Chapter 3 2
Methods for Dissociation, Fractionation, and Selective Reconstitation o f Chromatin G . S. STEIN
AND
J. L. STEIN
Departmew of Biochemistry and Molecular Biology and Department of Immunology and Medial Microbiology, University of Floriab, Gainesville, Florida
I. Introduction Chromatin reconstitution provides a direct approach for assaying the contribution of various genome-associated macromolecules toward defining the availability of specific genetic sequences for transcription. This procedure, by which chromatin is dissociated, fractionated into its principal components, and then reconstituted in a selective manner, was developed by Bonner and co-workers in the early 1960s (Z,2). While there are several modifications of the technique, generally chromatin is dissociated in the presence of high salt-urea and then reconstituted by gradually removing the salt, followed by removal of the urea. In the present article we will describe the method used in our laboratory for chromatin dissociation and reconstitution and discuss evidence for the level of fidelity attained by the procedure.
11. Preparation of Chromatin The procedures employed for isolation of chromatin have a very direct bearing on the final product. In fact, chromatin could be appropriately defined by the isolation protocol. It therefore follows that the validity of chromatin as representative of the genome found in the nucleus of intact cells, as well as the feasibility of dissociating, fractionating, and reconstituting chromatin, is dependent on the method used for chromatin isolation. 379
380
G. S. STEIN AND J. L. STEIN
Although a broad spectrum of approaches has been utilized for isolation of chromatin, there is no single procedure that appears to be optimal for all organisms, tissues, and cell types. Rather, modifications in protocols are necessary to accommodate specific biological situations. The following are general considerations which should determine the acceptability of a particular technique: (a) It is best to utilize nuclei free of cytoplasmic material and stripped of the outer aspect of the nuclear envelope. This can be best achieved by treatment with a citric acid-buffer or with nonionic detergents, such as Triton X-100 or NP-40. The concentrations ofdetergent which can be tolerated vary depending on the cell type. (b) An effort should be made to eliminate material present in the nuclear sap. (c) Caution must be exercised to avoid extraction of proteins bound to chromatin. (d) An attempt should be made to shear the DNA as little as possible. (e) Nuclease and protease activity should be minimized. Described below are the procedures which we use for preparation of chromatin from two lines of tissue culture cells, HeLa S, cells (human cervical carcinoma cells grown in suspension culture) (3) and WI-38 human diploid fibroblasts (embryonic lung cells grown in monolayer culture) (4), as well as the procedure we utilize for the preparation of chromatin from mouse and rat liver (5).
A. Tissue Culture Cells All procedures are carried out at 4OC. Cells grown in suspension culture are harvested by centrifugation at 1000 g for 5 minutes, and cells grown in monolayers are scraped from the culture vessel with a rubber policeman and then collected by centrifugation. Treatment with trypsin should be avoided since the enzyme can utilize chromosomal proteins as substrates. The harvested cells are washed three times with Earle's balanced salt solution to remove serum proteins-ach wash step followed by centrifugation at 1000 g for 5 minutes. Cells are lysed by resuspension in 80 volumes of 80 mM NaCl-20 m M EDTA-1% Triton X-100 (pH 7.2) and agitated with a Vortex mixer for 20 seconds at maximal speed. Nuclei are pelleted by centrifugation at 1000 g for 5 minutes in aswinging-bucket rotor and then washed twice in 80 mM NaCl-20 mM EDTA-1% Triton X-100. The nuclei should now be free of visible cytoplasmic material when examined by phase contrast microscopy-this should be a routine procedure. Electron microscopic examination of the nuclei should reveal the absence of the outer aspect of the nuclear envelope, and often the inner component of the nuclear envelope is also removed by detergent treatment. Nuclei are washed twice with 80
32.
