Eur. J. Biochem. 194, 811 -823 (1990) 6 FEBS 1990

Factors affecting nucleosome structure in transcriptionally active chromatin Histone acetylation, nascent RNA and inhibitors of RNA synthesis Lidia C. BOFFA', Janis WALKER', Thelma A. CHEN', Richard STERNER', Maria R. MARIANI' and Vincent G. ALLFREY'

' Laboratory of Cell Biology, Rockefeller University, New York, USA Instituto Nazionale por la Ricercd sul Cancro, Genoa, Italy (Received March 21/July 5, 1990) - EJB 90 0321

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury-agarose (Hg-agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non-histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg-agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt-labile associations with SH-reactive non-histone proteins, such as the high-mobility-group proteins, HMG-1 and HMG-2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg-agarose-bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing a-globin DNA, pass through the column. The histones of both Hg-agarose-bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri- and tetra-acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N-acetyl groups is also far greater in the Hg-agarose-bound nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg-agarose column. However, the ability of Hgagarose chromatography to detect localized changes in chromatin structure is evidenced by an 11-fold increase in the Hg-agarose binding of nucleosomes containing the DNA of the butyrate-inducible alkaline phosphatase gene, compared to the Hg-agarose-bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg-agarose-bound nucleosomes released by dithiothreitol, binding of the SH-reactive nucleosomes to the Hgagarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment. The inhibition of RNA polymerase I1 by a-amanitin results in a loss of the affinity for Hg of nucleosomes containing c-myc and histone H4 gene sequences, but there is no corresponding loss of Hg affinity with nucleosomes containing 28s ribosomal DNA sequences, which are transcribed by amanitin-resistant RNA polymerase I. When RNA synthesis is inhibited by actinomycin D, which we find to intercalate preferentially into the DNA of transcriptionally active DNA sequences, there is no loss of Hg affinity of nucleosomes containing the histone H4 or c-myc DNA sequences. The results indicate that the intercalation of actinomycin D into the DNA of transcribing chromatin can lock the nucleosomal transcription complex in the SH-reactive configuration that is characteristic of the template-active state.

It is now generally accepted that the chromatin of transcriptionally active genes must undergo rapid changes in structure and composition for gene expression to take place. In recent years, substantial progress has been made to elucidate the means by which the expression of specific classes of genes is regulated in response to trans-acting factors involved in initiation and repression, and in the characterization of those proteins and their modes of action [l - 81. However, the precise mechanism by which the structural changes common to all genes are initiated and maintained still remain obscure. What Correspondence to V. G. Allfrey, Rockefeller University, 1230 York Avenue, New York, NY 10021 USA Enzymes. Micrococcal nuclease (EC 3.1.31.l); ribonuclease A (EC 3.1.27.5); proteinase K (EC 3.4.21.14); restriction enzymes, EcoR1, HindIII, BumHl (EC 3.1.21.4).

are the molecular events that facilitate progression of RNA polymerases through the nucleosomes along the length of a gene? It is clear that the nucleosomal organization of chromatin is a key factor in controlling the accessibility and topology of the DNA template. Transcriptionally active DNA sequences exist in association with nucleosomal proteins, and this assembly is now known to be a dynamic structure (9 - 161. The technique of Hg-agarose affinity chromatography developed in this laboratory not only separates the nucleosomes of active and inactive genes [14- 171; it also has been shown to reveal the timing and extent of gene expression [16, 181. The procedure fractionates the nucleosomes released during limited micrococcal nuclease digestion of isolated nuclei by chromatography on Hg-agarose columns. The modified

812 nucleosomes of transcriptionally active DNA sequences are retained bv the column, while the compactly - beaded nucleosomes of inactive genes, such as the pre-proenkephalin gene of rat liver 1141, or the a-globin gene of HeLa cells [15], are rejected. The bound nucleosomes are retained on the Hgagarose column for two reasons: (a) they become associated with thiol-containing non-histone proteins recruited to the transcribing DNA; (b) the unfolding of transcribed nucleosomes to uncover the previously shielded thiol groups of histone H3 molecules located at the center of the nucleosome core particle. These two classes of active nucleosomes can be separated by a two-stage elution procedure [17]. It has been shown that both classes of nucleosomes contain oncogenic DNA sequences being transcribed at the time, and that those sequences no longer bind to the Hg-agarose column when transcription is normally repressed [16], or when it is blocked by inhibitors of RNA polymerase 11 activity [18]. As changes in chromatin structure might be expected to be a prerequisite to gene expression, this technique provides a means whereby transcription can be studied in relation to the structure of the nucleosome, its component proteins and their postsynthetic modifications. In the present study, this procedure was employed to fractionate the nucleosomes of HeLa cells in logarithmic growth, to study the changes induced by inhibitors of RNA synthesis, and to establish that the nascent transcripts associated with the active nucleosome fractions are not involved in the Hgbinding reaction. We also explore the relationship between acetylation of the nucleosomal core histones and the binding of nucleosomes to the Hg-agarose column, and present evidence that the degree of histone hyperacetylation achieved by exposing HeLa cells to 5 mM sodium butyrate is not sufficient to cause a major change in the Hg-binding properties of HeLa nucleosomes. However, localized changes in the chromatin of butyrate-treated cells are detectable, as evidenced by the Hg binding of nucleosomes containing DNA of the butyrateinducible alkaline phosphatase gene.

Eflects of inhihitors on R N A synthesis in intact and permeabilized cells 200-mi suspensions of intact HeLa cells containing 3 7 x lo5 cells/ml in complete medium were divided into two equal volumes. Actinomycin D (Sigma) was added to one aliquot to give a final concentration of 10 pg/ml. The cells were incubated at 37°C in the dark for the indicated exposure periods before adding [5-3H]uridine (28 Ci/mmol; New England Nuclear). After 10 min, 0.5-ml aliquots of each cell suspension were precipitated with 10% trichloroacetic acid, 30 mM sodium diphosphate, and collected on nitrocellulose filters (PH79; Schleicher & Schuell). The filters were washed twice with 10% trichloroacetic acid, 30 mM sodium diphosphate and dried, and the incorporated 3H was measured by scintillation counting. The remainder of each cell suspension was employed for the isolation of nuclei and separation of the nucleosomes as described below. In studies using a-amanitin as the RNA synthesis inhibitor, the cells were permeabilized as described and suspended in buffer A at a concentration of 3 x lo7 cells/ml. Aliquots of the suspension were exposed to 20 pg/ml a-amanitin for 8 min at O'C, warmed to 37°C for 2 min, and nucleotides were added to give final concentrations of 2.5 mM ATP. 0.2 mM GTP, 0.2 mM CTP and 1.0 pM [5,6-3H]uridine triphosphate (36.9 Ci/mmol; New England Nuclear). Incubation was continued at 37 "C for 10 min, at which time the cells were precipitated with 10% trichloroacetic acid, 30 mM sodium diphosphate, collected on nitrocellulose filters and washed prior to measurement of 3H. For studies comparing the effect of amanitin on RNA synthesis and nucleosome structure, the permeabilized cells were resuspended in buffer B [15 mM Tris/ HCI (pH 7.4), 25 mM NaC1, 25 mM KCl, 5 mM sodium butyrate, 5 mM or 1 mM MgClz and 0.35 M sucrose], containing 0.1 mM 1,2-epoxy-3-(4-nitrophenoxy)propaneand 0.1 mM phenylmethylsulfonyl fluoride as protease inhibitors, and exposed to 20 pgiml a-amanitin for 10, 20 or 30 min prior to isolation of the nuclei and preparation of nucleosomes for Hgagarose affinity chromatography.

