Cell, Vol. 15.945-954, November 1979, Copyright 8 1979 by MIT

Studies on Histone Organization in the Nucleosome Using Formaldehyde as a Reversible Cross-Linking Agent

Vaughn Jackson* MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England

Summary

A new procedure is described which allows selec- tive reversal of formaldehyde cross-linking in both histone-histone and histone-DNA of nuclei isolated from calf thymus. All ten possible dimers of the four non-H1 histones, H3, H26, H2A and H4, are observed, the major dimers being H3-H3, H3- H2A, H2B-H2A, H2a-H2A and two separate dimers of H2B-H4. Although oligomers of the non-H1 histones are formed by prolonged treatment with this reagent, 50% of the histones continue to remain resistant to cross-linking with each other. For those histones which cross-link, the site of cross-linking within the molecules is located in the “core” (trypsin-resistant) region and therefore indicates proximities for these molecules within the nucleosome. The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA.

Introduction

Several lines of evidence, both morphological and biochemical, have established that the eucaryotic chromosome exists as a repetitive structure. Elec- tron microscopy of the chromatin fiber presents a picture of beads (nucleosome) relatively uniformly spaced along a string of DNA (Olins and Olins, 1974; Woodcock, Safer and Stanfield, 1976). Stud- ies involving digestion of isolated nuclei with nu- cleases have indicated that each repeat structure represents approximately 200 base pairs (bp) of DNA (Hewish and Burgoyne, 1973; Nell, 1974a): 140 bp supercoiled within the beaded structure (nucleosomes) and approximately 60 bp located between the beads (Simpson and Whitlock, 1976). The DNA between beads is also believed to be super-coiled under physiological conditions (Finch, Noll and Kornberg, 1975).

Several attempts have been made to define the role which each histone plays in supercoiling the DNA and producing the repeat structure. The 140. bp DNA has been found associated with the four non-H1 histones, H4, H2A, H2B and H3 (Shaw et al., 1976), and histone Hl is believed to be involved in the packaging of DNA between the nucleosomes (Noll and Kornberg, 1977). Reconstitution studies have indicated that all four of the non-H1 histones

* Present address: Department of Biochemistry, University of Iowa. Iowa City, Iowa 52242.

are required for generation of nucleosomal struc- ture (Germond et al., 1975) and suggest that inter- actions between these histones may have a major role in maintaining the structural organization. Preliminary evidence for histone-histone interac- tions has come from in vitro studies of histone solutions by using physical probes such as fluores- cence anisotrophy (D’Anna and lsenberg, 1974) and nuclear magnetic resonance (Lewis, Bradburg and Crane-Robinson, 1975). These studies indicate that all interactions between the non-H1 histones are observed; certain dimers, however, are much preferred - H2A-H2B, H3-H4 and H2B-H4. Verifica- tion for these types of interactions occurring within the nucleosome comes from cross-linking studies with tetranitromethane (H4-H2B, H2A-H2B) (Mar- tinson and McCarthy, 1975), ultraviolet light (H4- H2B, H2A-H2B) (Martinson, Shetler and McCarthy, 1976), carbodimides (H3-H4) (Bonner and Pollard, 1975) and formaldehyde (H2B-H4, H2A-H2B) (Van Lente, Jackson and Weintraub, 1975). Studies with the reversible cross-linking agent 4-methyl mer- captobutyrimidate (Hardison, Eichner and Chalk- ley, 1975; Hardison et al., 1977) revealed a much more extensive distribution of dimers. The most frequent dimers observed were H3-H2B, H2B-H2A, H3-H2A and, to a much lesser extent, H3-H4, H2B- H2A and H3-H3. Again as was seen in the previous studies, there was very little indication of the pres- ence of homodimers except for the small percent- age of H3-H3 observed in cross-linking with 4- methyl mercaptobutyrimidate. Methyl 4-mercapto- butyrimidate is a lysine cross-linking agent which bridges a distance of 14 A (Aizawa, Kurimoto and Yokono, 1977). If another lysine cross-linking agent could be found to bridge much shorter distances, then a comparison of the dimers produced be- tween these two reagents would present a clearer picture of short and long distance interactions between histones. Formaldehyde is such a reagent (it bridges distances of 2 A).

