aggregation. However, while it would appear unlikely that a tetramer of the .io
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histones could, by mishandling, be induced to associate into a highly specific octamer apparently identical to the one found in intact chromatin (Thomas & Kornberg, 1975a,b), the possibility of the accidental breakdown of such material is rather great. We would therefore conclude that, as previously proposed, the core protein of the nucleosome exists as an octamer in its most native state in high-salt solutions, although this structure may be relatively labile and readily damaged. We are grateful to Valerie Furber for expert technical assistance, to Graham Parr for the amino acid analyses and to the Science Research Council for a grant (B/RG/3508.3) to one of us (J. 0. T.). We thank Dr A. Klug for helpful comments on the manuscript.
REFERENCES Campbell, A. M. & Cotter, R. I. (1976). FEBS Letters, 70, 2099211. Casassa, E. F. & Eisenberg, H. (1964). Advan. Protein Chem. 19, 287-395. Cohn, E. J. & Edsall, J. ‘IL’. (1943). Proteins, Amino Acids and Peptides, Reinhold Publishing Corp., New York. Edelstein, S. J. & Gibson, Q. H. (1971). In Probes of Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T. & Mildvan, A. S., eds), vol. 2, pp. 417-429, Academic Press, New York. Elgin, S. C. R. & Weintraub, H. (1975). Annu. Rev. Biochem. 44, 725-774. Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. & Klug, A. (1977). Nature (London), 269, 29-36. Hewish, D. R. & Burgoyne, L. A. (1973). Biochem. Biophys. Res. Commun. 52, 504510. Hjelm, R. P., Kneale, G. G., Suau, P., Baldwin, J. P., Bradbury, E. M. & Ibel, K. (1977). CeEZ,10, 139-151. International Critical Tables (1933). Natn. Acad. Sci., U.S.A. McGraw-Hill, New York. Joffe, J., Keene, M. & Weintraub, H. (1977). Biochemistry, 16, 1236-1238. Kellett, G. L. (1971). J. Mol. Biol. 59, 401-424. Kornberg, R. D. (1974). Science, 184, 868-871. Kornberg, R. D. (1977). Annu. Rev. Biochem. 46, 931-954. Kornberg, R. D. & Thomas, J. 0. (1974). Science, 184, 865868. Kratky, O., Leopold, H. & Stabinger, H. (1973). Methods Enzymol. 27 (D), 98-110. Noll, M. & Kornberg, R. D. (1977).J. Mol. BioZ. 109, 393-404. Noll, M., Thomas, J. 0. & Kornberg, R. D. (1975). Science, 187, 1203-1206. Noren, I. B. E., Ho, C. & Casassa, E. F. (1971). Biochemistry, 10, 3222-3229. Olins, A. L., Carlson, R. D., Wright, E. B. $ Olins, D. E. (1976). NucZ. Acida Res. 3, 3271-3291. Oudet, P., Gross-Bellard, M. & Chambon, P. (1975). Cell, 4, 281-300. Oudet, P., Germond, J. E., Bellard, M. & Chambon, P. (1977). Phil. Trans. Roy. SOC. London, in the press. Pardon, J. F., Worcester, D. L., Wooley, J. C., Tatchell, K., Van Holde, K. E. & Richards, B. M. (1975). NucZ. Acids Res. 2, 2163-2176. Sanger, F. & Thompson, E. 0. P. (1963). Biochim. Biophys. A&, 71,468-471. Shaw, B. R., Herman, T. M., Kovacic, R. T., Beaudreau, G. S. & Van Holde, K. E. (1976). Proc. Nat. Acad.Sci., U.S.A. 73, 505-509. Svedberg, T. & Pederson, K. 0. (1940). The Ultracentrijuge, Oxford University Press, London. Tanford, C. (1969). J. Mol. BioZ. 39, 539-544. Thomas, J. 0. (1977). In Molecular BioZogy of the Mammalian Genetic Apparatus (Ts’o, P. 0. P., ed.), vol. 1, pp. 1999209, Elsevier/North-Holland Biomedical Press, Amsterdam. Thomas, J. 0. & Kornberg, R. D. (1975a). Proc. Nat. Acad. Sk., U.S.A. 72, 2626-2630. Thomas, J. 0. & Kornberg, R. D. (1975b). FEBS Letters, 58, 353-358. Thomas, J. 0. t Kornberg, R. D. (1977). Methods Cell BioZ. 18,429-440.
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Thomas, J. 0. & Thompson, R. D. (1977). Cell, 10, 633-640. van der Westhuyzen, D. R. & von Holt, C. (1971). FEBS Letters, 14, 333~-342. Weintraub, H., Palter, K. & Van Lente, F. (1975). Cell, 5, 85--l 10.
Note added in proof. Pardon et al. (1977) consider the core protein in 2 M-NaCl to be H. heterotypic tetramer and the cross-linked core protein to be an octamer. However, they find that uncross-linked and cross-linked core protein have very similar radii of gyrat,ion as measured by neutron scattering. They explain this apparent paradox by suggestsing t,hat, the heterotypic tetramer is a flat disk and that in the cross-linked octamer two such disks are st.acked directly on top of each other. From our results both the untreated and t.hr (aross-linked core protein would be expected to have essentially the samr radius of gyration, since both are octamers. REFERENCE Pardon, J. F., Worcester, D. L., Wooley, J. C., Cotter, R. M. (1977). NucZ. Acida Res. 4, 3199-3214.
R. I., Lilley,
D. M. J. & Richards,
