Proc. Natl. Acad. Sci. USA
Vol. 75, No. 4, pp. 1680-1684, April 1978 Biochemistry
Characterization of the histone core complex (histone tetramer/histone octamer/equilibrium/crosslinking)
SU-YUN CHUNGt, WALTER E. HILLY, AND PAUL DOTYt t Department of Biochemistry and Molecular Biology, Harvard University, Cambridge,
Montana, Missoula, Montana 59812
Massachusetts 02138; and t Department of Chemistry, University of
Contributed by Paul Doty, January 16, 1978
ABSTRACT A stable histone core complex containing equimolar ratios of H2A, H2B, H3, and H4 has been isolated from chicken erythrocyte chromatin in high salt. This complex has been characterized by sedimentation and chemical crosslinking studies. In velocity ultracentrifugation, only one single sharp sedimentation boundary has been observed with sedimentation coefficient S = 3.8 L 0.1. The molecular weight of the histone core com has been investigated by low-speed sedimentation equilibrium studies over a wide range of protein concentration. Analysis of the apparent weight-average molecular weight as a function of concentration indicates that the histone core complex in 2 M NaCl, pH 9.0, is in equilibrium between a tetramer (H2AXH2BXH3XH4) and an octamer species with a tetramer molecular weight I(H2AXH2BXH3XH4)12 of 55000. The equilibrium constant K is approximately 1.2 X 10-5 liter per mol at 100. Evidence of such a tetramer-octamer equilibrium in solution is also supported by the results of our chemical crosslinking experiments on the histone core complex. A well-characterized entity within the basic subunit of chromatin, the nucleosome, is the "core particle," which has a molecular weight of 200,000 and contains approximately 140 base pairs of DNA and two molecules each of the histones H2A, H2B, H3, and H4 (1, 2). Electron microscopy (3, 4), neutron scattering (5, 6), and x-ray diffraction (7) studies indicate that the "core particle" is a disk 110 A wide by 55 A high with two turns of DNA localized around the compact histone center. The arrangement of histones in the center and along the DNA within a nucleosome is not known. Proximity of certain histones within the nucleosome has been established by protein crosslinking studies; in particular, the use of zero-length crosslinking agents, ultraviolet light, tetranitromethane, and carbodiimide, has demonstrated close contact between H2B and H4, H2B and H2A, and H3 and H4, respectively (8, 9); furthermore, H2B is probably bound simultaneously to both H2A and H4 in vvo (8). Solution studies on mixtures of isolated histones have shown that histones H2A, H2B, H3, and H4 interact pairwise and that each possesses at least two distinct domains for interaction with other histones (9). Their interactions can be roughly divided into two categories: strong interactions between the pairs H2A-H2B, H2B-H4, and H3-H4, and weak interactions between the pairs H2A-H3, H2B-H3, and H2A-H4 (9). Along with dimeric complexes found in mixtures of histone pairs, the arginine-rich histones form a tetramer [(H3)(H4)]2 (10-13). In addition, more complex oligomeric structures are found in solutions of high ionic strength (2 M NaCl) at neutral to alkaline pH (pH 7-10) after gentle extraction of the chromatin with high salt, although the precise nature of the oligomers is not clear (6, 14-17). The elevated ionic strength and pH reduce the repulsions between the highly charged histones, possibly providing an environment similar to that provided by DNA in the nucleosome. In fact, the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
configuration of the histone complex in high salt (but not in low salt) is at least very similar to that of histones in nucleosome (6, 14, 17), suggesting that the protein species present in the high salt solution is the histone core complex within nucleosomes. In view of the complexity of histone-histone interactions and the potential implications of the oligomeric structure of the histone core complex in the internal structure of the nucleosome, we have critically investigated the histone core complex at high ionic strength by physical and chemical techniques. We find that the histone core complex contains equimolar amounts of H2A, H2B, H3, and H4 and exists in a tetramer-octamer equilibrium with a tetramer molecular weight of 55,000. MATERIALS AND METHODS Preparation