Cell, Vol. 6, 85-110,

September

1975,

Copyright

\z

1975by

MIT

Histones H2a, H2b, H3, and H4 Form a Tetrameric Complex in Solutions of High Salt Harold Weintraub’, Karen Palter, and Frederick Van Lente Frick Laboratory Department of Biochemical Sciences Princeton University Princeton, New Jersey 08540

Summary In 2 M NaCI, hlstones H2b, H2a, H3, and H4 form a heterotyplc tetrameric complex made up of one chain of each hlstone. This complex has been analyzed by hydrodynamic techniques. It Is lndistlngulshable from histones In chromatln by Its resistance to trypsin, pattern of reactivity with 125l. and ability to form speclftc crossllnked products after treatment with formaldehyde. It is proposed that this complex is responsible for protecting the small DNA fragments produced by exhaustive nuclease digestion of nuclei and that on the average two of these complexes protect the larger 180-200 base pair unit produced by partial treatment of nuclei with nuclease. Introduction Two extreme mechanisms could explain how histones become assembled with newly replicated DNA during chromosome replication. The first is an “induced-fit” model where a particular histone conformation is dependent upon specific types of interactions with the DNA. The alternative mechanism is that the histones first adopt a particular configuration and then the newly synthesized DNA passively winds about them. Experimentally, the two mechanisms lead to very different predictions. While the induced-fit hypothesis predicts that the chromosomal conformation of histones cannot exist in the absence of DNA, the second explanation predicts the existence of a solution form of histones that is identical to that present in chromosomes. In solution, all pairs of histones can interact (D’Anna and Isenberg, 1974; Kornberg and Thomas, 1974; Roark, Geoghegan, and Keller, 1974; Kelley, 1973; Bradbury and Crane-Robinson, 1971; Rubin and Moudrianakis, 1975; Bonner and Pollard, 1975; Martinson and McCarthy, 1975). [The nomenclature we used was histone H2a = llbl = f2a2; histone H2b = llb2 = f2b; histone H3 = III = f3; histone H4 = IV = f2al.l How these associations in solution might relate to those in chromatin is not known. One homotypic histone pair, the argininerich histones H3 and H4. can form a tetramer in ‘To

whom

communications

should

be addressed

solution (Kornberg and Thomas, 1974) which is in equilibrium with dimers of H3-H4 (Roark et al, 1974). [For convenience, we define interactions involving only arginine-rich histones or only slightly lysine-rich histones as homotypic interactions, and interactions involving arginine-rich histones and slightly lysine-rich histones as heterotypic interactions.] The other homotypic histone pair, the slightly lysine-rich histones H2b and H2a, seem to form either dimers (Kelley, 1973) or multiples of dimers in solution (Kornberg and Thomas, 1974). Specific associations between all 4 histones (involving heterotypic interactions) in solution have yet to be described; indeed, crosslinking evidence has been presented against these types of interactions (Kornberg and Thomas, 1974). The failure to show heterotypic complexes between all 4 histones in solution is paradoxical, since it is known from a large number of X ray studies that all 4 of the major histones are required to generate the characteristic X ray pattern of chromatin when histones are reconstituted with DNA (Kornberg and Thomas, 1974; Pardon and Wilkins, 1972; Richards and Pardon, 1970). In addition, after nuclease digestion of chromatin, subsequent treatment with trypsin generates a series of discrete particles containing protein and DNA (Weintraub, 1975). An analysis of the proteins present in most of these particles shows that all histones are present in stretches of DNA between 45 and 145 base pairs long. This might indicate that all 4 histones interact at a very local level in order to fold the DNA in the chromosome. Such a notion is supported by the finding that a crosslinked product between the heterotypic histones, H2b (slightly lysine-rich) and H4 (arginine-rich), could be identified after treatment of chromatin with formaldehyde (Van Lente, Jackson, and Weintraub, 1975) or tetranitromethane (Martinson and McCarthy, 1975). In solution, these two heterotypic histones form strong associations (D’Anna and Isenberg, 1974); however, this type of interaction has yet to be demonstrated when all 4 histones are present. The apparent failure to demonstrate solution complexes of all 4 histones in light of the growing evidence that all 4 histones are intimately associated over short stretches of DNA would appear to favor an inducedfit mechanism of chromosome assembly. In this paper, we describe a heterotypic complex containing all 4 histones. In solution, this complex behaves as a tetramer containing one each of the 4 major histones. The existence of this complex and the additional finding that many of its properties are similar to histones in chromatin indicate that DNA is not uniquely required for histones to adopt their “native” configuration.

Cell 86

A Heterotypic

Histone

Tetramer

a7

Results Sensitivity of Hlstones to Trypsin When chromatin is treated with trypsin, approximately 30 amino acid residues are cleaved from the N terminal ends of histones H3, H4, H2b, and (possibly) H2a (Weintraub and Van Lente, 1974). [Also see the experiments of Brandt, Bohm, and Von Holt (1975), who have sequenced the cleavage sites where the N terminals of H3 and H4 are cleaved by a similar protease.] All of histone Hl and the red cell-specific histone H5 appear to be completely digested to small peptides. The resistance to trypsin of 70-90 amino acid residues containing histone C terminals was attributed to their specific inter- or intramolecular interactions since these fragments become sensitive to trypsin when digested in the presence of 6 M urea (even though these conditions do not remove histones from DNA), and since these fragments remain resistant to trypsin after being removed from the DNA in 2 M NaCI. Figure la shows the trypsin digestion pattern for histones in chromatin. At high concentrations of trypsin, a stable, high molecular weight, trypsin-resistant histone limit digest is obtained. It was previously shown (Weintraub and Van Lente, 1974) that these high molecular weight, trypsin-resistant histone fragments contained the tyrosine-containing, C terminal tryptic peptides from histones H4 and H2b, but were missing tyrosine-containing peptides located near the N terminus of histone H3. This indicated that for at least 3 of the 4 major histones in chromatin, trypsin cleaves the basic N terminals from a trypsinresistant C terminal segment. In Figure 1 b, the same preparation of chromatin was suspended in 2 M NaCl and DNA was pelleted by centrifugation. The histones in the supernatant (2 M histones) were then digested with trypsin in a solution containing 2 M NaCl and 0.2 mM EDTA (pH 7.4). The digestion of histones in 2 M NaCl is indistinguishable from the digestion in chromatin, except that in 2 M NaCI, the rate is about 20 times faster than in chromatin. Undigested histone and the total histone limit-digest produced in 2 M NaCl were eluted from the gel, iodinated with 1251,digested with trypsin, and fingerprinted. Figure 2 shows that the iodinatable peptides are very similar. As Figure

1. Digestion

of Chromatin

(A) Chromatin (1 mgm/ml in loaded directly onto 15% SDS (B) Histones (1 mg/ml) in 2 The resistant histone products in 2 M NaCI) disappears, and

and Histones

with the limit-digest from chromatin, the only peptides that are missing are those present at the N terminus of histone H3 near stretches of basic residues (Hnilica, 1972). From the known sequence (Hnilica, 1972) of each histone, the remaining tyrosine-containing peptides are found either in the C terminal peptide, near it, or in the hydrophobic middle of the molecule. We conclude that trypsin cleaves histones in 2 M NaCl in a manner that is very similar if not identical to the way the histones are digested in chromatin. In order to monitor the loss of histone N terminals directly, mature erythrocytes (from 14 day-old embryos) which do not synthesize histones were labeled with ‘4C-acetate. Under these conditions histones H3, H2a, H2b, and H4 became acetylated, but not Hl or H5. After trypsin digestion of chromatin, over 90% of the incorporated acetate was absent from the trypsin limit-digest, but present in trichloracetic acid (20%) soluble material. The same results apply to histones in 2 M NaCI. Since histones are acetylated at their N terminal residues (Louie, Candido, and Dixon, 1973), this experiment indicates directly that the N terminals from these histones are digested by trypsin. An alternative possibility is that acetylated histones represent a small subpopulation of histones that are completely digested by trypsin; this is unlikely, however, since approximately 40% of all histone H3 and H4 molecules are acetylated (Ruiz-Carrillo et al., 1974) and previous work (Weintraub, 1975) has shown that over 90% of the histone molecules in the population are represented in high molecular weight, trypsin-resistant fragments. This followed from the observation that over 90% of the 35S-methionine incorporated into histones was present in the high molecular weight, trypsin limit-digest. The large amount of methionine recovered in this fraction after trypsin treatment of chromatin (or 2 M histones) further emphasizes the fact that histone C terminal segments are resistant, since methionine is present only in this part of these proteins (Hnilica, 1972). These results suggest that tryptic digestion of histones might be used to test whether a particular conformation of histones in solution corresponds to the particular conformation(s) of histones in chromatin. Experimentally, the simplest parameter

with Trypsin

histone) in 0.2 mM EDTA (pH 7.1) was digested with increasing concentrations of trypsin and the sample gels. M NaCI, 0.2 mM EDTA (pH 7.1) were digested with trypsin and also loaded directly onto the same gel. are labeled pl -p5. At very high trypsin concentrations the cleavage product, pl (formed from histones p5 increases in intensity. pl is thought to be a product of histone H3.

Trypsin concentration (left to right) in pg/ml: Chromatin: 0; 1; 5; 50; 100; 200; 300; 400; 2 M histones: 0; .Ol; .l; 1; 5; 10; 100; 200. Over 90% of the input protein enters these gels

as assayed

using

iodinated

chromatin.

Cell 88

tions at low ionic strength and render the histones completely sensitive to trypsin. Table 1 shows that 2 M histones reconstituted with DNA by stepwise dialysis to low ionic strength (in the presence or absence of 5 M urea) yield a trypsin limit-digest. Unlike the other preparations, these reconstituted histones are digested at about the same rate that trypsin cleaved histones in intact chromatin. Finally, if histones are extracted from nuclei with 0.5 M H2S04, precipitated with ethanol, redissolved in water, and dialyzed into 2 M NaCI, a stable limit-digest is obtained, indicating that acid extraction does not denature histones irreversibly by these criteria. In contrast, individual histones or histone pairs (Van Der Westhuyzen and Von Holt, 1973) (the arginine-rich pair or the lysine-rich pair), which had been isolated after acid extraction and similarly digested with trypsin in 2 M NaCl (or after reannealing to DNA), fail to show the characteristic pattern of trypsin resistance (Figure 4, Table 1). When the individual histone pairs were mixed together in equimolar quantities, denatured in acid, renatured by dialysis into 2 M NaCI, 0.5% DTT, 0.01 M Tris-HCI (pH7.4), and treated with trypsin in high salt, a stable limit-digest was produced. These experiments indicate that the rate of renaturation of the homotypic pairs in our 2 M NaCl conditions is very slow, but can be accelerated if all 4 histones are allowed to interact.

of digestion to monitor is whether or not the histones yield a limit-digest after extensive treatment with trypsin. The data in Table 1 summarize results from trypsin digestions in a variety of different conditions. In particular, high salt concentrations are required if histones are to remain resistant to trypsin in the absence of DNA. If salt is removed, the histones become sensitive to trypsin. This can be observed in Figure 3, which shows a dose response to trypsin in 0.2 M NaCI. In conditions of low ionic strength, the characteristic limit-digest is not observed. If DNA is merely added to the 0.2 M NaCl preparation, a limit-digest is obtained (Table I), but the rate of digestion is still 20 fold faster than observed with chromatin. Alternatively, if DNA is omitted and if the salt concentration is raised from 0.2 M to 2 M, trypsin resistance is restored. Thus either high salt or DNA is sufficient to induce a trypsin-resistant conformation in histones. (Other polyanions have not been tested in this assay.) To test whether the sensitivity to trypsin at low ionic strength is a consequence of repulsive forces generated by the highly charged histone N terminals, histones were digested in high salt to remove the N terminals, dialyzed down to low salt, and redigested with trypsin. A stable limit-digest was observed even though redigestion occurred at low salt. This indicates that repulsive forces between histone N terminals may distort histone comforma-

Figure

2. Tryptic

Peptides

Present

in High Molecular

The high molecular weight, trypsin-resistant an SDS gel, precipitated, iodinated, digested

Weight,

Trypsin-Resistant

(A) Whole histone. (B) Limit-digest from trypsin digestion of histones in 2 M NaCI. (C) Key identifying the origins of each tryptic peptide as previously The open circles in (6) indicate is from top to bottom.

the missing

Histone

Complexes

cleavage products obtained by digesting histones with with trypsin at low ionic strength, and fingerprinted.

peptides

in 2 M NaCl trypsin

in 2 M NaCl were

eluted

determined.

from the N terminus

of histone

H3. Electrophoresis

is from

left to right.

