Chapter 20 Fractionation of Nonhistone Chromosomal Proteins Utilizing Hydroxyapatite Chromatography

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Fractionation of N onhistone Chromosomal Proteins Utilieng Hydroxyapatite Chromatography A. J. MAcGILLIVRAY* Beatson Institute for Cancer Research, Wolfon Laboratory for Molecular Pathology. Bearsden. Glasgow, Scotland

I. Introduction Much interest has centred recently on the nonhistone proteins of chromatin since they have been found to contain many of the biological activities of the nucleus, e.g., enzymes of nuclear metabolism such as the nucleic acid polymerases. However, perhaps more biological significance has been attached to the findings that these proteins have activities which appear to control the synthesis of RNA. These properties include the ability to affect the template activity of both DNA and chromatin in vitro and, in particular, the control of the expression of specific genes (Z.2). Work in this laboratory has been directed toward the study of eukaryotic gene-regulatory proteins, and one of our first steps involved the development of procedures to isolate and fractionate the nonhistone proteins of chromatin. Perhaps the most efficient of the methods currently available for the preparation of nonhistone proteins involves their isolation from chromatin which has been dissociated in solutions of high ionic strength containing denaturants such as urea (3).Our approach has been to conduct such a fractionation of chromatin using a column of hydroxyapatite, whereby at nearneutral pH the application of salt-urea dissociated chromatin results in the basic histones being unretained and the nonhistone proteins (and RNA) being adsorbed by the column. By using appropriate concentrations of phosphate buffer in the eluting medium, the nonhistone proteins can be *Present address: Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex BN1 9QG, England. 329

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eluted while the DNA is still retained by the hydroxyapatite. Moreover, a preliminary fractionation of the nonhistone proteins can be obtained by using stepwise increase in phosphate concentrations (3-5).

11. Preparation of Hydroxyapatite Hydroxyapatite is prepared from Na,HPO, . 2 H,O (Merck 6580) and CaC1, (Merck 2382) according to the method of Bernardi (6) and stored at 4 " in 1 mM sodium phosphate @H 6.8) containing a few drops of chloroform. It has been our experience that hydroxyapatite prepared by other procedures or obtained from some commercial sources either possesses slow flow rates or gives unsatisfactory separations. However, another laboratory (7) has reported satisfactory results with hydroxyapatite from a supplier. Prior to use the hydroxyapatite is washed and defined by sedimentation after gentle suspension in 2 M NaCl-5 M urea-1 mM sodium phosphate (pH 6.8) containing 2 mM Tris. * After repeating the procedure several times columns of hydroxyapatite are packed and equilibrated at room temperature with the same solution.

111. Preparation of Nuclei and Chromatin For most purposes nuclei are prepared from tissues and cells by sucrose and Triton X-100 procedures described by Birnie (7u). Except where stated all procedures are carried out at 4".Chromatin is prepared by extraction of nuclei by means of homogenization in 0.14 M NaC1-0.05 M Tris-HC1 (PH 7.9-5 mM ethlenediaminetetraacetic acid (EDTA)a.l Mm phenylmethylsulforylfluoride (PMSF)., After stirring for 20 minutes the suspension is centrifuged at 15,000 g for 15 minutes. This procedure is repeated twice.

IV. Chromatography on Hydroxyapatite The final pellet of chromatin is homogenized in sufficient 2 A4 NaC1-5 M urea-1 mM sodium phosphate (pH 6.8)-2 mM T r i ~ ~ 4mM . l PMSF to give approximately 0.5 mg of DNA/ml. After centrifugation at 15,000 g for 15 'The pH of the final solution is adjusted to 6.8 using concentrated HCI. *PMSF acts as a protease inhibitor (76). A 10mMstock solution is prepared in isopropanol. lThe pH of the final solution is adjusted to 6.8 using concentrated HCI.