CHROMATIN RECONSTITUTION
381
volumes of 0.15 M NaC1-10 mM Tris (pH 8.0) and centrifuged at lo00 g for 5 minutes to remove the detergent. It is important to remove the supernatant completely following the last wash step since the salt will interfere with nuclear lysis. Nuclei are lysed by resuspension in double-distilled water (at a concentration of 250-500 pg of DNA per milliliter) by gentle agitation with a Vortex mixer. Nuclear lysis results in a marked increase in viscosity, and after swelling in an ice bath for 20 minutes, the material is clear and gelatinous. Incomplete removal of cytoplasm during nuclear isolation, incomplete nuclear lysis, and protein denaturation will be reflected by a “cloudy” or “milky” appearance of the gel. Chromatin is pelleted by centrifugation at 12,000g for 20 minutes in a fixed-angle rotor, and then the chromatin is again washed in distilled water and pelleted at 12,000g. The chromatin pellets should be clear to slightly opalescent and extremely gelatinous. Chromatin prepared by this method has a protein:DNA ratio of 1.8-2.0. The histone: DNA ratio is 1.1, and the nonhistone chromosomal protein :DNA ratio is 0.89. All procedures can be carried out in the tube in which cells were initially harvested, resulting in 85-95% recovery of chromatin. Avoiding sonication or homogenization steps results in minimal shearing of the DNA. We have observed that shearing can result in a significant increase in chromatin template activity assayed with exogenous RNA polymerase.
B.
Mouse and Rat Liver
Animals are sacrificed by cervical dislocation. All lobes of the liver are excised, placed in a plastic boat on ice, and weighed. Nuclei are prepared by a modification of the method of Cheveau et al. (6).The liver is immediately minced with surgical scissors and suspended in 20 volumes of 2.2 M sucrose-4 mM MgCl, . All procedures are carried out at 4OC. The liver is homogenized to homogeneity in a motor-driven Potter-Elvehjem homogenizer with a wide-clearance Teflon pestle. The homogenate is filtered through one layer of Miracloth (Chicopee Mills, New York)and centrifuged in a Beckman SW-27 rotor for 60 minutes at 25,000 rpm. The supernatant is discarded and, after removal of material adhering to the walls of the centrifuge tube, the nuclear pellet is resuspended in 80 volumes of 0.15 M NaCl10 mM Tris (pH 8.0). After centrifugation at 1500 g in a swinging-bucket rotor for 3 minutes, the nuclei are again washed in 0.15 MNaCl-10 mM Tris (pH 8.0). The latter two washing steps deplete the nuclei of sucrose. Nuclear lysis and recovery of chromatin are carried out as described above for tissue culture cells.
382
G. S. STEIN AND J. L. STEIN
111. Dissociation, Fractionation, and Reconstitution
A.
Dissociation
All procedures are camed out at 4°C. Solid NaCl and urea are added to the chromatin preparation to final concentrations of 3 M and 5 M, respectively. The salt-urea gel mixture is briefly agitated on a Vortex mixer, and then the appropriate volume is achieved by addition of 10 mMTris (pH 8.0). The presence of NaCl should promote dissociation of chromatin components which are held together by electrostatic bonds, and urea should promote dissociation of components held together by hydrophobic and hydrogen bonding. Dissociation is achieved by intermittent agitation with a Vortex mixer for 2-3 hours. We have found that the optimal concentration of chromatin for dissociation under these conditions is 500 pg/ml DNA; higher concentrations of “minimally sheared chromatin” result in a solution which is unmanageably viscous. The dissociated chromatin is centrifuged in a fixed-angle rotor at 180,000g for 36 hours, after which time 95% of the chromosomal proteins are recovered in the supernatant. The supernatant should contain less than 0.1% nucleic acid. If the chromatin is excessively sheared, the supernatant may contain a high level of nucleic acid, which can in some instances be removed by additional centrifugation. Oligonucleotides which may result from extreme shearing or nuclease digestion will not pellet, and, if the level of nucleic acid in the supernatant remains high, the preparation may best be discarded, Sodium chloride is removed from the chromosomal proteins prior to ion exchange chromatography by dialysis against 100 volumes of 5 M urea10 mM Tris (pH 8.3); three changes of the dialysis buffer is adequate. The urea (before addition of Tris buffer) should be run over a mixed-bed ion exchange resin (for example, Bio-Rad resin AG501-X8), and the pH should be checked just prior to use. This is necessary to ensure the absence of cyanates and ammonia, which can accumulate during storage, as well as to remove other impurities from the urea. In reconstitution experiments in which purified DNA is used, the DNA is isolated from intact cells by the method of Marmur (7).The DNA preparations are then treated with RNase (50 pg/ml, heated for 10 minutes at 80°C to eliminate DNase activity) and Pronase (50 pg/ml, self-digested at 37°C for 60 minutes), followed by a series of phenol and chloroform-isoamyl alcohol (24: 1) extractions to remove the enzymes. We have not observed proteolytic activity during isolation, dissociation, fractionation, and reconstitution of HeLa S , cell and WI-38 chromatin. However, in systems where protease activity occurs, fidelity of reconstitution can be achieved by carrying out these procedures in the presence of
32.