This paper is a detailed reanalysis of formalde- hyde cross-linking in nuclei. Procedures are de- scribed which allow selective reversal of either histone-DNA or histone-histone cross-linking. Uti- lizing these procedures, it was determined that instead of just two dimers (Van Lente et al., 1975), all 10 possible dimers of the four non-H1 histones are produced by formaldehyde. The most frequent dimers are H3-H3, H2A-H2A, H2A-H2B, H3-H2A and H2B-H4. This is the first reported instance of homo- dimers representing a significant percentage of the dimers observed in nucleoprotein. Also observed was the presence of two separate dimers of H2B- H4, one of which is produced at a slower rate than the other dimers. This paper also describes exper- iments which locate the general area of the cross- link in both histone-histone and histone-DNA.

cell 946

Results

When nuclei are treated with 1% formaldehyde at 4°C and the cross-linking of the histones is subse- quently assayed on SDS-polyacrylamide gels, it is found that with increasing time, formaldehyde cross-links the histones to apparent dimers, and at the same time, both dimer and histone monomers are cross-linked to very high molecular weights which do not penetrate the gel (Figure l)..These high molecular weights must be >llO,OOO daltons, the apparent molecular weight of the octamer de- scribed by Thomas and Kornberg (1975a) (Figure 1A). Since there appear to be at least two stages in the fixation process-cross-linking to dimers and cross-linking to high molecular weight polymers- these two processes were assayed separately.

Electrophoretic Separation of Histone Dimers To quantitate the various dimers produced by the fixation process, it is necessary to remove Hl histone from the sample. Its molecular weight is similar to the dimers and therefore overlaps them in SDS gel (Figure 1). Hl can be selectively re- moved by extraction with 0.5 M perchloric acid (PCA) (Phillips and Johns, 1965), which selectively

C-1

0 1 5 15 30 60 2 4 6 12 16 24 A

MINS. HRS.

Figure 1. Formaldehyde Cross-Linking of Histones in Nuclei

Nuclei at 1 mg/ml in DNA are fixed in 1% formaldehyde at 4°C for increasing times. The fixation process is stopped by adjusting the samples to 0.4 N l-@O,. After sonication, the samples are centri- fuged at 20,000 x g for IO min and the supernatant is dialyzed to remove excess formaldehyde. The acid-insoluble pellet is ana- lyzed in Figure 5. The samples (1 ml) are adjusted to 1% SDS, 30 mM Tris (pH 8) and electrophoresed on 18% SDS-acrylamide gels at 130 V for 18 hr at 4”C, 30 pl per channel. Sample (A) containing the cross-linked octamer is produced by treatment of nuclei in 2.0 M NaCI. 100 mM NaB,O, (pH 9.0) with dimethylsuberimidate (1 mg/ml) at 25°C for 60 min (Thomas and Kornberg, 1975a). The reaction is stopped by adjusting the solution to 0.4 N &SO,, and the acid-soluble fraction is processed as above.

precipitates the non-H1 histones, H3, H2A, H2B and H4. By repeated solubilization and reprecipi- tation of the non-H1 histone perchlorates, Hl can be totally removed from the solution (Figures 2a and 2b). If this same procedure is applied to his- tone samples isolated from nuclei treated with formaldehyde, then a much clearer picture emerges of the distribution of histone dimers (Fig- ures 2c and 2d). If these same samples are electro- phoresed on 36 cm SDS gels, eleven dimers are resolved (Figures 2e and 2f).

There are ten possible dimers that can be formed by the combination of the four non-H1 histones. If C-1 (+I

1

I 1

11111IIII 23456 7 8 910 1’1

*. I. -5. .a,’ -. *, : *. *. :

C-1 ‘. ,a*’ (+I

Hi & i ,,&“.. DIMERS l-/3

‘/-14 H2A

Figure 2. Selective Removal of Hl and the Resolving of Histone Dimers

(a) The acid-soluble fraction of unfixed nuclei; (b) the same fraction after the removal of Hl by treatment with 0.5 N PCA (see Experimental Procedures); (c) the acid-soluble fraction of a 1 min fixation of nuclei with formaldehyde followed by removal of Hl with 0.5 N PCA; (d) the same as (c). except that the fixation is for 30 min; (e and f) the same as samples (c) and (d), except that electrophoresis is on 36 cm SDS gels (see Experimental Proce- dures).

Formaldehyde Cross-Linking of Chromatin 947

one assumes that the site of fixation in each his- tone is located in the same relative position in each molecule, then by knowing the molecular weight and relative mobility of the monomer histones in SDS gels, an estimate can be made of which histones are present in each dimer. These predic- tions are shown in Table 1. To verify the soundness of these predictions, it is necessary to develop a procedure which will define the histones present within the dimers.