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minutes the pellet is treated in the same way and the two extracts combined. The solution is sonicated4fortwo periods of 15 seconds each and then centrifuged at 15,000 g for I 5 minutes to remove traces of residual material. The supernatant (50-100 ml; OD,,, = 5-10) is then applied to the equilibrated column of hydroxyapatite.5 Solutions containing up to I5 mg of DNA are normally applied to a 25 x 1.6 cm column, and those containing between 15 and 50 mg are normally applied to a column of dimensions 25 x 2.2 cm. The flow rate is set between 5 and 10ml/hour, and with concentrated chromatin solutions it is sometimes necessary to occasionally gently stir the top of the column with a glass rod. After the sample has entered the column a small volume of the salt-urea solution is added to the top of the hydroxyapatite and allowed to sinkin. This solution is then applied to the column until, as seen by monitoring fractions at 280 nm, the histone fraction, hydroxyapatite fraction 1 (HAPI), is eluted. The nonhistone proteins are then obtained by passing the following solutions through the column: 2 M NaCI-5 M urea-2 mM Tris-50 mM phosphate (pH 6.8) (HAP2) 2 M NaCI-5 M urea-2 mM Tris-200 mM phosphate (pH 6.8) (HAP3) 2 M guanidine hydrochloride-2 mM Tris-200 mM phosphate (PH 6.8) (HAP4) If necessary a fraction containing DNA as the major constituent can then be eluted by increasing the phosphate concentration in the last solution to 500 mM (HAPS). Analyses of the fractions prepared from mouse leukemic Friend cells are given in Table I. Fraction HAP1 represents the bulk of the chromatin protein which electrophoretic and amino acid analyses show to consist of histones (4,5). However, labeling of nonhistone proteins with [3H]tryptophan shows that some 15% of the labeled proteins are eluted in this fraction. Twodimensional gel electrophoresis confirms the presence of high-molecularweight basic proteins in fraction HAP 1 (5). Fraction HAP 2 consists of the major part of the nonhistone proteins, accounting for some 12%ofthe total chromatin proteins, together with a small quantity of RNA. Both fractions HAP 3 and 4 contain protein and a considerable quantity of RNA, the former constituent representing 9 and 12% of the tryptophan-labeled nonhistone proteins, respectively. The DNA is strongly retained by the column, only traces being found in fractions HAP3 and 4 after thymidine labeling. Hence the bulk of the DNA is eluted along with RNA and a small quantity of protein in fraction HAPS. This fractionation of chromatin proteins appears to depend largely on the degree of posttranslational modification of the proteins by, e.g., phospho'An M.S.E. Ultrasonic Power Unit set at 1.5 A is used. 'The column is normally run at room temperature. However, by substituting potassium for sodium salts we have achieved identical separations and recoveries at (5). 4

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TABLE I ANALYSES OF HYDROXYAPA-IITE FRACTIONS OF MOUSELEUKEMIC FRIENDCELLCHROMATIN Protein

RNA

DNA

[I'PJADPR~ Phosphate concentraChemicaP Fraction tion (a) HAP1 HAP2 HAP3 HAP4

HAP5

1 50 200 200

500

72 12 3 4 1

"HI tryptophanaSb

15 41 9 12 3

3

Acididbasic amino acids

0.5 1.3 1.3 1.2 nd g

P

(cpm/mg (cpm/mg x x lo-.

0.1 3.3 13.5 5.2 4.5

1 .o 21.6 38.3 81.2 nd

Mean chain length

Chemicale

1.5 5.8 11.5 8.0 nd

0 0.03 0.5 0.75 1

aPercentage in each fraction. 'Isotope labeling for one generation time. 'Isotope labeling for nuclei with r p P ] A T P in v i m . 'Isotope labeling of nuclei with p2P]nicotinamide adenine dinucleotide (NAD) in vitro. Ratio using protein concentration as 1. fIsotope for 30-minute pulse. End = not determined.