CHROMATIN RECONSTITUTION
383
protease inhibitors such as phenylmethylsulfonylfluoride and diisopropylfluorophosphonate. Appropriate protease inhibitors have been summarized by Carter and Chae (8).
B. Fractionation of Chromosomal Proteins While a number of approaches have been pursued for the fractionation of chromosomal proteins into histones and nonhistone chromosomal proteins, the procedure that we most often utilize is ion-exchange chromatography on QAE Sephadex (9,ZO) a technique reported by Gilmour and Paul ( I 1 ) .
1. BATCHSEPARATION QAE Sephadex A-25 or A-50 is equilibrated against 5 M urea-10 mM Tris (pH 8.3). The Sephadex is hydrated in 50 volumes of buffer and at least three changes of buffer are made over a period of 24 hours. A-25 and A-50 will yield similar separation; however, we have found the A-25 to be preferable since A-50 undergoes significant osmotic shrinkage during salt fractionation. This is particularly a problem in the column separation procedure described below. The amount of Sephadex required is 1 gm/25 mg of chromosomal proteins. Chromosomal proteins in 5 M urea-10 mM Tris (pH 8.3) are added to the QAE Sephadex and mixed intermittently for approximately 45 minutes at 4 "C. The slurry is then poured into a porcelain Biichner funnel lined with Whatman No. 1 filter paper or into a sintered-glass filter (medium porosity). The filtrate, consisting of greater than 98% of the histones and approximately 5% of the nonhistone chromosomal proteins, is collected by vacuum filtration. The slurry is then washed with 50 volumes of 5 Murea-10 mM Tris (pH 8.3) to elute any residual histones. Nonhistone chromosomal proteins are eluted as a single fraction by washing the QAE Sephadex with 3 M NaCl-5 M urea-10 mM Tris (pH 8.3). Alternatively, several nonhistone chromosomal protein fractions can be collected by eluting the resin with increasing concentrations of NaCl in 5 M urea-10 mM Tris (pH 8.3). If it is necessary to concentrate the chromosomal protein fractions, this can be readily achieved by any one of several methods: (a) transferring the protein solution to a dialysis bag and then covering the dialysis tubing with dry Sephadex; a rapid decrease in the fluid volume can be achieved by this method if the Sephadex directly in contact with the dialysis tubing is changed every 30 minutes; (b) dialyzing against a concentrated sucrose solution, followed by subsequent dialysis against 5 M urea-10 mM Tris to remove the sucrose; (c) concentrating in an Amicon pressure filtration apparatus using a UM-10 filter.
3 84
G. S. STEIN AND J. L. STEIN
2. COLUMN CHROMATOGRAPHY A column of QAE Sephadex can be prepared, and the chromosomal proteins equilibrated against 5 M urea-10 mM Tris (pH 8.3) can be fractionated as in the batch procedure described above. It is strongly recommended that only QAE Sephadex A-25 be used with column fractionation because the osmotic shrinkage of QAE Sephadex A-50 will break the continuity of the column. The sample is applied to the column in 5 M urea-10 mM Tris (pH 8.3), and histones are eluted in the void volume. The nonhistone chromosomal proteins are eluted with a NaCl gradient in a buffer of 5 M urea-10 mM Tris (pH 8.3) or by a stepwisebatch procedure.