Identification of Dimers Procedures have recently been developed whereby histones which have been fixed with formaldehyde can be reversed such that the monomer histones are again obtained. Evidence for this type of rever- sal is shown in Figure 3. When acid-extracted histones are adjusted to pH 7-2.0 M KCI, formal- dehyde treatment results in a rapid cross-linking of the histone to a molecular weight so large that the protein falls out of solution and will not enter a 2% acrylamide gel (Figure 3a). If the excess formalde- hyde is removed by dialysis and the cross-linked protein is heated at 100°C in the presence of 2.0 M guanidine hydrochloride, 0.5 M e-mercaptoetha- nol, then the cross-linking is totally broken within 30 min (Figure 3b). This same procedure can be applied to cross-linked histones when immobilized

Table 1. tdentification of the Histones Present in the Formaldehyde Cross-Linked Dimers

Dimer Molecular Number Predicted Weight

% Dimer Frequency (60 Min

Observed Fixation)

1 H3-H3 30,648 H3-H3 6

2 H3-H2B 29,098 H3-H2B 1

3 H3-H2A 29,284 H3-H2A 12

4 H2B-H2B 27,540 H2-H2B 3

5 H2B-H2A 27,734 H2B-H2A 10

6 H2A-H2A 27,920 H2A-H2A 13

7 H3-H4 26,606 H3-H4 2

8 - H2B-H4 25

9 H2B-H4 25,056 H2B-H4 25

10 H2A-H4 25,242 H2A-H4 2

11 H4-H4 22.564 H4-H4 1

The prediction for histones present in the dimers is based on two factors-molecular weight and relative mobility of the monomer histones in SDS gels. The molecular weights of the four non-H1 histones are: H3-15,324; H2A-13,960; H2B-13,774; H4- 11,282 (Bostock and Sumner, 1978). The relative mobility is a significant factor with respect to H2A and H2B. H2B migrates more slowly in SDS gels, although its molecular weight is larger than H2A (see Figure 1). Because of this anomaly, HZB-containing dimers will electrophorese more slowly than H2A-containing di- mers when cross-linked to the same type of histone.

in an acrylamide gel. In this case, however, the 2.0 M guanidine-hydrochloride is no longer necessary. Thus when a slice of the dimer region of a 36 cm gel (Figures 2e or 2f) is heated at 95°C for 30 min at either pH 6.8 or pH 8.5 (see Experimental Proce- dures) and electrophoresed in the second dimen- sion on SDS gels, the histones present in the dimers can be identified. The reversal at pH 6.8 (Figure 4a) allows clear identification of which histones are present in each dimer, and the rever- sal at pH 8.0 (Figure 4b) allows quantitation of the histones present in each dimer. Table 1 shows the results of this analysis. The first observation to be made is that our predictions for the histones pres- ent in the dimers are correct. In other words, the location of cross-linking by formaldehyde, whether in the N terminal region or core region of each histone molecule, is similar for all non-H1 histones. Second, all ten possible dimers are observed, with certain dimers being much preferred-H3-H3, H3-

a b

(+I

0 1 30 1 5 lo 20 30 60

(MINS.) (MINS.1 TIME OF TIME OF REVERSAL FIXATION

Figure 3. Formaldehyde Cross-Linking and the Reversal of Cross-Link for Histones in Solution

(a) Calf thymus histones (Hi removed by extraction with 0.5 M PCA) are adjusted to 2.0 M KCI. 10 mM TEA (pH 7.4) and made 1% formaldehyde. At 1 and 30 min, samples are taken and adjusted to 0.4 N H,SO,. dialyzed for 16 hr at 4°C and then electrophoresed as described in Figure 1. (b) The histone sample cross-linked for 30 min and dialyzed is adjusted to 2.0 M guanidine hydrochloride, 0.5 M Pmercaptoeth- anol, 50 mM TEA (pH 7.4), and heated at 95°C for up to 60 min. Samples are taken at 1,5,10.20,30 and 60 min, and after dialysis against 0.4 N H,.SO, for 18 hr at 4-C, are electrophoresed on SDS gels as described in Figure 1.

Cell 946

a b IST DIMENSION <, IST DIMENSION

H3 em__ _-mm m_ _______ 0-m H2B ._______ ---Q--- H2A m-----_----- ~---~-------------------

H4------ ----- --------- ------- 0 I

------ a(-J mmmaas-m-m-mm-._ _

IIII IIIII I 11 1098 7 65432 1

Figure 4. Identification of the Histones in the Dimers A histone sample isolated from nuclei cross-linked with formaldehyde for 30 min is electrophoresed on a 36 cm SDS gel (Figure 21). After staining and destaining the gel, a 1 cm strip is cut through the dimer region perpendicular to the bands and incubated for 60 min at 25°C in either 1% SDS, 0.25 M Tris at pH 6.6 (a) or at pH 6.0 (b). The slices are heated in a fresh bath of the same solution, but also containing 0.5 M &mercaptoethanol, for 20 min at 95°C. The acrylamide strip is then polymerized into an 16% SDS-acrylamide gel and electrophoresed in the second dimension.

