THE Jowwar. OF Bmmcrc~r. CHRM~TW Vol. 253, No. 12, Issue of June 25, pp. 4446-4450, 1978 Printed in U.S.A.

Fractionation and Characterization of Chromosomal Proteins by the Hydroxyapatite Dissociation Method*

(Received for publication, October 3, 1977)

Kerry S. Bloom+ and John N. Anderson

From the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

A method was developed which enables the charac- terization and fractionation of chromosomal proteins according to their chromatin binding properties. The method is based on the ability of hydroxyapatite to bind native chromatin in solutions which do not disso- ciate chromosomal proteins from the DNA. The pro- teins are then selectively dissociated from the immo- bilized chromatin by treatment with NaCl, urea, or guanidine HCl. The hydroxyapatite dissociation method represents a rapid one-step fractionation pro- cedure which results in the quantitative recovery of chromosomal proteins devoid of nucleic acids and is suitable for large preparations.

The hydroxyapatite dissociation method provides a versatile procedure for the study and preparation of chromosomal proteins. The patterns of dissociation of both histones and nonhistone chromosomal proteins by NaCl and urea from chicken oviduct chromatin were characterized by this method. In addition, this tech- nique enabled the purification of the major histone species in a single operation. Partial purification of specific nonhistone proteins, including the estrogen receptor, was also achieved. We suggest that this method will be a useful tool in elucidation of the chem- ical and biological properties of the proteins from chro- matin.

DNA of eukaryotes is associated with a fixed level of histone protein and a variable amount of nonhistone chromosomal protein and RNA to form a complex which is defined as chromatin. Chromatin proteins have received considerable attention in recent years because of their roles in the regula- tion of chromosome structure and function. However, resolu- tion of these proteins, and in particular the nonhistones, has proven particularly difficult because of the high affinity of these proteins for DNA, their propensity to aggregate in the absence of DNA or strong denaturants, and proteolytic deg- radation during prolonged isolation procedures used in their preparation.

Fractionation of chromatin proteins has generally followed one of three basic schemes. The first method employs strong acid (HCl, or H2S04) for extraction of histones from chromatin (l-4). This procedure has limited application since acid treat- ment affects structural and functional properties of both his- tone and nonhistone proteins (5). The second approach in- volves initial dissociation of proteins from DNA by treatment

* This work was supported by Grant NP-214 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a Predoctoral National Institutes of Health Traineeship.

of chromatin with NaCl (22 M) and urea (25 M) (6, 7). The DNA is removed by prolonged ultracentrifugation (6) or hy- droxyapatite chromatography (7). In the latter approach, dis- sociated chromatin in 2 M NaCl, 5 M urea, 1 mM sodium phosphate is applied to hydroxyapatite columns and the DNA and nonhistone proteins are retained. The bulk of the nonhis- tones are eluted by increasing phosphate concentration to 50 InM. Proteins are further resolved by ion exchange chroma- tography, gel filtration, and electrophoresis. The third frac- tionation method takes advantage of the differential DNA binding properties of the chromosomal proteins (6, 8-10). Proteins are sequentially dissociated from DNA by treatment of chromatin with increasing amounts of salt or urea (or both) at various levels of pH. Each dissociation step is followed by centrifugation to pellet the nondissociated protein. DNA com- plex. Histones are generally removed from nonhistone pro- teins by ion exchange chromatography or acidification.

There are several limitations to the available methods for chromatin fractionation. First, these procedures are multistep, laborious, and time-consuming. This becomes particularly important because of possible proteolysis during the isolation steps. Second, many of these methods cannot easily be per- formed on a preparative scale. In addition, overall protein recovery is often low and selective loss of chromosomal pro- teins cannot be excluded. Finally, the number of different nonhistone fractions that can be practically obtained by these procedures is limited. In this report, we describe a simple one- step method for the fractionation of chromosomal proteins that circumvents most of these difficulties.