Chromatography

from

A Heterotypic 89

Histone

Tetramer

Crosslinking by Formaldehyde C/womatin It was previously shown (Van Lente et al., 1975) that both formaldehyde and glutaraldehyde treatment of chromatin or nuclei results in the appearance of two crosslinked dimers, one composed of histones H2b and H4, and the other containing histones H2b and H2a. As the formaldehyde concentration was increased, additional products up to tetramers could be observed. Crosslinked products larger than tetramers were not found, since at high concentrations of formaldehyde all of the histones were in very high molecular weight material that failed to enter the acrylamide gel. Figures 5a,b show the accumulation of crosslinked products up to tetramers as chromatin is treated with increasing concentrations of formaldehyde. The appearance of crosslinked histones is correlated with the rate of disappearance of individual histones; thus H2b disappears faster than H2a and H4, while H3 is the most resistant to crosslinking by formaldehyde.

Table

1. Summary

Histone

of Results

from

Trypsin

Digestions

of Histone

2 M Histones Figure 5c demonstrates that histones in 2 M NaCl (pH 7.1) are crosslinked by formaldehyde into products up to tetramers which comigrate with the crosslinked products obtained from chromatin (Figure 5b). Moreover, the sequential disappearance, with increasing concentrations of formaldehyde, of H2b, followed by H2a and H4, and finally by H3, is also observed for histones in 2 M NaCI. At higher formaldehyde concentrations a very stable and reproducible tetramer band is generated (Figure 5d,e). Some dimer is also present. Virtually the same products are produced over a wide range of formaldehyde concentrations (between 0.08% and 2%) and histone concentrations (between 25 pg/ml and 5 mg/ml), over a wide range of temperatures (between 0°C and 22°C) and reaction times (between 1 hr and 24 hr), and between pH 6.5 and pH 9.0. The generation of the tetramer band is independent of the presence of histones Hl and H5, since they are clearly very resistant to crosslink-

in a Variety

Condition

Chromatin,

0.2 mM EDTA

2 M NaCI,

(pH 7.1)

0.2 mM EDTA

(pH 7.1)

0.8 M NaCI. 0.2 mM EDTA

(pH 7.1)

of Conditions Presence of Histone Limit-Digest

Approximate of Digestion Relative to Chromatin

+

1

+

20

+

20

0.2 M NaCl (pH 7.1) 0.01

M NaCI,

2 M NaCl

20

0.2 mM EDTA

+

0.2 M NaCI,

(pH 7.1)

0.2 M NaCl --+ 0.2 mM EDTA

2 M NaCI, 0.2 mM EDTA

(pH 7.1)

0.05 M Na Acetate

Chromatin,

5 mM Na Acetate

20 2 M NaCI, 0.2 mM EDTA

(pH 7.1)

2 M NaCI,

(pH

+ DNA (equal

histones

Acid-purified

H2b

w

+

1

+

1

+

1

+

20

“”

dia’“ri>

(pH 7.1) 0.2 mM EDTA

2 M NaCl (pH

(pH 7.1)

7.1)

2 M NaCl (pH 7.1)

+ DNA “”

Acid-purified

H3 + H4 w H3 + H4 -0.1

Acid-purified

H2b

Acid-purified

H2b + H2a + DNA “”

+ H2a -

m

20

20

d’O’“ris> 0.1 M NaCl (pH 7.1)

Acid-purified

2 M NaCl (pH 7.1) !

20

(pH 5.5)

+ DNA

H2b -

20 20

+

2 M NaCl Acid-extracted

+ +

5.5)

+ DNA ‘roD diOly*i’> 0.2 mM EDTA + 6 M urea

(pH 7.1)

weight)

+ 6 M urea

2 M NaCl

Acid-purified

Rate

20

2 M NaCl (pH 7.1)

20

M NaCl (pH

20

7.1)

2 M NaCl (pH 7.1)

20

di@‘siS> 0.2 mM EDTA

0.2 M NaCl (pH 7.1) -

20 +

Histone concentrations were 1 mg/ml; trypsin digestion was for 30 min at 37°C. Concentrations were from 10 pg/ml to 200 pg/ml. All solutions contained 0.2 mM EDTA, usually adjusted to pH 7.1, “Step-dialysis” indicates reconstitution with DNA as described in Experimental Procedures. Acid-purified H2b was isolated as described by Johns (1967). Acid-purified H2b and H2a or H3 and H4 were isolated after acid extraction according to the methods of Von Holt and Van Der Westhuyzen. The approximate rate of digestion was determined in relation to chromatin from the trypsin concentration needed to yield a limit-digest or, where applicable, complete digestion,

Cell 90

ing under these conditions and since the pattern in Figure 5e can be generated from 2 M histones obtained from chromatin that had previously been depleted of Hl and H5 by prior digestion with trypsin. Tryptic peptide analysis (Van Lente et al., 1975) of the iodinatable tyrosine residues in this broad tetramer peak show the presence of H2b, H2a, H3, and H4. In summary, the pattern of formaldehyde crosslinking (like the pattern of trypsin digestion) for 2 M NaCl histones is very similar to the pattern obtained with chromatin or nuclei. The predominant tetramer band obtained by crosslinking 2 M histones indicates that in these conditions the majority of histones (except Hl and H5) are present in complexes up to tetramers.

Histone Conformation in 2 M NaCI: Trypsin-Resistant Complexes pH 7.1 The previous experiments suggest that the conformation of isolated histones in high salt is similar to that of histones in chromatin. In order to characterize the state of histones in high salt, 2 M histones were layered over a 5-20% sucrose gradient containing 2 M NaCI. The sample was centrifuged for 48 hr, and aliquots were collected and precipitated with 25% TCA. The precipitable material in each fraction was analyzed on 15% SDS gels. Figure 6 shows the results from a typical experiment. Approximately 80% of the major histones migrate as an apparent single species containing H3, H4, H2b, and H2a. It is assumed for the present time that in 2 M NaCl all 4 of these histones are complexed to each other. This complex shall be referred to as a heterotypic complex. In contrast, previously described complexes of the arginine-rich histones H3 and H4 (D’Anna and Isenberg, 1974; Kornberg and Thomas, 1974; Roark et al., 1974) or the slightly lysine-rich histones H2b and H2a (D’Anna and Isenberg, 1974; Kornberg and Thomas, 1974; Kelley, 1973) will be referred to as homotypic complexes. About 10% of the 4 major histones applied to these

Figure

Figure

3. Trypsin

Digestion

of Histones

in Low Ionic

Strength

2 M NaCl histones were dialyzed into 0.2 M NaCI, 0.2 mM EDTA (pH 7.1). A slight precipitate formed which contained histones H3 and H4 (see Figure 6). The low ionic strength histones (1 mg/ml) were digested with increasing concentrations of trypsin and loaded (left to right: 0; 0.01; 0.1; 1; 5; 10; 100; 200 gg/ml trypsin) directly onto 15% SDS gels. Although not observed in this preparation, a resistant band corresponding to pl is often observed. Conditions were the same as in Figure 1 and the two experiments can be compared directly.

4. Sensitivity

of Homotypic

Histone

Pairs

to Trypsin

After extraction in 0.5 M Hz SO4 from chromatin. the individual histone pairs were obtained according to the method of Van Der Westhuyzen and Von Holt (1973). (A) Lysine-rich histones, H2b, and H2a. (B) Arginine-rich histones, H3 and H4. (C) Total histone. Each series in (A) and (B) shows from left to right: (1) control, (2) control, (3) histone (1 mg/ml) in 2 M NaCl digested with 10 pg/ml trypsin for 30 min at 37°C (4) histone (1 mg/ml) plus DNA (1 mg/ml) mixed directly in 0.2 mM EDTA (pH 7.1) and digested with 10 pg/ml trypsin for 30 min at 37°C. No characteristic limitdigest is observed from the acid-denatured homotypic pairs.

A Heterotypic 91

Histone

Tetramer

gradients appears as a more slowly sedimenting complex of H2b and H2a. The remaining histone consists of the slowly sedimenting histones Hla, Hl b, and H5. The apparent “excess” of H2b and H2a is matched by an enrichment of H3 and H4 in material, presumably aggregated, eluted from the bottom of the centrifuge tube with SDS. Resedimentation of the isolated heterotypic complex (Figure 7) results in the appearance of a major peak containing this complex and a trailing H2b-H2a homotypic peak. Again, some H3 and H4 could be recovered at the bottom of the centrifuge tube. This indicates that the heterotypic complex dissociates into the slightly lysine-rich homotypic complex and the aggregated H3-H4. The experiment described in Figure 7 proves that the heterotypic complex dissociates. In order to show that this dissociation occurs because of a re-

versible equilibrium between the heterotypic complex and homotypic complexes, tracer amounts (approximately 1 pg) of iodinated histone H2b isolated according to the method of Johns (1967) (approximately 106 cpm/pg) were added to 500 pgm of 2 M histone (pH 7.1). The mixture was run in sucrose gradients, and the specific activity of iodinated H2b was determined in the heterotypic complex and in the trailing region of the gradient containing H2b and H2a. The specific activity in the heterotypic complex was determined to be 4.1 x 103 cpm/mg of H2b; the specific activity in the trailing homotypic complex was 4.5 x 103 cpm/ mg of H2b. This experiment shows that tracer amounts of added H2b are incorporated into the heterotypic complex and that the specific activity of H2b is about the same in heterotypic and homotypiccomplexes.Thusthesedifferentcomplexesare, in fact, in equilibrium with each other.

