Downloaded from symposium.cshlp.org on June 25, 2016 - Published by Cold Spring Harbor Laboratory Press
Scattering Studies of Chromatin Subunits J. F. PARDON,* R. I. COTTER,* D. M. J. LILLEY,* D. L. WORCESTER,~"A. M. CAMPBELL,:~ J. C. WOOLEY,*wAND B. M. RICHARDS* *Scarle Research Laboratories, Lane End Road, High Wycombe, Bucks HP12 4HL, England; ~f Materials Physics Division, AERE Harwell, and Physics Department, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England; $ Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ Scotland
senting DNA, protein, nucleoprotein, and s o l v e n t we have compared the scattering calculated for both spherical and cylindrical models with the experimental data. The cylindrical models were also made to approximate both prolate and oblate ellipsoids. Spherical models were unable to account for the high-angle scattering profiles; prolate ellipsoids were also found to be unsatisfactory. However, the scattering data can be accounted for by a compact cylindrical model with the protein approximating an oblate ellipsoid, axial ratio about 2, and with the DNA preferentially positioned as a partial loop at the top connecting to a second partial loop at the bottom of the structure. The model for the core particle is supported by scattering measurements made on solutions of histone core protein, which consists of a stable complex of histones H2A, H2B, H3, and H4 in 2.0 M NaC1 at pH 9.0 (Thomas and Kornberg 1975; Weintraub et al. 1975; Campbell and Cotter 1976; Lilley et al. 1977). We have studied the core protein itself (Wooley et al. 1977), chemically cross-linked core protein (Pardon et al. 1977), and trypsinized coreprotein complexes (D. M. J. Lilley et al., in prep.). X-ray-scattering profiles of concentrated gels of individual chromatin subunits contain the features of the profiles from intact chromatin. In particular, the characteristic reduction in intensity of the firstorder maximum at ~110/~ is observed as the concentration is increased, together with the increase in the intensity of the 55-A reflection. Dilute solutions of subunits produce a shoulder at ~ 6 0 / ~ and maxima at 37, 27, 22, and 18/k - similar to the profiles from dilute solutions of intact chromatin (Richards et al. 1976; Olins et al. 1977). It would appear from these data that the integrity of the intercore particle DNA is not essential for the formation of higher order structures, at least at the high chromatin concentration with which we have worked. Studies of the radii of gyration of small oligomers have been made to determine the separation of the core particles which are spaced by linker DNA. The results suggest that extended "beads-on-a-string" structures, as are sometimes visualized in the electron microscope, are unlikely to occur in solution, even in the absence of divalent ions.
Since the Cold Spring Harbor Symposium of 1973, the value of scattering techniques for the study of chromatin has increased significantly as a result of two developments: the discovery of new biochemical methods for isolating discrete chromatin subunits (Noll 1974; Shaw et al. 1976; Varshavsky et al. 1976) and the emergence of neutron-scattering facilities (Haywood and Worcester 1973; Ibel 1976) and techniques (Stuhrmann 1974; Engleman and Moore 1975; Stuhrmann et al. 1976) for the study of biochemical macromolecules. Neutron techniques have proved especially appropriate for the study of chromatin as they allow the relative scattering power of the DNA and the protein components to be varied with respect to the solvent (HzO/D20 mixtures) (Bram et al. 1974; Baldwin et al. 1975; Pardon et al. 1975). The difference between the neutron-scattering amplitude densities of H20 and D~O is sufficiently large to provide both positive and negative contrast, or a scattering density of the solvent that is comparable to the average scattering density of the histones or of the DNA. In addition to the new neutron-scattering techniques, we have also used X-ray and laser light scattering to study chromatin subunits. Initial neutron-scattering measurements of chromatin core particles enabled us to determine the radii of gyration of the core particles under conditions where either the protein or the DNA dominates the scattering, and thus to demonstrate unambiguously that the DNA is predominantly on the outside of the particle (Pardon et al. 1975). Recently we have extended these studies to include neutron measurements made at higher scattering angles and also X-ray-scattering profiles. Data collected at higher angles provide new information on the shape of the particle and on the relative locations of the DNA and protein within the particle. These data enable us to eliminate various possible models for the core particle and to consider the acceptability of others. Using a computer program which calculates scattering profiles for models defined by separate domains of different scattering density - reprew Present address: Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138.