C. Reconstitution Chromosomal proteins and DNA are combined in 3 M NaCl-5 M urea10 mM Tris (pH 8.3). We have found that the optimal range of DNA concentration is between 200 and 800 pg/ml, and the protein :DNA ratio should not exceed 4: 1. The reconstitution mixture is dialyzed for 3 hours against 20 volumes of 3 M NaCl-5 M urea-10 mM Tris (pH 8.3). Then every 3 hours the NaCl concentration is progressively decreased. The sequential NaCl steps which we utilize in our gradient dialysis procedure are 3 M, 2.5 M, 2 M, 1.5 M, 1 M, 0.8 M, 0.7 M,O.6 M, 0.5 M, 0.4 M, 0.2 M, 0.1 M. TheNaCl is then completely removed by dialysis against several changes of 5 M urea-10 mM Tris (pH 8.3), and the reconstituted chromatin is pelleted by centrifugation at 12,000 g for 30 minutes. The reconstituted chromatin preparation is washed twice in distilled water or 10 mM Tris (pH 8.0)-each wash followed by centrifugation at 12,000g. Recovery of DNA as chromatin is 75%.
IV.
Fidelity of Reconstitution
Several lines of evidence have suggested the equivalence of native and reconstituted chromatin. We have compared native and reconstituted chromatin preparations from HeLa S, cells and found them to be indistinguishable with respect to the following parameters (12): (a) The banding patterns of histones and nonhistone chromosomal proteins when fractionated electrophoretically according to charge and molecular weight on a high-resolution polyacrylamide gel; (b) The binding of histones and nonhistone chromosomal proteins in chromatin when assayed by extractability with dilute mineral acids and ionic detergents; (c) The extent of prayinduced thymine damage in the DNA; (d)The availability of sites for binding
32.
CHROMATIN RECONSTITWION
385
of “reporter molecules” which exhibit specificity for intercalculation in the major or minor grooves of the DNA helix; (e) the circular dichroism spectra; (f) the in vitro transcription under conditions which prohibit reinitiation. Additional evidence for fidelity of chromatin reconstitution can be gleaned from studies which have demonstrated that transcription of globin (13-Z5), histone (10,16-19), and ovalbumin (20) genes from chromatin remains unaltered following dissociation, fractionation, and reconstitution. Other studies in which fidelity of transcription was assayed by hybridization to DNA complementary to total poly(A)-containing polysomal RNA suggest the similarity of native and reconstituted chromatin (21 ;C. B. Chae, private communication). However, to view the question of fidelity of chromatin reconstitution from a realistic perspective, one must continue to assay additional parameters of the native and reconstituted material. ACKNOWLEDGMENT These studies were supported by grants from the National Institutes of Health (GM 20535, CA 18875, DA 01188) and the National Science Foundation (BMS 75-18583).
REFERENCES I. 2. 3. 4. 5.
Bekhor, I., Kung, G. M., and Bonner, J. J. Mol. Biol. 39, 351 (1969). Huang, R. C., and Bonner, J., Proc. Natl. Amd. Sci. U.S.A. 54, 960 (1965). Stein, G. S., and Borun, T. W., J. Cell Biol. 52,292 (1972). Stein, G. S., Chaudhuri, S. C., and Baserga, R., J. Biol. Chem. 247, 3918 (1972). Thomson, J. A., Stein, J. L., Kleinsmith, L. J., and Stein. G. S., Science 194, 428 (1 976).
6. Cheveau, J., Moule, Y.,and Rouiller, C., Exp. Cell Res. 11, 317 (1956). 7. Marmur, J., J. Mol. Biol. 3,208 (1961). 8. Carter, D. B., and Chae. C.-B., Biochemistry 15, 180 (1976). 9. Stein, G. S., and Farber, J. L., Proc. Natl. Amd. Sci. U S A . 69, 2918 (1972). 10. Stein, G. S., Park, W. D., Thrall, C. L., Mans, R. J., and Stein, J. L., Nature (London) 257, 764 (1975). 11. Gilmour, R. S., and Paul, J., FEBS Lett. 9, 242 (1970). 12. Stein, G. S., Mans, R. J., Gabbay. E. J., Stein, J. L., Davis, J.. and Adawadkar, P. D., Biochemistry 14, 1859 (1975). 13. Paul, J., Gilmour, R. S., Affara, N., Birnie, G., Harrison, P., Hell, A., Cold Spring Harbor. Symp. Quanr. Biol. 38. 885 (1974).