4 :3 .::

Figure

5. Formaldehyde

Crosslinking

of Histones

in Chromatin

and in 2 M NaCl

(A) Control of chromosomal proteins. concentrations of formaldehyde for 60 min at 0°C. Formaldehyde concentrations (8) Treatment of chromatin (OD 2L0 = 20) with increasing left to right: 0.005%; 0.01%; 0.015%; 0.05%; 0.1%; 0.25%; 0.5%; 1 .O%; 2.0%; 5.0%. Arrows mark crosslinked products, X1 is a dimer of H2b and H4. X2 is a dimer of H2b and H2a. X3 contains H2b and H4 (Van Lente et al., 1975). Buffer is 0.2 mM EDTA (pH 7.1). (C) Treatment of 2 M histones (1 mg/ml) with formaldehyde at pH 7.1 for 60 min at O’C in 0.2 mM EDTA (pH 7.1) (or 0.01 M triethanolamine). Formaldehyde concentrations from left to right: 0; 0.05%; 0.08%; 0.1%; 0.15%; 0.2%). The crosslinked products Xl, X2, X3, and X4 correspond exactly to the products obtained from chromatin. (D) Limit crosslinking pattern obtained after treatment of 2 M histones with formaldehyde. Conditions were the same as in (C) except that the formaldehyde concentration was 1% (right) and 0% (left). The predominant product is a tetramer, although some dimer and hexamer is also present. Over 70% of the Coomasie blue staining material that is loaded onto the gel is present as a tetramer. (E) Densitometer tracing of the gel obtained from an experiment similar to the one shown in D (bottom scan). The unmarked peak at the top of the gel represents very high molecular weight material that does not enter the gel. The top scan is from a control gel. Tracings were done with a Joyce-Loebl MKIIIB microdensitometer. Migration is from right to left. Arabic numbers indicate the approximate the molecular weights from the crosslinked

position dimers

of histone dimers, Xl and X2, whose

tetramers, composition

and so on, as standardized has been determined.

using

histones

H3, H4, and

Cell 92

In relation to either Hb, bovine serum albumin, or myoglobin markers, the heterotypic complex has an average sedimentation coefficient of 3.8 .f 0.3.S, the homotypic, slightly lysine-rich complex has an average sedimentation coefficient of about 2.3S, and the lysine-rich histones have sedimentation coefficients of about 1.4s (Table 2). Densitometer tracings of the histones in the heterotypic peak show that their relative ratios are the same as those obtained for unfractionated histones isolated directly from whole nuclei. Virtually the same sedimentation pattern is obtained when l/10 the amount of

histone is loaded onto the gradient. In all of the 18 sucrose gradients done at high salt, the leading edge of the peak containing the heterotypic complex is hyper-sharp, while the lagging edge usually trails behind. Almost all gradients display a rather broad peak. This behavior can be explained if the heterotypic complex is in equilibrium with a slower sedimenting species as demonstrated in Figure 7. The general features of these gradients are unchanged when Mg+z, Ca+2, or PO 4 are added to concentrations up to 0.1 M. Formation of the gradient in 3 M NaCl or 2 M NH4S04 also has no effect. The pattern is independent of pH between the ranges of 7-9; it is also unchanged if the 2 M histones are obtained from nuclei instead of chromatin, or if dithiothreitol is included in the gradient or during all of the steps in the isolation procedure.

Figure

6. Histone

Associations

in Different

Solutions

0.4 ml of histones (2 mg/ml) were layered over a 5-20% sucrose gradient and sedimented for 46 hr. Direction of migration was from right to left in A, 6, and C. Recovery of material in the gradient was over 65%. (A) and (D): Histones in 2 M NaCl (pH 7.1). (6): Histones in 2 M NaCl (pH 5.5). (C): Histones in 0.2 M NaCl (pH 7.1).

A Heterotypic 93

Histone

Tetramer

The 3.8s heterotypic complex is also reconstituted after histone purification by acid extrqction and subsequent dialysis of histones into 2 M NaCI, 0.01 Tris-HCI, (pH 7.1). This shows that pure histones have the capacity to reform the heterotypic complex in the absence of DNA, RNA, or nonhistone proteins. In order to measure the concentration dependence of the sedimentation velocity, 2 M histones were iodinated and run on sucrose gradients at

Figure 7. Dissociation Complexes

of

Heterotypic

Complex

into

concentrations of 10 mg/ml, 1 mg/ml, 500 pg/ml, 100 pg/ml, and 10 pg/ml. Fractions were precipitated, run on SDS gels as described in Figure 6, and then autoradiographed. Between 10 mg/ml and 500 pg/ml, no difference in sedimentation velocity could be detected by our methods, the heterotypic complex running at 3.8 * 0.3s. At 100 pg/ml, the sedimentation velocity dropped to about 3.OS, and at 10 pg/ml, the sedimentation velocity was about 2.5s. Since we can only speculate on the nature of the type of equilibrium occurring with 2 M histones, a rigorous analysis of these “S compared with C” results is impossible. Nevertheless, they clearly indicate that the heterotypic complex is in equilibrium with more slowly sedimenting species. They also provide a clear indication that one must be extremely cautious in interpreting data obtained from this type of complex equilibrium system. Since the sedimentation velocity of 3.8s measured in sucrose gradients is stable over a very wide range of histone concentrations, it is likely that this value represents a major species present over this concentration range. pH 5.5, 2 M NaCl If the pH of the 2 M NaCl gradient is lowered to 5.5, then the heterotypic complex gives rise to two different sedimenting homotypic complexes (Figure 6b). The arginine-rich complex migrates at about 3.2S, and the slightly lysine-rich complex at about 2.4s. These two forms probably correspond to the fractions which were originally isolated by Van Der Westhuyzen and Von Holt (1973) on Sephadex and which were further characterized by Kornberg and Thomas (1974), as well as other workers (D’Anna and Isenberg, 1974; Roark et al., 1974). If the pH is brought from 5.5 back to 7.1, the heterotypic complex is regenerated as assayed by sucrose gradient analysis.

Homotypic

The heterotypic complex was isolated from a gradient similar to the one shown in Figure 7A and run on a second gradient in 2 M NaCl (pH 7.1). About 15% of the total arginine-rich histone aggregated and was recovered on the bottom of the tube. Sedimentation is from right to left.

Table 2. Summary of Physical Gradient Sedimentation

Parameters

of Histones

M OPP

and Histone

Complexes

from

Data from

Gel Permeation

s

M

Ro

Chromatography f/f,

and Sucrose a/b

Heterotypic Complex

96,000

3.6

60,400

30.5

A

1 .49

9

Hla

96,000

1.4

22,180

38.4

A

2.07

23

Hlb

84,000

1 .4

21,200

36.7 A

2.10

23

H2b-H2a

32,000

2.3

25,250

26.6 A

1.37

7

H5

36,000

1 .4

16,000

27.7 A

1.69

11

40.8 A

1.64

12

H3-H4

tetramer

115,000

M,,,-Apparent molecular weight as determined S-Sedimentation coefficient. M-Molecular weight calculated from combined R,-Stokes radius. f/f.-Frictional ratio. a/b-Axial ratio assuming prolate ellipsoid.

3.2

53.800

by gel filtration data

from

chromatography

gel permeation

in daltons.

chromatography

and sucrose

gradient

sedimentation.

Cell 94

Histone Conformation in 0.2 M NaCl (pH 7.1): Trypsin-Sensitive Complexes When the ionic strength of the sucrose gradient is reduced to 0.2 M, keeping the pH at 7.1, the sedimentation pattern observed in Figure 6c is obtained. In addition, approximately 30% of histones H3 and H4 are recovered as aggregated material on the bottom of the centrifuge tube. The gradient in Figure 6c is complex. The arginine-rich histones are observed as broadly sedimenting material, while the slightly lysine-rich histones appear as a discrete peak cosedimenting with nonstoichiometric amounts of the arginine-rich histones at about 2.5s. This sedimentation behavior can be explained if the two homotypic complexes have a very weak affinity for each other in these low ionic strength conditions, and if histones H3 and H4 form heterogeneous aggregates with each other after dissociating from H2b and H2a during the sedimentation. To test this, the peak containing H2b, H2a, and smaller

amounts of H3 and H4 was resedimented under the same conditions. The gradient that was obtained was identical to the one shown in Figure 6c, except that all of H3 and H4 either aggregated or sedimented faster than H2b and H2a. This shows that at 0.2 M NaCI, associations between the two homotypic complexes are relatively weak ones. We do not know the exact conformation of the arginine-rich histones in these conditions; however, it is clearly not the same as the tetramer that is observed at pH 5.5, since the former, but not the latter, aggregate. Additional experiments have shown that the heterotypic tetramer begins to dissociate when the NaCl concentration is reduced to 1M. In gradients parallel to those shown in Figure 6, each fraction was digested with trypsin and assayed for the presence or absence of trypsin-resistant histone cleavage fragments. The results show that in contrast to digestion in 0.2 M NaCI, in 2 M NaCl at either pH 5.5 or pH 7.1, each histone fraction

.4 .3

iii 0” .2 0

.1

46

47

48

49

50

51

r* (cm*) Figure

8. Molecular

Weight

of Heterotypic

Complex

Approximately 0.3 ml of the heterotypic complex (700 bgm/ml) isolated from sucrose gradients as m Figure 7a was centrrfuged to equrlrbrrum in 2 M NaCI. 0.01 M Tris-HCI (pH 7.1), as described in Experimental Procedures. The graph is a plot of the logarithm of the optical density at 280 versus rz(cm2) the distance from the center of the rotor. The methods used to determine weight average molecular weight are described in Experimental Procedures. The plot yields a straight line except for a slight deviation near the meniscus, possibly indicating species of lower molecular weight. Insert: Schlieren pattern of a velocity sedimentation experiment of whole histone In 2 M NaCI, 0.01 M Tris-HCI (pH 7.1) Two peaks are apparent. These probably correspond to the faster sedimenting heterotypic complex and the slower sedimenting lysine-rich histones. Histone concentration was 4 mgm/ml.

A Heterotypic 95

Histone

Tetramer

displayed high molecular weight trypsin-resistant fragments containing polypeptide chains that were indistinguishable from those obtained from chromatin. This clearly demonstrates that homotypic interactions are sufficient to give trypsin resistance, since each of the homotypic complexes isolated from the sucrose gradients in 2 M NaCl at pH 5.5 yielded trypsin-resistant histone fragments. (As expected, no resistant fragments were obtained in the region of these gradients where histones Hl and H5 migrated.) Although the homotypic slightly lysine-rich complexes sediment similarly in 0.2 M and 2.0 M NaCI, they are clearly different conformations since the latter, but not the former, gives trypsin-resistant fragments. In summary, sucrose gradient analysis shows that in 2 M NaCl (pH 7.1) histones H3, H4, H2b, and H2a migrate together as an apparent complex with a sedimentation coefficient of 3.8s. In low ionic strength or at low pH, the complex dissociates. As would be predicted, in both these conditions formaldehyde does not crosslink H2b and H4, although the H2b-H2a dimer is produced (unpublished results). (The failure to observe this heterotypic dimer at pH 5.5 is probably not a consequence of the pH per se, since treatment of chromatin with formaldehyde at pH 5.5 generates this product, possibly indicating that the DNA in chromatin stabilizes H2b-H4 interactions when the pH of the chromatin solution is decreased to 5.5) Further Characterization of the Heterotypic Complex Molecular Weight The molecular weight of the heterotypic complex containing all 4 histones was determined in 2 M NaCl (pH 7.1) by low speed equilibrium centrifugation after purifying the complex (700 pg/ml) on sucrose gradients. The histone concentration across the centrifuge cell was determined by ultra violet optics at a wavelength of 280 nm. Figure 8 is a plot of the logarithm of the optical density compared with the corresponding square of the distance from the center of the rotor. The plot yields a straight line which corresponds to a weight average molecular weight of 51,000 daltons, equivalent to a tetramer containing one each of the 4 histones. (It should be pointed out that this is a weight average molecular weight with contributions from all the forms present in equilibrium with the heterotype tetramers.) At 10 mg/ml, the complex had a 2 average molecular weight of 57,000 daltons as analyzed by Schlieren optics. Higher concentrations of histone could not be analyzed under these conditions, since the complex tended to precipitate during the course of the equilibrium run. The insert to Figure 8 shows an analytical velocity sedimenta-

tion analysis of the combined 2 M histone preparation. The faster sedimenting major peak has a sedimentation coefficient of 3.838, and probably corresponds to the heterotypic complex of 3.8s shown in Figure 6a. The slower sedimenting material is probably derived largely from histone Hl and H5. The sedimentation coefficient of the faster peak is independent of concentration between 1 mg/ml and 10 mg/ ml when analyzed in the analytical ultracentrifuge. In fact, it decreases by about 15% as the concentration increases. This behavior is explained in terms of the increase in viscosity and the backward flow of solvent that occurs at high protein concentrations (Schachman, 1959). Analysis of protein concentrations below 1 mg/ml was not performed using the analytical ultracentrifuge. In contrast to the hypersharp leading edge observed by sucrose gradient analysis, the 3.83s peak seen in the analytical ultracentrifuge is very symmetric at the leading edge. This difference is most readily explained if the complex dissociates at low concentrations. In the sucrose gradients, the leading edge of the peak is at low concentration and as a result, the complex dissociates into slower sedimenting homotypic forms. Thus a hypersharp leading edge forms. In the analytical ultracentrifuge, low concentrations of protein are not present at the leading edge. Consequently, a more symmetrical peak is obtained. Gel Filtration Figure 9 shows the elution of the heterotypic tetramer from agarose in 2 M NaCl (pH 7.1). Fractions were precipitated with TCA and analyzed on SDS gels as described in Figure 6. As in Figure 6, the majority of protein eluted in a broad peak as a complex containing all 4 histones. When a 10 fold higher histone concentration is loaded onto the column, an identical elution pattern is obtained. The frictional coefficient of the peak containing the heterotypic complex corresponds to a globular protein with molecular weight of about 100,000 daltons (the range in apparent molecular weight across the peak is between 50,000 and 104,000 daltons). Given this value and a sedimentation coefficient of 3.8s (Figure 6), it is possible to calculate a molecular weight of 60,400 daltons for the heterotypic complex (Table 2; see Experimental Procedures). This is consistent with the molecular weight determined by sedimentation equilibrium. Assuming that the shape of the heterotypic complex is prolate, the hydrodynamic data suggest an axial ratio for the heterotypic complex of approximately 9:l (Table 2). As was the case for the sucrose gradients, the leading edge of the heterotypic complex is hyper-sharp in gel filtration and the peak is very broad. Although histone Hl sediments like H5 in these conditions, histone H5 is retarded by