H2A, H2B-H2A, H2A-H2A and H2B-H4. Third, note that there are two dimers of H2B-H4 (dimer 8 and 9). Dimer 8 differs from dimer 9 not only in its apparent molecular weight (site of fixation is differ- ent), but it is produced at a much slower rate than dimer 9 (compare Figures 2e and 2f). In addition the production of dimer 8 by formaldehyde fixation is extremely dependent upon the integrity of the nucleoprotein. If the nucleoprotein in nuclei is solubilized by either shearing in low ionic strength (Bartley and Chalkley, 1973) or with staphylococcal

nuclease. (Nell, Thomas and Kornberg, 1975), the production of dimer 8 is greatly decreased (V. Jackson, manuscript in preparation).

Cross-Linking of Histone Molecular Weight Products It was shown in Figure 1 that formaldehyde rapidly cross-links histones in nuclei into a form which is not acid-soluble and will not penetrate the gel. It has been previously reported (Jackson and Chalk- ley, 1974) that formaldehyde cross-links histone to

Formaldehyde Cross-Linking of Chromatin 949

-13\ -128 im -l4-

0 1 5 '15 30 60 2 4 8 I

12 18 24 A MINS. HRS.

Figure 5. Histone Products Cross-Linked to DNA by Formaldehyde

The acid-insoluble pellets of Figure 1 which contain histones cross-linked to DNA by the formaldehyde fixation are resuspended in SDS, 50 mM Tris (pH 8) and incubated at 3PC for 2 days. The samples were sonicated to decrease the molecular size of the DNA and tl directly electrophoresed on 18% SDS acrylamide gels as described in Figure 1. Sample (A) contains the octamer which is produced cross-linking nuclei in 2.0 M NaCI. 100 mM NaS,O, (pH 9) as described in Figure 1.

1% hen 1 by

DNA in sheared nucleoprotein, and a procedure was described to reverse this particular cross-link allowing identification of both histone and DNA. If the acid-insoluble fractions of Figure 1 are sub- jected to these reversal conditions, which involve incubation of the samples at 37°C for 2 days in 1% SDS at pH 7 (see Experimental Procedures), then one can observe the results shown in Figure 5. This treatment has indeed caused reversal of formalde- hyde cross-links, presumably the reversal of his- tone-DNA. Since, however, there are higher molec- ular weight oligomers in the SDS gel (Figure 5), it is possible that a significant percentage of the monomer histone may result from the breakdown of histone-histone cross-links. We have carried out control experiments which include incubations of cross-linked histones under these conditions and subsequent analysis on two-dimensional gel elec-

trophoresis (as in Figure 4). These experiments demonstrate that histone-histone cross-links are not broken by incubations of 37°C for 2 days (unpublished observations).

Analysis of the Dimer and Oligomer Distribution in Cross-Linked Nuclei The data of Figures 1 and 5 taken together allow one to analyze the dimer distribution throughout 24 hr of fixation. Figure 6a shows that the rate of formation of dimers 8 and 9 (the two H2B-H4 dimers) are not the same. Dimer 8 is 3 times slower than dimer 9, and yet after 24 hr of fixation, there is an equivalent amount of each. Clearly the H2B and H4 histones in these two dimers are organized differently. Both the rate of fixation (apparent mo- lecular weight) and rate of formation are different. It is not apparent whether these differences exist

cell 950

within any given nucleosome or represent hetero- geneity of nucleosomes. The remaining nine di- mers do not show this heterogeneity. For example, there is only one H3-H3 dimer. The remaining nine dimers also have a rate of formation similar to dimer 9 (unpublished observations). This hetero- geneity is characteristic of only one dimer of H2B- H4-dimer 8.

The data of Figures 1 and 5 can be used to analyze the rate of conversion of non-H1 histones

100

50

RATE 4o OF

FORMATlOP?

20

10

a I I I ,,I, I I I 1 2 3 4 '8 12 16 20

HOURS

4

o-o-

HOURS Figure 6. Raie of Formation of Dimers and Higher Oligomers by Formaldehyde Cross-Links

(a) The rate of formation of dimer 8 and 9 (HZBH4); data are taken from Figures 1 and 5.