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Downloaded from symposium.cshlp.org on June 25, 2016 - Published by Cold Spring Harbor Laboratory Press
12
PARDON ET AL. EXPERIMENTAL PROCEDURES
Preparation and purification of chromatin particles. Chromatin particles were prepared using two different procedures. In the first, the chromatin was digested in the nucleus with staphylococcal nuclease, and core particles were purified by column chromatography using the methods described by Shaw et al. (1974). In the second method, chromatin was prepared by lysing the nuclei first and then digesting it with staphylococcal nuclease. Nuclei from 24 ml of chicken blood were lysed in 50 ml 10 mM Tris, pH 7.2, and then made 0.75 mM in calcium. After 10 minutes of digestion at 37 ~ C with 100 units/ml nuclease, the chromatin was adjusted to 10 mM EDTA and placed on ice. The chromatin subunits were purified by centrifugation for 18 hours at 48,000 rpm using a Beckman Ti14 zonal rotor (Varshavsky et al. 1976) containing a 10-45% w/v sucrose gradient. The core particles were selected from the fractionated gradient, dialyzed into 10 mM Tris, 0.7 mM EDTA (pH 7.5), and concentrated using an Amicon PM10 ultrafiltration membrane, followed by centrifugation into a 40% w/w sucrose cushion. The sample (0.5 ml) was subsequently dialyzed for 30 hours against 3 • 500 volumes of 10 mM Tris, 0.7 mM EDTA (pH,pp 7.1) in the required percentage of D20, using redistilled D20. Chromatin oligomers were prepared using the second of the above methods followed by a further purification by zonal centrifugation. Preparation of histone core protein. Nuclei from chicken erythrecytes were prepared by the procedure described by Shaw et al. (1976) and lysed into distilled water at 0 ~ C. The chromatin gel was extracted twice with 0.6 M NaC1, 10 mM sodium bisulrite, 12.5 mM sodium borate (pH 9.0) and washed further in 0.65 M NaC1 to release H1 and H5, which were separated by centrifugation for 1 hour at 30,000 r p m in a Beckman T30 rotor. The core protein was then dissociated from the DNA by addition of solid NaCI to a final concentration of 2.0 M and separated from the DNA by centrifuging the gel for 16 hours at 43,000 rpm in a Beckman TS0 rotor. To minimize dissociation of the protein complex, the volume of the chromatin in 2.0 M NaC1, pH 9.0, was chosen to give a final solution of core protein with concentration in the range of 2-5 mg/mL Scattering measurements. Neutron-scattering profiles were obtained using both the small-angle diffractometer (Haywood and Worcester 1973) at the PLUTO reactor of the Atomic Energy Research Establishment, Harwell, as described previously (Pardon et al. 1975), and the D l l instrument of the Institut Laue-Langevin, Grenoble (Ibel 1976). Data were collected using the Harwell diffractometer for scattering angles 0.7 to 16 degrees in increments of 0.1 degree. The D l l instrument was used with a sample-to-detector distance of 1.71 m for recording
high-angle scattering profiles, 2.56 m for radius of gyration measurements on core-protein and coreparticle solutions, and 10.56 m for chromatin oligomers. X-ray-scattering measurements were obtained using a Searle X-ray camera equipped with Franks double-mirror optics mounted on an Elliott GX6 rotating anode generator. Light-scattering measurements were performed using a helium-neon laser (Campbell 1976). Samples were extensively dialyzed to chemical equilibrium before measurement of the light scattering or of the refractive increment. The refractive increments of the DNA and protein were 0.165 m l / g and 0.176 ml/g, respectively, and the refractive increments of the chromatin particles did not vary with concentration over the ranges studied. Samples were filtered through Schleicher and Schull filters of pore size 0.45 #m.