Cell 96

agarose to a much greater extent. This probably indicates that histone HI is more extended and histone H5 more compact (Table 2). The different shape of histone H5 may be related to its biological function during red cell development. Table 2 also shows the hydrodynamic data for the arginine-rich tetramer and the lysine-rich dimer in 2 M NaCI. velocity To summarize, by both sedimentation and gel filtration, the 4 major histones in 2 M NaCl (pH 7.1) run together as an apparent tetramer. When the pH is lowered to 5.5 (keeping the NaCl at 2 M), the heterotypic complex gives rise to both arginine-rich and slightly lysine-rich homotypic complexes as reported by Van Der Westhuyzen and Von Holt (1973) and extended by Kornberg and Thomas (1974). In 0.2 M NaCl at pH 7.0, only homotypic complexes are observed on agarose (data

Figure

9. Gel Filtration

of Heterotypic

not shown). This is in keeping with the sedimentation studies (Figure 6c), which suggest that in low ionic strength there is a decreased affinity of arginine-rich histones for lysine-rich histones, and consequently, the heterotypic complex begins to dissociate and the arginine-rich complex appears to aggregate. Is the Complex Really Heterotyplc? By both sedimentation and gel filtration, H3, H4, H2b, and H2a migrate together as an apparent complex. Nevertheless, it is possible that each histone is really involved in homotypic interactions, and that it is fortuitous that homotypic complexes in 2 M NaCl cofractionate by these two different methods. Some evidence against this possibility comes from crosslinking experiments. Thus formaldehyde

Complex

The apparent molecular weight for histones and histone complexes was determined on Bio-Gel Agarose A 0.5 M. K., was calculated from the elution volume and plotted versus molecular weight (on a logarithmic scale). Protein standards shown are: (1) alkaline phosphatase, (2) ovalbumin, (3) myoglobin (horse heart), (4) lactate dehydrogenase (rabbit muscle), (5) hexokinase (yeast). Arrows indicate the range over which the complex eluted. Insert: Each fraction Note the hypersharp

from the agarose column was precipitated with TCA and analyzed leading edge and the broad peak of the heterotypic complex.

on 15% SDS

gels.

Elution

is from

left to right.

A Heterotypic 97

Histone

Tetramer

crosslinking demonstrates that H2b and H2a are associated, and that H2b and H4 are associated. These crosslinked dimers appear as very sharp, discrete bands on SDS gels. In contrast, the crosslinked tetramer migrates very broadly. Consequently, even though the tetramer band contains all 4 major histones, it does not necessarily follow that all of the histones are actually associated, since, for example, a tetramer (or octamer) of pure H2b would be expected to migrate in a similar way as a tetramer (or octamer) of H2a. In fact, almost any combination of histones that produces a tetramer (or octamer) might be expected to migrate like any other combination. We believe, then, that crosslinking experiments with histones will yield ambiguous conclusions when the products are not sharp, discrete bands, as indeed they are not for the tetramer generated by formaldehyde treatment of 2 M histones. Interactions between H2a-H2b and H3-H4 To demonstrate that the presumed heterotypic complex results from interactions between homotypic histone pairs, one can show that the elution pattern of H2b-H2a on Sephadex G-100, in 2 M NaCl (pH 7.4), depends upon the presence of H3H4. Pure H2a-H2b elutes from these columns with an apparent molecular weight of 32,000 daltons (Table 2). In contrast, pure H3-H4 elutes with an apparent molecular weight of 115,000 daltons, and the heterotypic complex with an apparent molecular weight of 96,000 daltons. In order to show that H2aH2b interacts with H3-H4, we modified the technique of “sedimentation partition chromatography” as first described by Yamamoto and Alberts (1974) to gel permeation columns. In essence, the procedure allows H2a-H2b to flow through a Sephadex G-100 column in an environment that contains a constant concentration of H3-H4. The experiment was done as follows: Sephadex columns (40 x 0.5 cm) were first loaded with a 1 ml layer of H3-H4 at either 0, 100, or 1000 pg/ml; then 0.05 ml of a sample containing ‘251-labeled H2a-H2b, and the stated concentration of unlabeled H3-H4 was loaded. This was followed by another 3.5 ml layer of only unlabeled H3-H4 at the given concentration. After each layer was applied, it was allowed to run into the column bed before the next layer was introduced. Once the top layer had entered the gel bed, a reservoir containing buffer was attached to the column. All solutions contained 2 M salt and were at a pH of 7.4. The elution profile of H2a-H2b was determined by counting the elution of 1251.The loading regime was designed to assure that during the separation, H2a-H2b was always in an environment that contained a constant amount of H3-H4. As can be seen in Figure 10, the H2a-H2b peak elutes at lower volumes (corresponding to a higher apparent

molecular weight) as the H3-H4 concentration in the column is raised. By 100 pg/ml of H3-H4, the elution volume appears to reach a limit that corresponds to the position of the heterotypic tetramer. The simplest interpretation of these experiments is that the H2a-H2b dimer interacts with some conformation of H3-H4 to form a heterotypic tetramer. When 2 M histones are sedimented in sucrose gradients or eluted from agarose columns, about 20% of H2a-H2b is observed as a homotypic dimer; in addition, the shape of the peak containing the heterotypic tetramer is distinctly nongaussian. This was interpreted in terms of an equilibrium between the heterotypic complex and homotypic forms. In contrast, when H2a-H2b is eluted from Sephadex columns (Figure 10) under conditions where there

P

c

32,OOOr 26.000

H3-H4

I $

(1 mgm/ml)

ii

+

.wgm/ml -

! y kH2b-H2a’(20

II

24.000 f

1

i /(100~gm/ml)

+

(3O~gm/ml)

H3-H4 H2b-H2a

H2b + H20

(100 yzgm/ml)

0

0

2

4

6

ELUTION Figure

10. Interaction

between

IO

8 VOLUME

HZb-H2a

12

14

(ml)

and H3-H4

Calf thymus histones were used for this experiment. Chromatin was extracted in 0.25 M H2S04 for 1.5 hr at 4°C and precipitated in 6 vol of absolute ethanol at -20°C. After dissolving in 0.1 M acetic acid, the histones were dialyzed against 0.2 M NaCI, 20 mM Tris (pH 7.4). H2b-H2a was separated from H3-H4 and HI on Sephadex G-100 in the same buffer, and pure H3-H4 was obtained as a precipitate when the H3-H4 peak was made 70% saturated ammonium sulfate. These histones were found to behave identically to chick histone on sucrose gradient and gel permeation experiments as described in Figures 6 and 9. They also have the same iodinatable tryptic peptides. H2a-H2b was iodinated as discussed in Experimental Procedures. Sephadex columns (40 x 0.5 cm) were first loaded with a 1 ml layer of H3-H4 at either 0, 100, or 1000 pg/ml, then 0.05 ml of a sample containing ‘251-labeled H2a-H2b and the stated concentration of unlabeled H3-H4 were loaded. This was followed by another 3-5 ml layer of only unlabeled H3-H4 at the given concentrations. After each layer was applied, it was allowed to run into the column bed before the next layer was introduced. Once the top layer had entered the gel bed, a reservoir containing buffer was attached to the column. Columns were run at 4”C, at a flow rate of 1.2 ml/hr in 2 M NaCl 20 mM Tris (pH 7.4).

Cell 98

is always a constant environment of excess H3-H4, the peak of H2a-H2b is gaussian and there is little, if any, material migrating as a dimer. This behavior is most easily interpreted in terms of an equilibrium between heterotypic forms and homotypic forms. The high concentrations of the homotypic H3-H4

that is included in the column pushes the distribution of the equilibrium toward the heterotypic tetramer; consequently, the small amount of H2b-H2a present is driven into the heterotypic conformation and a gaussian peak with no trailing dimer of H2bH2a is observed.

H3 H2b H2a H4

Figure

11, Sedimentation

Properties

of Protease-Treated

Heterotypic

Complex

(A) Sucrose gradient sedimentation of heterotypic complex from 2 M histones. Analysis of each fraction on SDS gels is described in Figure 6a. Sedimentation is from left to right. (B) The 2 M histones were treated with trypsin to digest histones HI and H5 and partially digest histones H3 and H4. The complex was layered over 5-20% sucrose gradients made up in 2 M NaCl at pH 7.1 and sedimented in parallel with the 2 M histones in Figure 12A. The trypsinized complex and the untrypsinized complex sedimented indistinguishably. Slightly more and slightly less digestion of 2 M histones gave the same results. The slower sedimenting material to the left represents cleavage fragments from Hi and H5 and nonhistone proteins. (C) Digestion of H2b by an endogenous protease found in rabbit serum. 2 M histones were incubated for 2 hr with increasing concentrations of a crude immunoglobulin fraction from rabbit serum. The samples were loaded directly onto 15% SDS gels as described in Figure 1. Note that the cleavage product (arrow) produced by digestion of H2b, and that the protease preparation has some activity for H5. The immunoglobulins (brackets) migrate in the upper part of the gel. (D) Sedimentation properties of 2 M histones treated with the immunoglobulin protease specific for H2b. After digestion of H2b the 2 M complex was analyzed on sucrose gradients. Top:control gradient; bottom: 2 M histones treated with H2b protease. The sedimentation coefficients of the two complexes ware indistinguishable. Sedimentation was for 24 hr instead of the 48 hr used for (A) and (B). The immunoglobulins are sedimenting at 6.6s. Note that the H2b cleavage fragment (arrow) cosediments with the complex.