PHI L"C1 uridinee,f Chemicale thymidine"'

0 0.02 1.15 2.9 nd

0 0 0 0 100

0 0 1.2 1.2 92

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rylation and adenosine diphosphoribosylation (5,8).As indicated by specific activities and the adenosine diphosphoribose (ADPR) chain length, fractions H A P 1 3 appear to elute in an order of increasing modification. Being intermediate between HAP2 and 3 fractions, HAP4 proteins are an exception to the rule, and it is possible that these are aggregated polypeptides requiring the denaturing powers of guanidine hydrochloride for their removal from the column. The small quantity of protein eluting with the DNA in fraction HAP5 also appears to be highly phosphorylated, but the large amount of nucleic acid has prevented further characterization of this material. Yields of protein from the hydroxyapatite column are high. The total recovery from the type of experiment shown in Table I in which chromatin equivalent to 10 mg of DNA was applied to a 25 x 1.6 cm column was over 90%. Lower yields are experienced when the procedure is scaled up. On the whole this affects the recovery of the nonhistone proteins, a factor perhaps associated with losses due to adsorption-desorption effects on hydroxyapatite.

V.

Removal of Polynucleotides

As described above nucleic acids, particularly RNA, contaminate the nonhistone protein fractions obtained from hydroxyapatite columns. In addition, free poly-ADPR of chain length up to 20 units also coelutes with the nonhistone proteins (8). It is often necessary to remove these polynucleotides since they can interfere with certain separation techniques and also with nucleic acid hybridization procedures. The following procedure removes nucleic acids and allows some degree of concentration of the proteins. Hydroxyapatite column fractions are concentrated to not more than 1 mg of protein/ml by dialysis against Carbowax6 and then dialyzed at room temperature against 8 M urea-0.2 M Tris = HCl (PH 8)-0.2 mM PMSF. The samples are mixed with an equal volume of 60% (w/w) CsCl, and 5-ml volumes are centrifuged at 120,000gavgfor 3 5 4 minutes at 4OC. The proteins, free of nucleic acids, are found in the top 1.5 ml of the gradient. After this volume has been collected by unloading the gradient the CsCl is removed by dialysis against a suitable buffer, e.g., 8 M u r e a 4 3 M Tris-HC1 (pH 8.3) which is the medium used for reduction of the proteins prior to isoelectric focusing (9). 6Carbowax (mol wt 15,000-20,OOO) is supplied by G. T. Gurr, High Wycombe, Bucks, England.

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VI. A.

Characterization of Hydroxyapatite Fractions

Electrophoresis

We have characterized the proteins in the hydroxyapatite fractions by two-dimensional gel electrophoresis employing a combination of isoelectric focusing and sodium dodecyl sulfate (SDS)-electrophoresis in polyacrylamide gels ( 2 , 5 , 9). Figure 1 shows the polypeptide pattern of HAP1-4 fractions of mouse liver chromatin after nucleic acids have been removed by centrifugation in CsC1-urea and the proteins subsequently reduced. The HAP1 fraction is seen to consist of the very basic low-molecularweight histones together with a few species of high-molecular-weight nonhistone proteins, one group of which has almost neutral isoelectric points (Fig. 1A). The HAP2 proteins, on the other hand, show a wide range of both molecular weights and isoelectric points, amounting to some 40 distinct polypeptide species (Fig. 1B). The HAP3 proteins consist of a more homogeneous group, all with molecular weights greater than 30,000. The major polypeptide species have isoelectric points near pH 6 (Fig. 1C). A similar situation exists in relation to the HAP4 fraction, except that the major protein species have isoelectric points near neutrality (Fig. 1D). An interesting feature of the technique is that there appears to be little overlap of the major species from one fraction to another. Other experiments (2,5.9) show that the HAP2 proteins consist of a mixture of phosphorylated and nonphosphorylated species, the major phosphoproteins being acidic and basic components, both of which are of low molecular weight. On the other hand, in the HAP3 and 4 fractions the major protein species appear to be the most highly phosphorylated.

B. Biological Assay We have attempted to locate the activity responsible for the expression of the globin genes by reconstituting mouse embryo liver chromatin from DNA, histones, and individual hydroxyapatite fractions of nonhistone proteins (10). After transcription of the chromatin by E. coli RNA polymerase, the RNA produced was tested for the presence of globin gene transcripts using complementary DNA prepared from mouse globin mRNA as aprobe. The most efficient expression of the globin genes was given by chromatin containing HAP2 proteins, followed by that containing HAP3 proteins. This result indicates that the proteins which regulate the expression of the globin genes are found among the bulk of the nonhistone proteins in the HAP2 fraction with a small amount also being eluted with the HAP3 proteins.