and Characterization of Histone Core Complex. Red blood cells were obtained from adult white Leghorn chickens and the nuclei were isolated according to Shaw et al. (18). The histone core complex was prepared at 40 by the method of Wooley et al. (17; personal communication). The purified nuclei were lysed against an excess of distilled water; the histones H1 and H5 and nonhistone proteins were removed from the chromatin by raising the salt to 0.6 M NaCl/10 mM sodium borate, pH 9.0. The chromatin was then pelleted at 24,000 rpm for 90 min in a Beckman 30 rotor. The pellet was washed twice in 0.6 M NaCl/10 mM sodium borate, pH 9.0, and centrifuged as before. The histone core complex was dissociated from DNA by adding NaCl to 2 M at pH 9.0 and isolated by pelleting the DNA at 45,000 rpm for 14 hr in a Beckman 65 rotor. Throughout the preparation, 0.1 mM phenylmethylsulfonyl fluoride was used to inhibit protease activity. The histone core complex was free of DNA, as indicated by an A2W/A2W ratio of 17-18, and was stable for more than a month stored at 40. To determine the extinction coefficient of the histone core complex in 2 M NaCl, pH 9.0, we measured the absorption spectra in a Cary 118 spectrophotometer and determined the protein concentrations independently by both amino acid analysis (AAA Lab, Seattle, WA) and dry-weight measurement. Both methods yield an extinction coefficient of 4.2 ± 0.1 liter per g-cm at 230 nm. Sedimentation Experiments. For sedimentation studies, all protein solutions were in dialysis equilibrium. The sedimentation experiments were performed in a Spinco model E analytical ultracentrifuge equipped with a photoelectric scanner. An ultraviolet absorption optical system and 12-mm double sector cells filled with solution and dialysate were routinely used. Absorbances of protein solutions across the ultracentrifuge cells were converted into concentrations in g/liter by multiplication by conversion factors, which were determined by measuring the absorbances of tryptophan solutions in a Cary 118 spectrophotometer and in the ultracentrifuge. In some experiments at high protein loading concentrations (>5 g/liter), Abbreviation: CHES, cyclohexylaminoethane sulfonic acid.
1680
Biochemistry: Chung et al. both Rayleigh interference and schlieren optical systems were used in addition. Sedimentation Equilibrium Analysis. For the analysis of a nonideal tetramer-octamer association, we followed a simple graphical procedure developed to evaluate the association constant and the second virial coefficient (19). From conservation of mass, the equilibrium constant K is described as c = cl + KcI2 and (1 - fl)fl = Kcfl, in which c is the total concentration, cl is the tetramer concentration, and fi = cl/c is the weight fraction tetramer. The apparent average molecular weights, MwcPP and MnCaPP, are related to the true average molecular weights, MWC and Mm, by [1] MI/MwcaPP = MI/Mwc + B1Mlc [2] Mu/Mncapp = MI/Mnc + BIMjc/2 in which M1 is the monomer molecular weight and Mncapp is evaluated by trapezoidal numerical integration from the plot of MwcaPP against c following the definition C/MncaPP = SO dC/MwcaPP (20). The second virial coefficient B1 can be eliminated from Eqs. 1 and 2 by introducing a new parameter 4 = 2 Ml/MncaPP - M/MwcaPP = 2 MI/Mnc - MI/Mwc, or = (1 + fl) -1/(2-fl). [3] At a given concentration c, 4 can be obtained from the experimental data (MWCaPP and MnCaPP) and f can be obtained as the solution to the quadratic Eq. 3. A plot of (1 - fI)/f against cfh should yield a straight line with the slope equal to the equilibrium constant K. The second virial coefficient B1 can be obtained from the slope of the linear plot of MI/MwcaPP - 1/(2 - fl) against c. RESULTS Composition and Stoichiometry of Histone Core Complex. The composition of the histone core complex, analyzed in polyacrylamide/urea gels, is shown in Fig. 1. Comparison of the densitometric tracings of the gels for the histone core complex and the total histone extract from erythrocyte nuclei indicates that histones HI and H5 are completely absent in the histone core complex. The relative ratios of H2A, H2B, H3, and H4 obtained from the area under each resolved peak in Fig. 1
H2B H3 H5
Proc. Natl. Acad. Sci. USA 75 (1978)
1681
