THE JOURNAL OF BIOLOGICAL CHEM~TRY Vol. 252, No. 8, Issue ofApn1 25, pp. 2630.2639, 1977

Printed tn U.S A.

Chromatin Fractionation Related to Cell Type and Chromosome Condensation but Perhaps Not to Transcriptional Activity*

(Received for publication, May 13, 1976, and in revised form, December 6, 1976)

MICHAEL M. SEIDMAN AND R. DAVID COLE

From the Department ofBiochemistry, University of California, Berkeley, Berkeley, California 94720

A chromatin fractionation procedure has been developed that involves the shearing of swollen chromatin and the separation of chromatin fragments by sucrose gradient cen- trifugation. When chromatin from rapidly growing HeLa cells was fractionated, four partially resolved peaks were obtained. Partial characterization of the chromatin frac- tions indicated that each contained similar lengths of DNA formed in a complex with histones in nucleosomes. The most likely difference between the fractions is the degree of intra- or interstrand association of the fibers of nucleosomes. Chromatins from chicken erythrocytes and mitotic chromo- somes were analyzed by the procedure. These chromatins yielded gradient profiles that were distinctly different from each other and from interphase chromatin. These results suggested that the fractionation procedure reflected at least some of the differences in the structure of chromatin known to exist in vivo.

The association of pulse-labeled RNA with a particular chromatin fraction is frequently used to support claims of successful separation of transcriptionally active chromatin from inactive chromatin. Since our data show that the slowly sedimenting fractions preferentially bind RNA whose synthesis was clearly not in progress at the start of the fractionation, this criterion is suspect. The presence of equal amounts of satellite DNA in all fractions of mouse L- cell chromatin indicated that the method did not fractionate on the basis of the in vivo transcriptional activity.

The organization and structure of eukaryotic chromosomes has been the subject of many studies (1). DNA is formed in a complex with chromosomal proteins in such a manner that a large amount of DNA is confined in a small volume. Chromo- somal structure varies from one cell type to another and from one stage of the cell cycle to another. This variation in packag- ing is accompanied by changes in activities and functions of

DNA metabolism such as transcription and replication. In interphase cells, chromatin is organized such that some DNA sequences are transcribed and all sequences can be replicated.

* This work was supported by Grants GB 38658 from the National Science Foundation, GM 20338 and GM 0031 from the National Institutes of Health, and by Contract NOl-CB-43866 from the Na- tional Cancer Institute, and by the Agricultural Experimental Sta- tion at the University of California, Berkeley.

The chromatin in nucleated erythrocytes is more condensed and supports a very low level of transcription and no replica- tion. Mitotic chromosomes are even more highly condensed and no transcription or replication occurs.

Thin section electron microscope studies show interphase chromatin to be a network of nucleoprotein fibers. These fibers are now known to consist of DNA packaged in o bodies or nucleosomes (2, 3). Both transcribed and nontranscribed DNA sequences are found in nucleosomes (4). It has been accepted for a long time that the fibers containing DNA that is tran- scribed in viuo are loosely organized (often referred to as extended chromatin, or euchromatin), while nontranscribed DNA is present in fibers which are tightly packed (often called condensed chromatin, or heterochromatin). In this paper we shall refer to chromatin that was transcribed in uivo as active

chromatin and that which was not, as inactive chromatin. Since one of the fundamental current questions in biology concerns the mechanism of gene regulation in eukaryotes, the separation and characterization of active and inactive chroma- tin is of interest.

Chromatin fractionation procedures have been described by Frenster et al. (51, Yasmineh and Yunis (6), Reeck et al. (7), Bonner et al. (81, and McCarthy et al. (9) among others. Although there is some variation in procedure, each method involves the fragmentation of chromatin and the separation of the fragments on the basis of physicochemical differences ex-

pected of active and inactive chromatin. The major goal of these studies was to separate chromatin components that were transcriptionally active in vivo from those that were inactive. Criteria such as satellite DNA enrichment (lo), in vitro tem- plate activity, and localization of pulse-labeled RNA (9, 11) were used to support claims for successful fractionation.

A procedure developed by McCarthy et al. (9) involved me- chanical shearing of purified chromatin. The components in the sheared chromatin were separated by centrifugation on sucrose gradients. When chromatin from cultured Drosophila cells was fractionated this way two peaks of chromatin ap- peared in the gradient. The chromatin that sedimented more slowly was a much better template for Escherichia coli RNA polymerase than the faster sedimenting fraction. Similar re- sults were obtained by Murphy et al. (11) with fractionated chromatin from mouse myeloma cells. Both groups reported that nascent RNA was localized in the slower sedimenting fraction. These and other data seemed to identify the slower sedimenting chromatin fraction as active chromatin. Since

2630

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Chromatin Fractionation 2631

then Doenecke and McCarthy have presented further evidence microscopy. There were no nuclei visible in the final chromosome

to support this assignment (12). preparations.

All chromatin fractionation procedures employ as a starting hypothesis that the structural distinctions between active and inactive chromatin will survive the solubilization and frag- mentation of the chromatin, but a study by Howk et al. (13) suggests that this hypothesis may be invalid. They reported that their use of the procedure used by Murphy et al. (11) as well as the method described by Reeck et al. (7) had failed to fractionate chromatin on the basis of authentic in vivo tran- scriptional activity. They found that all chromatin fractions contained equal amounts of DNA sequences that were tran- scribed in uivo and also contained sequences that were not transcribed in vivo. Similar results have been reported by Krieg and Wells (14).

Shearing of Chromatin

Washed chromatin was suspended in the appropriate buffer and loosely homogenized in a Dounce glass homogenizer with a B pestle. The chromatin was adjusted to the desired concentration and then sheared with a Sorvall Omni-Mixer microattachment homogenizer in the mini cup. Usually 3 to 6 ml of chromatin suspension was sheared. A few drops of 1-octanol were added just before shearing to reduce foaming. The voltage was controlled by a Variac powerstat.