RESULTS AND DISCUSSION’

The electrophoretic patterns of chromatin proteins pre- sented in Figs. 1 and 2 illustrate the utility of the hydroxyapa- tite dissociation method for fractionation of chromosomal proteins. Sheared hen oviduct chromatin was applied to hy- droxyapatite columns in a solution which does not dissociate chromosomal proteins from DNA (10 InM Nap, pH 7.0). Chromatin proteins were subsequently dissociated from the immobilized DNA or other chromatin components still bound to the DNA by stepwise elution with NaCl, urea, or combi- nations of the two. This procedure yields essentially quanti- tative recovery of chromosomal proteins free from nucleic acids (Fig. 3).

This method can be used to study the dissociation of histones from chromatin. The dissociation pattern of histone

’ The experimental procedures, supporting data, and references are presented in a miniprint format immediately following this paper. Figs. 3,4,5 and 10 and Table I will be found on p. 4450. Miniprint can easily be read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 77M-1578, cite author(s) and include a check or money order for $1.35 per set of photocopies.

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Fractionation of Chromosomal Proteins 4447

from hydroxyapatite-bound chromatin by NaCl is shown in Fig. 1. The very lysine-rich histone (Hl) was completely dissociated in 0.5 to 0.75 M NaCl. The moderately lysine-rich species (H2A and H2B) were eluted with 0.75 to 1.25 M NaCI,

ABCDE GWIJKL

Fro. 1 (top). SDS-polyacrylamide gel electrophoretic patterns of proteins dissociated from hydroxyapatite-bound chromatin; salt elu- tion followed by salt and urea. Unfractionated chromatin (U) proteins are shown in Lane A. Twenty AM units of chromatin at 2 AMI units/ ml of NaP (IO mM, pH 7.0) were applied to a hydroxyapatite column. Chromatin which failed to bind to the matrix is shown in Lane B. The column was washed with 80 mM NaP (Lane C) and the proteins selectively dissociated from the immobilized chromatin by NaCl (D to ZZ) followed by NaCl and urea (I to K). Solutions were 80 ITIM in NaP (pH 7.0) until Lane L, where phosphate molarity was made 500 mM to elute nucleic acid. Twenty milliliters of each solution were applied to the column. The concentrations of Nap, NaCl, and urea are shown below. Identification of histone species was determined by comparison to commercially prepared calf thymus histones.

ABCD EF G HI J KL

U 10 80 80 80 80 80 80 80 80 80 500 NaP (mM) U - - 0.25 0.5 0.75 1.25 2 2 2 2 2 NaCl (M) U----we-- --248 5 urea (M)

FIG. 2 (bottom). SDS-polyacrylamide gel electrophoretic patterns of proteins dissociated from hydroxyapatite-bound chromatin; urea elution followed by salt and urea. Lane A, unfractionated chromatin proteins; Lane B, unbound chromatin proteins; Lane C, proteins dissociated by application of 80 mM NaP (see Fig. 1). Proteins were selectively dissociated from hydroxyapatite-immobilized chromatin prepared as described in Fig. 1 by urea (D and E) followed by salt and urea (F to ZQ. Phosphate molarity was 80 mM (pH 7.0) in all solutions until the last fraction (L) when phosphate molarity was 500 mM.

ABCDEF GH I J KL

U 10 80 80 80 80 80 80 80 80 80 500 NaP (mM)

U - - - - 0.25 0.5 0.75 1.25 2 2 2 NaCl (M)

U--255 5 5 5 58 5 urea (M)

whereas the arginine-rich histones (H3 and H4) were disso- ciated only at the higher salt concentrations (1.25 to 2 M NaCl). Reduction of nonelectrostatic interaction by 5 M urea drastically altered the salt dependence of histone dissociation (Fig. 2). The moderately lysine-rich histones (H2A and H2B), as well as the arginine-rich histone species (H3 and H4),

FIG. 6 (top). Isolation and fractionation of histone proteins. Twenty Astio units of chromatin in 1 mM NaP (pH 6.0) were applied to hydroxyapatite. Columns were washed, then equilibrated, with 20 ml of 4 M urea, 70 mM NaP followed by 10 ml of 4 M urea, 1 mM NaP. Elution was carried out with a linear gradient (125 + 125 ml) of NaCl (0.0 to 2.0 M) in 4 M urea, 1 mM NaP (pH 6.0). Five-milliliter fractions were collected and dialyzed against 0.1% SDS. Representative frac- tions of the gradient were applied to the SDS-polyacrylamide gel lanes: 0.1 to 0.6 M NaCl in Lanes A to Hand greater than 1.0 M NaCl in Lanes Z to L.