14. Barrett, T., Maryanka, D., Hamlyn. P., and Could, H.,Proc. Naif. Amd. Sci. U.S.A. 71, 5057 (1974). 15. Chiu, J.-F., Tsai, Y.-H.. Sakuma, K., and Hnilica, L. S., J. Biol. Chem. 250, 9431 (1975). 16. Park, W. D., Stein, J. L., and Stein, G. S., Biochemistry 15, 3296 (1976). 17. Stein, J. L., Reed, K..and Stein, G. S., Biochemistry 15, 3291 (1976). 18. Jansing, R. L., Stein, J. L., and Stein, G. S . Proc. Natl. Amd. Sci. U.S.A. 74, 173 (1977). 19. Stein, G. S., Stein, J. L., Kleinsmith. L. J., Thomson, J. L., Park, W. D., and Jansing, R. L., Cancer Res. 36,4307 (1976). 20. Tsai, S., Tsai, M.-J., Harris, S., and OMalley, B. W., J. Biol. Chem. 251, 6475 (1976). 21. Biessmann, H., Gjerset. R. A., Levy, W. B.. and McCarthy, B. J., Biochemistry 15, 4356 (1976).
Methods for Dissociation, Fractionation, and Selective Reconstitation o f Chromatin G . S. STEIN
AND
J. L. STEIN
Departmew of Biochemistry and Molecular Biology and Department of Immunology and Medial Microbiology, University of Floriab, Gainesville, Florida
I. Introduction Chromatin reconstitution provides a direct approach for assaying the contribution of various genome-associated macromolecules toward defining the availability of specific genetic sequences for transcription. This procedure, by which chromatin is dissociated, fractionated into its principal components, and then reconstituted in a selective manner, was developed by Bonner and co-workers in the early 1960s (Z,2). While there are several modifications of the technique, generally chromatin is dissociated in the presence of high salt-urea and then reconstituted by gradually removing the salt, followed by removal of the urea. In the present article we will describe the method used in our laboratory for chromatin dissociation and reconstitution and discuss evidence for the level of fidelity attained by the procedure.
11. Preparation of Chromatin The procedures employed for isolation of chromatin have a very direct bearing on the final product. In fact, chromatin could be appropriately defined by the isolation protocol. It therefore follows that the validity of chromatin as representative of the genome found in the nucleus of intact cells, as well as the feasibility of dissociating, fractionating, and reconstituting chromatin, is dependent on the method used for chromatin isolation. 379
380
G. S. STEIN AND J. L. STEIN
Although a broad spectrum of approaches has been utilized for isolation of chromatin, there is no single procedure that appears to be optimal for all organisms, tissues, and cell types. Rather, modifications in protocols are necessary to accommodate specific biological situations. The following are general considerations which should determine the acceptability of a particular technique: (a) It is best to utilize nuclei free of cytoplasmic material and stripped of the outer aspect of the nuclear envelope. This can be best achieved by treatment with a citric acid-buffer or with nonionic detergents, such as Triton X-100 or NP-40. The concentrations ofdetergent which can be tolerated vary depending on the cell type. (b) An effort should be made to eliminate material present in the nuclear sap. (c) Caution must be exercised to avoid extraction of proteins bound to chromatin. (d) An attempt should be made to shear the DNA as little as possible. (e) Nuclease and protease activity should be minimized. Described below are the procedures which we use for preparation of chromatin from two lines of tissue culture cells, HeLa S, cells (human cervical carcinoma cells grown in suspension culture) (3) and WI-38 human diploid fibroblasts (embryonic lung cells grown in monolayer culture) (4), as well as the procedure we utilize for the preparation of chromatin from mouse and rat liver (5).