Separation of Chromatin Fractions

In this paper we describe a chromatin fractionation proce- dure based on the method developed by McCarthy et al. (9) and employed by Murphy et al. (11). This procedure appears suc- cessful in reflecting some aspects of endogenous chromatin structure and organization, but like the results of Howk et al.

(13), and Krieg and Wells (14), ours suggest that the fractiona- tion is not to be correlated with in uiuo transcriptional activ- ity

Sheared chromatin was applied to 12.5 ml, 0.35 to 1.7 M sucrose, 10 mM TrisiHCl, pH 8.0, gradients. The gradients were centrifuged in an SW 41 rotor (Beckman) at 41,000 rpm for 195 min. The tempera- ture was set at 4”. The gradient was collected from the top by pumping 2 M sucrose in through the bottom of the cellulose nitrate tube. The gradients were pumped through a turbulence free flow cell (Molecular Instruments Co.) mounted in a Gilford 2000 continuously recording spectrophotometer. The absorbance was measured at 260 nm.

DNA Molecular Weight Determination

MATERIALS AND METHODS

Cells

HeLa S:, and mouse LAS-cells were grown in suspension culture in Joklik modified minimal Eagle’s medium supplemented with peni- cillin, streptomycin, and 5% calf serum (Gibco).

Erythrocytes were obtained from fresh rooster blood. Clotting was prevented by the addition of a few milliliters of 5 mM Na,EDTA, pH 7.5, and the cells collected by centrifugation at 1500 x R. The cells were washed with Dulbecco’s phosphate-buffered saline (15) and nuclei isolated as described for HeLa and LAS-cells.

Chromatin (150 ~11, isolated from cells which had been labeled with [:‘H]thymidine was mixed with 10 ~1 of 10 mM Na,EDTA, pH 7.5, 10 ~1 of 20% sodium dodecyl sulfate and 15 ~1 of nuclease-free pronase (Calbiochem) 10 mg/ml (18). The mixture was incubated at 37” for 1 h and applied to linear 5 to 20% sucrose gradients (in 10 mM TrisiHCl, 250 mM NaCl, 5 rnM Na,EDTA). The gradients were cen- trifuged in an SW 41 rotor at 41,000 rpm for 240 min at 23”. fd[14C]DNA, a gift from Dr. Alex Karu of the University of Califor- nia, Riverside, was used as an internal molecular weight marker. The molecular weight of the DNA was determined using the proce- dure of Studier (19).

Labeling of Cells

Isolation of Nuclei

Nuclei were isolated using a modification of a method described by Wray and Stubblefield (16). All cells were washed with Buffer A (1.0 rnM calcium chloride, 0.5 rnM Pipes (1,4-piperazinediethanesulfonic acid) (Calbiochem), pH 6.8, 0.1 M hexylene glycol). The cells were suspended at a concentration of 10’iml in Buffer A and allowed to swell for 15 to 20 min. The swollen cells were lysed in a Dounce glass homogenizer with 10 strokes of the A pestle. The nuclei were pelleted by centrifugation at 1500 x g and washed once with Buffer A. There were very few whole cells at the end of this procedure and nuclear recovery was around 95%. The nuclei were free of cytoplasmic tabs, and nucleoli were plainly visible when viewed with a phase contrast microscope.

RNA Labeling- [:‘H]Uridine (New England Nuclear), 1 mCi/ml, 40 &i/mmol was added to exponentially growing cells to a final concentration of 5 &i/ml. The cells were stirred for the indicated times, and then the incorporation was stopped by the addition of an equal volume of ice-cold 10 rnM sodium azide in phosphate-buffered saline solution. The cells were collected and washed once with cold phosphate-buffered saline/sodium azide.

DNA Labeling- [methyl-3HlThymidine (Amersham, 1 ,&i/ml, 50 Ciimmol) was added to exponentially growing cells to a final concen- tration of 0.5 pCi/ml. The cells were grown in the presence of the label for 48 h to insure extensive labeling.

RNA Extraction

The nuclei were washed with Buffer A containing 0.5% Triton X- 100, in order to remove nuclear membranes, and then washed twice with 75 rnM NaCl, 24 rnM EDTA, pH 8.0. All buffers were kept at 4”.

Chromatin was prepared from [“Hluridine-labeled cells. The chro- matin suspension was adjusted to 10 mM TrisiHCl, 1% sodium Sarko- syl, and 1 mM Na,EDTA, pH 8.0. The mixture was phenol-extracted and the aqueous phase was treated with alcohol. The precipitate was collected by centrifugation and dissolved in a small volume of 10 mM TrisiHCl, 1 rnM Na,EDTA, pH 8.0.

Isolation of Chromatin RNA Polymerase Assay

Chromatin was isolated from all cells by suspension of the nuclei in 10 rnM TrisiHCl, 1 rnM Na,EDTA, pH 8.0, followed by centrifuga- tion at 10,000 x g for 15 min. The chromatin was washed by gentle teasing of the chromatin gel in the appropriate buffer (see “Re- sults”).

The RNA polymerase activity of chromatin and chromatin frac- tions was assayed according to the method of Roeder and Rutter (20). The assays were incubated at 37” for 20 min. The reactions were terminated by spotting 100 ~1 of the assay mixture onto DE81 paper circles (Whatman) which were processed for counting as described by Blatti et al. (21).