Rate of formation = OO,, of dimer at time (t) ODS, of dimer after 24 hr

x 100

Rate of formation of dimer 8 (LO). rate of formation of dimer 9 (60). (b) The percentage of histones present as monomer, dimer and higher oligomers; data are taken from Figures 1 and 5.

% cross-linked OD,, of monomer or dimer or oligomer at time (t) x ,oo

= OD,, of monomer + dimer + oligomer at time (t)

Percentage of histones as monomer (O--O), dimer (C-- 0) and higher oligomers (L-0). Higher oligomers represent all cross-linked products from trimers to octamers.

from monomer+ dimer-, higher oligomers. These results are summarized in Figure 6b. The first point to be made is that not all the monomer histones are cross-linked to dimers or higher oligomers. Approximately 50% of the total non-H1 histones remain resistant to cross-linking. This is true even if the conditions for cross-linking include high concentrations of formaldehyde and high temper- ature-that is, 37°C (unpublished observations). The second point is that the conversion to higher oligomers is a very slow process with no tendency to form an octameric unit selectively (see Figure 5). One might argue that this slow rate is reflected in the chemistry of the formaldehyde cross-linking where the excess formaldehyde competitively blocks the E-NH, group of the lysines in the his- tones (potential sites for cross-linking). This does not appear to be the case, for when histones are allowed to interact extensively (2.0 M KCI acid- extracted histone), as shown in Figure 3, the rate of formaldehyde cross-linking is very rapid (80% cross-linked within 1 min). The rapid increase in rate of cross-linking in 2.0 M KCI is caused by the increased binding and aggregation between the histones. Thus one explanation for the slow rate of conversion of histones to higher molecular weight oligomers in the formaldehyde fixation of nuclei is that the cross-linking sites in the histones are simply not close enough, and only after an exten- sive amount of random motion within the nucleo- some can one detect oligomers. This explanation can also be applied to the conversion of monomer histones to dimers. Whereas there is an initial 4- 5% that is cross-linked within 1 min (see Figures 2 c and 6b), the remaining cross-linking has a slow and measurable rate. It would appear that the majority of the histones are not in a position to be cross-linked to one another, but will so give the random motions within the nucleosome. A second explanation is that there is a subclass of nucleo- somes which have a structure that does not permit formaldehyde cross-linking of the histones. The 50% of the histones which do not cross-link may be in this class. Metabolic modifications, such as acetylation and phosphorylation, may have a role in this packaging. These same modifications may also cause differential cross-linking within the nu- cleosome.

Localization of Cross-Linking Sites in Histone- Histone and Histone-DNA The type of localization with which this paper is concerned relates to whether the cross-linking is occurring in the N terminal or core region of the histone molecules. Weintraub and Van Lente (1974) have reported that if nucleoprotein is sub- jected to trypsin treatment, there is a selective

Formaldehyde Cross-Linking of Chromatin 951

digestion of the N terminal region (- 25 amino acids) leaving what has been described as the “core” region intact. It is commonly believed that this core region contains the sites for histone- histone interactions. The N terminal (rich in basic amino acids) is considered to be interacting pri- marily with the DNA, leaving it more sensitive to trypsin activity. The probable site for cross-linking of histone to DNA would be this region.

Nuclei were fixed for 5 hr in 1% formaldehyde, and after extensive washing to remove excess reagent, they were digested with trypsin. Unfixed nuclei were digested with trypsin under the same conditions. The digestion was stopped by treat- ment with acid, and the acid-soluble fraction was applied to SDS gels. As shown in Figure 7, the tryptic digest of unfixed nuclei (Figure 7a) contains histones with increasing conversion to the resistant form (core), while the fixed nuclei (Figure 7b) have none at all. The acid-insoluble fraction from the unfixed nuclei also does not show the presence of histone (data not shown). If this same acid-insolu- ble fraction is incubated 37°C for 2 days (conditions

a C-1

Hl-

TRYPSIN-

H3 - H2B- H2A’ H4-

? g/ml 0 2 10 50 200

b

for reversal of histone-DNA cross-links) and ana- lyzed on SDS gels, the tryptically digested histones are present (Figure 7~). These results indicate that the core region of the histones cross-links to DNA. Does the N terminal region also cross-link to DNA? The preliminary answer to the question is yes. 3H- lysine-labeled nucleoprotein which has been de- pleted in Hl by treatment in Cl.6 M NaCl has twice as much 3H-lysine fixed to the DNA than what can be accounted for by lysine present in the core region (unpublished observations).