A. Tissue Culture Cells All procedures are carried out at 4OC. Cells grown in suspension culture are harvested by centrifugation at 1000 g for 5 minutes, and cells grown in monolayers are scraped from the culture vessel with a rubber policeman and then collected by centrifugation. Treatment with trypsin should be avoided since the enzyme can utilize chromosomal proteins as substrates. The harvested cells are washed three times with Earle's balanced salt solution to remove serum proteins-ach wash step followed by centrifugation at 1000 g for 5 minutes. Cells are lysed by resuspension in 80 volumes of 80 mM NaCl-20 m M EDTA-1% Triton X-100 (pH 7.2) and agitated with a Vortex mixer for 20 seconds at maximal speed. Nuclei are pelleted by centrifugation at 1000 g for 5 minutes in aswinging-bucket rotor and then washed twice in 80 mM NaCl-20 mM EDTA-1% Triton X-100. The nuclei should now be free of visible cytoplasmic material when examined by phase contrast microscopy-this should be a routine procedure. Electron microscopic examination of the nuclei should reveal the absence of the outer aspect of the nuclear envelope, and often the inner component of the nuclear envelope is also removed by detergent treatment. Nuclei are washed twice with 80
32.
CHROMATIN RECONSTITUTION
381
volumes of 0.15 M NaC1-10 mM Tris (pH 8.0) and centrifuged at lo00 g for 5 minutes to remove the detergent. It is important to remove the supernatant completely following the last wash step since the salt will interfere with nuclear lysis. Nuclei are lysed by resuspension in double-distilled water (at a concentration of 250-500 pg of DNA per milliliter) by gentle agitation with a Vortex mixer. Nuclear lysis results in a marked increase in viscosity, and after swelling in an ice bath for 20 minutes, the material is clear and gelatinous. Incomplete removal of cytoplasm during nuclear isolation, incomplete nuclear lysis, and protein denaturation will be reflected by a “cloudy” or “milky” appearance of the gel. Chromatin is pelleted by centrifugation at 12,000g for 20 minutes in a fixed-angle rotor, and then the chromatin is again washed in distilled water and pelleted at 12,000g. The chromatin pellets should be clear to slightly opalescent and extremely gelatinous. Chromatin prepared by this method has a protein:DNA ratio of 1.8-2.0. The histone: DNA ratio is 1.1, and the nonhistone chromosomal protein :DNA ratio is 0.89. All procedures can be carried out in the tube in which cells were initially harvested, resulting in 85-95% recovery of chromatin. Avoiding sonication or homogenization steps results in minimal shearing of the DNA. We have observed that shearing can result in a significant increase in chromatin template activity assayed with exogenous RNA polymerase.
B.
Mouse and Rat Liver
Animals are sacrificed by cervical dislocation. All lobes of the liver are excised, placed in a plastic boat on ice, and weighed. Nuclei are prepared by a modification of the method of Cheveau et al. (6).The liver is immediately minced with surgical scissors and suspended in 20 volumes of 2.2 M sucrose-4 mM MgCl, . All procedures are carried out at 4OC. The liver is homogenized to homogeneity in a motor-driven Potter-Elvehjem homogenizer with a wide-clearance Teflon pestle. The homogenate is filtered through one layer of Miracloth (Chicopee Mills, New York)and centrifuged in a Beckman SW-27 rotor for 60 minutes at 25,000 rpm. The supernatant is discarded and, after removal of material adhering to the walls of the centrifuge tube, the nuclear pellet is resuspended in 80 volumes of 0.15 M NaCl10 mM Tris (pH 8.0). After centrifugation at 1500 g in a swinging-bucket rotor for 3 minutes, the nuclei are again washed in 0.15 MNaCl-10 mM Tris (pH 8.0). The latter two washing steps deplete the nuclei of sucrose. Nuclear lysis and recovery of chromatin are carried out as described above for tissue culture cells.
382
G. S. STEIN AND J. L. STEIN
111. Dissociation, Fractionation, and Reconstitution
A.