Mimic Chromosomes

Mouse LAS-cells were treated with vinblastine sulfate (Eli Lilly) at a concentration of 0.5 pg/mi. The drug was added directly to growing suspension cultures at a cell concentration of 2 X lO”/ml. The per cent of mitotic figures was determined by Giemsa staining of fixed cultures (17). The yield of cells arrested in metaphase was highly variable. On some occasions 30 to 40% mitotic arrest was obtained and these preparations were used in the experiments de- scribed here. The chromosomes were purified by the method of Wray and Stubblefield (16) except that the 37” incubation step was omitted. The purity of the chromosomes was monitored by phase contrast

Satellite DNA Analysis

DNA was extracted from mouse LA9 chromatin fractions by the method of Cech et al. (22). Analytical CsCl centrifugation was per- formed as described by these authors except centrifugation was at 44,000 rpm for 24 h in the Spinco model E ultracentrifuge. Optical grade CsCl (Harshaw) was used. Buoyant densities were measured relative to Micrococcus lysodeikticus marker DNA with a density of 1.733 g/ml (a gift of Dr. J. E. Hearst of the University of California, Berkeley). Density differences between bands were calculated ac- cording to the method of Schmid and Hearst (23).

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2632 Chromatin Fractionation

Thermal Denaturation Analyses

Chromatin fractions and unfractionated chromatin were dialyzed ver,sus 5 rnM Tris, pH 8.0. Thermal denaturation analyses were performed in a Gilford model 2000 recording spectrophotometer. The range of the analysis was from 30-100” with a temperature increase of 15”/h. Absorbance was read every 2 min. The data were corrected for the expansion of the buffer.

region peak is much broader than the others, suggesting a greater constituent heterogeneity.

-Nuclease Digestion

Micrococcal nuclease digests of chromatin and chromatin fractions were performed as described by Axe1 et al. (24).

Polyacrylamide Gels

Electrophoresis of chromatin proteins on polyacrylamide gels con- taining sodium dodecyl sulfate was as described by Ferro-Luzzi Ames (25).

Protein and DNA Determinations

Shearing to Limit-Fig. 1 also illustrates an experiment in which the shearing time was varied while the voltage was held constant. When unsheared chromatin was centrifuged most of the material pelleted. The soluble portion forms a low broad peak in the central part of the gradient. After 90 s of shearing the basic elements of the profile were apparent (Fig. la), however 20 to 30% of the chromatin pelleted. The A,,;,, pattern was essentially constant across the range of 3 to 6 min of shearing and there was no pellet. These results indicate that shearing is necessary for the generation of the chromatin profile. That is, endogenous nuclease activity is not responsi- ble for the general appearance of the pattern. In any case a “limit shear” condition is reached after 3 min of shearing.

The protein content of the chromatin samples was determined by amino acid analysis (26). The method of Raynaud and Ohlenbusch was used to determine DNA content (27). Histone:nonhistone ratios were determined by densitometer analysis of Coomassie blue- stained, sodium dodecyl sulfate containing polyacrylamide gels of chromatin proteins.

The relationship between the limit shear chromatin profiles and the molecular weight of the DNA isolated from the chro- matin at various shear times was determined in the experi- ment illustrated in Fig. 2. DNA was isolated from unfraction- ated chromatin after the indicated shearing times. The sedi- mentation coefficient of the DNA was determined by centri-

RESULTS -

Application of Modified Procedure to HeLa Cell Interphase Chromatin -One of our initial goals was to study structures in active chromatin, such as transcriptional and replicative com- plexes, that might be more fragile than the bulk of the chro- matin structure. In the procedure used by McCarthy et al. (9)

chromatin was purified by centrifugation through concen- trated sucrose (1.7 M), but because the structures of interest might not survive this step we dispensed with it. In adapting the fractionation procedure of McCarthy et al. (9) to chromatin from HeLa cells we used the following criterion for chromatin solubility; sheared chromatin that was not pelleted from 10 mM Tris/HCl, pH 8.0, when centrifuged at 10,000 x R for 30 min was considered soluble. When such chromatin was centri- fuged on a sucrose gradient, a well resolved profile of absorb- ance at 260 nm was obtained and all chromatin was contained in the gradient. This behavior is characteristic of chromatin that had been purified by sucrose washing (9). However, when chromatin was prepared by lysis of nuclei in 10 mM Tris/HCl and sheared directly, more than 50% of the chromatin was found in a 10,000 x g pellet. When the sheared chromatin was centrifuged on a 0.35 to 1.7 M sucrose gradient again more than 50% was found in the pellet. Consequently attention was given to preparing chromatin that would be solubilized by shearing.

Preparation of Soluble Chromatin -After considerable ex- perimentation a solubilization method, based on that of Shaw and Huang (28), was devised. Chromatin was prepared from nuclei and then washed by pelleting at 10,000 x ,g from the following buffer series: 1 x 5 mM Tris/HCl, 0.5 mM Na,EDTA; 1 x 2.5 mM Tris/HCl, 0.25 mM Na,EDTA; 2 x 1 mM Tris/HCl; 1 x 0.5 mM Tris/HCl. All buffers were at pH 8.0.

The chromatin was visibly swollen after the wash proce- dure. The washed chromatin was suspended in 0.5 mM Tris/ HCl at a concentration of 15 A,,,, units/ml and sheared in the Omni-Mixer at 40 V for 5 min. The sheared chromatin was completely soluble.

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When the sheared chromatin was centrifuged on a 0.35 to 1.7 M sucrose gradient (in 10 mM Tris, pH 8.0) at 205,000 x R for 195 min the absorbance profile shown in Fig. Id was obtained. The profile might be described as a series of overlap- ping peaks, A, B, C, and D as indicated in the figure. The D

FIG. 1. Profiles of fractionated chromatin; effect of shearing time. Chromatin was prepared from HeLa cells and adjusted to 15 A.&ml and sheared at 40 V for the times indicated in the panels. The sheared chromatin was fractionated on sucrose gradients, and ana- lyzed as described under “Materials and Methods.”

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Chromatin Fractionation 2633

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Fraction Number FIG. 2. Sedimentation analysis of DNA extracted from sheared

chromatin at different shearing times. Chromatin was prepared from HeLa cells which had been incubated with [“Hlthymidine, and sheared for the times indicated. Aliquots were removed after each shearing time and the sedimentation properties of the DNA analyzed as described under “Materials and Methods.”