Particle characterization. The distribution of DNA sizes in the various chromatin-particle preparations was determined using 3.5% polyacrylamide gels with HaeIII restriction fragments of PM2 DNA as markers. The histene composition of the particles and core protein was determined using 15% SDSpolyacrylamide gels (Laemmli 1970). Analytical ultracentrifugation was performed using both Beckman model E and MSE analytical ultracentrifuges equipped with ultraviolet (UV) and schlieren optics. X-ray- and neutron-scattering calculations. Calculations of the scattering from spheres were made using the formula taken from Guinier and Fournet (1955). The calculations for nonuniform cylinders were made using a computer program developed for models in which a cylinder is subdivided into coaxial cylindrical domains of different scattering density. The two outer domains could additionally be subdivided such that each included a central zone with one scattering density sandwiched between zones with a different density. Having defined the domains and assigned scattering densities, the computer program divided the structure into a large number (typically 700) of small elements of equal volume and used the coordinates of the centers of the elements to calculate the spherically averaged scattering profile from the expression derived by Debye (1915). The zones representing different molecular components were assigned arbitrary scattering densities relative to a unit scattering density for DNA. Calculations were made using an IBM 370/135 computer. RESULTS Characterization o f Chromatin Subunits and Core Protein
A polyacrylamide gel analysis of the core-particle DNA extracted using pronase and phenol and run on 3.5% polyacrylamide gels, alongside HaeIII re-
Downloaded from symposium.cshlp.org on June 25, 2016 - Published by Cold Spring Harbor Laboratory Press
CHROMATIN SUBUNIT STRUCTURE
Figure 1. Polyacrylamidegels (3.5%) (Loening 1967) showing (a)HaeIII restriction endonuclease digest of PM2 DNA and (b)coelectrophoresis of core particle DNA and HaeIII digest ofPM2 DNA. Sizing of HaeIII fragments (Noll 1976; Lohr et al. 1977) is given at the left. Polyacrylamide gel (3.5%) analysis of DNA extracted from (c)total nuclear digest, (d)dinucleosomes, and (e)trinucleosomes. H#eIII fragments from PM2 DNA for calibrating the oligomer DNAs are also shown (f).
striction fragments ofPM2 DNA, is shown in Figure 1 a and b. A similar gel indicating the DNA length heterogeneity of DNA molecules isolated from chromatin oligomers is shown in Figure lc-f. Data from physical-chemical analyses of the chromatin subunits are summarized in Table 1. Ultracentrifugation analysis and data taken from laser light-scattering studies of histone core protein are shown in Figure 2. The light-scattering data are consistent with a histone complex of molecular weight 56,500-60,000 (Campbell and Cotter 1976), having a sedimentation coefficient of 3.8 + 0.1S. Polyacrylamide gel analysis of histone core protein, also shown in Figure 2, shows histone bands corresponding to H2A, H2B, H3, and H4, with very little
13
Figure 2. Characterization of hiztone core protein. (a) Sedimentation velocity ultracentrifuge data from chicken erythrocyte core protein. Plot ofln r(r = boundary position) against time with rotor speed 52,000 rpm using the Beckman model E ultracentrifuge. The plot remains linear for a run of over 2 hr. (b) Laser light-scattering data showing the relationship between apparent molecular weight and concentration for chicken erythrocyte core protein giving an extrapolated molecular weight of 56,500 + 3400 (Campbell and Cotter 1976). (c) 15% SDS-polyacrylamide geI analysis (Laemmli 1970) of core protein showing virtual absence of nonhistone proteins, histone H1, and histene HS. Histones H2A, H2B, H3, and H4 appear to be present in equal proportions. contaminating H1 or H5. Core protein has less t h a n 0.1% DNA contamination as judged by diphenylamine reaction (Burton 1956) and lack of dissymmetry of scattered laser light (Campbell and Cotter 1976).