Dissociation
All procedures are camed out at 4°C. Solid NaCl and urea are added to the chromatin preparation to final concentrations of 3 M and 5 M, respectively. The salt-urea gel mixture is briefly agitated on a Vortex mixer, and then the appropriate volume is achieved by addition of 10 mMTris (pH 8.0). The presence of NaCl should promote dissociation of chromatin components which are held together by electrostatic bonds, and urea should promote dissociation of components held together by hydrophobic and hydrogen bonding. Dissociation is achieved by intermittent agitation with a Vortex mixer for 2-3 hours. We have found that the optimal concentration of chromatin for dissociation under these conditions is 500 pg/ml DNA; higher concentrations of “minimally sheared chromatin” result in a solution which is unmanageably viscous. The dissociated chromatin is centrifuged in a fixed-angle rotor at 180,000g for 36 hours, after which time 95% of the chromosomal proteins are recovered in the supernatant. The supernatant should contain less than 0.1% nucleic acid. If the chromatin is excessively sheared, the supernatant may contain a high level of nucleic acid, which can in some instances be removed by additional centrifugation. Oligonucleotides which may result from extreme shearing or nuclease digestion will not pellet, and, if the level of nucleic acid in the supernatant remains high, the preparation may best be discarded, Sodium chloride is removed from the chromosomal proteins prior to ion exchange chromatography by dialysis against 100 volumes of 5 M urea10 mM Tris (pH 8.3); three changes of the dialysis buffer is adequate. The urea (before addition of Tris buffer) should be run over a mixed-bed ion exchange resin (for example, Bio-Rad resin AG501-X8), and the pH should be checked just prior to use. This is necessary to ensure the absence of cyanates and ammonia, which can accumulate during storage, as well as to remove other impurities from the urea. In reconstitution experiments in which purified DNA is used, the DNA is isolated from intact cells by the method of Marmur (7).The DNA preparations are then treated with RNase (50 pg/ml, heated for 10 minutes at 80°C to eliminate DNase activity) and Pronase (50 pg/ml, self-digested at 37°C for 60 minutes), followed by a series of phenol and chloroform-isoamyl alcohol (24: 1) extractions to remove the enzymes. We have not observed proteolytic activity during isolation, dissociation, fractionation, and reconstitution of HeLa S , cell and WI-38 chromatin. However, in systems where protease activity occurs, fidelity of reconstitution can be achieved by carrying out these procedures in the presence of
32.
CHROMATIN RECONSTITUTION
383
protease inhibitors such as phenylmethylsulfonylfluoride and diisopropylfluorophosphonate. Appropriate protease inhibitors have been summarized by Carter and Chae (8).
B. Fractionation of Chromosomal Proteins While a number of approaches have been pursued for the fractionation of chromosomal proteins into histones and nonhistone chromosomal proteins, the procedure that we most often utilize is ion-exchange chromatography on QAE Sephadex (9,ZO) a technique reported by Gilmour and Paul ( I 1 ) .
1. BATCHSEPARATION QAE Sephadex A-25 or A-50 is equilibrated against 5 M urea-10 mM Tris (pH 8.3). The Sephadex is hydrated in 50 volumes of buffer and at least three changes of buffer are made over a period of 24 hours. A-25 and A-50 will yield similar separation; however, we have found the A-25 to be preferable since A-50 undergoes significant osmotic shrinkage during salt fractionation. This is particularly a problem in the column separation procedure described below. The amount of Sephadex required is 1 gm/25 mg of chromosomal proteins. Chromosomal proteins in 5 M urea-10 mM Tris (pH 8.3) are added to the QAE Sephadex and mixed intermittently for approximately 45 minutes at 4 "C. The slurry is then poured into a porcelain Biichner funnel lined with Whatman No. 1 filter paper or into a sintered-glass filter (medium porosity). The filtrate, consisting of greater than 98% of the histones and approximately 5% of the nonhistone chromosomal proteins, is collected by vacuum filtration. The slurry is then washed with 50 volumes of 5 Murea-10 mM Tris (pH 8.3) to elute any residual histones. Nonhistone chromosomal proteins are eluted as a single fraction by washing the QAE Sephadex with 3 M NaCl-5 M urea-10 mM Tris (pH 8.3). Alternatively, several nonhistone chromosomal protein fractions can be collected by eluting the resin with increasing concentrations of NaCl in 5 M urea-10 mM Tris (pH 8.3). If it is necessary to concentrate the chromosomal protein fractions, this can be readily achieved by any one of several methods: (a) transferring the protein solution to a dialysis bag and then covering the dialysis tubing with dry Sephadex; a rapid decrease in the fluid volume can be achieved by this method if the Sephadex directly in contact with the dialysis tubing is changed every 30 minutes; (b) dialyzing against a concentrated sucrose solution, followed by subsequent dialysis against 5 M urea-10 mM Tris to remove the sucrose; (c) concentrating in an Amicon pressure filtration apparatus using a UM-10 filter.