FIG. 3. High voltage shearing profile of HeLa cell chromatin. Chromatin was prepared from HeLa cells and adjusted to the stan- dard concentration of 15 A,,,,,/ml. It was sheared in the Omni-Mixer at 80 V and applied to a gradient under the standard conditions. The positions of the chromatin fractions from the 40-V shearing are shown.

same speed (29). The data from the experiments with high speed shear and low concentration indicate that the chroma- tin profiles of Fig. 1 are sensitive to factors which affect the extent of fragmentation of the chromatin.

fuging the DNA on linear 5 to 20% sucrose gradients. The DNA from unsheared chromatin (Fig. 2~) contains sequences of high molecular weight: > 40 x 10”. However, after shearing has begun, the molecular weight distribution is quite similar from one time to the next, although by 6 min of shearing there is some reduction in sedimentation coefficients of the DNA. Sedimentation coefficients and molecular weights at each time interval are listed in Table I.

Variability of Profile -In the course of this work chromatin was fractionated many times. A range of variability in the amount of material in each region of the profile is given in Table II. An interesting feature of these data is that while the amounts of Fractions A and D did vary from preparation to preparation, it is as if each did so at the expense of the other, for the relative amounts of Fractions B and C were always quite similar.

The stability of the gradient profile and relative constancy of DNA molecular weight over the range of shearing times sug- gest that the peaks in the chromatin gradient profile represent discrete structures rather than intermediates in the shearing process, some of which would disappear when shearing was complete.

Not only was the chromatin fractionation profile reproduci- ble under the standard conditions but a number of factors could be varied without significantly affecting the pattern. These include the use of either rapidly growing or stationary phase cells, the omission of the NaCl: NasEDTA and Triton washes of the nuclei, the isolation of the nuclei at pH 10.5 (to inhibit nucleases) (30) and centrifugation of the gradients at room temperature instead of 4”.

Variation in Shearing Conditions -Shearing the chromatin Molecular Weight of DNA from Each Fraction - Chromatin at higher speed resulted in a loss of resolution in the gradient was prepared and fractionated and the molecular weights of profile. When the chromatin was sheared at 80 V for the usual the DNA from the A, B, and D regions of the gradient were 5 min only a broad peak in the A region appeared. This is determined. The DNA from all fractions appeared heteroge- shown in Fig. 3. A similar result was obtained when chroma- neous in size with a sedimentation profile similar to Fig. 2~. tin concentration was reduced by a factor of 3 prior to shearing The mean molecular weight of the DNA from Fraction A was at the standard speed and time (40 V for 5 min). This last 2.7 x lo”, that from Fraction B, 3.0 x 10” and that from observation suggests that there is a self-protecting effect of the Fraction D, 3.3 x 10”. While there was some fractionation on chromatin which may be similar to the results obtained when the basis of DNA length, the size of the DNA was plainly a DNA solutions of varied concentrations are sheared at the very minor factor in the fractionation process.

TABLE I

Sedimentation coefficients and approximate molecular weights of

DNA extracted from chromatin sheared for different times

Sedimentation coeffF5ent Time

Molecular weight

LOW Peak High” LOW Peak High

min x10-"

2 10.2 15.2 23.5 0.92 3 10.5 3 10.6 15.2 23.5 1.0 3 10.5 4 10.6 15.2 23.5 1.0 3 9.3 6 9.6 14.8 23.5 0.8 2.7 8.9

n Low and high refer to measurements taken at 20% of maximum peak height on either side of the peak in the 5 to 20% linear sucrose gradient.

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2634 Chromatin Fractionation

Proteins and Protein Content of Fractions-The pro- tein:DNA ratios (w/w) of the fractions were: Fraction A, 1.5, Fraction B, 1.7; Fraction D, 1.4. Histones were extracted from each of the chromatin fractions and analyzed by electrophore- sis on sodium dodecyl sulfate containing polyacrylamide slab gels. There were no differences between the histone patterns of the fractions and little difference in the amount of total his- tone (relative to DNA). All five histones were present in all fractions. These results are similar to those reported by Mc- Carthy et al. (9). Total proteins in each fraction were analyzed in other experiments. Each fraction gave a number of nonhis- tone bands on the gels. However, there did not appear to be any significant variation in the spectrum of proteins from one fraction to the next. There were differences in the nonhis- tone:histone protein ratios of the fractions as reflected in the protein:DNA ratios cited above. The nonhistone:histone ratio of Fraction A was 1.0; that of B, 1.3; that of D, 0.6. The possibility of a significant degree of proteolytic degradation was ruled out by the appearance of well stained lysine-rich histone bands in electrophoretic gels along with the absence of the characteristic products of their degradation.

Thermal Denaturation Analysis -When chromatin is melted and the hyperchromicity plotted uersus temperature, a profile is obtained that has been interpreted as demonstrating the multiplicity of DNA:protein structures in chromatin. Since procedures, like the one described in this paper, are designed to fractionate such structures, melting analyses are frequently used to characterize chromatin fractions (9, 10, 14). Active chromatin is thought of as less constrained and-thus its DNA is expected to be less thermally stable than that of inactive chromatin. This view of chromatin, when related to the chro- matin fractionation procedure, predicts that the slowly sedi- menting chromatin will have a lower melting temperature than that of the rapidly sedimenting chromatin.