As shown in Figure 7c, the dimers when digested with trypsin do not disappear, as might be ex- pected if one or both of the cross-linked sites occurred in the N terminal region. Since the cross- linked dimers are resistant to trypsin and have apparent molecular weights of cross-linked cores, the cross-link must be in the core region of the histone. Further evidence that the dimers have been digested to “core” histone can be made by reversal of the cross-link and analysis in the second dimension on SDS gels, as shown in Figure 4 (unpublished observations). These observations do

C d

Figure 7. Localization of Cross-Linking Sites by Tryptic Digestion

(a) Unfixed nuclei at 1 mg/ml in DNA and 10 mM TEA, 10 mM MgCI, (pH 7.4) are digested with trypsin at 4°C for 60 min. Samples are treated with 0.4 N f-&SO, and left at 4°C for 46 hr. This treatment is necessary to prevent renaturation of trypsin when the pH is readjusted to 7. Solutions are then centrifuged at 20,000 x g for 10 min, and the acid-soluble supernatant is dialyzed and applied to SDS gels as described in Experimental Procedures. (b) Same as (a), except that the nuclei are cross-linked for 5 hr in 1% formaldehyde, washed 5 times with 10 mM TEA, 10 mM MgCI, (pH 7.4) and then digested with trypsin. The acid-soluble supernatant is analyzed on SDS gels. (c)The acid-insoluble pellets of (b) containing histones cross-linked to DNA are resuspended in 1% SDS, 50 mM Tris (pH 6) and incubated at 37°C for 46 hr. Samples are then sonicated and applied directly to SDS gels. (d) Same as (b), except that the nuclei are cross-linked for 30 min. After tryptic digestion, the acid-soluble fraction is applied to SDS gels.

DIGESTED DIMERS

DIGESTED MONOMER

Cell 952

not preclude the possibility .of N terminal cross- linking, but if such cross-linking occurs, it must include the same two histone molecules cross- linked in the core region. These data confirm our previous observation that because of the predicta- bility of dimer location in SDS gels (Table l), the cross-linking must be occurring in a similar loca- tion for all histones. This location is in the core region.

Discussion

Van Lente et al. (1975) previously identified the existence of two dimers, H2B-H4 and H2A-H2B, produced by formaldehyde fixation. Their proce- dure for identification of the histones in the dimers included elution of the dimer from the gel, iodina- tion and tryptic mapping. No other dimers were identified perhaps because of the masking effect of Hl, which co-electrophoreses in the dimer region, and the difficulty of resolving the dimers on SDS gels. This paper describes procedures which in- volve selective extraction of Hl and, after electro- phoresis on 36 cm SDS gels, the resolving of eleven dimers produced by formaldehyde cross- linking of the four histones H3, H2A, H2B and H4 in nuclei. It also describes a simple procedure for reversal of the formaldehyde cross-link such that the histones present in each dimer can be identi- fied. These results indicate that all ten possible combinations of the four non-H1 histones are pres- ent as dimers. There appear to be two classes of dimers: the more frequent (6-25%) includes H3-H3 (6%), H3-H2A (12%), H2A-H2B (lo%), H2A-H2A (13%) and H2B-H4 (25% dimer 8; 25% dimer 9), and the less frequent (l-3%) includes H3-H2B (l%), H2B-H2B (3%), H3-H4 (2%) H2A-H4 (2%) and H4-H4 (1%). Can these frequencies be utilized to describe histone organization within the nucleosome?

The primary site of formaldehyde cross-linking is the E-NH* group of lysine in the histones. This

4 group has a pk of 11.3 and thus at a pH of 7, it is 99.9% protonated (positively charged). The repul- sive forces between two of these groups in two histones makes it highly improbable that a 2 A cross-link (methylene bridge of formaldehyde) could be produced unless there were binding forces between those two histones. The cross-link- ing site may or may not be located near the actual binding site of the histones. Wherever it is located, it is a result of intimate interactions somewhere in the two histone molecules. It is of interest that, as reported in this paper, the cross-linking site occurs in the core region of the histones and that all the dimers appear to be cross-linked in the same gen- eral area within the core. Since the core region is the presumed site for binding between histones,

these results suggest that the lysine may be located near the binding site.

A two-dimensional model for histone organiza- tion within the nucleosome is shown in Figure 8. This model emphasizes the presence of the homo- dimers H3-H3 and H2A-H2A. It also emphasizes the presence of two dimers of H2B-H4, possibly dimer 8 and dimer 9 described in this paper. Included in this figure are the observed dimer frequencies for four other cross-linking reagents which have been used to study histone organization in the nucleo- some. These are tetranitromethane (Martinson and McCarthy, 1975), ultraviolet light (Martinson et al., 1976) and carbodiimide (Bonner and Pollard, 1975), all three of which cross-link at distances <2 A, and methyl 4-mercaptobutyrimidate (Thomas and Kornberg, 1975b; Hardison et al., 1975, 1977), which cross-links distances of 14 A (Aizawa et al., 1977). The dimer frequencies observed with these reagents are consistent with this model. Any three- dimensional model of histone organization in the nucleosome must be consistent with the histone proximities shown in Figure 8.