FIG. 7 (bottom). Comparison of the dissociation of loosely and tightly bound nonhistone proteins. Two separate experiments are shown. Lanes B to D, hydroxyapatite-bound chromatin fractions selectively dissociated by salt, followed by salt and urea. Lanes H to J, proteins from a hydroxyapatite-bound chromatin column which was initially eluted with urea, followed by salt and urea. Both columns were then eluted with 4 M GdnHCl, 5 M urea, 80 mM NaP (E and ZQ followed by 500 mM Nap, 2 M NaCl, 5 M urea (F and L): Lanes A and G are unfractionated chromatin. Fifty milliliters of each indicated solution were applied to the columns. The first 10 ml were collected for analysis. Between Lanes Hand Z, the column was washed with 10 ml of 5 M urea, 1 mM NaP to equilibrate the column with low phosphate. Histones would then be removed with a minimum amount of nonhistone by elution with 5 M urea, 2 M NaCl, 1 mM NaP (Lane Z). Arrows were utilized to indicate three protein bands which exhibit different modes of dissociation (see text).

A BCDEF GHI J KL U 80 80 80 80 500 U 80 1 80 80 500 NaP (mM) U 0.25 2 2 - 2u-2 2-2 NaCl (M) u -- 5 5 5u5 5 55 5 urea (M) u --- 4 - U - - - 4 - GdnHCl (M)

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FIG. 8. Dissociation of the estrogen receptor from oviduct chro- matin; salt extraction followed by salt and urea. Freshly prepared nuclei were isolated, and endogenous estradiol was exchanged with [3H]estradiol as described under “Experimental Procedures.” Nuclei were then digested with micrococcal nuclease and 4 ml of each chromatin preparation (approximately 1.5 mg of DNA) were applied to hydroxyapatite columns in 5 mM NaP (pH 7.0), 1.5 mM EDTA. Extensive washing of the columns with application buffer enabled removal of free [3H]estradiol. Proteins were selectively dissociated by elution of the columns with 10 ml of the solutions indicated in the

exhibited a shift in the ease with which they were dissociated from chromatin by NaCl in the presence of urea. Dissociation of the lysine-rich histone (Hl) by NaCl was unaffected by urea.

The salt-dependent dissociation patterns of histones from hydroxyapatite-bound chromatin in the presence or absence of urea (Figs. 1 and 2) were essentially identical with those observed in previous studies where histones were dissociated from chromatin in solution (11, 12). This observation suggests that the hydroxyapatite dissociation method enables the frac- tionation of histones according to their chromatin binding properties. Figs. 4 and 5 demonstrate that the specific disso- ciation patterns of greater than 95% of the total chromosomal proteins result from their differential chromatin binding prop- erties and not from protein-hydroxyapatite interactions.

The hydroxyapatite dissociation method is a useful tool for the isolation of specific chromatin proteins since the protein fractions obtained by dissociation of chromatin in higher ionic strengths are devoid of proteins dissociated at lower NaCl levels. Fig. 2 shows that substantial purification of the three major histone groups (lysine-rich, moderately lysine-rich, and arginine-rich) can be obtained in a single operation. We have optimized conditions for histone purification and the results are shown in Fig. 6. Histones H2A (A to D), H2B (E and F), and Hl (G and H), respectively, were free of most other histones and nonhistones. Fractio,ns I and J, containing H3 and H4, were completely devoid of other histone and nonhis- tone proteins as determined by electrophoretic analysis.