The A, B, and D chromatin fractions were melted simulta- neously to give the data shown in Fig. 4. The A and B fractions are quite similar, but differ from the D chromatin in two important respects. The first is the greater amount of the DNA in the A and B fractions which melts in the 40-65” range. The second is the melting temperature of the most thermally sta- ble component of the chromatin fractions. The DNA in chro- matin which melts below 65” (in this buffer) is thought to be either free of proteins or formed in a complex with nonhistone proteins (31). The hyperchromicity of the A and B chromatins in the 40-65” range is 25 and 23%, respectively; that of D, 6%. At the other end of the profiles the A and B chromatins have a melting temperature of 79-80” while that of the D chromatin is 83-84”. Although the interpretation of the higher temperature melting transitions has been discussed, a precise interpreta- tion is still unavailable (31). It is clear, however, that the D region chromatin contains a larger proportion of DNA with a melting temperature greater than 65”. There also appears to be some structure(s) characterized by the 83-84” melting tem-

TABLE II

Relative per cent absorbance (260 nm) in each HeLa cell chromatin fraction

Fraction I

Preparation

II III IV

A 17 24 28 33 B 29 28 29 28 C 13 15 15 13 D 42 31 28 25

perature which is not found in the A and B chromatin frac- tions.

In another experiment, A and D chromatin fractions and sheared, unfractionated chromatin were melted simultane- ously. The temperatures at which the highest transitions oc- curred were: A, 80”, unfractionated chromatin, 82”, and D, 83.5”. This indicates that the fractionation procedure segre- gated components of differing thermal stability. The charac- teristics of the fractions are summarized in Table III.

N&ease Digestion of Chromatin Fractions -The pro- tein:DNA ratios and the histone content of the chromatin fractions seemed to rule out the possibility that the fractiona- tion was based simply on protein content. However, the slowly sedimenting chromatin contained a significant amount of DNA that melted in the 40-65” range. This raised the possibil- ity that the arrangement of proteins, particularly histones, might be different in the A fraction compared to the D chroma- tin. To test this possibility, micrococcal nuclease digests of the chromatin fractions were performed as described by Axe1 et al. (24). Digests of nuclei or unsheared chromatin yield nuclease- resistant DNA fragments of about 200 base pairs (32). When sheared chromatin is digested a more complicated pattern is obtained (33). The 200 base pair fragments are observed, but

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FIG. 4. Thermal denaturation analysis of chromatin Fractions A, B, and D, performed as described under “Materials and Methods.” h,, = A,,,, at 30”; h,,, = A,,<,, at loo”, !z,~ = A,;,, at the variable tempera- ture.

TABLE III

Characterization of chromatin fractions Analysis Fraction A Fraction B - Fraction D

DNA Protein:DNA Nonhistone:histone Hyperchromicity

(40-65") Highest T,,,

2.7 x 10” 3.0 x 106 3.3 x 10” 1.5 1.7 1.4 1.0 1.3 0.6

25% 23% 6%

79-80 79-80” 83-84"

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Chromatin Fractionation 2635

also fragments of approximately 145, 130,90, and 80 base pairs (and smaller as well). The differences in digestion pattern have been attributed to the disruptive action of shearing on the nucleosomes that give rise to the 200 base pair fragments (34). A possible consequence of washing and shearing of chro- matin during the fractionation procedure is the generation of a mixed population af DNA. protein complexes. There might be DNA loosely formed in a complex with histones in non-nucleo- somal configurations, as well as intact nucleosomes. Differ- ences in structure at this level could be the basis for the

distribution of chromatin in the gradient. The results of the nuclease digestion of the chromatin frac-

tions ruled out this explanation. There were no significant differences in the patterns of nuclease-resistant DNA frag- ments of the fractions. All fractions contained 200, 145,130,90, 80, and smaller base pair fragments. The patterns were alike quantitatively and they were essentially identical with those of other investigators (24, 33).

These results indicated that the chromatin fractions did not differ at the level of the histone. DNA complexes, as assayed by the nuclease digestion, and that nucleosomes were present in all fractions. Interestingly, we observed identical patterns when swollen but unsheared chromatin was digested, indicat- ing that the washing procedure itself greatly affects the native structure of the ZJ body.

The results of these experiments suggest that the fractiona- tion is related to differences in the conformation of fibers of nucleosomes at a level above that of the individual nucleo- some.

Extension of Method to Other Chromatins -One of the fun- damental working assumptions in the fractionation of chroma- tin is that at least some elements of the in vivo structure and organization of chromatin fibers survive the process of solubil- ization, scission, and separation. One way of testing this as- sumption is to ask if chromatins known to differ from inter- phase chromatin in their in uivo structure would yield differ-

ent profiles when analyzed by the fractionation procedure. Accordingly, the chromatins from chicken erythrocytes and mitotic chromosomes were chosen for study. Clearly, if these chromatins yielded profiles similar to that from interphase chromatin it would be difficult to argue that significant struc- tural elements characteristic of the particular chromatin had survived the manipulation during the fractionation protocol.

Chicken erythrocytes were obtained from fresh rooster blood. The cells were washed and the nuclei and chromatin prepared exactly as described for the HeLa cell interphase chromatin. The chromatin was visibly swollen after the wash series, as the HeLa chromatin had been. When the chromatin was sheared (40 V for 5 min) and centrifuged on a gradient, more than 80% of the material pelletted. There was a small peak before the A region of the gradient. This was a highly reproducible observation. When the erythrocyte chromatin was sheared for 5 min at 80 V, a procedure which rendered all of the material soluble, the profile shown in Fig. 5 was ob- tained. There are two well resolved peaks, one in front of the A region of the gradient and one in the D. It will be recalled that shearing interphase chromatin at 80 V did not produce any material in the D region although it did convert all the A,,,, to A region material (Fig. 3).

Mitotic chromosomes were obtained from vinblastine- treated mouse LAS-cells and purified as described by Wray

and Stubblefield (16). The purified chromosomes were run through the standard wash procedure. The chromosomes did not swell to form a gel during the wash procedure as did the

interphase and erythrocyte chromatins. Instead, they had a granular appearance and were readily dispersed in each wash buffer. The profile of the sheared chromosomes is shown in Fig. 6. There was only a small shoulder in Region I where the A peak was observed with interphase chromatin. This was followed by a sharp Region II where the B peak runs, a broad

Region III corresponding to C, and Regions IV and V where the D peak runs. The relative amounts ofA,,j,, in each region are as follows: 15%, II 17%, III 24%, IV 25%, V 29%. The profile shown in Fig. 6 was from chromatin sheared for 6 min, but there were no significant differences in the profiles from 4 to 7 min of shearing indicating that this was the limit shear pro- file.