Neutron Scattering from Dilute Solutions of Histone Core Protein Neutron-scattering profiles obtained from solutions of histone core protein and chromatin core particles are shown in Figure 3. The low-angle region of the scattering profile for histone core protein has been studied within a wide range of protein concentrations (2.5-40 mg/ml). These data, presented in the form of Guinier plots in Figure 4, are linear over the range 0.0005 < Q2
Scattering Studies of Chromatin Subunits J. F. PARDON,* R. I. COTTER,* D. M. J. LILLEY,* D. L. WORCESTER,~"A. M. CAMPBELL,:~ J. C. WOOLEY,*wAND B. M. RICHARDS* *Scarle Research Laboratories, Lane End Road, High Wycombe, Bucks HP12 4HL, England; ~f Materials Physics Division, AERE Harwell, and Physics Department, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England; $ Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ Scotland
senting DNA, protein, nucleoprotein, and s o l v e n t we have compared the scattering calculated for both spherical and cylindrical models with the experimental data. The cylindrical models were also made to approximate both prolate and oblate ellipsoids. Spherical models were unable to account for the high-angle scattering profiles; prolate ellipsoids were also found to be unsatisfactory. However, the scattering data can be accounted for by a compact cylindrical model with the protein approximating an oblate ellipsoid, axial ratio about 2, and with the DNA preferentially positioned as a partial loop at the top connecting to a second partial loop at the bottom of the structure. The model for the core particle is supported by scattering measurements made on solutions of histone core protein, which consists of a stable complex of histones H2A, H2B, H3, and H4 in 2.0 M NaC1 at pH 9.0 (Thomas and Kornberg 1975; Weintraub et al. 1975; Campbell and Cotter 1976; Lilley et al. 1977). We have studied the core protein itself (Wooley et al. 1977), chemically cross-linked core protein (Pardon et al. 1977), and trypsinized coreprotein complexes (D. M. J. Lilley et al., in prep.). X-ray-scattering profiles of concentrated gels of individual chromatin subunits contain the features of the profiles from intact chromatin. In particular, the characteristic reduction in intensity of the firstorder maximum at ~110/~ is observed as the concentration is increased, together with the increase in the intensity of the 55-A reflection. Dilute solutions of subunits produce a shoulder at ~ 6 0 / ~ and maxima at 37, 27, 22, and 18/k - similar to the profiles from dilute solutions of intact chromatin (Richards et al. 1976; Olins et al. 1977). It would appear from these data that the integrity of the intercore particle DNA is not essential for the formation of higher order structures, at least at the high chromatin concentration with which we have worked. Studies of the radii of gyration of small oligomers have been made to determine the separation of the core particles which are spaced by linker DNA. The results suggest that extended "beads-on-a-string" structures, as are sometimes visualized in the electron microscope, are unlikely to occur in solution, even in the absence of divalent ions.
Since the Cold Spring Harbor Symposium of 1973, the value of scattering techniques for the study of chromatin has increased significantly as a result of two developments: the discovery of new biochemical methods for isolating discrete chromatin subunits (Noll 1974; Shaw et al. 1976; Varshavsky et al. 1976) and the emergence of neutron-scattering facilities (Haywood and Worcester 1973; Ibel 1976) and techniques (Stuhrmann 1974; Engleman and Moore 1975; Stuhrmann et al. 1976) for the study of biochemical macromolecules. Neutron techniques have proved especially appropriate for the study of chromatin as they allow the relative scattering power of the DNA and the protein components to be varied with respect to the solvent (HzO/D20 mixtures) (Bram et al. 1974; Baldwin et al. 1975; Pardon et al. 1975). The difference between the neutron-scattering amplitude densities of H20 and D~O is sufficiently large to provide both positive and negative contrast, or a scattering density of the solvent that is comparable to the average scattering density of the histones or of the DNA. In addition to the new neutron-scattering techniques, we have also used X-ray and laser light scattering to study chromatin subunits. Initial neutron-scattering measurements of chromatin core particles enabled us to determine the radii of gyration of the core particles under conditions where either the protein or the DNA dominates the scattering, and thus to demonstrate unambiguously that the DNA is predominantly on the outside of the particle (Pardon et al. 1975). Recently we have extended these studies to include neutron measurements made at higher scattering angles and also X-ray-scattering profiles. Data collected at higher angles provide new information on the shape of the particle and on the relative locations of the DNA and protein within the particle. These data enable us to eliminate various possible models for the core particle and to consider the acceptability of others. Using a computer program which calculates scattering profiles for models defined by separate domains of different scattering density - reprew Present address: Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138.