are identical for both gel tracings, indicating that the four histones are present in essentially equimolar quantities. Since it is well established that the four inner histones are present in equimolar ratio in nuclei (22), we conclude that the histone core complex isolated in this work consisted of histones H2A, H2B, H3, and H4 in equimolar ratio. Apparent Specific Volume. In order to accurately evaluate the sedimentation coefficient s2Ow and molecular weight of the histone core complex in 2 M NaCl/10 mM cyclohexylaminoethane sulfonic acid (CHES), pH 9.0, the apparent specific volume (4*) at constant chemical potential was determined experimentally. The protein solutions were prepared by exhaustive dialysis against the solvent. The measured density of the histone core complex in 2 M NaCl/10 mM CHES, pH 9.0, as a function of protein concentration is presented in Fig. 2. The density increment ?p/?c obtained from the slope of a linear least-squares regression of the data points is 0.210. Substituting op/ac = 0.210 and the measured solvent (dialysate) density po into the expression (23), O* = (1 - bp/1c)/po, yields an apparent specific volume of 0.730 ± 0.005 ml/g. This experimentally determined value is comparable to the values of v = 0.733 (H3 and H4) and i = 0.732 (H2A and H2B) which are estimated from the amino acid composition of calf thymus histones and corrected for the Donnan effect (24). In a similar manner, the apparent specific volume of the histone core complex in 1 M NaCl/10 mM CHES, pH 9.0 was determined to be 0.733 + 0.005 ml/g. Sedimentation Velocity. Boundary sedimentation velocity of the histone core complex in 2 M NaCl/10 mM CHES, pH 9.0, was measured at various protein concentrations (0.2-18 g/liter). A single symmetric sedimentation boundary was observed in all cases. A plot of s2.w as a function of concentration is shown in Fig. 3. Except at very low concentration, s20,w decreases very slightly with increasing concentration and extrapolates to so20, of 3.8 +0.1. The same sedimentation coefficient was obtained in the presence of magnesium ions (10-100 mM MgCl2) and at pH 7.0. However, we observed that at low salt (1 M NaCl), low pH (pH 5.0), or low protein concentrations (
Vol. 75, No. 4, pp. 1680-1684, April 1978 Biochemistry
Characterization of the histone core complex (histone tetramer/histone octamer/equilibrium/crosslinking)
SU-YUN CHUNGt, WALTER E. HILLY, AND PAUL DOTYt t Department of Biochemistry and Molecular Biology, Harvard University, Cambridge,
Montana, Missoula, Montana 59812
Massachusetts 02138; and t Department of Chemistry, University of
Contributed by Paul Doty, January 16, 1978
ABSTRACT A stable histone core complex containing equimolar ratios of H2A, H2B, H3, and H4 has been isolated from chicken erythrocyte chromatin in high salt. This complex has been characterized by sedimentation and chemical crosslinking studies. In velocity ultracentrifugation, only one single sharp sedimentation boundary has been observed with sedimentation coefficient S = 3.8 L 0.1. The molecular weight of the histone core com has been investigated by low-speed sedimentation equilibrium studies over a wide range of protein concentration. Analysis of the apparent weight-average molecular weight as a function of concentration indicates that the histone core complex in 2 M NaCl, pH 9.0, is in equilibrium between a tetramer (H2AXH2BXH3XH4) and an octamer species with a tetramer molecular weight I(H2AXH2BXH3XH4)12 of 55000. The equilibrium constant K is approximately 1.2 X 10-5 liter per mol at 100. Evidence of such a tetramer-octamer equilibrium in solution is also supported by the results of our chemical crosslinking experiments on the histone core complex. A well-characterized entity within the basic subunit of chromatin, the nucleosome, is the "core particle," which has a molecular weight of 200,000 and contains approximately 140 base pairs of DNA and two molecules each of the histones H2A, H2B, H3, and H4 (1, 2). Electron microscopy (3, 4), neutron scattering (5, 6), and x-ray diffraction (7) studies indicate that the "core particle" is a disk 110 A wide by 55 A high with two turns of DNA localized around the compact histone center. The arrangement of histones in the center and along the DNA within a nucleosome is not known. Proximity of certain histones within the nucleosome has been established by protein crosslinking studies; in