The difference between the mouse mitotic chromatin profile and that from HeLa cell interphase chromatin was not due to the use of cells of different species. This was demonstrated by the limit shear profile of chromatin from interphase LAS-cells shown in Fig. 7. There was a large A peak, a small B peak, and a broad D region; there was no C peak. The profile differed in both peak position and material distribution from the mi- totic chromatin profile of the same cells. The LA9 interphase profile did show the same peaks seen in the HeLa chromatin profile; however, there were substantial and highly reproduci- ble quantitative differences. The relative amounts of Ap6,, in

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FIG. 5. Profile of chicken erythrocyte chromatin. Erythrocyte chromatin was sheared at 80 V in the Omni-Mixer and centrifuged on a sucrose gradient. The positions of the chromatin peaks from the standard HeLa cell chromatin fractionation are shown.

I I I I I

FIG. 6. Profile of mitotic chromosomes from mouse L-cells. Mi- totic chromosomes were prepared from mouse L-cells as described under “Materials and Methods.” The purified chromosomes were washed and sheared as described for HeLa cell chromatin. The sheared chromosomes were centrifuged on a sucrose gradient under the standard conditions. The positions of the chromatin fractions obtained from interphase mouse L-cells (Fig. 7) are shown.

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2636 Chromatin Fractionation

OL I I I I I I I

0 4 8 12 IS 20 24

Fraction Number

FIG. 7. Fractionation of mouse L-cell chromatin. Chromatin was isolated from exponentially growing mouse L-cells and fractionated exactly as described for HeLa cell chromatin. The designation of the chromatin fractions follows the pattern established for HeLa cell chromatin.

each region of the LA9 interphase profile were A, 29%; B, 18%; C, 11%; D, 41%.

The results of these experiments with erythrocyte and mi- totic chromatin indicate that the fractionation procedure is sensitive to differences in endogenous chromatin structure and organization and these differences are reflected in the gradient profile. It is also apparent that the procedure reflects differ- ences between interphase chromatins from different cell types.

Nascent RNA -The procedure developed by McCarthy et al. (9) and employed by Murphy et al. (11) was reported to sepa- rate transcriptionally active chromatin from the inactive form. It seemed appropriate to apply to the HeLa chromatin fractions some of the criteria used to support these claims. One method of identifying a chromatin fraction as having been transcriptionally active in uiuo is the localization of nascent RNA in the chromatin fraction (11, 35, 36). The experimental design is straightforward. Cells are labeled for a short time (10 to 15 min) with radioactive uridine and then chromatin from these cells is isolated and fractionated. The radioactive RNA associated with the chromatin is considered nascent RNA, i.e. RNA that was being transcribed at the time of the chromatin isolation and is in a hybrid structure with the template DNA for at least part of its length. The presence of the labeled RNA in a particular chromatin fraction suggests that chromatin was transcriptionally active in viuo.

The HeLa cell chromatin fractions were examined for the presence of nascent RNA in the following experiment. Cells were labeled with [JHluridine for 15 min at 37”. Chromatin was isolated, run through the usual washing procedure, sheared, and centrifuged on the sucrose gradients. Fractions were collected from each gradient and the radioactivity in each fraction determined. The results are shown in Fig. 8a. The peak of the radioactivity was coincident with t,he B fraction. There was a shoulder of label in the A region and no signifi- cant radioactivity in the D region. When radioactive uridine was mixed with unlabeled chromatin and run on the gradient the label was found to sediment more slowly than the in uiuo label as shown in Fig. 8a. This result might suggest that the chromatin in the B region and perhaps in A as well, was transcriptionally active in uiuo. This result is similar to those reported by several authors (9, 11, 36).

06

7 0

2-G&

15 min I .l. Q)

Fraction Number

FIG. 8. Localization of putative nascent RNA. Exponentially growing HeLa cells were incubated with I”Hluridine for the times indicated in the figures, and the chromatin fractionated in the usual way. The absorbance profile (~1 of the chromatin and the oresence of mcorporated [“HI (. ) are shown in the figure. Also ind&ated is the position of free 13Hluridine (- - -1 when mixed with unlabeled chromatin and centrifuged on a gradient.

The location of nascent RNA in the A-B region was further tested by labeling the cells for 1 h with [3Hluridine. In this time period a considerable amount of completed RNA chains would be synthesized (37). At the time of chromatin isolation from these cells, label will be found in mature RNA species as well as nascent chains. If the RNA associated with the chro- matin is exclusively nascent RNA, then the label should ap- pear in the same position in the gradient as in the short time label experiment. The results are shown in Fig. 86. The label again appears in the A and B regions but there is more label in the A region than the B. One possible explanation for this is that the B region might really be the site of nascent RNA formation and that the label in the A region represents RNA that is not nascent but that is nonetheless chromatin-associ- ated. The level of this RNA would be expected to rise as a function of labeling time. This explanation raises the possibil- ity that the label in the B region also represents chromatin- bound RNA that is not nascent. A number of RNA species are known to be associated with chromatin (381, but are not pres- ent as RNA-DNA hybrids (39). The obvious control experi- ment is to mix labeled RNA with unlabeled chromatin and determine the position of the label in the gradient after cen- trifugation of the mixture.