MATERIALS AND METHODS

Isolation of' nuclei and preparation of nucleosomes

Cell culture rrnd pprmeahili~ation

All procedures were carried out at 4 C unless otherwise specified. Cells were harvested by centrifugation at 600 x g for 15 min, washed extensively with 0.14 M NaCl, 5 mM sodium butyrate, 0.1 mM 1,2-epoxy-3-(4-nitrophenoxy)propane, 0.1 mM phenylmethylsulfonyl fluoride, and resuspended at lo7 cells/ml in 80 mM NaCl, 5 mM sodium butyrate, 0.1 mM 1,2-epoxy-3-(4-nitrophenoxy)propane, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.2. After 10 min Nonidet P-40 was added to give a final concentration of 0.5% and the cells were broken by shearing in a Dounce-type glass homogenizer with a tight-fitting pestle (type B, Kontes, Inc., Vineland NJ). The homogenate was centrifuged at 2000 x g for 10 min and the nuclear pellet washed extensively with buffer B. The nuclei were suspended in buffer B at a concentration of 1 mg DNA/ ml, Micrococcal nuclease (Boehringer, Mannheim) was added at 10 U/ml and digestion was started by the addition of CaCl, to 0.5 mM. After 5 min at 3 7 T , digestion was stopped by adding EGTA to give a final concentration of 5 mM and rapid cooling to 0°C. The suspension was centrifuged at 10000 x g for 20 min and the supernatant (S fraction) containing the released nucleosomes was analyzed for DNA content and employed for chromatographic fractionation of the nucleosomes.

HeLa S3 cells were maintained in suspension culture at a density of 3 - 7 x 10s cells/ml in Joklik-modified minimal essential medium supplemented with antibiotics, 2 mM glutamine and 5 % calf serum (Gibco, Grand Island, New York). To test the effects of sodium butyrate on nucleosomal structure and histone acetylation, cells were cultured for 15 h in the above medium containing 5 mM sodium butyrate. In order to study changes in nucleosome structure following inhibition of RNA polymerase I1 by a-amanitin, the cells were first permeabilized to the inhibitor, modifying the method of Miller et al. [19]. Cells were collected by centrifugation at 600 x g for 15 min, washed twice in 150 mM sucrose, 80 mM KCl, 35 mM Hepes (pH 7.4), 5 mM MgCl,, 0.5 mM CaCI, and 5 mM sodium butyrate (buffer A), then resuspended in buffer A at 10' cells/ml. A one-third volume of lysolecithin solution at I mg/ml (Sigma, Type I) was added, and the cells maintained for 1 min at 0 'C. The process was stopped by the addition of 5 vol. icecold buffer A, followed by centrifugation. At this point, over 95'10 of the cells were permeable to a 0.4% solution of Trypan blue.

813 Ajfinity chromatography of nucleosomes

Transcriptionally active and inactive nucleosome fractions were separated by Hg-agarose affinity chromatography, using a modification [I71 of the method described previously [14161. Briefly, the S fraction was adjusted to 5 mM EDTA and applied to a column of Affi-Gel 501 (Bio-Rad, Richmond, CA) which had been washed with buffer C [lo mM Tris/HCl (pH 7.5),25 mM KC1,25 mM NaCl, 5 mM sodium butyrate, 5 mM EDTA, 0.1 mM 1,2-epoxy-3-(4-nitrophenoxy)propane and 0.1 mM phenylmethylsulfonyl fluoride]. Elution with buffer C to remove the unbound nucleosomes preceded a twostage fractionation of the Hg-bound nucleosomes. A subset of the bound nucleosomes was eluted with buffer C containing 0.5 M NaCl [17]. The remaining nucleosomes were then displaced from the column in buffer C containing 10 mM dithiothreitol (Bio-Rad) in addition to 0.5 M NaCl. Elution was monitored by measuring the DNA content of each fraction by the Hoechst dye-fluorescence assay [20] and by its absorbance at 260 nm. In several experiments, nucleosomes were further purified, either before or after Hg-agarose affinity chromatography, by gel-filtration chromatography on 1 cm x 90 cm columns of Sephacryl S-200 (Pharmacia) in order to remove smaller chromatin fragments, adventitious proteins and unassociated RNA fragments [14]. The columns were equilibrated and eluted with buffer C. To test whether proteins associated with nascent RNA chains participate in the binding of active nucleosomes to the Hg-agarose column, the RNA was first 3H labeled by incubating 100 ml of a HeLa cell suspensions containing lo7 cells/ml with 2.5 mCi [5-3H]uridine (30 Ci/mmol) for 30 min at 37°C. The nuclei were isolated and digested with micrococcal nuclease to release 7% of their DNA into the S fraction, which was then divided into two equal aliquots. One was treated with 50 pg/ml ribonuclease A (Sigma) for 45 min at 4"C, while the other served as a control. Both were chromatographed on parallel columns of Sephacryl S-200 and Affigel-501, and nucleosomes were separated as described measuring the recovery of DNA and [3H]RNA radioactivity of each fraction. ( 3 H1Actinomycin

D binding to nucleosome fractions

A log-phase HeLa cell suspension (900 ml) containing 5 x 10' cells/ml was pelleted by centrifugation and resuspended in 150 ml fresh complete medium containing 0.1 pM [3H]actinomycin D (4 Ci/mmol; Amersham). After 20 min at 37"C, the cells were harvested and washed. Nuclei were isolated and the nucleosomes were fractionated by Hg-agarose affinity chromatography, as described [14]. Free actinomycin D was removed by chromatography on Sephacryl S-200, and aliquots of the unbound and Hg-bound nucleosomes were analyzed for DNA content and [3H]actinomycin D radioactivity, as determined by scintillation counting. D N A purification and sizing

DNA was prepared from the unbound and Hg-bound nucleosome fractions as described by Maniatis et al. [21]. Following phenol/chloroform extraction, the DNA in the aqueous phase was precipitated in ethanol, and resuspended in 10 mM Tris/HCl, pH 8.0, 1 mM EDTA. RNA and protein contaminants in the samples were digested with 50 pg/ml RNase A for 1 h at 3 7 T , followed by 100 pg/ml proteinase

K for 2 h at 37°C in the presence of 0.1% SDS. The DNA was extracted, precipitated in ethanol [21], and dissolved in 10 mM Tris/HCl (pH 7.4), 1 mM Na EDTA for hybridization experiments. DNA concentrations were determined by the Hoechst assay [20]. DNA sizes of the nucleosome fractions were determined by electrophoresis in polyacrylamide gels, as described by Maniatis et al. [22], using $X 174 DNA restriction fragments as standards. D N A hybridization procedures

Nucleosomdl DNA (2 pg) from each sample was applied to a nylon membrane (Nytran) in a slot-blot apparatus (Schleicher & Schuell). Membrane-bound DNA were hybridized to random-primed 32P-labeled probes for rDNA, histone H4, c-myc and alkaline phosphatase DNA using the Southern procedure, as described in [21]. The ribosomal DNA probe was the plasmid 1-19 [23] containing 4.8 kb mouse genomic DNA, including the gene for 28s rRNA (kindly supplied by Dr. Edward M. Johnson of the Mount Sinai School of Medicine). The probe for histone H4 was a 710-bp DNA fragment from plasmid pHU4A, containing a human H4 gene and its flanking sequences [24]. The probe for histone H2A was an 866-bp DNA fragment containing the entire human H2A gene and several hundred base pairs of 3'- and 5'-flanking sequences [24]. As a control for a gene not expressed in HeLa cells, we employed a 0.3 - 0.4 kb fragment of human a-globin cDNA cloned in PMB9 (JW101) [25]. The probe for c-myc was a 9-kb EcoRl - Hind111 fragment in plasmid pHSR-1 which encodes the full human c-myc gene comprising three exons and two introns [26]. The probe for the alkaline phosphatase gene was a 0.7-kb EcoRl - BamHl fragment of the human gene in plasmid pUC18 [27, 281. The filters were washed [21], dried and exposed to Kodak X-OMat AR5 film for 48 h with a Dupont Cronex I-G Plus intensifying screen at -70°C. After development in Kodak X-OMat M4 developer, the autoradiograms were scanned with a laser densitometer (LKB Ultroscan 2202), and peak areas corresponding to each slot blot were integrated. Protein analyses