particular, the use of zero-length crosslinking agents, ultraviolet light, tetranitromethane, and carbodiimide, has demonstrated close contact between H2B and H4, H2B and H2A, and H3 and H4, respectively (8, 9); furthermore, H2B is probably bound simultaneously to both H2A and H4 in vvo (8). Solution studies on mixtures of isolated histones have shown that histones H2A, H2B, H3, and H4 interact pairwise and that each possesses at least two distinct domains for interaction with other histones (9). Their interactions can be roughly divided into two categories: strong interactions between the pairs H2A-H2B, H2B-H4, and H3-H4, and weak interactions between the pairs H2A-H3, H2B-H3, and H2A-H4 (9). Along with dimeric complexes found in mixtures of histone pairs, the arginine-rich histones form a tetramer [(H3)(H4)]2 (10-13). In addition, more complex oligomeric structures are found in solutions of high ionic strength (2 M NaCl) at neutral to alkaline pH (pH 7-10) after gentle extraction of the chromatin with high salt, although the precise nature of the oligomers is not clear (6, 14-17). The elevated ionic strength and pH reduce the repulsions between the highly charged histones, possibly providing an environment similar to that provided by DNA in the nucleosome. In fact, the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
configuration of the histone complex in high salt (but not in low salt) is at least very similar to that of histones in nucleosome (6, 14, 17), suggesting that the protein species present in the high salt solution is the histone core complex within nucleosomes. In view of the complexity of histone-histone interactions and the potential implications of the oligomeric structure of the histone core complex in the internal structure of the nucleosome, we have critically investigated the histone core complex at high ionic strength by physical and chemical techniques. We find that the histone core complex contains equimolar amounts of H2A, H2B, H3, and H4 and exists in a tetramer-octamer equilibrium with a tetramer molecular weight of 55,000. MATERIALS AND METHODS Preparation and Characterization of Histone Core Complex. Red blood cells were obtained from adult white Leghorn chickens and the nuclei were isolated according to Shaw et al. (18). The histone core complex was prepared at 40 by the method of Wooley et al. (17; personal communication). The purified nuclei were lysed against an excess of distilled water; the histones H1 and H5 and nonhistone proteins were removed from the chromatin by raising the salt to 0.6 M NaCl/10 mM sodium borate, pH 9.0. The chromatin was then pelleted at 24,000 rpm for 90 min in a Beckman 30 rotor. The pellet was washed twice in 0.6 M NaCl/10 mM sodium borate, pH 9.0, and centrifuged as before. The histone core complex was dissociated from DNA by adding NaCl to 2 M at pH 9.0 and isolated by pelleting the DNA at 45,000 rpm for 14 hr in a Beckman 65 rotor. Throughout the preparation, 0.1 mM phenylmethylsulfonyl fluoride was used to inhibit protease activity. The histone core complex was free of DNA, as indicated by an A2W/A2W ratio of 17-18, and was stable for more than a month stored at 40. To determine the extinction coefficient of the histone core complex in 2 M NaCl, pH 9.0, we measured the absorption spectra in a Cary 118 spectrophotometer and determined the protein concentrations independently by both amino acid analysis (AAA Lab, Seattle, WA) and dry-weight measurement. Both methods yield an extinction coefficient of 4.2 ± 0.1 liter per g-cm at 230 nm. Sedimentation Experiments. For sedimentation studies, all protein solutions were in dialysis equilibrium. The sedimentation experiments were performed in a Spinco model E analytical ultracentrifuge equipped with a photoelectric scanner. An ultraviolet absorption optical system and 12-mm double sector cells filled with solution and dialysate were routinely used. Absorbances of protein solutions across the ultracentrifuge cells were converted into concentrations in g/liter by multiplication by conversion factors, which were determined by measuring the absorbances of tryptophan solutions in a Cary 118 spectrophotometer and in the ultracentrifuge. In some experiments at high protein loading concentrations (>5 g/liter), Abbreviation: CHES, cyclohexylaminoethane sulfonic acid.