A Heterotypic 99

Histone

Tetramer

Pfofease Treatment of fhe Complex Figure 11 b shows the sedimentation behavior of 2 M histones isolated after the digestion of chromatin with limited concentrations of trypsin, so that only H3 and H4 are cleaved. In a parallel experiment, the sedimentation pattern of the undigested histones is shown (Figure 11 a). The sedimentation coefficients of the heterotypic peaks for the controls and the trypsin-treated histones were hardly distinguishable under these conditions. Whereas complete digestion with trypsin yields a complex of resistant fragments that sediments at 1.9s (data not shown), the cleavage fragments produced as a consequence of partial trypsin treatment comigrate with the complex at 3.8s. This indicates that they are associated with the undigested histones H2b and H2a, since if they were complexed with themselves, their sedimentation constant would have decreased relative to that of the undigested histones. These characteristics can be shown for histone H2b as well. We have found that the gammaglobulin fraction of rabbit serum contains a protease activity that is very specific for H2b in 2 M NaCI. Figure llc shows that when 2 M histones are incubated with increasing concentrations of this fraction, only H2b is digested. A cleavage fragment from H2b is also produced. If, after cleavage of H2b, the 2 M preparation is sedimented down a sucrose gradient, the cleavage fragment migrates with the other histones at a rate of about 3.8s (Figure lld). In conclusion, after specific cleavage of particular histones by proteases, the resistant fragments from these digestions comigrate with the undigested histones. Although explanations of these observations can be much more intricate, the simplest one is that all 4 histones are indeed associated with each other in a heterotypic complex. Hydrophobic Chromatography A separation method based on the hydrophobic character of proteins has recently been described (Rimerman and Hatfield, 1973). In this technique, proteins are loaded onto a column of norleucine coupled to sepharose by CNBr activation. The loading is usually accomplished by raising the ionic strength in order to promote interactions of the protein with norleucine (or, in principle, with any other hydrophobic amino acid coupled to sepharose). When the ionic strength of the solution is decreased, the interaction with norleucine also decreases and the protein is eluted. Initial experiments showed that in 2 M NaCI, histone would not adhere to these columns. If, however, 1 M potassium phosphate (pH 7.5) was included, then the histones (still in 2 M NaCI) were bound. (Histones did not bind to valine-sepharose in similar conditions). The histones were then eluted from the norleucinesepharose by a stepwise decrease in the phosphate

concentration (keeping the solution 2 M in NaCl and at pH 7.5 in order to preserve the integrity of the heterotypic complex). Figure 12a shows the elution profile. The first histones to be removed are histones Hl a and Hl b; these are followed by histone H5, and then by some H2b and H2a. At about 0.5 M phosphate, a complex of all 4 histones is eluted, and finally at zero phosphate a complex of histones H3 and H4 is eluted. Typically, about 60% of the major histones are found in the heterotypic tetramer by this procedure. This contrasts to about 80-90% for the sedimentation and gel filtration fractionations. It is possible that the presumed “hydrophobic” interactions with the column weaken the interactions that keep the heterotypic complex intact. In contrast to this elution profile observed at pH 7.5 in 2 M NaCI, when the pH is decreased to 5.5 (keeping the NaCl at 2 M), the elution profile shown in Figure 12b is obtained. Here the heterotypic complex breaks down and all the histones run as homotypic complexes. Controls showed that in the absence of potassium phosphate all of the histones came through the column in the first fraction. These results support previous findings that in 2 M NaCl a large fraction of histone is present as a heterotypic complex and that this complex dissociates when the pH is dropped from 7.1 to 5.5. In summary, the simplest structure for 2 M histones at pH 7.1 that is consistent with all of our data is a tetramer containing one each of the 4 histones. While this is certainly the most reasonable structure, certain other conformations cannot be excluded. For example, if half of the 2 M complexes consisted of tetramers of H2b-H2a-2H4, and the other half of tetramers of H2b-H2a-2H3, our experiments would not detect this. Our current work is aimed at clarifying these points, although the particular complexes mentioned above are not likely since W. Bonner (personal communication) has demonstrated an H3-H4 dimer after crosslinking 2 M histones with a carbodimide. Is the Heterotypic Complex Present in Chromatin? Reconsfifufion and Nuclease Digestion Axel et al. (1974) have shown that dissociated chromatin can be reconstituted by stepwise dialysis from 5 M urea, 2 M NaCl down to low ionic strength. The criteria for “proper” reconstitution are that subsequent treatment with nuclease yields an identical pattern of nuclease resistant DNA fragments (Axe1 et al., 1974) as obtained from chromatin, and that roughly 50% of the reconstituted chromatin is sensitive to nuclease, as is the case for native chromatin. In extending these experiments, it was reasoned that if a particular histone conformation was similar to that present in chromatin, then reconstitu-

Cell 100

tion should succeed in the absence of urea, and that only the gentle reannealing achieved by salt dialysis would be necessary. To test this hypothesis histones in 2 M NaCl were reannealed to an equal amount of DNA by stepwise dialysis (in the absence of urea) into lower salt concentrations. This. was done at pH 5.5 and at pH 7.1 to test whether the very different histone forms present in these two different conditions reannealed differently. The results from subsequent nuclease digestion (performed at pH 5.5 and 7.1, respectively) are shown in Figure 13. The nuclease-resistant fragments obtained after reconstitution at either pH were identical to those obtained from native chromatin. In addition, the amount of DNA made acid soluble was 47 i 3% for both conditions. Simply mixing histones and DNA at low ionic strength led to no resistant DNA fragments. Not surprisingly, after reconstitution the trypsin-resistant histone fragments were also identical at pH 5.5 and pH 7.1 (Table 1). Thus by the criteria mentioned previously both the pH 5.5 homotypic forms and the pH 7.1 heterotypic form can be successfully reconstituted to DNA at the respective pH and in the absence of urea. Clearly, little

is known about the ability of DNA to shift one histone form into another form. fodfnatfon Figure 14 is an autoradiograph of an acrylamide gel showing histones that were iodinated with 1251 in chromatin or free in solution at low ionic strength after acid denaturation. The most striking difference is that histone H2a is not significantly iodinated in chromatin or in 2 M NaCl (pH 7.1). When 2 M histones were iodinated at pH 5.5, however, H2a became labeled. Figure 15 is a peptide map of the iodinated trypsin peptides from histones treated with 1251 in 2 M NaCl (pH 7.1) and 2 M NaCl (pH 5.5). They were compared to the maps from histones iodinated in chromatin or after denaturation. The heterotypic complex iodinates more like chromatin than the homotypic complexes. These differences will be analyzed in more detail in a separate communication. For the present, only the reactivity of H2a will be discussed. Only 1 tyrosinecontaining peptide from H2a is reproducibly resolved in these procedures, and its reactivity increases at the lower pH. Of the total cpm of 1251 that are incorporated into chromatin, less than 0.5%

Figure

Columns

12. Separation

of Heterotypic

Complex

on Norleucine-Sepharose

Histones were loaded in 2 M NaCI, 1 M potassium phosphate; (A) pH 7.5 and (B) pH 5.5. The phosphate concentration was lowered in a stepwise fashion keeping the NaCl concentration at 2 M. Phosphate elution in (A) from left to right: 1 M; 1 M; 0.6 M; 0.6 M; 0.4 M; 0.2 M; 0.1 M; 0 M phosphate. Phosphate elution in (8) from left to right: 1 M; 0.8 M; 0.6 M; 0.4 M; 0.2 M; 0 M. Histones H3 and H4 usually elute first on uncoupled Sepharose columns prepared in the absence of norleucine; histone Hi usually elutes next, together with the heterotypic complex: finally H2b and H2a elute in conjunction with H5 (see Figure 9).

A Heterotypic 101

Histone

Tetramer

go into the H2a peptide; in 2 M NaCl at pH 7.1, again less than 0.5% are in the H2a peptide; at pH 5.5, however, there is a 5 fold increase in reactivity and over 2.5% of the total incorporation is in this peptide. In all of these conditions, about 50% of the total 1251reacts with histones by the time the incubation is stopped. These experiments support the idea that the heterotypic complex present in 2 M NaCl (pH 7.1) resembles the configuration of histones in chromatin. Bivoc and Reeder (manuscript in preparation) have come to very similar conclusions using essentially the same type of analysis with 1251. Formaldehyde Cross/inking For the most part, the pattern of crosslinking for chromatin and 2 M histones (Figure 5) is independent of concentration of histone between 25 pg/ml and 5 mg/ml; it is independent of temperature of the reaction between 4°C and 22°C pH between 6.5 and 9.0, and the time of the reaction between 1 hr and 24 hr. These control experiments make it probable that the observed products come from histones that are complexed to each other very specifically. Recently we have found that in very defined conditions, secondary (and presumably

Figure 13. Reconstitution with DNA

of Heterotypic

and Homotypic

weaker) interactions can be detected. Figure 16 shows that when low concentrations of chromatin are fixed overnight at 22”C, crosslinked products larger than tetramers can be observed. About 30% of the histone is found in a broad dimer band, about 5% as a tetramer, 20% as a hexamer, 30% as an octamer, and 15% as a decamer. An analysis of the iodinatable tryptic peptides found in the octamer (according to the methods of Van Lente et al., 1975) shows the presence of histones H2a, H2b, H3, and H4. As mentioned previously, these higher order interactions are only detected when crosslinking is done at 22°C for 16 hr with concentrations of chromatin less than 200 pg/ml. While this work was in progress, Hyde and Walker (1975) reported that chromatin crosslinked with formaldehyde for 16 hr at 22°C in the presence of 2 M NaCl gave a pattern of products significantly larger than tetramers and, by comparison, very similar to our pattern (shown in Figure 16a) for chromatin. In contrast, the pattern that we obtained for pure histones in 2 M NaCl yielded predominantly tetramers and very little material larger than tetramers, even when crosslinked at 22°C for 16 hr. Only after adding DNA back into our 2 M histone preparations can we obtain a pattern similar to the one obtained by Hyde and Walker for chromatin in 2 M NaCI. The pattern that we obtained (Figure 16) is virtually identical to the pattern of dimers, hexamers, octamers, and decamers obtained from

H3 H2b H2a

H3 H2b H2a

H4

H4

Complexes

Heterotypic complexes were reconstituted at pH 6.8; homotypic complexes at pH 5.5. No urea was present in these experiments. Subsequent nuclease digestionswereat pH 6.8 or 5.5. respectively. Nuclease resistant fragments from reconstituted chromatin are shown from the pH 6.8 reconstitution (left), the pH 5.5 reconstitution (middle), and control chromatin (right). Both reconstitutions are within normal variation of digests from native chromatin. The analysis is on 6% acrylamide gels that have been stained with ethidium bromide. The bottom 3 bands run ahead of the tracking dye. They have not been discussed in our previous communications (Weintraub, 1975). but more recent work shows that they are released as free DNA after trypsin digestion of nuclease particles. The smallest fragment is about 20 base pairs.

Figure

14. Failure

of Histone

H2a to be lodinated

Histones were iodinated with 1251as described in cedures and then analyzed on 15% SDS gels. autoradiograph of iodinated (1) acid-denatured water; (2) chromatin in 0.2 mM EDTA (pH 7.1); M NaCI, 0.2 mM EDTA (pH 7.1). The amount into each well was about 20 $g in each case.

Experimental ProFrom left to right: histone in distilled (3) histones in 2 of protein loaded

Cell 102

chromatin crosslinked at low concentrations in low ionic strength for 16 hr at 22’C. The simplest interpretation of these crosslinking studies is that in 2 M NaCl and in the absence of DNA, histones are present as a heterotypic tetramer. The addition of DNA to histones in 2 M NaCl allows

Figure

15. Peptide

Map of Histones

lodinated

in Different

the heterotypic tetramers to become “oriented” such that they are crosslinked into the higher order hexamers, octamers, and decamers that are also observed when chromatin is crosslinked in similar conditions.

Solvents

lodination of (A) chromatin, 0.2 mM EDTA (pH 7.1); (B) 2 M NaCl histones. 0.2 mM EDTA (pH 7.1); (C) 2 M NaCl histones. 0.2 mM EDTA, 0.005 M sodium acetate (pH 5.5); (D) acid-extracted histones in 0.2 mM EDTA (pH 7.1). The circles in (A) and (B) show the uniodinated peptide from H2a. The many differences between the intensity of iodination of particular peptides will be more fully documented in a subsequent paper.