The total protein content of each nucleosome fraction was determined by the method of Ohnishi and Barr [29]. Protein complements of the run-through and Hg-bound nucleosome fractions were compared by electrophoresis in 15% or 414% polyacrylamine gels containing 0.1% SDS [30]. For comparison of histone acetylation in the nucleosome fractions, each fraction was dialyzed successively against 5 mM sodium butyrate, 0.2 mM sodium butyrate and 0.1% acetic acid, then lyophilized. Each fraction was extracted in 5 % (massjvol.) guanidinium chloride, 0.1 M potassium phosphate buffer (pH 6.8), and the histones were purified by ion-exchange chromatography on Bio-Rex 70, as described in [14]. Electrophoretic separations of the acetylated forms of the histones were performed in gels containing 15% polyacrylamide, 5.5% (by vol.) acetic acid, 8 M urea, 0.2% Triton X100, as described in [14, 311. The gels were stained with 0.25% Coomassie brilliant blue R250, or with silver [32], prior to quantitation of the acetylated forms by laser-scanning densitometry. (3H]Acetyl uptake and turnover in nucleosomal histones

HeLa S3 cells were suspended in 80 ml growth medium at a concentration of 4 x lo7 cells/ml. Sodium [2-3H]acetate

814 ( 5 mCi; 3.6 Ci/mmol; New England Nuclear) was added and the cells were incubated at 37°C for 20 min. After centrifugation, the cells were resuspended in 1.5 l fresh medium. A 500-ml aliquot was adjusted immediately to 5 mM sodium butyrate to block histone deacetylase activities during subsequent steps in the preparation of the nucleosome fractions. Nuclei were isolated and digested with micrococcal nuclease to release 7.8% of their total DNA into the S fraction which was then used to separate the nucleosomes by Hg-agarose affinity chromatography, as described above. A second 500ml aliquot was incubated in non-radioactive medium in the absence of sodium butyrate for 15 min at 37 "C to allow turnover of the incorporated histone acetyl groups. At that time, sodium butyrate was added to 5 mM, nuclei were digested to release 8.1% of the DNA and the nucleosome fractions were prepared. The third 500-ml aliquot was incubated for 30 min at 37 "C, before addition of sodium butyrdte. The nuclei were isolated and digested to release 8.0% of the DNA and the nucleosome fractions were prepared as described. To each fraction was added 0.1 vol. 3 M sodium acetate, pH 4, and 2.5 vol. ethanol. After storage at -20°C for 12 h, the samples were centrifuged at 20000 x g for 30 min and the pellets were lyophilized. Histones were extracted in 0.25 M HC1 and precipitated in acetone. The pellets were redissolved in 1% SDS and 3H radioactivity determined by scintillation counting. RESULTS AND DISCUSSION Separation qf t w o clusses of nucleosomes containing reactiw thiol groups by Hg-ugarose gffi'nitji chrornutogruphy

The chromatographic separation of transcriptionally active nucleosomes has its origins in two earlier observations: (a) the finding that the nucleosomes of ribosomal genes 'unfold' during transcription to reveal the previously shielded SH groups of histone H3, and that this reactivity of the H3 thiols is lost when rDNA transcription ceases [9]; (b) that histone H3 can be readily separated from other histones by chromatography on Hg-agarose columns [33]. A similar unfolding of nucleosomes during the transcription of other genes is indicated by H3 thiol reactivity in the non-nucleolar chromatin of Physuvuni [34], as well as in the active chromatin of mammalian [15. 171 and avian cells [35]. The accessibility of H3 thiol groups in transcribing nucleosomes, as contrasted to their non-reactivity in nontranscribing nucleosomes, suggested that the nucleosomes of active genes co~ild be isolated by Hg-agarose affinity chromatography. A procedure for the separation of transcriptionally active and inactive nucleosomes has been described [14]. It is based on the selective retention of the thiol-reactive nucleosomes by organomercury-agarose columns when mixtures of active and inactive nucleosomes are passed through the column. In the original method [14], nucleosomes released from isolated nuclei during a limited digestion with micrococcal nuclease were applied to the Hg-agarose column, which retained the nucleosomes containing the transcribed DNA sequences of the cell type examined, while the compactly beaded nucleosomes of transcriptionally inert genes passed through the column. The retained nucleosomes were then eluted in one step by displacement with 10 mM dithiothreitol. By using DNA probes for genes known to be transcribed in the liver, such as the albumin and transferrin genes, it was shown that those DNA sequences were recovered in the Hgbound nucleosome fraction, while the nucleosomes of a gene

not transcribed in the liver, such as pre-proenkephalin, did not bind to the column [14]. This one-step elution technique for separating active (Hg-bound) and inactive (unbound) nucleosomes was used to demonstrate that nucleosomes along the c-fos and c-myc genes assumed an altered, SH-reactive structure during transcription, but quickly reverted to a compact non-SH-reactive configuration when transcription of each gene was terminated [16]. More recent studies have revealed that the retention of transcriptionally active nucleosomes by the Hg-agarose column involves two separable modes of binding [17]. The procedure has been modified accordingly to elute the active nucleosomes in two stages. The first uses 0.5 M NaCl to release nucleosomes retained on the column because of salt-labile associations with SH-reactive non-histone high-mobilitygroup proteins (such as HMG-1 and HMG-2) located in the active chromatin. The second stage employs 1 0 m M dithiothreitol to displace nucleosomes in which a conformational change in the nucleosome core has made the thiol groups of histone H3 accessible to SH reagents [I 71. Here we apply the new method to recover two classes of transcriptionally active nucleosomes from HeLa cells and to explore changes in nucleosome structure when transcription is blocked by a-amanitin or actinomycin D. Evidence is also presented that transcriptionally active nucleosomes differ from inactive nucleosomes in their extent of histone acetylation and in the turnover of the N-acetyl groups. In addition, we show that ribonucleoproteins associated with the transcription complex are not responsible for the Hg binding of the active nucleosomes. Finally, the ability of Hg-agarose chromatography to detect localized differences in HeLa chromatin structure is demonstrated by the easy detection of the activation of a specific gene (alkaline phosphatdse). The first step in the procedure is to degrade the chroindtin of isolated cell nuclei by limited endonuclease digestion. HeLa nuclei were treated with micrococcal nuclease to release 6 10% of the total DNA, and the released nucleosomes were applied to an Hg-agarose column. After washing the column to remove unbound nucleosomes, the Hg-bound chromatin subunits were eluted successively with buffers containing 0.5 M NaCl and 10 mM dithiothreitol, as shown in Fig. 1 A. Under these experimental conditions, about 12.0% of the applied nucleosomal DNA is retained by the Hg-agarose column (average of 14 experiments). Salt-labile associations with thiol-reactive non-histone proteins are responsible for the retention of about 60% of the total Hg-bound nucleosomal DNA (Table 1). All of the remaining Hg-bound nucleosomes are released from the column when 10 mM dithiothreitol is added to the eluting buffer (Fig. 1A). If the S fraction is adjusted to 0.5 M NaCl before application to the Hg-agarose column, there is no appreciable fraction of the Hg-bound nucleosomes which elutes in 0.5 M NaCI, due to the loss of non-histone proteins involved in the binding reaction; under these conditions, all of the Hg-bound nucleosomes are released in 10 mM dithiothreitol (data not shown). The relative proportions of the salt-labile and dithiothreitol-eluted nucleosome fractions vary depending upon the extent of endonuclease digestion. The more extensive the digestion, the lower the proportion of dithiothreitol-eluted nucleosomes. For example, the proportion of the dithiothreitol-eluted fraction drops from 60% of the total Hgbound nucleosomes (when 8% of the DNA is released by endonuclease digestion) to 40% (at 12% digestion) and to 20% (after 16% of the DNA is digested). The greater susceptibility to endonuclease attack of the unfolded nucleosomes