If formaldehyde causes extensive distortion of structure, then the significance of these results becomes questionable. Several previous studies, including low angle X-ray diffraction (Olins, Carl- son and Olins, 1975), circular dichroism (Senior and Olins, 1975) and electron microscopy (Senior, Olins and Olins, 1975), indicate that distortion is minimal. Perhaps even more sensitive measures of distortion would involve using nucleases such as staphylococcal nuclease (Nell, 1974a) and DNAase I (Nell, 1974b), which cleave at selected locations in nucleoprotein. Since reversal procedures for separating histone from DNA have been described in this paper, it is now also possible to analyze the DNA from cross-linked chromatin. Such studies have been carried out with both staphylococcal nucleases and DNAase I. In both cases, the cleav- age products are the same as observed in unfixed nuclei (unpublished observations). These observa- tions suggest that fixation with this reagent does not alter appreciably the nucleoprotein structure.

\MMB/ \MMB’ \MMB/

Figure 8. Two-Dimensional Model of Histone Organization in the Nucleosome Based on Cross-Linking Data

(MMB) methyl 4-mercaptobutyrimidate; (TNM) tetranitromethane; (UV) ultraviolet light; (CDM) carbodiimide; (HCHO) formaldehyde.

Formaldehyde Cross-Linking of Chromatin 953

In fact, once the fixation has been completed, the structure becomes resistent to denaturation by 0.1 M HCI, 0.1 M NaOH, 2.0 M NaCl and 5 M urea as assayed by nuclease specificity (unpublished ob- servations). This resistance to denaturants appears to be a result of extensive histone-DNA cross-links (100% of the histones cross-link to DNA). Cross- linking agents such as dimethyl suberimidate, methyl 4-mercaptobutyrimidate and tetranitro- methane do not extensively cross-link histones to DNA, but do cross-link histones to histones. These agents do not provide this resistance (unpublished observations). Formaldehyde is known to react with both the imino (McGee and von Hippel, 1975a) and amino (McGee and von Hippel, 1975b) groups in the bases of DNA. Its ability to produce a 2 A bridge between these bases and the lysines from both the N terminal and core region of the histones indicates the importance of these two regions in stabilizing nucleosomal structure through DNA- histone interactions.

One additional point should be made concerning the advantages in using formaldehyde to study protein proximities. By using the reversal condi- tions described in this paper, one can reobtain an intact and unmodified protein (no methylene group associated with the protein). These conditions of reversal also do not destroy metabolic modifica- tions, such as acetylation and phosphorylation, which may be present on the protein (unpublished observations). It is thus possible to isolate a group of cross-linked proteins and, after reversing the cross-link and separating the proteins, to complete an unhindered physical characterization of these proteins.

Experimental Procedures

Preparatbn of Nuclei and Solubillzed Chromatin Calf thymus nuclei were prepared by homogenization of the tissue in 0.25 M sucrose, 10 mM MgC&. 10 mM Tris, 50 mM NaH SO, (pH 6.5) and subsequent washing of the nuclear pellet with three washes of the same buffer containing 1% Triton X-100. This nuclear pellet was then washed twice in 10 mM Mg Cl,, 10 mM TEA (Triethanolamine) (pH 7.4) (TM buffer) and suspended in the same at a concentration of 1 mg/ml in DNA (OD,,, = 20). This nuclear suspension could then be directly cross-linked with form- aldehyde, or the nuclei could be solubilized by either nuclease treatment (Noll et al., 1975) or mechanical shear (Bartley and Chalkley, 1973).

Reaction wlth Formaldehyde Formaldehyde from three sources was used: a 37°C solution (Fischer Scientific) which is neutralized before use; an alkaline reflux and distillation of paraformaldehyde (Grossman, 1968); and direct vaporization of formaldehyde from paraformaldehyde. Formaldehyde from all three sources gave identical results. For convenience, the 37% solution was routinely used. The nuclei suspension or solubilized chromatin at a concentration of 1 mg/ ml was made 1% formaldehyde and cross-linked at 4°C for the desired time. The reaction was stopped by taking aliquots and adjusting them to 0.4 N H,SO, followed by sonication of the

sample. Samples were centrifuged 20,000 x g for 10 min and the acid-soluble and insoluble fractions were collected. The acid- soluble fraction containing histones free of DNA was dialyzed against 0.4 N &SO,, 50 mM Pmercaptoethanol to remove excess formaldehyde, and then dialyzed against 10 mM HCI, 50 mM 2- mercaptoethanol to remove excess acid. This sample was then ready for electrophoresis or for further processing such as rever- sal of the fixation or selective extraction of Hl (see below).