A Heterotypic 103

Histone

Tetramer

Table 3 is a qualitative summary comparing the properties of histones in chromatin to those of histones in the various solvents examined in these studies. The heterotypic complex is indistinguishable from the type of complexes present in chromatin by 4 independent criteria: -sensitivity to trypsin, -pattern of formaldehyde crosslinking, -pattern of iodination with 1251, -capacity to be reconstituted with DNA. It should also be pointed out that Kornberg and Thomas (1974) have successfully reconstituted the chromatin X ray pattern from DNA and a mixture of the arginine-rich tetramer and the slightly lysinerich complexes. To do this it was necessary to take the histones and DNA into 2 M NaCl at neutral pH. In these conditions, it is probable that the histones went into the heterotypic form and that it was the heterotypic complex that actually produced the re-

6 4

constituted structure as analyzed by X ray analysis. In conclusion, a large number of assays suggest that the properties of the heterotypic complex are very similar to the properties of histone complexes in chromatin. None of these experiments rule out the possibility that other types of histone-histone interactions are also present in chromatin. Discussion The complexity of Histone Structures Hydrodynamic Data The aim of this work has been to correlate a variety of histone conformations present in solution with those that are present in chromatin. The most effective assay in screening a large number of conditions for histones in solution was to monitor the sensitivity of the histones to trypsin. The generation of a high molecular weight trypsin limit-digest is at least

IO8 -. 64-

2-

Figure

16. Crosslinking

of Secondary

Associations

by Formaldehyde

for 16 hr at 22OC in 0.2 mM EDTA (or 0.01 M triethanolamine) (A) Treatment of low concentrations of chromatin (00 zL0 = 2) with formaldehyde (pH 7.1). Formaldehyde concentrations from left to right: 0%; 0.2%; 0.5%. Arabic numbers indicate the approximate positions of histone dimers, tetramers, and so on, as calculated according to the method described in the legend to Figure 5. This is clearly an approximation, since the histones migrate anomalously on SDS gels; it is also not clear how formaldehyde-treated histones should migrate in these conditions. (B) “Orientation” of 2 M histones by addition of DNA. Histones were at 200 pg/ml and DNA was added to a concentration of 200 pglml. Fixation was at 22°C for 16 hr in 0.2 mM EDTA (pH 7.1) [or 0.01 M triethanolamine (pH 7.1)]. In the absence of DNA, a tetramer is the predominant species in these conditions. Formaldehyde concentrations right to left: 0%; 0.1%; 0.2%; 0.5%. (C) Densitometer tracing of the gel obtained from an experiment similar to the one shown in (6). Bottom scan: tryptic peptide analysis of the iodinatable tyrosine residues in the broad band in the dimer region of the gel indicates the presence of H2b, H2a, Hl, and H5, with trace amounts of H3 and H4. The top scan is from a control gel. Migration is from right to left.

Cell 104

Table

3. Summary

of the Behavior

of Histones

in Various

Solutions

Histones in 2 M NaCl Criteria

(PH 3.3)

Correspondence physiological

pH +

Reconstitution with DNA in absence of urea

+

lodination

of Histones

in Chromatin

Histones in 0.2 M NaCl

Chromatin 0.2 mM EDTA

(PH 7.1)

(PH 7.1)

+

+

+

+

crosslinking

probes of histone react in chromatin.

+

+

+

+

+

+

of H2a

The different way histones

to the Behavior

Histones in 2 M NaCl (PH 7.1)

to

Pattern of trypsin digestion

Formaldehyde

Compared

conformation

are

listed,

+

and the response

one necessary but not sufficient criterion, indicating that the histones may be in a solution conformation that resembles the one(s) present in chromatin. The results of the trypsin experiments indicated that high salt is required for the production of a trypsin limit-digest. Presumably the high salt substitutes for the high local charge density offered by DNA in chromatin. In analyzing this further, it was shown that trypsin resistance in 2 M NaCl was independent of pH over the range between 5.5 and 7.5. Nevertheless, 3 different fractionation schemes showed that the conformation of histones in high salt at pH 5.5 was different than that at pH 7.1. The former pH yielded what Kornberg and Thomas (1974) and others (D’Anna and Isenberg, 1974; Roark et al., 1974) described as a tetramer of histones H3 and H4, and what we and others (Kelley, 1973) think is a dimer of histones H2b and H2a. The more physiological pH yielded a heterotypic complex containing stoichiometric numbers of all 4 histones; in addition, about 20% of the histones could also be found in the arginine-rich and slightly lysine-rich homotypic complexes at pH 7.1. These were shown to be in equilibrium with the heterotypic complex. Conditions of pH and ionic strength appear to shift these equilibria one way or another. What is the molecular weight of the heterotypic complex? Sedimentation equilibrium shows that the complex has a weight average molecular weight equivalent to that of a tetramer. In addition, a combination of gel filtration and sedimentation indicates a molecular weight that is also consistent with a tetramer. For all of these methods, it is important to consider the effect an equilibrium might have on the molecular weight estimates. This is most readily evaluated by doing the analysis as a function of concentration. The sedimentation coefficient remained constant between 500 yg/ml and 10 mg/ml; the apparent molecular weight on agarose was also

of histones

in the indicated

solutions

is given

relative

to the

constant between 1 mg/ml and 20 mg/ml; and the molecular weight was unchanged between 700 pg/ml and 10 mg/ml. These relatively constant hydrodynamic parameters indicate that over the range of concentrations that have been analyzed, the predominant form present in 2 M histones is a heterotypic tetramer. This is not to say that other complexes cannot be in equilibrium with this one. A particularly intriguing possibility is that various forms of the same tetramer may occur. Finally our analysis does not exclude the possibility that an octamer is in equilibrium with the heterotypic tetramer; if this is the case, however, then the association constant must be too low to be detected by the hydrodynamic techniques. Our main conclusion from the hydrodynamic studies is that the conformation of histones in 2 M NaCl is very complex, but that the predominant species is a tetramer containing one each of the 4 major histones. Crosslinking studies, although more ambiguous, also support this type of structure. As far as we are aware, such a tetramer containing one each of 4 different protein chains is without precedent in protein chemistry. Stability and Fine Structure of Heterotypic Complex The heterotypic complex dissociates when the pH is lowered. That this occurs at about pH 5.5 may indicate that the protonation of a histidine is involved. In addition, the complex breaks down when the ionicstrength is lowered. It is possible that this is a consequence of the repulsive interactions between the basic histone N terminals, since the prior removal of these basic residues in 2 M NaCl allows trypsin resistance to remain at low ionic strength (Table 1). In chromatin, the tendency of the heterotypic complex to dissociate at low ionic strength is probably diminished by the interaction of basic residues with DNA phosphates. This is supported by the observation that addition of DNA to histones in low ionic strength conditions renders them resis-

A Heterotypic 105

Histone

Tetramer

tant to trypsin. In this regard, the high ionic strength conditions used in our characterizations may not be as unphysiological for histones as one might otherwise think. In 2 M NaCl the majority of histones are present as a heterotypic tetramer. As in nuclei, this complex is only partially accessible to trypsin and most evidence indicates that only about 20-30 residues in the basic histone N terminal regions are digested, and the remainder of these molecules are resistant, presumably because of histone-histone interactions, although intramolecular interactions cannot be excluded. The implication of this data is that the basic N terminals extend from a trypsin-resistant histone complex comprised of interacting histone C terminals. The orientation of these extended basic groups may then determine the path of the DNA fiber about the histone complex (Weintraub and Van Lente, 1974). Minor Hisfone Forms in Equilibrium with the Heterotypic Tetremer The heterotypic complex is in equilibrium with homotypic forms. In a variety of solution conditions, H2a and H2b appear to form a dimer. A similar if not identical dimer is one of the homotypic forms in equilibrium with the heterotypic tetramer. The nature of the arginine-rich form, however, is elusive. When 2 M histones are run on sucrose gradients or agarose columns, the H3 and H4 that are not present in the heterotypic tetramer appear to aggregate. In contrast, in a variety of conditions, no tendency for H2a and H2b to form aggregates larger than dimers has been observed by hydrodynamic methods. While H3 and H4 seem to dissociate from the heterotypic complex and form aggregates, the mixture experiments (Figure 10) show that the purified H3-H4 tetramer can associate with the H2aH2b dimer and form a heterotypic complex. The relationship between the aggregated form of H3-H4 and the tetrameric form of H3-H4 is clearly not understood. Since H3-H4 dissociates from a heterotypic tetramer, the equilibrium may involve the release of a dimer of H3-H4 as well as a dimer of H2a-H2b per heterotypic tetramer, although an alternative scheme may involve the transient formation of a heterotypic octamer which dissociates into an H3-H4 tetramer and two dimers of H2a-H2b. The Relations between the Heterotypic Complex and Chromosomal Histones The finding that a tetramer of all 4 histones can occur in solution and the additional finding that so many characteristics of this tetramer resemble those of chromosomal histones (see Table 3) support previous results demonstrating that all 4 major histones are intimately associated with each other and are bound to many of the discrete fragments

of DNA (between 145 and 45 base pairs in length) produced by staphylococcal nuclease digestion of chromatin or nuclei (Weintraub, 1975). The correspondence between the behavior of histones in 2 M NaCl (pH 7.1) and the behavior of histones in chromatin emphasizes the fact that DNA is not uniquely required for achieving “proper” conformations. In low ionic strength, however (and presumably in ionic conditions similar to those present in the cell), the histones are present in a trypsin-sensitive configuration. It is probable that in these conditions they must interact with DNA in order to adopt the “proper” conformations. Supporting this hypothesis is the finding that the pattern of formaldehyde crosslinking is very different when 2 M histones are crosslinked at pH 7.1 or pH 5.5. Yet when chromatin is crosslinked at either pH, the pattern of products is the same and virtually identical to that obtained with 2 M histones at pH 7.1. These findings also suggest that DNA can stabilize particular histone conformations. The Complexity of Nucleohistone InteractlonsFundamental Units Based upon a Heterotypic Tetramer Partial nuclease digestion of nuclei yields DNA fragments that are integer multiples of a unit length of about 180-200 base pairs (Hewish and Burgoyne, 1973; Nell, 1974a). This, together with the observation of a tetramer that contained the arginine-rich histones and several arguments about histone stoichiometries led to the proposal by Kornberg that the basic repeating unit in chromatin was homogeneous and that it contained 2 each of the 4 major histones (Kornberg, 1974). Additional information, however, suggests that an even smaller unit may exist and that even this is heterogeneous (Weintraub and Van Lente, 1974; Axel, Cedar, and Felsenfeld, 1974). Thus extensive digestion of chromatin or nuclei results in the production of a series of discrete resistant DNA fragments with an average size of about 100 base pairs (Weintraub, 1975; Axel et al., 1974; Clark and Felsenfeld, 1971; Sahasrabuddhe and Van Holde, 1974; Oosterhof, Hozier, and Rill, 1975). As illustrated in Figure 17, the heterotypic tetramer described here may be one integral component of the structure responsible for generating these smaller DNA fragments. How can a fundamental unit based upon a heterotypic tetramer explain the 180-200 base pair repeat observed after partial nuclease treatment? The stoichiometry of histone to DNA in the nucleus would require an average of 8 of the 4 major histones for every 200 base pairs (Kornberg, 1974). This suggests the simple explanation that two heterotypic tetramers form a repeating unit of 200 base pairs, while each individual tetramer forms a