Accordingly chromatin was isolated from cells which had been labeled with c”Hluridine for 10 min and from cells labeled for 60 min. The chromatins were washed and sheared and then the RNA was extracted from each preparation with phenol. These RNA samples were mixed with unlabeled, sheared chro- matin, and centrifuged on the gradients. For comparison an aliquot of each RNA sample was run on a gradient in the absence of unlabeled chromatin. The results are shown in Fig. 9. Both RNA samples when run alone sediment less rapidly

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Chromatin Fractionation 2637

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FIG. 9. Fractionation of unlabeled chromatin mixed with the [“Hluridine-labeled RNA. HeLa cells were labeled with [:‘Hluridine for the indicated times. Chromatin was prepared and sheared and the RNA extracted. The labeled RNA was mixed with unlabeled, sheared chromatin and the mixtures centrifuged on sucrose gra- dients (b, d). The positions of the RNAs isolated from chromatin after lo-min and 60-min incubations when run alone in the sucrose gradients are shown in a and c. The positions of the peaks (- - -) of the carrier chromatin (measured by absorbance at 260 nm) are shown in b and d, superimposed on the radioactive profile (---).

than the A position in the gradient. When mixed with unla- beled chromatin the label sediments in the A and B regions in both the lo-min and 60-min gradients.

RNA Polymerase Activity -A more direct way to analyze the relationship of the chromatin fractions to the site of the in vivo transcriptional activity would be to assay the fractions for RNA polymerase activity. Such an experiment has been re- ported by Doenecke and McCarthy (40). Accordingly, chroma- tin was prepared as described except that 1 InM dithiothreitol was added to all buffers. Nuclei and chromatin were assa%yed for RNA polymerase activity. Although the nuclei displayed significant levels of oc-amanitin-sensitive RNA polymerase and some a-amanitin-insensitive activity, the activity in the sheared unfractionated chromatin was approximately 1% that of the nuclear activity. The omission of Na,EDTA in the wash buffers did not enhance the activity significantly nor did the addition of exogenous DNA. No attempt was made to distin- guish RNA polymerase III activity (sensitive to high levels of oc-amanitin) from RNA polymerase I activity (insensitive to LY- amanitin). When the chromatin was fractionated and the fractions assayed, no activity could be detected in any of the chromatin regions in the gradient. Since a single wash of the nuclei significantly reduced activity (40%), it is likely that the loss of polymerase activity from the chromatin is a conse- quence of the extensive washing. Additionally, the polymerase activity is sensitive to shearing (41).

Localization of Satellite DNA -In order to test the relation- ship between the in uivo transcriptional activity and the chro- matin fractions, the latter were analyzed for the presence of satellite DNA. Mouse satellite DNA is rarely if ever tran- scribed (42). I f the method fractionated chromatin on the basis of the in viuo transcriptional activity, then the presence of this DNA species in a chromatin fraction(s) would strongly suggest that this chromatin had not been transcriptionally active. DNA was extracted from the chromatin fractions from LA9 interphase cells. For comparison DNA from unfractionated chromatin was prepared. The DNA samples were analyzed in CsCl gradients by equilibrium centrifugation. The results are shown in Fig. 10. In all samples the main band of DNA appears as a broad peak with a well defined shoulder. The

FIG. 10. Analysis of satellite DNA in chromatin fractions from mouse L-cells. The chromatin was as isolated from mouse L-cells (see Fig. 7). The DNA from each fraction was isolated and analyzed on neutral CsCl gradients in a Spinco model E. The positions of the Micrococcus lysodeikticus marker DNA (p = 1.733 g/ml) and the mouse satellite DNA (p = 1.691) are indicated.

shoulder peak is at a density of 1.691 g/cc, the known density of mouse satellite DNA (42). The lack of clear resolution between the main band and satellite DNA is probably due to the low molecular weight of the DNA. Nevertheless it is apparent that satellite DNA is found in all fractions and in comparable amounts relative to the main band.

DISCUSSION

The chromatin fractionation procedure described in this paper yields four partially resolved fractions which appear as a series of overlapping peaks when the sheared chromatin is centrifuged on a sucrose gradient. The stability of the chroma- tin profile over a range of shearing times suggests that the chromatin fractions are discrete structures and do not arise as the result of incomplete shearing of the chromatin. The char- acteristics of the chromatin fractions indicate that the frac- tionation is most likely based on structural differences in nucleosome-containing fibers other than length of DNA. For example, interstrand or intrastrand associations could yield chromatin particles that would be different in sedimentation constant and perhaps melting temperature, but still be similar in DNA length, protein:DNA ratio, etc. The relationship of such structures to the structure of chromatin in uivo is un- clear.

To test whether the chromatin fractions represent struc- tures found in vivo, rather than artifacts of washing and shearing, the procedure was extended to erythrocyte and mi- totic chromatins. They are well known to differ substantially from interphase chromatin in degree of condensation and com- paction of structure. Like the interphase chromatin, they con- tain nucleosomes (43, 44), and therefore, the structural differ- ences between the chromatins would appear to be in the higher order organization of the fibers of nucleosomes. It seemed possible that the manipulation, shearing, and solubili- zation of the chromatin, which constitute the fractionation procedure, would destroy the higher order chromatin organi-

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2638 Chromatin Fractionation

zation above the level of the nucleosomes, and if so, the results of the fractionation of these chromatins would be expected to be quite similar. However, as the results in Figs. 1, 5, and 6 indicate, this is clearly not the case. Each chromatin yields a distinctly different profile. It seems likely then, that at least some aspects of the organization of chromatin fibers survive the fractionation procedure and that the gradient profiles reflect endogenous differences in chromatin structure.