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Fig. 1. Fractionation of HeLa cell nucleosomes by Hg-agarose uffinity chromatography. Nucleosomes released by a limited micrococcal nuclease digestion of isolated nuclei were prepared from control cells (A), and from cells after treatment with a-amanitin (C), actinomycin D (D) and sodium butyrate (F). In some experiments the S fraction containing the released nucleosomes was subjected to gel-filtration chromatography on Sephacryl S-200 (B), or treated with ribonuclease A (E) prior to chromatography on Hg-agarose columns. After elution of the unbound nucleosome fraction (peak I), the Hg-bound nucleosomes were eluted in two stages, using 0.5 M NaCl (0.5) to release nucleosomes bound through salt-labile linkages to non-histone proteins containing reactive SH groups (peak 2), and 10 mM dithiothreitol (DTT) to release nucleosomes with reactive histone H3 thiols (peak 3). DNA distributions in the eluates were measured by absorbance at 260 nm and by the Hoechst DNA assay [20]

cated close to the nucleosome core. It will be shown that such nucleosomes contain transcriptionally active DNA sequences (Fig. 6). Under the conditions of endonuclease digestion which we have employed to study gene distribution in the various nucleosome fractions, over 90% of the DNA occurs in monomeric nucleosome lengths, and few dimers, trimers or oligomers are present in the nucleosome fractions (Fig. 2). This uniformity of DNA lengths in the different fractions avoids complications due to DNA length variability in measuring the DNA sequence content of each fraction by Structural differences in chromatographicully quantitative slot-blot hybridizations. sepurated nucleosome fractions Histone stoichiometry in active and inactive nucleosome D N A size differences. DNA prepared from the unbound fractions. The histone composition of the nucleosomes in the fraction and each of the Hg-bound nucleosome fractions were unbound, 0.5 M NaC1-eluted and 10 mM dithiothreitol-eluted sized electrophoretically [22]. Densitometric analyses of the fractions was analyzed by densitometry of the stained histone DNA-banding patterns show that monomeric nucleosomes bands separated by SDS/polyacrylamide gel electrophoresis. predominate in all fractions (Table 1). However, in limited Typical histone patterns are evident in all nucleosome fracdigestions, there is a significant difference in the amount of tions (Fig. 3A). The proportions of the individual histones, DNA present in the monomers of the different fractions. The relative to the total histone content of the nucleosome fracaverage DNA length of the monomers eluted in 0.5 M NaCl tions, were as follows. Unbound nucleosomes: H2A, 22.3% ; (170 bp) is about 15 bp longer than of the unbound H2B, 23.8% ; H3, 25.5% ; H4, 25.1%. 0.5-M-NaC1-eluted nucleosomes or of those eluted in 10 mM dithiothreitol (Table nucleosomes: H2A, 22.8%; H2B, 24.1%; H3, 25.2%; H4, 1). The longer DNA lengths indicate the presence of ‘linker’ 24.8%. Dithiothreitol-eluted nucleosomes: H2A, 23.9% ; DNA sequences in those nucleosomes that are retained on the H2B, 25.1%; H3, 24.8%; H4, 23.9%. The occurrence of all four nucleosomal core histones, Hg-agarose column because of salt-labile associations with non-histone proteins containing reactive SH groups. If H2A, H2B, H3 and H4, in stoichiometric proportions in each endonuclease digestion is more effective, the linkers are of the fractions, together with the DNA-sizing results, contrimmed and the difference in DNA length between the vari- firms the presence of intact nucleosomes in both the active ous nucleosome fractions is no longer apparent (as in Fig. 2). and inactive DNA sequences of the HeLa cell. This agrees Since such trimming of the DNA linkers does not reduce the with earlier observations on the stoichiometry of the core proportion of Hg-bound nucleosomes elutable in 0.5 M NaCI, histones in nucleosomes derived from the transcribing dowe conclude that the thiol-reactive protein(s) responsible for mains of the ribosomal genes of Physavum [34]. Such evidence the Hg binding of the salt-labile nucleosome fraction are lo- for histone stoichiometry argues against the view that tranin the dithiothreitol-eluted fraction is in accord with earlier observations of the rapid and preferential degradation by micrococcal nuclease of the SH-reactive nucleosomes of ribosomal genes [9, 361. Therefore, to minimize variability in the following experiments involving comparisons of nucleosome structures in cells exposed to inhibitors of RNA synthesis, we have selected conditions that provide a reproducible release of DNA from all nuclear preparations tested.

816 Table 1 . Proportiom and monomeric D N A lengths of nucleosome,fructions separated by two-stage Hg-agarose alfinity chrornatogruplzy Nucleosomes released during a limited micrococcal nuclease digestion of HeLa nuclei were fractionated by Hg-agdrose affinity chromatography to yield an unbound fraction (peak 1) and two Hg-bound fractions which were eluted successively in 0.5 M NaCl (peak 2) and 10 mM dithiothreitol (peak 3). The DNA of each fraction was isolated and sized electrophoretically, and the proportions of the monomeric, dimeric, trimeric and oligomeric nucleosomes were determined by laser-scanning densitometry of the ethidium-bromide-stained DNA bands. Distribution of DNA is presented as a proportion of total DNA applied to an Hg-agarose column, recovered in each peak. Nucleosomes are presented as a proportion of peak DNA present as monomeric, dimeric, trimeric and oligomeric nucleosomes ~~~~~