Cell 106

Figure 17. A Fundamental Unit of Chromosome upon the Heterotypic Tetramer

Structure

Based

A basic unit of chromosome structure is a tetramer containing all 4 histones. This defines the positions at which staphylococcal nuclease digests nuclear DNA in the limit-digest. Two heterotypic complexes may come together to form a larger unit which defines the points at which the nuclease attacks during partial digestion. As discussed in the text, the forces holding the two heterotypic complexes together may be those which stabilize the homotypic slightly lysine-rich interactions and the homotypic arginine-rich interactions. Removal of histones with 2 M NaCl at pH 7 leads to breakage of homotypic interactions and preservation of heterotypic interactions, while removal of histones with 2 M NaCl at pH 5.5 discourages heterotypic interactions, but promotes homotypic interactions. The model is clearly a first approximation since it does not account for the heterogeneity of the chromosomal subunits. Both the N terminal histone tails and the C terminal histone complexes bind to DNA and therefore fold the chromosome by crosslinking the DNA as previously described (Weintraub, 1975). (0) H2b; (X) HZa; (0) H3; (A) H4

more fundamental unit defined by about 100 base pairs (Weintraub, 1975; Axe1 et al., 1974; Weintraub and Van Lente, 1974; Van Holde, Sahasrabuddhe, and Shaw, 1974; Sahasrabuddhe and Van Holde, 1974). Although this explanation is satisfactory to a first approximation, it is probably too simple for two reasons. First, nuclease experiments (Weintraub, 1975; Axel et al., 1974; Weintraub and Van Lente, 1974) indicate that there is some degree of heterogeneity in the nuclease-resistant DNA fragments generated by complete digestion of chromatin; second, the experiments described here and elsewhere show that histones have the potential to form a great number of possible conformations. Thus while it is clear from electron microscopic (Olins and Olins, 1974), X ray (Kornberg and Thomas, 1974; Bradbury and Crane-Robinson, 1971; Pardon and Wilkins, 1972; Richards and Pardon, 1970), neutron diffraction (Baldwin et al., 1974), and nuclease experiments (Hewish and Burgoyne, 1973; Nell, 1974a; Clark and Felsenfeld, 1971; Sahasrabuddhe and Van Holde, 1974; Oosterhof et al., 1975) that a regular spacing is present in nuclei, there is really no evidence showing that the repeating units responsible for this regularity are identical; indeed, most evidence indicates that some differences do occur. The nature of these differences, however, is presently not known. Is the arginine-rich tetramer first described by Kornberg and Thomas(1974) present in chromatin? We feel that although there is no strong published evidence demonstrating either its presence or ab-

sence in chromatin, its stability in certain conditions (D’Anna and Isenberg, 1974) makes its biological importance very probable. One very intriguing possibility is that some of the bonds used to stabilize H3 and H4 as a homotypic tetramer (and H2b and H2a as a homotypic dimer) are involved in forming self-complementary interactions between two heterotypic tetramers in chromatin. This is illustrated in Figure 17. Although we view it as an oversimplification inasmuch as it ignores the heterogeneity observed in chromosomal subunits, the model in Figure 17 explains several other important experimental findings. First, it provides very accessible regions every 180 base pairs for staphylococcal nuclease attack and somewhat less accessible zones within the 180 base pair repeat for further attack by either staphylococcal nuclease or pancreatic DNAase (Nell, 1974b). Second, it maximizes the types of histone-histone interactions that have been described here and elsewhere. Thus although it is clear that the heterotypic tetramer is the most stable solution form of histones in 2 M NaCl at pH 7.1 and that its conformation is similar to histones in chromatin, it is equally clear that the heterotypic tetramer is in equilibrium with homotypic forms. The model shown in Figure 17 is designed to maximize these “minor” states by satisfying certain binding domains between homotypic histones. This is most easily done if two heterotypic complexes associated with each other. The postulated self-complementary associations would be stabilized by some of the bonds that are used to generate dimers of H2b and H2a and tetramers of H3 and H4. Third, in the model shown in Figure 17, the histones have been arranged diagramatically as a linear array of 4 different histones per 80-100 base pairs. An alternative conformation, also see D’Anna and lsenberg (1974), would be the more symmetric arrangement below: H:a--H3 H h b---H14 This particular configuration has the advantage that the strong binding domains between all the histones can be satisfied in one tetrameric unit. It also accounts for the fact that dimers of H2b-H2a and H2b-H4 are obtained after treatment of 2 M histones with formaldehyde. On the other hand, the linear arrangement that is shown in the diagram has the advantage that it easily accounts for the asymmetry of the tetrameric unit (see Table 2) and also explains how two asymmetric heterotypic tetramers can form a rather symmetric nuclease particle as determined by Sahasrabuddhe and Van Holde

A Heterotypic 107

Histone

Tetramer

(1974). It also provides a larger number of possible interactions between the smaller subunits determined by the heterotypic tetramers. This may be very important for stabilizing sharp bends in the folded DNA while still allowing the chromosome enough flexibility to replicate. Thus there are reasons to suppose that either of these conformations is preferred and the actual details clearly require further experimental investigation. Fourth, the model in Figure 17 predicts that each repeating unit of 180-200 base pairs contains two loops of folded DNA. Recent work with the SV40 “mini-chromosome” has shown that SV40 closed circular DNA is packaged into about 20 “nu” bodies, each of which is associated with about 200 base pairs of DNA (Griffith, 1975; Germond et al., 1975). Since it is clear that the histones in each “nu” body are responsible for generating the supercoils in the purified SV40 DNA molecule (Germond et al., 1975), and since there may be 40 supercoils per molecule (Wang, 1974), then each “nu” body is associated with an average of 2 superhelical turns. This is consistent with the idea that the 180-200 base pair repeat contains 2 heterotypic tetramers and that each tetramer is responsible for producing 1 superhelical turn as shown in Figure 17. It is also consistent with recent interpretations of “nu” body structure obtained from neutron diffraction studies (Baldwin, et al., 1974). Finally, the model accounts for previous findings (Weintraub, 1975) indicating that histone N terminals bind to some regions of DNA and histone C terminals bind to other regions of DNA, and that the N terminals extend outward from trypsin-resistant C terminal histone complexes (Weintraub and Van Lente, 1974). The model makes no attempt to deal with the molecular basis for the heterogeneity found in chromosomal subunits. Formalistically it is very similar to models proposed by Olins and Olins (1974), Kornberg (1974), Baldwin et al. (1974), Van Holde et al. (1974), and Weintraub (1975); however, in postulating a smaller subunit based upon a heterotypic tetramer and in suggesting the binding domains of the various histone-histone interactions, it presents a more complete account of the data that has accumulated since these models were originally formulated. One apparent failure of the model is that it predicts that a very stable octamer should be present in solution. Using hydrodynamic and crosslinking techniques, we have not been able to demonstrate such an octamer in the absence of DNA. This must mean that the postulated homotypic interactions holding the two heterotypic tetramers together (Figure 17) are relatively weak. This is paradoxical since in a variety of conditions the interactions promoting the tetrameric form of H3-H4 are extremely strong

(D’Anna and Isenberg, 1974; Kornberg and Thomas, 1974). A reasonable explanation for the failure to see an octamer is that some (but not all) of the binding regions used to stabilize the tetramer between H3 and H4 are in fact also used to stabilize the heterotypic tetramer described here. In some sense, the heterotypic tetramer and the homotypic arginine-rich tetramer appear to be mutually exclusive conformations in solution. Presumably the DNA in chromatin must stabilize certain bonding domains between histone complexes and, as a result, promote the postulated self-complementary interactions between the two heterotypic tetramers in each “nu” body. Some support for this comes from the observation that the addition of DNA to histones in 2 M NaCl allows them to be crosslinked by formaldehyde into products as large as hexamers, octamers, and decamers. This variety of sizes may form the basis for the heterogeneity of chromosomal subunits previously described (Weintraub, 1975; Axel et al., 1974). Experlmental

Procedures

Materials Trypsin-TPCK and staphylococcal nuclease were obtained from Worthington Biochemicals. Sodium 1251 (17 Ci/mg) and sodium ‘4C-acetate (58.8 mCi/mM) were purchased from New England Nuclear. Formaldehyde was obtained from J. T. Baker as a 37.1% aqueous solution with 10% methanol added as a preservative. Bovine pancreatic protease was purchased from Aldrich Chemicals. F12 tissue culture media was obtained from Grand Island Biological Company. Cell and Chromatin Preparation Chromatin was prepared from mature erythrocytes obtained from 16 day-old chicken embryos. Erythrocytes were collected in SSC [0.14 M NaCI, 0.01 M Tris-Cl (pH 7.1) 0.015 M Na Citrate], centrifuged, and washed several times in SSC. The cells were lysed with 0.5% Nonidet P40 in 0.01 M NaCI, 0.005 M MgCb, 0.01 M Tris-Cl (pH 7.4), and the nuclei pelleted by centrifugation. The nuclei were washed free of hemoglobin with lysis buffer and then washed once with 0.075 M NaCI, 0.2 mM EDTA, 0.01 M Tris-HCI (pH 7.4). The purified white nuclear pellet was suspended in 0.2 mM EDTA (pH 7.1) and sonicated for 20 set with a Branson sonicator (setting 5). The crude sonicate was centrifuged at 10,000 x g for 30 min, and the supernatant taken as isolated chromatin. Chromatin (DNA) concentration was determined by absorbance at 260 nm. The molecular weight of the DNA in the chromatin preparation was about lo& daltons. Hlstone Complex Preparatlon Chromatin in 0.2 mM EDTA (pH 7.1) at a DNA concentration of 4 mg/ml was diluted with an equal volume of 4 M NaCI, 5 mM EDTA. 1 mM Tris-HCI (pH 7.4), and centrifuged for 18 hr at 48,000 rpm in a SW 50.1 rotor to remove DNA. The supernatant fraction was removed and stored in solution as -2O’C. This 2 M NaCl extract exhibited a Az~,,/AzLo ratio of at least 6. and was stable at -2OOC for l-2 weeks. The solution also remained perfectly clear and there was no evidence of precipitation. Sodium dodecyl sulfate (SDS) gel electrophoresis showed no differential loss of any histone class and no signs of histone degradation. A small volume of sample in 2 M NaCl could be loaded directly onto 15% SDS gels without significant loss of resolution.

Cell 108

Histone complex-containing histones acetylated with ‘“C-acetate were prepared from chromatin isolated from mature 14 day-old erythrocytes incubated overnight with 25 &i/ml sodium r4C-acetate in F12 media (GIBCO) in the presence of 50 PM cycloheximide. Trypsln Digestions Chromatin and histone complexes were digested with trypsin at doses indicated in the figure legends for 30 min at 37OC. The reaction was stopped with the addition of 0.5 vol of SDS gel sample buffer, boiled for 1 min. and loaded onto 15% sodium dodecyl sulfate polyacrylamide stacking gels. Polyacrylamide Gel Electrophoresis 15% polyacrylamide sodium dodecyl sulfate gels were made using a modification of the procedure of Laemmli (1970). The separating gel was made with an acrylamide to bisacrylamide ratio of 300:4 in 0.1% sodium dodecyl sulfate, 0.375 M Tris-Cl (pH 8.8). The stacking gel was made 3% polyacrylamide with an acrylamide to bisacrylamide ratio of 300:8 in 0.1% sodium dodecyl sulfate, 0.125 M Tris-Cl (pH 6.8). Gels were electrophoresed at 130 V for 6 hr using a buffer system of 0.38 M glycine, 0.05 M Tris (pH 8.8). Gels were stained overnight with 0.1% coomassie brilliant blue in 50% methanol, 10% acetic acid, and diffusion destained with 5% methanol, 7% acetic acid. Samples were mixed with an equal volume of buffer containing 4% SDS, 20% glycerol, 10% mercaptoethanol, 0.001% bromphenol blue, 0.125 M Tris-Cl (pH 6.8) and boiled for 1 min prior to loading onto the gel. 6% polyacrylamide-ethidium bromide gels for DNA electrophoresis were made with an acrylamide to bisacrylamide ratio of 300:8 in 2 pg/ml ethidium bromide, 0.02 M sodium acetate, 2 mM EDTA, 0.05 M Tris-Cl (pH 7.8). Samples were diluted with 1 vol of 25% glycerol, 5% bromphenol blue, 0.02 M sodium acetate, 2 mM EDTA, 0.04 M Tris-Cl (pH 7.8) prior to loading on the gel. Gels were preelectrophoresed for 15 min, and samples were electrophoresed for 1.5 hr at 150 V. Gels were stained with 2 mg/L ethidium bromide in water, and bands visualized with ultraviolet illumination and photographed through a red filter. Formaldehyde Crosslinking Chromatin and histone complexes were reacted at 4’C or 20°C for 2-16 hr with varying concentrations of formaldehyde (which had been adjusted to pH 7.8 with NaOH) (Van Lente, et al., 1975). The crosslinking of histone complexes was accomplished either in 2 M NaCI, 2.0 mM EDTA (pH 7.4), or 2 M NaCI, 2.0 mM EDTA, 5 mM sodium acetate (pH 5.5). Chromatin was crosslinked in 0.2 mM EDTA (pH 7.1) or in 0.01 M Triethylamine(pH 8.0). After incubation with formaldehyde, the proteins were precipitated with 25% trichloroacetic acid and washed with acetone: the precipitate was redissolved in SDS gel sample buffer and loaded onto 15% SDS stacking gels. Sucrose Gradients Solutions of histone complexes in 2 M NaCI, 2 mM EDTA, 1 mM Tris-Cl (pH 7.4) were layered onto 5-20% (w/v) sucrose gradients containing either 2 M NaCI, 2 mM EDTA (pH 7.1). or 2 M NaCI, 5 mM sodium phosphate (or sodium acetate) (pH 5.5). The gradients were centrifuged for 48 hr at 48,000 rpm in an SW 50.1 rotor at 5°C. Fractions were collected and the protein precipitated with 25% trichloroacetic acid, washed with acetone, dried, dissolved in SDS gel sample buffer, and loaded onto 15% SDS stacking gels. In trypsin digestion experiments, fractions from the sucrose gradients were incubated directly with 20 gg/ml trypsin-TPCK for 30 min at 37OC, precipitated with 25% trichloroacetic acid, and applied to gels as described. Chromatin Reconstltutlon Histones were removed from the DNA in chromatin or nuclei with 2 M NaCI. 2 mM EDTA. 1 mM Tris-Cl (pH 7.4), and reconstituted (Axel. Cedar, and Felsenfeld. 1973) with chick DNA at a mass ratio