Reversal of Formaldehyde Fixatbn For reversal of histone cross-links in solution, the solution was adjusted to 2.0 M guanidine hydrochloride, 0.5 M P-mercaptoeth- anol and heated at 95°C for up to 60 min. The solution was cooled to 4°C and dialyzed against 10 mM HCI, 50 mM P-mercaptoethanol at 4°C for 16 hr. The sample was now ready for analysis.

For reversal of histone cross-links in an acrylamide gel. the stained gel slice from the first dimension electrophoresis was incubated at 25°C for 60 min in 1 .O% SDS, 0.25 M Tris at either pH 6.8 or pH 8.8. This slice was then heated in the same solution containing 0.5 M 2-mercaptoethanol at 95°C for 30 min and directly polymerized into the separating gel - 18% acrylamide, 0.6% bisactylamide, 0.1 SDS, 0.75 M Tris (pH 8.8). The histones were then electrophoresed in the second dimension.

For reversal of histoneDNA cross-links, the samples were adjusted to 1% SDS, 50 mM Tris (pH 8.0) and incubated at 3PC for 2 days. The samples were then directly electrophoresed to examine the histones.

Selective Removal of Hl Hlstone (Phillips and Johns, 1965) The acid-soluble solution containing 10 mM HCI, 50 mM 2- mercaptoethanol was adjusted to 0.5 N perchloric acid (PCA). After 30 min at 4°C. the sample was sonicated and centrifuged at 20,000 x g for 10 min. The supernatant containing Hl was discarded and the pellet was redissolved in 10 mM HCI. The cycle was then repeated twice, and the final pellet was dissolved in 10 mM HCI, 50 mM 2-mercaptoethanol. This solution was then prepared for electrophoresis by adjusting to 1% SDS, 10% su- crose, 20 mM Tris, 0.002% BPB (pH 7) from a 5 x concentrate.

Condltbns for SDS-Acrylamlde Electrophoresis The electrophoresis conditions are a modification of the Laemmli (1970) procedure. The electrophoresis buffer is 0.1% SDS, 0.025 M Tris, 0.20 M glycine (pH 8.3), and the separating acrylamide gel is 18% acrylamide, 0.09% bisacrylamide. 0.1% SDS, 0.75 M Tris (pH 8.8). For SDS-acrylamide gels of 35 cm length, the bisacryl- amide concentration was increased to 0.6%. The stacking gel was 2.5% acrylamide, 0.125% bisacrylamide, 0.1% SDS, 0.125 M Tris (pH 6.6). Electrophoresis was at 150 V for 18 hr with the 15 cm gels, and at 250 V for 48 hr for the 35 cm gels. All electrophoresis was at 4°C. After electrophoresis, the gels were stained in 0.1% Coomassie brilliant blue R, 40% methanol, 10% acetic acid for 12 hr. destained in 40% methanol, 10% acetic acid, and scanned in a CAMAG microdensitometer.

Conditions for Enxymatic Analysis of Nuclei Cross-Linked by Formaldehyde Nuclei were cross-linked with 1% formaldehyde, and at the appro- priate time, they were washed 3 times with 10 mM TEA, 10 mM MgCI, (pH 7.4) and suspended in the same solution at 1 mg/ml in DNA. The nuclei suspension was then digested with either the nucleases or trypsin at 25°C.

To analyze the trypsin digests, the digest was first stopped by adjusting the solution to 0.4 N H,SO,. It was then sonicated and stored for 48 hr prior to centrifugation at 20,000 x g for 10 min. The acid-soluble fraction (supernatant) containing the histones free of DNA could be directly electrophoresed after dialysis against 10 mM HCI, 50 mM P-mercaptoethanol to remove excess acid. The acid-insoluble fraction containing histones cross-linked to DNA was resuspended in 1% SDS, 50 mM Tris (pH 8), and

cell 954

incubated at 37°C for 2 days to reverse histone-DNA cross-links. The histones were directly analyzed on SDS gels.

Acknowledgments

This work was supported by an NIH postdoctoral fellowship and the Medical Research Council.

The cost of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 26, 1978; revised July 31,1978

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