Analysis of the nonhistone proteins of chromatin is more difficult than histone analysis because of the marked hetero- geneity of these proteins. The loosely bound nonhistone chro- mosomal proteins have been defined as those proteins which are solubilized from the chromatin complex by treatment with low concentrations of NaCl or KC1 (0.15 to 0.5 M) or urea (5 M) in the absence of salt (13, 14). The tightly bound nonhis-

figure. One milliliter of each fraction was added to 10 ml of scintillation fluid (toluene (1 liter): 2,5-diphenyloxazole (PPO) (5 g): 1,4-bis[2-(5. phenyloxazolyl)]benzene (POPOP) (0.5 g)) and radioactivity was de- termined in a liquid scintillation counter. Ninety-nine per cent of the charcoal-resistant [3H]estradiol applied to each column was re- covered. The remainder of each fraction was analyzed for protein content. Protein recovery was 106%. Open histograms, nuclei ex- changed with [3H]estradiol; shaded histograms, nuclei exchanged with [3H]estradiol plus a 100-fold excess of diethylstilbestrol.

tones represent those proteins which remain associated with the chromatin under these conditions. These proteins can be dissociated from the DNA by treatment with NaCl (~-2 M) and urea (~5 M) ‘or GdnHCl’ (6, 9). The hydroxyapatite dissociation method can be used to prepare loosely and tightly bound fractions of nonhistones, free of most histone proteins, in a single operation (Fig. 7). The oviduct nonhistone chro- matin proteins can be classified into three groups according to their patterns of dissociation from immobilized chromatin (Fig. 7, A to F). A few of the nonhistones appeared to be completely dissociated from the chromatin by treatment with 0.25 M NaCl (Fig. 7B). An example of such a polypeptide is designated (I -). The second group of nonhistones was apparently not dissociated from the chromatin complex by NaCl treatment, but appeared in the column effluent only after addition of urea (Fig. 70) or GdnHCl (Fig. 7E). See (2 -+) for an example of this group. The remaining species, which comprised the majority of nonhistones, exhibited bimodal patterns of dissociation. These polypeptides were detectable in the loosely bound fraction (Fig. 7B) as well as the more tightly bound fractions (Fig. 7, D and E). An example of this group is designated (3 +). Fractionation and analysis of chromatin proteins from chicken lung, liver, and brain (optic lobes) by the procedures described in Fig. 7 (A to E) also revealed these three nonhistone distribution patterns and the majority of nonhistones in these tissues, like the nonhistones in the oviduct, exhibited the bimodal pattern of dissociation (data not shown).

Estrogen regulates the growth, differentiation, and synthe- sis of the egg white proteins in the chicken oviduct and these events are presumably mediated by the chromatin-bound

’ The abbreviations used are: GdnHCl, guanidine HCl; TKM, 50 rnM Tris-HCl (pH 7.5), 25 mM KCl, 5 mM MgClr; NaP, mixture of NaH2P04 and NaZHPOr; SDS, sodium dodecyl sulfate.

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Fractionation of Chromosomal Proteins 4449

[Phosphate] mM 1 80 80 80 80 80 80 80 80 80 80 80 80 [N&l] H 80 80 80

80 - - - - - - - - - - - [Urea] M

.25 .25 .5 .5 .75 .75

28: 2.0

28: - - - 2.0 3.5

:"o" 3.5 5.0 5.0 8.0 8.0 5.0 5.0 5.0 5.0 5.0 5.0 8.0 8.0 5:0

FRACTION NUMBER FIG. 9. Dissociation of estrogen receptor from oviduct chromatin; urea extraction followed by salt and urea. Preparation of chromatin and

chromatographic procedure were as described in Fig. 8. These columns were initially eluted with urea, followed by salt and urea. Open histogram, nuclei exchanged with [“Hlestradiol; shaded histograms, nuclei exchanged with ]‘H]estradiol plus a loo-fold excess of diethylstil- bestrol.