Nucleosome fraction pedk

~~~~~~~

Distribution of DNA

~

~

Nucleosomes present as monomers

1 2 3

% total

%

88 7 5

75 37 93

dimers

Monomeric DNA lengths trimers

oligomers bP

15 33 6

3 30 1

7 trace -

155 170

155

histones of the Hg-bound nucleosome fractions differ from the histones of unbound nucleosomes in their post-translational modifications. The acetylation of 1-4 lysine residues in the amino-terminal domains of histones H3 and H4 results in a stepwise reduction in positive charge which permits electrophoretic separation and densitometric quantitation of each modified form, as shown for histone H4 in Fig. 3 B. The proportions of the various acetylated isoforms of histone H4 are shown for the unbound, 0.5-M-NaC1-eluted and dithiothreitol-eluted nucleosomes in Table 2. The two Hg-bound nucleosome fractions of HeLa cells grown in the absence of the deacetylase inhibitor, sodium butyrate, show considerable enrichment in their content of the most acetylated isoforms of histone H4, compared to the unbound nucleosome fraction. In the dithiothreitol-eluted fraction, over 32% of the H4 molecules occur in their tri- and tetra-acetylated forms; this is considerably higher than the total tri- and tetra-acetylated H4 content of nucleosomes eluted in 0.5 M NaCl (12.5%), and the 7.6% observed in the unbound nucleosomes (Table 2). A similar enrichment of the hyperacetylated forms of histone H3 was also detected in the Hg-bound nucleosome fractions (data not shown). Given the evidence that both of the Hg-bound fractions contain the transcribed DNA sequences of HeLa cells (Fig. 6), these results place the hyperacetylated forms of histone H4 in 4 5 6 both classes of transcriptionally active chromatin subunits. 1 2 3 Fig. 2. D N A sizing ofnuc.leosotne,fractions. DNA was prepared from This confirms earlier observations on the high levels of histone each of the HeLa cell nucleosome fractions and sized electrophore- acetylation in the total Hg-bound nucleosome fractions of tically. The positions of the DNA bands, as revealed by staining with liver [14] and HeLa cells [15]. The link between hyperacetylaethidium bromide, are shown: lane 1, DNA of total nucleosomes tion of the core histones and transcription is further released by a limited micrococcal nuclease digestion of HeLa nuclei; strengthened by the observation that nucleosomes containing lanes 2 and 3, DNA of nucleosomes that did not bind to the Hg- the transcribed a-D-globin DNA sequences of avian red cells agarose column; lane 4, DNA of nucleosomes eluted in 0.5 M NaCl; are immunoprecipitated by antibodies specific for hyperlane 5 , DNA of nucleosomes eluted in 10 mM dithiothreitol; lane 6, acetylated histone H4 [37]. All these findings are consistent Hue111 restriction fragments of 4x174 DNA employed as size with the hypothesis that acetylation of the histones facilitates markers. Note that monomeric nucleosomes predominate in all fractions when endonuclease digestion is optimal. These nucleosomes transcription by increasing DNA accessibility to RNA polymerases [38] and with the finding that hyperacetylation of the were used Tor the DNA-hybridization studies shown in Fig. 6 histones increases the DNase I sensitivity of HeLa chromatin P91. scription is accompanied by a selective loss of any histones Histone ucetyl-group t u r n o w in uctive and inactive from the nudeosome core. In agreement with earlier findings nucleosomes. In order to compare the turnover of histone [14,15], the Hg-bound nucleosomes do not contain significant acetyl groups in the unbound and Hg-bound nucleosome fracamounts of histone H1. tions, HeLa cells were incubated in the presence of 3H-labeled Histone hypperuwt,vlution in the Hg-hound nucleosome.frac- acetate for 20 min, then placed in non-radioactive medium. tions. Although all of the nucleosome fractions contain stoi- Aliquots of the cell suspension were withdrawn after 0,15 and chiometric amounts of histones H2A, H2B, H3 and H4, the 30 min for preparation of the nuclei and chromatographic

817 A B Butyrate

Control

t

a:

1

2

3

4

5

Direction of migrotion

Fig. 3. Histones, histone H4 isoforms and non-histone proteins of nucleosome fractions. (A) Each of the nucleosome fractions was analyzed for its protein composition by electrophoresis in SDS/polyacrylamide gels. The positions of the protein bands, as revealed by staining with Coomassie brilliant blue R-250, are shown. Lane 1, proteins of the total nucleosome fraction released by a limited micrococcal nuclease digestion of HeLa nuclei; lane 2, proteins of the total nucleosome fraction after gel-filtration chromatography on Sephacryl S-200; lane 3, proteins of the unbound nucleosome fraction; lane 4, proteins of nucleosome fraction eluted in 0.5 M NaC1; lane 5, proteins of nucleosome fraction eluted in 10 mM dithiothreitol. Note the presence of the core histones, H2A, H2B, H3 and H4 in all fractions. These are not removed by Sephacryl S-200 chromatography, unlike many of the non-histone proteins (compare lanes 1 and 2, and the corresponding densitometric tracings in Fig. 5). (B) Electrophoretic separation of histone H4 isoforms containing 0, 1, 2, 3 and 4 W-acetyllysine groups. Densitometric tracings of the histone H4 bands of control (A) and butyrate-treated cells (B) are compared. In both cases, the patterns are shown for, in descending order, histone H4 of total nuclei; histone H4 of unbound nucleosomes (peak 1); histone H4 of nucleosomes eluted in 0.5 M NaCl (peak 2); and histone H4 of nucleosomes eluted in 10 mM dithiothreitol (peak 3). The proportions of the various acetylated forms are given in Table 2

Table 2. Histone H4 acetylation levels in nucleosome fractions separated by two-stage Hg-agarose affinity chromatography Histones were prepared from the whole nuclei and from the unbound and Hg-bound nucleosome fractions of HeLa cells cultured in the presence and absence of 5 mM sodium butyrate. The acetylated forms of the histones were separated by electrophoresis on acid/urea/Triton/ polyacrylamide gels and stained with Coomassie blue. The proportions of the histone H4 isoforms were determined by laser-scanning densitometry of the five H4 bands (see Fig. 3 B) Conditions of experiment

Source of histone H4

N-Acetyl content of histone 0

1

2

3

4

%

Control cells

nuclei peak 1 peak 2 peak 3

40.0 f 2.0 33.3 f 1.4 21.3 f 0.9 30.2 f 0.6

28.7 f 0.7 41.5 f 2.1 40.9 f 0.4 17.1 f 0.2

15.1 f 1.4 11.7 0.05 19.2 f 1.3 19.8 f 0.3

9.0 f 1.0 5.6 f 0.1 9.8 0.4 19.2 f 0.4

6.8 i:0.7 2.0 +_ 0.1 2.7 f 0.7 13.2 f 0.2

Butyrate-treated cells

nuclei peak 1 peak 2 peak 3

2.6 f 0.3 5.3 f 0.3 1.2 f 0.1 1.4 f 0.2

32.3 f 0.8 23.5 f 0.5 20.8 f 1.4 5.3 0.4

25.5 f 0.5 28.4 f 0.2 29.4 f 0.3 16.1 f 2.5

27.5 f 0.8 25.9 f 1.3 29.3 0.5 53.4 f 1.4

11.5 f 1.8 14.7 & 0.7 19.3 f 0.5 23.2 f 1.4

separation of the nucleosome fractions. Analyses of the 3H radioactivity of their histone complements (Fig. 4) show striking differences, not only in the initial incorporation of [3H]acetateinto the Hg-bound and unbound nucleosome frac-

tions, but also in the rapidity of acetyl group turnover. Since the Hg-bound nucleosomes are known to contain the transcriptionally active DNA sequences, it is clear that transcription is accompanied by dynamic modifications of histone N-

818 500,

0 A0

A 0.5 DTT

1'0 1'5 20 30 Time in13H]-acetate-free medium (min)

Fig. 4. Hi.~toiic-ac.(,t.~Igroul) turnover in nuc.leosome,fructionsseparated hy Hg-ugarosc, &nit), chroinatogruphy. HeLa cells were incubated for 20 min iii the presence of ['Hlacetate to label the N'-acetyllysine residues of thc nucleosomal core histones. After washing to remove the radioactive acetate, the cells were incubaled in nonradioactive medium for 0. 15. and 30 min before isolation of the nuclei and chromatographic separation of the nucleosomes. The histones were extracted from each fraction and their 'H-radioactivities determined by scintillation spectrometry: ( 0 )unbound nucleosomes (RO); ( A ) nucleosomes eluted in 0.5 M NaCl (0.5); ( 0 )nucleosomes eluted in 10 mM dithiothreitol (DTT).Note that the highest initial radioactivity occurs in the dithiothreitol-eluted nucleosomes, which also show a very high rate of acetyl group turnover during the unlabelled chase