3 84
G. S. STEIN AND J. L. STEIN
2. COLUMN CHROMATOGRAPHY A column of QAE Sephadex can be prepared, and the chromosomal proteins equilibrated against 5 M urea-10 mM Tris (pH 8.3) can be fractionated as in the batch procedure described above. It is strongly recommended that only QAE Sephadex A-25 be used with column fractionation because the osmotic shrinkage of QAE Sephadex A-50 will break the continuity of the column. The sample is applied to the column in 5 M urea-10 mM Tris (pH 8.3), and histones are eluted in the void volume. The nonhistone chromosomal proteins are eluted with a NaCl gradient in a buffer of 5 M urea-10 mM Tris (pH 8.3) or by a stepwisebatch procedure.
C. Reconstitution Chromosomal proteins and DNA are combined in 3 M NaCl-5 M urea10 mM Tris (pH 8.3). We have found that the optimal range of DNA concentration is between 200 and 800 pg/ml, and the protein :DNA ratio should not exceed 4: 1. The reconstitution mixture is dialyzed for 3 hours against 20 volumes of 3 M NaCl-5 M urea-10 mM Tris (pH 8.3). Then every 3 hours the NaCl concentration is progressively decreased. The sequential NaCl steps which we utilize in our gradient dialysis procedure are 3 M, 2.5 M, 2 M, 1.5 M, 1 M, 0.8 M, 0.7 M,O.6 M, 0.5 M, 0.4 M, 0.2 M, 0.1 M. TheNaCl is then completely removed by dialysis against several changes of 5 M urea-10 mM Tris (pH 8.3), and the reconstituted chromatin is pelleted by centrifugation at 12,000 g for 30 minutes. The reconstituted chromatin preparation is washed twice in distilled water or 10 mM Tris (pH 8.0)-each wash followed by centrifugation at 12,000g. Recovery of DNA as chromatin is 75%.
IV.
Fidelity of Reconstitution
Several lines of evidence have suggested the equivalence of native and reconstituted chromatin. We have compared native and reconstituted chromatin preparations from HeLa S, cells and found them to be indistinguishable with respect to the following parameters (12): (a) The banding patterns of histones and nonhistone chromosomal proteins when fractionated electrophoretically according to charge and molecular weight on a high-resolution polyacrylamide gel; (b) The binding of histones and nonhistone chromosomal proteins in chromatin when assayed by extractability with dilute mineral acids and ionic detergents; (c) The extent of prayinduced thymine damage in the DNA; (d)The availability of sites for binding
32.
CHROMATIN RECONSTITWION
385
of “reporter molecules” which exhibit specificity for intercalculation in the major or minor grooves of the DNA helix; (e) the circular dichroism spectra; (f) the in vitro transcription under conditions which prohibit reinitiation. Additional evidence for fidelity of chromatin reconstitution can be gleaned from studies which have demonstrated that transcription of globin (13-Z5), histone (10,16-19), and ovalbumin (20) genes from chromatin remains unaltered following dissociation, fractionation, and reconstitution. Other studies in which fidelity of transcription was assayed by hybridization to DNA complementary to total poly(A)-containing polysomal RNA suggest the similarity of native and reconstituted chromatin (21 ;C. B. Chae, private communication). However, to view the question of fidelity of chromatin reconstitution from a realistic perspective, one must continue to assay additional parameters of the native and reconstituted material. ACKNOWLEDGMENT These studies were supported by grants from the National Institutes of Health (GM 20535, CA 18875, DA 01188) and the National Science Foundation (BMS 75-18583).
REFERENCES I. 2. 3. 4. 5.
Bekhor, I., Kung, G. M., and Bonner, J. J. Mol. Biol. 39, 351 (1969). Huang, R. C., and Bonner, J., Proc. Natl. Amd. Sci. U.S.A. 54, 960 (1965). Stein, G. S., and Borun, T. W., J. Cell Biol. 52,292 (1972). Stein, G. S., Chaudhuri, S. C., and Baserga, R., J. Biol. Chem. 247, 3918 (1972). Thomson, J. A., Stein, J. L., Kleinsmith, L. J., and Stein. G. S., Science 194, 428 (1 976).
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