The major interest in the field of chromatin fractionation is to relate the fractionated chromatin structures to the chroma- tin templates that were transcriptionally active in vivo. Al- though the criterion of nascent RNA localization at first ap- peared to be a reliable index of in vivo transcriptional activity, the results shown in Figs. 8 and 9 suggest that such an interpretation of the labeling pattern is subject to challenge. The data from the mixing experiment (Fig. 9) suggest that the appearance of the radioactivity in the A and B fractions may represent a capacity of those chromatins to bind RNA, rather than representing the site of in vivo transcription. Since the interaction of RNA with chromatin in vivo is likely to be more intimate than can be the case in these mixing experiments, these are conservative control experiments. The extensive washing of the chromatin does not remove the associated RNA. When labeled RNA was added to unlabeled nuclei, 10% of the label survived the chromatin isolation and wash proce- dure, and sedimented with the A and B chromatin fractions. These results, together with the observations of Monahan and Hall (381, challenge the validity of the “nascent RNA” crite- rion as a means of identifying a chromatin fraction as having been transcriptionally active in vivo. There is no way to elimi- nate the possibility that transcription in vivo took place in chromatin templates that are found in one or all chromatin fractions and that the work-up and centrifugation of the chro- matin results in the relocation of the once nascent RNA to the A and B regions. While it is not possible to rule out the presence of nascent RNA in the A-B regions, its presence cannot be convincingly demonstrated by this approach.

Although the requirements for a stable hybrid of nascent RNA and DNA template have not been ascertained for eukar- yotic chromatin, the situation has been analyzed in detail for the transcription of phage DNAs by Escherichia coli polymer- ase (45), and the transcription of superhelical viral DNA (46). The conclusion of these studies, based on the size of the RNA- DNA hybrid during transcription and the stability of RNA- DNA hybrids relative to DNA-RNA hybrids is that the pres- ence of the RNA polymerase is required to retain the nascent RNA on the DNA. If a similar situation exists in the eukar- yotic systems then the failure of our chromatin fractions to display measurable RNA polymerase activity is another dem- onstration of the difficulty of interpreting the data from the nascent RNA localization experiments. Doenecke and Mc- Carthy have prepared chromatin with measurable levels of endogenous RNA polymerase activity (40). Chromatin with endogenous RNA polymerase was fractionated and a peak of activity was found in the more slowly sedimenting chromatin, although some activity was found throughout the gradient. However, as noted by these authors, the problem of artifactual binding sites generated during the fractionation procedure renders these data inconclusive with respect to the in vivo-in vitro relationships of the fractions. The presence in the slowly sedimenting fraction of an appreciable amount of free DNA, or DNA loosely formed in a complex with protein, is indicated by the melting analysis in Fig. 4 (9). Such DNA could provide initiation sites for RNA polymerase in vitro.

A more reliable approach to the problem of the relevance of the chromatin fractions to transcription is that of the satellite DNA localization experiment. Our results show that the mouse satellite is present in comparable amounts in all frac- tions. Since the satellite DNA is not transcribed and since, quite obviously, some chromatin must be transcribed in. uivo, the results indicate that the fractionation procedure does not fractionate primarily on the basis of the in vivo transcriptional activity. Howk et al. (131, and Krieg and Wells (14), have reached a similar conclusion in their study of both transcribed and nontranscribed DNA sequences in chromatin fractions prepared as described by Murphy et al. (111, Reeck et al. (71, and Janowski et al. (47).

Many of the physical characteristics of the “active chroma- tin” fractions prepared by several groups (7-9, 11, 35, 48) are consistent with the notion that this chromatin was transcrip- tionally active in viuo, but the properties of this chromatin are also consistent with those of damaged (nicked, degraded etc.) chromatin fibers. No direct, unambiguous evidence has been obtained to support the claims for successful fractionation. Moreover, our results (particularly with satellite DNA) and those of Howk et al. (13), Lewis et al. (491, Varshavsky et al. (50), and Krieg and Wells (14), strongly suggest that these fractionation techniques are inappropriate for most studies of questions about the regulation of transcription in. vivo.

Irrespective of the distribution of any particular DNA se- quences, higher orders of chromatin structure might be too delicate to withstand the manipulations that are a part of all published fractionation procedures, and a rearrangement in one of the higher orders of chromatin structure might produce various states of aggregation, differing in sedimentation prop- erties. Lewis et al. (49) have demonstrated striking changes in the physical properties of chromatin exposed to even mild shearing. These included changes in the solubility and viscos- ity of the chromatin. They found that chromatin prepared so as to be soluble in 0.15 M buffer aggregated and precipitated when sheared. Their results suggest that the operations of the fractionation protocol may generate chromatin aggregates. Recently, Varshavsky et al. discussed the possible role of divalent cations in chromatin fractionation in light of the sedimentation of chromatin exposed to low concentrations of Mg”+ (50). They suggested that the cations might affect the association of chromatin fragments leading to aggregates with differing sedimentation properties; such aggregation might even explain differences in melting temperatures and variabil- ity of gradient profiles such as observed by us and by others. Whatever the mechanism of aggregation, it is plausible that the distribution of sheared chromatin that we have observed in sucrose gradients is an expression of different extents of aggregation. The A region chromatin, for example, might contain relatively nonaggregated chromatin fibers while the D region material would consist of chromatin aggregates of var- ious size. The amount of DNA in a given region would reflect the probability of formation of an aggregate of that S value, but the actual DNA sequence would not influence the aggrega- tion process and thus all DNA sequences would appear in all chromatin fractions. If aggregation is a major factor in deter- mining the distribution of chromatin fragments in sucrose density centrifugation, it is interesting that it is affected by endogenous factor(s) specific to cell type, mitotic stage, etc. that are not directly related to transcription. The present procedures might be useful in the identification of such fac- tors, and perhaps even particular structural features of chro- matin that are not concerned directly with gene expression.

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Chromatin Fractionation 2639

This prospect seems especially promising in the case of mitotic chromosomes and erythrocyte chromatin which may be pro- tected from physical damage by their highly condensed state.

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M M Seidman and R D Coleperhaps not to transcriptional activity.

Chromatin fractionation related to cell type and chromosome condensation but

1977, 252:2630-2639.J. Biol. Chem. 

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