acetyl content that involve both the rates of uptake and turnover of the acetyl groups. Butjr(ite-iniJucc(1 histone hyperacetylation in nucleosome ,fractions. Hyperacetylation of the nucleosomal core histones can be induced experimentally by exposing cells to 5 m M sodium butyrate [40]. This effect has been shown to result from inhibition by- butyrate of histone deacetylase activity without blocking the acetyltransferase activities of the nucleus [39, 41 -431. We have examined the distribution of the hyperacetylated forms of histone H4 in the Hg-bound and unbound nucleosome fractions of cells exposed to 5 mM sodium butyrate. The results (Fig. 3 B ; Table 2) again show that the highest levels of H4 acetylation occur in the Hg-bound nucleosoine fractions, but the amounts of tri- and tetra-acetylated H4 in the unbound nucleosorne fraction are significantly higher than those of the corresponding nucleosomes from untreated cells (Table 2). Despite the overall 2.5-fold increase in tri- and tetraacetylated histone H4 content of the nuclei from butyratetreated cells, there is no appreciable increase in the proportion of nucleosomes binding to the Hg-agarose column, nor is there any obvious change in thc relative proportions of the two Hgbound nucleosome fractions (Fig. 1 F). This is not due to a lack of sensitivity of the procedure, for it will be shown below that it can easily- detect the increase in the Hg binding of nucleosomes derived from a DNA sequence (the alkaline

phosphatase gene) which is activated in butyrate-treated HeLa cells [44]. It should also be emphasized that the absence of a direct correlation between levels of histone acetylation and Hg binding of nucleosomes from cells exposed to sodium butyrate should not be taken as proof that acetylation of the core histones is not required for the nucleosome structural changes that precede or accompany transcription, because butyrate treatment also has profound effects on other modifications of histone primary structure, including histone phosphorylation and methylation [45]. Nonhistone proteins of the Hg-hound nucleosome jkactions. Many non-histone proteins are released from HeLa nuclei during limited digestions with micrococcal nuclease. Many of those proteins contain reactive thiol groups and bind to the Hg-agarose column independently of the nucleosomes. Moreover, they will be eluted together with the nucleosomes in 10 mM dithiothreitol. To minimize this adventitious contamination, the nuclear S fractions are first chromatographed on columns of Sephacryl S-200, which passes the nucleosomes but retards proteins of molecular mass below 200 kDa. This type of chromatography does not alter the subsequent behavior of the nucleosomes during chromatography on the Hg-agarose column (Fig. lB), nor does it reduce the total yield of Hg-bound nucleosomes or alter the proportions of the 0.5-M-NaCl-eluted and dithiothreitol-eluted nucleosome fractions (Table 3). Comparisons of the electrophoretic patterns of the proteins of the nuclear S fraction before and after Sephacryl S200 chromatography show that the procedure removes many components of the mixture (Figs 3A and 5); but many nonhistone proteins of molecular mass below 200 kDa emerge with the chromatin subunits. A number of these proteins have been identified and will be described elsewhere; some are associated with nascent RNA chains. It has been shown that the Hg-bound nucleosome fraction of HeLa cells contains nascent RNA fragments which remain with the nucleosoines during subsequent centrifugation in sucrose gradients [15]. To determine whether RNA-binding proteins on nascent transcripts play a role in the retention of transcriptionally active nucleosomes on the Hg-agarose column, comparisons were made of nucleosome fractionations in control and ribonuclease-treated S fractions. Following chromatography on Sephacryl S-200 to remove nucleotides and RNA fragments, the S fractions were applied to parallel Hg-agarose columns and the Hg-bound nucleosomes were eluted in 0.5 M NaCl and 10 mM dithiothreitol. The elution diagram of the RNase-treated nucleosomes (Fig. 1 E) did not differ from that of the untreated nucleosomes (Fig. 1A), nor was there any change in the yields or proportions of the two HE-bound nucleosome fractions (Table 3). Since RNase treatment had removed more than 90% of the nascent RNA transcripts from the dithiothreitol-eluted nucleosome fraction (Table 3), it is highly unlikely that thiol-reactive RNA-binding proteins participate in the Hg-binding of active nucleosomes. Transcrihed D N A sequences in thc Hg-hound nucleosome ,fractions. We have shown that nucleosomes retained by the Hg-agarose column and eluted in one step by dithiothreitol contain the transcriptionally active DNA sequences of rat liver, HeLa and 3T3 cells; but they do not contain non-transcribed DNA sequences [14-161. Here we examine whether the two nucleosome fractions that differ in their mode of Hg binding both originate in the active genes of HeLa cells. We examined the DNA sequences of the 0.5-M-NaC1-eluted (Fig. 6, peak 2) and dithiothreitol-eluted (peak 3) nucleosomes

819 Table 3. DNA distribution in HeLu nttcleosornefractions,followin:: rihonuclease treatment and gel,filtration of the HeLa nuclear S,fruction Comparisons were made of nucleosome recoveries and DNA distribution during Hg-agarose affinity chromatography of S fractions (from cells labeled with [3H]uridine) which had or had not been treated with RNase A before passage through the Hg-agarose column. Similar comparisons are shown for S fractions which had, or had not, been chromatographed on Sephacryl S-200 prior to passage through the Hgagarose column. n, number of experiments Conditions

n

DNAin S fraction

DNA recovered after Scphacryl

DNA recovered in peak

1

Control RNase-treated Control Sephacryl-treated

4 4 5 3

% total

%

7.8 0.7 7.8 0.7 8.1 kO.7 7.0 f 0.5

95 96 -

86

Direction of migration

Fig. 5. Separation of nucleosomes from adventitious non-histone proteins by gel-filtration chromatography. Proteins of total nucleosome fraction S, released by a limited micrococcal nuclease digestion of HeLa nuclei, were separated electrophoreticdlly in SDS/polyaerylamide gels and stained as shown in Fig. 3 A. (A) Densitometric tracing of the proteins of the initial S fraction. (B) Tracing of proteins remaining in the total nucleosome fraction after gel-filtration on Sephacryl S-200. Note the persistance of the histones and elimination of many of the nonhistone proteins

by hybridizations to 32P-labeled DNA probes for the 28s ribosomal, histone H4, c-myc and alkaline phosphatase genes. Densitometric analyses of the slot-blots show a 2- 3-fold enrichment of all these genes in the Hg-bound nucleosome fractions, compared to the unbound nucleosomes (Fig. 6; Table 4). The degree of enrichment is higher in the nucleosomes eluted in 10 mM dithiothreitol than in nucleosomes eluted in 0.5 M NaCl. It should be emphasized that the appearance of the corresponding DNA sequences in the unbound nucleosome fraction is expected because these genes are not fully expressed in HeLa cells. When genes are not fully expressed, as in the case of the multiple ribosomal genes, the enrichment of rDNA sequences in the Hg-bound nucleosomes

[‘HIRNA content of peak

_ _ ~

-~

2

3

1

2

3

cpm/1012 nucleosomes ____

86 86 88 87

6.8 7.1 5.8 5.7

7.1 6.9 5.1 5.3

53 52

690 69

1380 85