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B 56

C

335

7 0

9PH

D

FIG. 1. Two-dimensional gel electrophoresis of protein fractions from mouse liver chromatin chromatographed on hydroxyapatite. The horizontal axis represents isoelectric focusing in the first dimension; the pH gradient is indicated as shown in (B). The vertical axis shows separation in the second dimension using gel electrophoresis in SDS; the position of marker proteins of known molecular weight is given in (B). Proteins stained with Coomassie blue are represented by lines or spots. (A) HAP1 ; (B) HAP2; (C) HAP3; (D) HAP4.

In collaboration with others we have also investigated certain immunoiogical properties of chromatin nonhistone proteins (11). It is well established that the infection of human lymphoid cells by Epstein-Barr (E.B.) virus is associated with the presence of a specific Epstein-Barr nuclear antigen (EBNA) to which the patient raises antibodies. Using IgG isolated from anti-

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EBNA human serum we have tested hydroxyapatite fractions of chromatin from an E.B. cell line for the presence of the antigen. In this case most of the immunological reactivity was detected in the HAP4 fraction, HAP3 and HAP2 proteins giving much-reduced reactions.

VII. Conclusion The hydroxyapatite procedure is a relatively simple one-column procedure which not only separates the nonhistone proteins from the histones in high yields, but also provides a preliminary fractionation of these proteins. Electrophoretic analyses show that the three subfractions of the nonhistone proteins consist of different polypeptide species and, moreover, other analyses indicate that different biological activities can be separated by this procedure. It should also be noted that other laboratories have used variations of the hydroxyapatite procedure to isolate nonhistone proteins.’

REFERENCES 1. MacGillivray, A. J., and Rickwood, D., in “Biochemistry of Differentiation and Develop-

ment” (J. Paul, ed.),Vol. 9, p. 301. Med. Tech. Publ. &., Oxford, 1974. 2. MacGillivray, A. J., in “The Organization and Expression of the Eukaryotic Genome” (E. M. Bradbury and K. Javaherian, eds.), Academic Press, New York, in press. 3. MacGillivray, A. J.. in “Subnuclear Components” (G. D. Birnie, ed.),p. 209. Butterworth, London, 1976. 4. MacGillivary, A. J., Cameron, A., Krauze, R. J., Rickwood, D., and Paul, J., Biochim. Biophys. Actu 277, 384 (1972). 5. Rickwood, D., and MacGillivray, A. J., Eur. J. Biochem. 51, 593 (1975). 6. Bernardi, G., in “Methods in Enzymology, Vol. 21: Nucleic Acids, Part D (L. Grossman and K. Moldave, eds.), p. 95. Academic Press, New York, 1971. 7. Appels, R., Bolund, L., and Ringertz, N. R., J. Mol. Biol. 87,339 (1974). 7u. Birnie, G., Methods Cell Biol. 17 (in press). 76. Nooden, L. D., van der Brock, H. W. J. and Sevall, J. S., FEBS Lett. 29, 326 (1973). 8. Rickwood, D., MacGillivray, A. J., and Whish, W. (in press). 9. MacGillivray, A. J., and Rickwood, D., Eur. J. Biochem. 41, 181 (1974). 10. Gilmour, R. S., and MacGillivray, A. J., Proc. Orono Meet. SOC.Dev. Biol. (in press). 11. Brown, T. D. K., Rickwood, D., MacGillivray, A. J. Klein, G., and Paul, J. (in press). 12. Bluthmann, H., Mrozek, S., and Gierer, A., Eur. J. Biochem. 58,315 (1975). ’Appels et al. ( 7 ) elute the nonhistone proteins in two fractions. The first is obtained by washing the column with 200 mM phosphate in salt-urea and would be equivalent to our HAP2 and HAP3 fractions. The second fraction is then eluted using 0.5 M NaCl-5 M urea200 mM phosphate-0.2% SDS and is probably similar to our HAP4 fraction. Bluthmann et al. (12) obtain two histone and four nonhistone protein fractions by eluting the hydroxyapatite column with 5 M urea containing different concentrations of both NaCl(0.45-2 M )and phosphate (1-175 mM).