11
Downloaded from symposium.cshlp.org on June 25, 2016 - Published by Cold Spring Harbor Laboratory Press
12
PARDON ET AL. EXPERIMENTAL PROCEDURES
Preparation and purification of chromatin particles. Chromatin particles were prepared using two different procedures. In the first, the chromatin was digested in the nucleus with staphylococcal nuclease, and core particles were purified by column chromatography using the methods described by Shaw et al. (1974). In the second method, chromatin was prepared by lysing the nuclei first and then digesting it with staphylococcal nuclease. Nuclei from 24 ml of chicken blood were lysed in 50 ml 10 mM Tris, pH 7.2, and then made 0.75 mM in calcium. After 10 minutes of digestion at 37 ~ C with 100 units/ml nuclease, the chromatin was adjusted to 10 mM EDTA and placed on ice. The chromatin subunits were purified by centrifugation for 18 hours at 48,000 rpm using a Beckman Ti14 zonal rotor (Varshavsky et al. 1976) containing a 10-45% w/v sucrose gradient. The core particles were selected from the fractionated gradient, dialyzed into 10 mM Tris, 0.7 mM EDTA (pH 7.5), and concentrated using an Amicon PM10 ultrafiltration membrane, followed by centrifugation into a 40% w/w sucrose cushion. The sample (0.5 ml) was subsequently dialyzed for 30 hours against 3 • 500 volumes of 10 mM Tris, 0.7 mM EDTA (pH,pp 7.1) in the required percentage of D20, using redistilled D20. Chromatin oligomers were prepared using the second of the above methods followed by a further purification by zonal centrifugation. Preparation of histone core protein. Nuclei from chicken erythrecytes were prepared by the procedure described by Shaw et al. (1976) and lysed into distilled water at 0 ~ C. The chromatin gel was extracted twice with 0.6 M NaC1, 10 mM sodium bisulrite, 12.5 mM sodium borate (pH 9.0) and washed further in 0.65 M NaC1 to release H1 and H5, which were separated by centrifugation for 1 hour at 30,000 r p m in a Beckman T30 rotor. The core protein was then dissociated from the DNA by addition of solid NaCI to a final concentration of 2.0 M and separated from the DNA by centrifuging the gel for 16 hours at 43,000 rpm in a Beckman TS0 rotor. To minimize dissociation of the protein complex, the volume of the chromatin in 2.0 M NaC1, pH 9.0, was chosen to give a final solution of core protein with concentration in the range of 2-5 mg/mL Scattering measurements. Neutron-scattering profiles were obtained using both the small-angle diffractometer (Haywood and Worcester 1973) at the PLUTO reactor of the Atomic Energy Research Establishment, Harwell, as described previously (Pardon et al. 1975), and the D l l instrument of the Institut Laue-Langevin, Grenoble (Ibel 1976). Data were collected using the Harwell diffractometer for scattering angles 0.7 to 16 degrees in increments of 0.1 degree. The D l l instrument was used with a sample-to-detector distance of 1.71 m for recording
high-angle scattering profiles, 2.56 m for radius of gyration measurements on core-protein and coreparticle solutions, and 10.56 m for chromatin oligomers. X-ray-scattering measurements were obtained using a Searle X-ray camera equipped with Franks double-mirror optics mounted on an Elliott GX6 rotating anode generator. Light-scattering measurements were performed using a helium-neon laser (Campbell 1976). Samples were extensively dialyzed to chemical equilibrium before measurement of the light scattering or of the refractive increment. The refractive increments of the DNA and protein were 0.165 m l / g and 0.176 ml/g, respectively, and the refractive increments of the chromatin particles did not vary with concentration over the ranges studied. Samples were filtered through Schleicher and Schull filters of pore size 0.45 #m.