is not as pronounced as has been observed for other genes; e.g. the fully active c-fos and c-myc genes of 3T3 cells [16]. The expression of c-myc, although continuous throughout the cell cycle [46], is complicated by the presence in HeLa cells of multiple copies of the gene, not all of which are transcribed. When a gene is not transcribed at all, as is the case for the aglobin gene in HeLa cells, its DNA sequences do not appear in the Hg-bound nucleosomes (Fig. 6). In synchronized HeLa cell cultures, DNA sequences of the H2A and H4 genes are readily detectable in the Hg-bound nucleosomes when they are transcribed during the S phase of the cell cycle, but little, if any, H2A or H4 DNA appears in the Hg-bound nucleosomes during the G-2 phase, when their transcription is repressed (Fig. 6). The enrichment of H4 DNA in the Hg-bound nucleosome fractions is not as evident in a randomly dividing HeLa cell population with fewer cells in the S phase (Fig. 6; Table 4). The alkaline phosphatase gene is expressed at very low levels in HeLa cells, but when it is induced in cells exposed to 5 mM sodium butyrate [44], the induction is readily evident in the enhanced recovery of its DNA sequences in the Hgbound nucleosomes (Fig. 6; Table 4). We conclude that two-stage Hg-agarose affinity chromatography provides direct access to two distinct nucleosome classes, both of which contain transcribing DNA sequences, and that the thiol reactivity of active nucleosomes is a complex phenomenon which can be attributed in part to non-histone proteins associated with the transcribed DNA sequences, and in part to conformational changes in active nucleosomes that expose the histone H3 thiols. This implies that when transcription ceases, two modifications of chromatin structure should take place: (a) whatever proteins are responsible for Hg binding of the salt-labile nucleosome fraction will be released; (b) the nucleosomes with accessible H3 thiol groups will revert to a compact configuration in which the SH groups are shielded. These expectations were confirmed in studies of the effects of u-amanitin on the Hg binding of HeLa nucleosomes.

Ejjects of u-amanitin of transcription and nucleosome structure What happens to the nucleosomes along active genes when their transcription is inhibited by the action of agents such as a-amanitin, which block RNA chain elongation by inactivating RNA polymerase I1 [47 - 51]? We began by comparing the effects of increasing concentrations of x-amanitin on total RNA synthesis in permeabilized HeLa cells. The cells were exposed to the drug for 10min and the RNA was pulse-labeled with [3H]UTP for

820

Fig. 6. Effects of 1-umonitin, ucririomycin D und sodium hutyrute on gene distribution in unbound and Hg-hound nuclmsotne fractions. HeLa cells exposed to x-amanitin (cc-am), actinomycin D (Act-D) and sodium butyrate (B) were employed for the isolation of nuclei and fractionation of the nucleosomes by Hg-agarose affinity chromatography. DNA was isolated from the unbound nucleosomes (peak 1) and from nucleosomes eluted in 0.5 M NaCl (peak 2) and in I 0 mM dithiothreitol (peak 3). The DNA samples were probed for sequence content by hybridization to 32P-labeledprobcs for the histone H4 gene, c-myc, rDNA and the alkaline phosphatase (AP) gene. The slot blots shown in the first three vertical panels are for rDNA. histone H4 and c-myc, respectively. They compare the distribution of those gene sequences in nucleosome peaks 1 - 3 from control, r-amanitin-treated and actinomycin-D-treated cells. The top two slot blots under AP (C and B) compare the content of alkaline phosphatasc gene sequences in the nucleosome fractions of control (C) and butyrate-treated (B) cells. Densitometric tracings are shown above each slot blot and analyzed in Table 4. The bottom set of slot blots under AP compares the distribution of histone H2A and H4 gene sequcnces in the unbound (U) and Hg-bound (B) nucleosomes prepared from S-phase HeLa cells (in which thc genes are actively transcribed) and from cells in the GZphase (when the genes are repressed). Note that both H2A and H4 DNA sequences appear in the Hgbound nucleosomes of S-phase cells, but neither appear in the Hg-bound nucleosomes of G 2 cells. The bottom set of slot blots under AP compares the distribution of a non-transcribed gene, a-globin, in the unbound (U) and Flg-bound (B) nucleosomes of S-phase HeLa cells. Note that no x-globin DNA is detected in the Hg-bound nucleosomes

30 min. Under these conditions, maximal inhibition was obtained at 20 pgiml (Fig. 7A). The effects of longer exposures to 25 pgjml x-amanitin on total RNA synthesis are summarized in Fig. 7 B. They show that exposures longer than 20 min do not further reduce the RNA synthetic capacity of the nuclei. These results were then compared with the effects of amanitin on nucleosome structure, as determined by the recovery of Hg-bound nucleosomes during Hg-agarose affinity chromatography. The binding studies (Fig. 7C) show a rapid decrease in the number of nucleosomes retained by the Hgagarose column following amanitin treatment. The change is clearly evident after only 10min exposure to the RNA polymerase I1 inhibitor and the decrease continues until 30 min. A comparison of the kinetics of inhibition of RNA synthesis (Fig. 7 B ) and the decrease in Hg binding of the

nucleosomes under the same conditions (Fig. 7 C) shows that the maximal effect on nucleosome structure is achieved about 10 min after the maximal suppression of transcription. This lag is presumably a reflection of the delay in restructuring the thiol-reactive nucleosomes to a non-reactive state. In considering molecular mechanisms that may be involved in the restructuring of the active chromatin, attention is directed to the kinetics of deacetylation of the Hg-bound nucleosome fractions. In both fractions, the histone N-acetyl groups have half-lives of 11 - 12 min (Fig. 4). Since hyperacetylation of histones H3 and H4 is known to increase the H3 thiol reactivity of nucleosomes [52],deacetylation of the histones of the dithiothreitol-elutable fraction would be expected to reduce the accessibility of the H3 thiol groups for Hg binding. The fact that the Hg-bound nucleosome fractions diminish by 50% after 30 min of amanitin treatment indicates that the altered nucleosomes of genes transcribed by RNA polymerase

821

A

A

r

-

Y

-o5

5

L-

100

10

25

20

a amanitin (pglrnl)

n

B Time (rnin) c

I

1

I

B

% I i , , , , , 10

20

30 40 Time (min)

50

60

Fig. 7. Effects of a-amanitin on total RNA synthesis and fractionation of nucleosomes by Hg-agarose affinity chromatography. (A) HeLa cells exposed for I0 min to the indicated concentrations of a-amanitin were pulse-labeled with [3H]UTP for I0 min, and the incorporation of the isotopic precursor into RNA was measured. Note that maximal inhibition was observed at 20 pg/ml. (B) HeLa cells were exposed to 20 Igg/ml a-amanitin. At the indicated times, aliquots of the cell suspension were pulse-labeled with [3H]UTP for 10 min, and the uptake of the precursor into total RNA was measured and expressed relative to that of cells pulse-labeled in the absence of the inhibitor. (C) Nucleosomes were prepared from control and a-amanitin-treated cells at the indicated times and passed through parallel Hg-agarose columns. The extent of nucleosome binding in the amanitin-treated cells is expressed relative to that observed in the control cells. Note that the maximal effect of a-amanitin on Hg binding of the nucleosomes is achieved about 10 min later than the maximal inhibition of RNA synthesis

I1 revert quickly to a compactly beaded configuration when amanitin binds to the polymerase and prevents further elongation of the nascent RNA chains. That such changes in nucleosome conformation have occurred in the histone H4 and c-myc genes is evident in comparisons of slot-blot hybridizations of those DNA probes to the DNA isolated from the Hg-bound nucleosome fractions of control and amanitintreated cells. Nucleosomes retained on the Hg-agarose column after amanitin treatment show a loss of over 50% of their cmyc and H4 DNA sequences (Fig. 6; Table 4). The nucleosomes retained on the Hg-agarose column following amanitin treatment would be expected to contain DNA sequences, such as those of ribosomal and tRNA genes, that are transcribed by the amanitin-resistant RNA polymerases I and 111. Changes in nucleosome conformation and SH reactivity were not observed in the ribosomal genes, whose transcription, mediated by amanitin-resistant RNA polymerase I, was not inhibited in the amanitin-treated HeLa cells. Slot-blot

Time (min)