of 1 :l by step gradient dialysis against 2.0, 1.2, 1 .O, 0.8, and 0.6 M NaCl containing 0.01% dithiothreitol. The salt dialysis solutions contained either 1 mM Tris-Cl (pH 7.4) or 5 mM sodium phosphate (pH 5.5). The 0.6 M NaCl solutions of nucleoprotein were dialyzed either against 1 x 10-S M CaCb, 5 mM sodium phosphate (pH 6.8) or 1 X 10-S M CaCb, 5 mM sodium phosphate (pH 5.5). The reconstituted nucleoproteins were digested with staphylococcal nuclease at a concentration of 20 pg/ml at a CaCIZ concentration of 1 x 1O-4 for 30 min at 37°C at either pH 6.8 or 5.5, respectively. The digestion was stopped with 10 mM EDTA. Sodium dodecyl sulfate was added to 0.1% and the mixtures digested with 100 pg/ml of bovine pancreatic protease (Aldrich) overnight at 37°C. An equal volume of ethidium bromide polyacrylamide gel sample buffer was added, and the samples loaded directly onto 6% polyacrylamide-ethidium bromide gels. The same results are obtained if the DNA is purified by phenol extraction and ethanol precipitation after protease treatment. lodlnatlon of Hlstones in Solution and In Chromatln Histones in chromatin and histone complexes were iodinated with trace amounts of carrier-free 1251 using the chloramine-T method (Weintraub and Van Lente, 1974). 100 pg of chromatin or histone complex were reacted with 50 pCi of carrier-free 1251(New England Nuclear, 17 Ci/mg) corresponding to an iodine to protein molar ratio of 0.01. The iodination was initiated with the addition of 0.1 mM chloramine-T and allowed to react at room temperature for 15 min. The reaction was stopped with the addition of sodium metabisulfite to 0.01 M, and the iodinated protein precipitated with 25% trichloroacetic acid, washed with acetone-(0.1 M) HCI, then with acetone, and then dried in vacua. lodination reaction volume was 100 gl, using buffer conditions indicated in the figure legends. The average efficiency of incorporation of added radioactivity was 5060%. lodination of specific stained bands from SDS gels of formaldehyde crosslinked products was performed according to the method of Bray and Brownlee (1973). Stained bands were cut from the 15% SDS stacking gel and eluted overnight with 0.1% SDS, 0.05 M sodium phosphate (pH 7.3). The eluted protein was precipitated with 25% trichloroacetic acid, washed with acetone, and iodinated with chloramine-T as previously described, using an iodine to protein molar ratio of 0.1. The iodinated protein was recovered by precipitation with 25% TCA after addition of 25 pg of bovine serum albumin. The TCA precipitate was washed with acetone-(0.1 M) HCI. acetone, and dried in vacua. The final iodinated protein represented 45% recovery of the protein initially on the gel. No differential loss of any histone class was observed when eluted material was rerun on gels. Peptide Mapping lodinated protein samples were taken up in 0.1 ml of 1% (w/v) ammonium bicarbonate at pH 8.5, and 1 pl of trypsin-TPCK (1 mg/ml in 1 mM HCI) was added. After incubation at 37’C for 2 hr, another 1 ~1 of trypsin solution was added and the digestion was continued for another 2 hr. The digestion was terminated by the addition of 1 drop of glacial acetic acid and the mixture lyophilyzed to dryness. The tryptic digests were taken up in 25 ~1 of distilled water and applied to 46 x 57 cm Whatman 3 MM paper. Descending chromatography was performed in the first dimension in 1-butanol-pyridine acetic acid water (15:10:3:12) for 16 hr. The paper was dried in an oven at 80°C and high voltage paper electrophoresis was performed in the second dimension at pH 3.5 (5% acetic acid and 0.5% pyridine) for 90 min at 2000 V. The paper was dried and the peptide “spots” visualized by exposing the map Kodak Royal X-omat X ray film. Identification of the origins of the peptides was made by comparison to maps of the individual histones. The radioactivity associated with the individual peptides was determined by cutting out the spots from the map and counting in a liquid scintillation counter. As discussed in more detail previously (Van Lente et al., 1975), the intensities of any particular

A Heterotypic 109

Histone

Tetramer

peptide spot depend upon the reactivity of the tyrosines in the peptide. Because the reactivity depends upon solvent conditions and neighboring residues, the intensities of particular residues are not necessarily a reflection of their molar ratios. Phosphate-tnduced Proteln Chromatography Histones in 2 M NaCI, 1 M potassium phosphate (pH 7.5) were loaded onto norleucine-sepharose columns prepared as described by Rimerman and Hatfield (1973). Approximately 0.5 ml of histone solution at 500 pg/ml was loaded onto 1 ml of packed sepharose preequilibrated in a Pasteur pipette. All steps were done at 37°C. Phosphate concentration was lowered by a stepwise procedure. 2 ml fractions were collected at 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 M phosphate concentration, while keeping the NaCl constant at 2 M. Flow rate was 0.25 ml per min. The fractions were precipitated with TCA and analyzed on SDS gels as described above. For pH 5.5 chromatography, the pH was adjusted using NaOH. All other procedures were identical to the pH 7.5 chromatography. Molecular Weight Determlnatlon The molecular weight of histones in 2 M NaCl (pH 7.1) was mined by equilibrium sedimentation in a Beckman Model E cal ultracentrifuge using ultraviolet optics at a wavelength nm. Centrifugation was at 8766 rpm at 5.2”C for 72 hr. The specific volume was taken at 0.725 cc/g and the density solvent at 1.08 g/cc. The slope of a plot of log C compared rz was used to determine the molecular weight according equation

M=2RT

2.303

(d log C)

S aM/f, deteranalytiof 280 partial of the with to the

(1)

(1 - ip) cJ2 d (W The sedimentation coefficient of the complex in 2 M NaCl was determined from the slope of a plot of the natural logarithm of the distance migrated for the schlieren peak compared with time. All values were corrected for temperature, solvent density, and viscosity.

Gel Flltration and Determination of Molecular Welght and Shape The molecular weight and shape of the heterotypic complex was calculated by a combination of data from gel permeation chromatography and sucrose gradient sedimentation (Siegel and Monte, 1966). Physical parameters were also determined for histones Hl , H5, the H2b-H2a complex, and the H3-H4 tetramer. Gel permeation chromatography was on Bio-Gel A 0.5 M (Bio-Rad Laboratories). Columns and gradients were run at 2 M NaCI, 20 mM Tris (pH 7.4). 2.5 x 100 cm columns were eluted at 4°C at a rate of 10 ml/hr. Void volume (V,) was determined with phage (OD& and total volume (V,) was calculated from the bed dimensions. The following protein standards were selected because their molecular weights and shapes were known (for these calculations it was assumed that proteins did not dissociate nor change shape in 2 M NaCI): alkaline phosphatase (E. coli), 77,500 daltons; ovalbumin, 43,500 daltons; myoglobin (horse heart), 16,900 daltons; lactate dehydrogenase (rabbit muscle), 150,000 daltons; hexokinase (yeast), 96,000 daltons. Fractions were precipitated with 20% TCA and analyzed on SDS gels. A standard curve was obtained by plotting the K,, for each protein (K.,

a gaussian distribution, Vs was taken to be the position at which the maximum amount of protein eluted. Histones Hla, Hlb, and H5 eluted separately. Apparent molecular weights (M.,,) were obtained for each histone species by calculating a K,, from each Vr and reading the Mapp (apparent molecular weight) from the standard curve. The elution volume of a protein in gel permeation chromatography depends upon its molecular (Strokes) radius. If the calibrating standards possess similar frictional ratios, waters of hydration, and partial specific volumes, the Vr will also correlate well with molecular weight. The elution volume gives an apparent molecular weight (Mar+,) based on the Stokes radius. By combining the results from a gel column calibrated with proteins known to be globular (for spherical proteins, M,,, = M) with a second physical method such as sucrose gradient sedimentation, one can accurately determine both the molecular weight and frictional coefficient of a particular protein species (Siegel and Monte, 1966). Sedimentation values for all histones were determined on 5-20% sucrose gradients. Since

= s)

t 0 against its known molecular weight (where Vr is the elution volume of the protein being studied). Chromatin was treated with 2 M NaCl and the DNA pelleted by centrifugation. 2 ml of the supernatant solution containing histones (25 mg/ml) was loaded onto a column. 4.5 ml fractions were collected. Fractions were precipitated with 20% TCA and analyzed on 15% SDS gels. The majority of protein eluted as a broad complex containing H3, H4, H2b, and H2a. This complex eluted with an ultra-sharp leading edge and trailed into a pure H2b-H2a complex. Since these proteins did not elute in

MI/M?

(2)

= S,f,/Szfz

(where f = frictional coefficient; script “1” refers to the sample to the standard) and since f, = 6II$t,

and M.,,

M = true molecular weight; subprotein, while subscript “2” refers

014 fIRi

(where R, = Stokes radius and 7 = the viscosity Equation (2) can be rewritten M,/M2

of the medium),

SI@L,,)“~

= ___ Wb,,)”

The standard used for our determinations was myoglobin with MI = 16,950 daltons and S.2 = 1.9 ( = Sz&. Using Equation (3) molecular weights for each of the histones were calculated from by gel chromatography and the sedimentation the bpp obtained coefficients obtained by sucrose gradient centrifugation, also using myoglobin as a standard. Finally, using the classical equations M = GvNR,S/(l

- Vp)

(4) (5)

[where M = molecular weight: R, = Stokes radius; S = tation coefficient; 5 = partial specific volume (0.725 f/f, = frictional ratio; 1) = viscosity of medium (0.01); p of medium (1.0 gm/cc); and N = Avogadro’s number], and a/b were calculated, where a/b is the axial ratio a prolate elipsoid.

sedimencm/gm); = density R,, f/f..,, assuming

Acknowledgments We thank Bruce Alberts and Marc Kirschner for their suggestions and criticisms; Jerry Beltz for some of the data shown in Figure 11 c, d; and the Whitehall Foundation and Richard Blumenthal for technical assistance. This work was supported by grants from the National Science Foundation and the American Cancer Society, Received

May 23, 1975;

revised

June

23, 1975

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