Particle characterization. The distribution of DNA sizes in the various chromatin-particle preparations was determined using 3.5% polyacrylamide gels with HaeIII restriction fragments of PM2 DNA as markers. The histene composition of the particles and core protein was determined using 15% SDSpolyacrylamide gels (Laemmli 1970). Analytical ultracentrifugation was performed using both Beckman model E and MSE analytical ultracentrifuges equipped with ultraviolet (UV) and schlieren optics. X-ray- and neutron-scattering calculations. Calculations of the scattering from spheres were made using the formula taken from Guinier and Fournet (1955). The calculations for nonuniform cylinders were made using a computer program developed for models in which a cylinder is subdivided into coaxial cylindrical domains of different scattering density. The two outer domains could additionally be subdivided such that each included a central zone with one scattering density sandwiched between zones with a different density. Having defined the domains and assigned scattering densities, the computer program divided the structure into a large number (typically 700) of small elements of equal volume and used the coordinates of the centers of the elements to calculate the spherically averaged scattering profile from the expression derived by Debye (1915). The zones representing different molecular components were assigned arbitrary scattering densities relative to a unit scattering density for DNA. Calculations were made using an IBM 370/135 computer. RESULTS Characterization o f Chromatin Subunits and Core Protein
A polyacrylamide gel analysis of the core-particle DNA extracted using pronase and phenol and run on 3.5% polyacrylamide gels, alongside HaeIII re-
Downloaded from symposium.cshlp.org on June 25, 2016 - Published by Cold Spring Harbor Laboratory Press
CHROMATIN SUBUNIT STRUCTURE
Figure 1. Polyacrylamidegels (3.5%) (Loening 1967) showing (a)HaeIII restriction endonuclease digest of PM2 DNA and (b)coelectrophoresis of core particle DNA and HaeIII digest ofPM2 DNA. Sizing of HaeIII fragments (Noll 1976; Lohr et al. 1977) is given at the left. Polyacrylamide gel (3.5%) analysis of DNA extracted from (c)total nuclear digest, (d)dinucleosomes, and (e)trinucleosomes. H#eIII fragments from PM2 DNA for calibrating the oligomer DNAs are also shown (f).
striction fragments ofPM2 DNA, is shown in Figure 1 a and b. A similar gel indicating the DNA length heterogeneity of DNA molecules isolated from chromatin oligomers is shown in Figure lc-f. Data from physical-chemical analyses of the chromatin subunits are summarized in Table 1. Ultracentrifugation analysis and data taken from laser light-scattering studies of histone core protein are shown in Figure 2. The light-scattering data are consistent with a histone complex of molecular weight 56,500-60,000 (Campbell and Cotter 1976), having a sedimentation coefficient of 3.8 + 0.1S. Polyacrylamide gel analysis of histone core protein, also shown in Figure 2, shows histone bands corresponding to H2A, H2B, H3, and H4, with very little
13
Figure 2. Characterization of hiztone core protein. (a) Sedimentation velocity ultracentrifuge data from chicken erythrocyte core protein. Plot ofln r(r = boundary position) against time with rotor speed 52,000 rpm using the Beckman model E ultracentrifuge. The plot remains linear for a run of over 2 hr. (b) Laser light-scattering data showing the relationship between apparent molecular weight and concentration for chicken erythrocyte core protein giving an extrapolated molecular weight of 56,500 + 3400 (Campbell and Cotter 1976). (c) 15% SDS-polyacrylamide geI analysis (Laemmli 1970) of core protein showing virtual absence of nonhistone proteins, histone H1, and histene HS. Histones H2A, H2B, H3, and H4 appear to be present in equal proportions. contaminating H1 or H5. Core protein has less t h a n 0.1% DNA contamination as judged by diphenylamine reaction (Burton 1956) and lack of dissymmetry of scattered laser light (Campbell and Cotter 1976).
Neutron Scattering from Dilute Solutions of Histone Core Protein Neutron-scattering profiles obtained from solutions of histone core protein and chromatin core particles are shown in Figure 3. The low-angle region of the scattering profile for histone core protein has been studied within a wide range of protein concentrations (2.5-40 mg/ml). These data, presented in the form of Guinier plots in Figure 4, are linear over the range 0.0005 < Q2