1680
Biochemistry: Chung et al. both Rayleigh interference and schlieren optical systems were used in addition. Sedimentation Equilibrium Analysis. For the analysis of a nonideal tetramer-octamer association, we followed a simple graphical procedure developed to evaluate the association constant and the second virial coefficient (19). From conservation of mass, the equilibrium constant K is described as c = cl + KcI2 and (1 - fl)fl = Kcfl, in which c is the total concentration, cl is the tetramer concentration, and fi = cl/c is the weight fraction tetramer. The apparent average molecular weights, MwcPP and MnCaPP, are related to the true average molecular weights, MWC and Mm, by [1] MI/MwcaPP = MI/Mwc + B1Mlc [2] Mu/Mncapp = MI/Mnc + BIMjc/2 in which M1 is the monomer molecular weight and Mncapp is evaluated by trapezoidal numerical integration from the plot of MwcaPP against c following the definition C/MncaPP = SO dC/MwcaPP (20). The second virial coefficient B1 can be eliminated from Eqs. 1 and 2 by introducing a new parameter 4 = 2 Ml/MncaPP - M/MwcaPP = 2 MI/Mnc - MI/Mwc, or = (1 + fl) -1/(2-fl). [3] At a given concentration c, 4 can be obtained from the experimental data (MWCaPP and MnCaPP) and f can be obtained as the solution to the quadratic Eq. 3. A plot of (1 - fI)/f against cfh should yield a straight line with the slope equal to the equilibrium constant K. The second virial coefficient B1 can be obtained from the slope of the linear plot of MI/MwcaPP - 1/(2 - fl) against c. RESULTS Composition and Stoichiometry of Histone Core Complex. The composition of the histone core complex, analyzed in polyacrylamide/urea gels, is shown in Fig. 1. Comparison of the densitometric tracings of the gels for the histone core complex and the total histone extract from erythrocyte nuclei indicates that histones HI and H5 are completely absent in the histone core complex. The relative ratios of H2A, H2B, H3, and H4 obtained from the area under each resolved peak in Fig. 1
H2B H3 H5
Proc. Natl. Acad. Sci. USA 75 (1978)
1681
are identical for both gel tracings, indicating that the four histones are present in essentially equimolar quantities. Since it is well established that the four inner histones are present in equimolar ratio in nuclei (22), we conclude that the histone core complex isolated in this work consisted of histones H2A, H2B, H3, and H4 in equimolar ratio. Apparent Specific Volume. In order to accurately evaluate the sedimentation coefficient s2Ow and molecular weight of the histone core complex in 2 M NaCl/10 mM cyclohexylaminoethane sulfonic acid (CHES), pH 9.0, the apparent specific volume (4*) at constant chemical potential was determined experimentally. The protein solutions were prepared by exhaustive dialysis against the solvent. The measured density of the histone core complex in 2 M NaCl/10 mM CHES, pH 9.0, as a function of protein concentration is presented in Fig. 2. The density increment ?p/?c obtained from the slope of a linear least-squares regression of the data points is 0.210. Substituting op/ac = 0.210 and the measured solvent (dialysate) density po into the expression (23), O* = (1 - bp/1c)/po, yields an apparent specific volume of 0.730 ± 0.005 ml/g. This experimentally determined value is comparable to the values of v = 0.733 (H3 and H4) and i = 0.732 (H2A and H2B) which are estimated from the amino acid composition of calf thymus histones and corrected for the Donnan effect (24). In a similar manner, the apparent specific volume of the histone core complex in 1 M NaCl/10 mM CHES, pH 9.0 was determined to be 0.733 + 0.005 ml/g. Sedimentation Velocity. Boundary sedimentation velocity of the histone core complex in 2 M NaCl/10 mM CHES, pH 9.0, was measured at various protein concentrations (0.2-18 g/liter). A single symmetric sedimentation boundary was observed in all cases. A plot of s2.w as a function of concentration is shown in Fig. 3. Except at very low concentration, s20,w decreases very slightly with increasing concentration and extrapolates to so20, of 3.8 +0.1. The same sedimentation coefficient was obtained in the presence of magnesium ions (10-100 mM MgCl2) and at pH 7.0. However, we observed that at low salt (1 M NaCl), low pH (pH 5.0), or low protein concentrations (