receptor. estrogen complex (15-18). To examine the dissocia- tion pattern of the estrogen receptor from oviduct chromatin, nuclease-sheared chromatin was preincubated with [“Hlestra- diol and the proteins fractionated by the hydroxyapatite dis- sociation method (Fig. 8). Approximately 70% of the specific binding sites were dissociated from the immobilized chromatin with the loosely bound chromosomal proteins in low ionic strength (0 to 0.5 M NaCl). Less than an additional 5% of the receptor sites were dissociated from the chromatin by increas- ing the NaCl concentration to 2 M. The remaining 30% of the receptors were eluted from the columns as tightly bound nonhistones by treatment with 2 M NaCl in the presence of urea. To ensure that the [“Hlestradiol in the eluted fractions shown in Fig. 8 was proportional to the amount of estrogen receptors dissociated from the immobilized chromatin, pro- teins were first fractionated by the hydroxyapatite dissociation method and then assayed for estrogen receptor content (Table I). Table I demonstrates that 75% of the receptor sites were dissociated in 0.25 M NaCl, whereas 25% of the receptor activity was eluted from the columns by treatment with 2 M NaCl, 8 M urea. These values are in excellent agreement with those shown in Fig. 8. The linearity of the double-reciprocal plots (not shown) and the similarities in the dissociation constants (Table I) indicated that loosely associated and tightly bound estrogen receptors are homogeneous in their affinity for estradiol.

The tightly bound estrogen receptors are thought to be resistant to salt extraction because of their direct association with DNA (19) or because of their association with acceptor proteins which in turn are bound tightly to the DNA (20-22). It has been suggested that the number of receptor binding sites on the chromatin is less than the number of chromatin receptors and, therefore, the loosely bound receptors may represent those molecules which are bound to the chromatin complex in a nonselective manner (20-24). The bimodal dis- sociation patterns exhibited by the majority of nonhistone chromosomal proteins (Fig. 7) may also result from a limited number of specific binding sites for these proteins. The pro-

portion of a given protein in the loosely or tightly bound fractions would then be dependent on the binding capacity and affinity of that protein for the DNA or for a protein which is tenaciously bound to the DNA.

Dissociation of the nonhistones from chromatin, like the histones, is dramatically altered by urea (Fig. 7, H to L). Greater than 80% of the nonhistone polypeptides were disso- ciated from the immobilized chromatin by 5 M urea (Fig. 7H) in the absence of NaCl, an observation which confirms earlier reports using conventional methods (14). The proteins disso- ciated in 5 M urea (Fig. 7H) contained all detectable polypep- tides eluted by 0.25 M NaCl (Fig. 7B) plus some of the proteins which were eluted with 2 M NaCl, 5 M urea (Fig. 70). A similar shift in the distribution of the estrogen receptor was observed following urea treatment (Fig. 9). Essentially all of the estro- gen receptor activity was eluted from the columns by treat- ment with urea in the absence of NaCl. Thus, the estrogen receptor, like the majority of nonhistone chromosomal pro- teins, can be dissociated from the chromatin by 8 M urea in low ionic strength. The very tightly bound nonhistone poly- peptides dissociated by treatment with GdnHCl (Fig. 7 E and K) exhibited similar banding patterns, regardless of whether the columns were initially treated with 0.25 M NaCl or 5 M urea.

In summary, the dissociation of chromosomal proteins from hydroxyapatite-bound chromatin represents a general method for the study and preparation of chromosomal proteins. The efficiency and selectivity of the hydroxyapatite dissociation technique enables the characterization, fractionation, and pu- rification of chromatin proteins by a single procedure. We suggest that this method will be a useful tool in elucidation of the chemical and biological properties of the proteins from chromatin.

Achnowledgment-We thank Bonnie Germain for her skillful tech- nical assistance.

REFERENCES

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Fractionation of Chromosomal Proteins

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K S Bloom and J N Andersonhydroxyapatite dissociation method.

Fractionation and characterization of chromosomal proteins by the

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