Fig. 8. EJfects of actinomycin D on total RNA synthesis andjractionation of nucleosomes by Hg-agarose affinity chromatography. (A) HeLa cells exposed to 10 pg/ml actinomycin D for the indicated times were pulse-labeled with [3H]UTPfor 10 min, and the uptake of the radioactive precursor into total RNA was measured and expressed relative to that of cells pulse-labeled in the absence of the inhibitor. (B) The nucleosomes were prepared from control and actinomycin-D-treated cells at the indicated times and passed through parallel Hg-agarose columns. The extent of nucleosome binding in the actinomycin-Dtreated cells is expressed relative to that observed in the control cells. Note that actinomycin D does not reduce the proportion of Hg-bound nucleosomes

hybridizations of the nucleosomal DNA to 32P-labeledrDNA probes clearly indicate that 28s ribosomal gene sequences were recovered equally in the Hg-bound nucleosome fractions of both control and amanitin-treated cells (Fig. 6; Table 4). The effects of actinomycin D on transcription and nucleosome structure

Actinomycin D was added to HeLa cells and its inhibitory effects on total RNA synthesis were measured 10 min and 20 min later (Fig. 8A). At the same times, we compared the proportions of nucleosomes in the Hg-bound and unbound fractions of the control and actinomycin-D-treated cells (Fig. SB). The results were unexpected. After 20 min the Hg-bound nucleosome fractions of the actinomycin-D-treated cells were not diminished, despite the fact that total RNA synthesis had been inhibited by 92% (Fig. 8A). The Hg-bound nucleosome fractions prepared from control and actinomycin-D-treated cells were then compared for DNA sequence content by hybridizations to DNA probes for histone H4 and c-myc DNA. The high recovery of those DNA sequences in the Hg-bound nucleosome fractions of the treated cells (Fig. 6; Table 4) contrasts with the results showing

822 Table 4. Effict.\ c!f treafinent ivith x-urnunitin, actinornycin D and sodium butyrate on distribution ofgcnes in Hg-bound and unbound nucleosome ,fractions HeLa S-3 cells, either untreated, or treated with a-amanitin, actinomycin D sodium Na butyrate, were used to prepare nucleosomes for Hgagarose affinity chromatography. D N A was isolated from the unbound nucleosomes (peak l), from thc Hg-bound nucleosome fraction eluted in 0.5 M NaCl (peak 2) and from the nucleosomes eluted in 10 mM dithiothreitol (peak 3). The D N A samples were probed for sequence content, using 32P-labeledprobes for the histone H4 gene, c-myc, rDNA, and the alkaline phosphatase gene, as shown in Fig. 6. The intensity of thc slot blots was determined by laser densitometry. Results with actinomycin D were from one experiment. No effect of actinomycin D on the Hg binding of nucleosomes containing rDNA sequences has been observed in four experiments on murine fibroblasts (BALB/c 3T3 cells). U, unit ~~~

~~

Conditions of experiment

Gene probe

Gene content of DNA in nucleosomes of peak

_ ~ _ _ _

1

2

3

UiPS (%)

Control Amanitin-treatcd Actinomyciii-D treated

histone H4

1.98 (100) 2.08 (105) 2.11 (107)

UIPg (Yo) 3.38 (100) 1.71 (51) 3.45 (102)

UlPLg (%I 4.64 (100) 1.98 (43) 4.32 (93)

Control Amanitin-treated Actinomyciii-D treated

c-rnyc

2.88 (100) 2.98 (103) 2.91 (101)

5.18 (100) 2.07 (40) 4.95 (96)

5.62 (100) 2.52 (47) 5.55 (99)

Control Amanitin-treatcd Actinomycin D

rDNA

2.42 (100) 2.66 (110) 2.63 (109)

5.17 (100) 4.72 (92) 1.50 (29)

Control Butyrate-treated

alkaline phosphatase

0.217 (100) 1.44 (664)

0.262 (100) 3.62 (1380)

5.19 (100) 5.52 (103) 1.63 (31) 0.381 (100) 4.31 (1130)

that actinomycin D had effectively blocked RNA synthesis (Fig. 8 A). Therefore, the nucleosomes in the transcribing cmyc and histone H4 genes had retained the altered conformations that characterize the template-active state, even though their transcription had been inhibited by actinomycin D. We conclude that the intercalation of actinomycin D into DNA prevented the conformational and structural changes that occurred in the nucleosomes along the c-myc and H4 genes when RNA polymerase I1 activity was blocked by aamanitin am fig. 6 ; Table 4). To confirm that the inhibitor had intercalated into the transcriptionally active nucleosomes, we added 3H-labeled actinomycin D to intact HeLa cells and, after 20min, the unbound and total Hg-bound nucleosome fractions were prepared and analyzed for their contents of [3H]actinomycin D. The results show that actinomycin D binds preferentially to the transcriptionally active nucleosomes retained by the Hgagarose column (25 160 dpm/pg DNA in the combined Hgbound nucleosome fractions, compared to 12680 dpm/pg DNA in the unbound nucleosomes). This preferential localization of the drug on active nucleosomes is in accord with earlier observations on the enhanced binding of [3H]actinomycin D to lymphocyte chromatin when RNA synthesis is stimulated by mitogens [53]. From the original specific activity of the added [3H]actinomycin D, the 3H radioactivities of the nucleosome fractions, the DNA content of each fraction and the average sizes of the nucleosomal DNA, an estimate was made of the number of actinomycin D molecules present/nucleosome. This figure was 2.0 for the nucleosomes of the total Hg-bound fraction, and 1.0 for the unbound nucleosome fraction. The presence of 2 molecules actinomycin D/nucleosome in the nucleosomes known to contain the transcribed DNA sequences indicates a substantial degree of DNA strand cross-linking in the transcriptionally active chromatin. It would appear that 2 cross-links/nucleosome are sufficient to effectively hinder progression of the transcription

complex through the H4 and c-nzyc genes. This is consistent with earlier evidence that the intercalation of actinomycin D blocks the elongation of nascent RNA chains [54, 551. The stabilization of the active configuration of nucleosomes along the histone H4 and c-myc genes is also in accord with evidence that the intercalation of actinomycin D into chromatin results in an increased local stiffening of the DNA [56].All these results lend support to models that invoke DNA strand cross-linking by actinomycin D in a manner that leads to entrapment of the RNA polymerases [57]. Conclusions It is clear that the nucleosomes along transcriptionally active DNA sequences exist in multiple forms, at least two of which can be separated by Hg-agarose affinity chromatography. Both classes of Hg-bound nucleosomes are characterized by high levels of acetylation of their core histones. Those high levels are maintained by a dynamic balance between rapid rates of acetate incorporation and a rapid enzymatic cleavage of the N-acetyl groups. The contrast in the kinetics of histone acetylation and deacetylation in the active (Hg-bound) and inactive (unbound) nucleosome fractions suggests that this modification is closely coupled to transcription of the associated DNA sequences. It is also clear that high levels of histone acetylation, per se, are not sufficient to cause nucleosome binding to the Hgagarose column, since exposure to the deacetylase inhibitor, sodium butyrate, does not result in any major shift in the proportions of the Hg-bound and unbound nucleosome fractions. However, localized changes in chromatin structure of genes induced by butyrate, such as the alkaline phosphatase gene, are easily detectable by the increase in Hg binding of its component nucleosomes. The effects of a-amanitin on nucleosome structure are of particular interest because they show that the active state of the nucleosomes is not stable when RNA polymerase I1 activity is blocked, despite the fact that amanitin would not be

823 expected to displace the transcription complex. The mechanism by which nucleosomes revert to the compact, inactive state is not known, but it is very likely that it involves a rapid deacetylation of the core histones. The effects of actinomycin D should provide further insights into the role of nucleosome structural changes in transcriptionally active chromatin. The intercalation and crosslinking of the DNA strands by actinomycin can stabilize the nucleosome in its template-active configuration. This opens the way to easy recovery and direct analyses of the proteins and their post-synthetic modifications in both the nucleosomes and the entrapped transcription complexes. Direct evidence that the RNA polymerases are entrapped in the actinomycin-D-modified nucleosomes and can be reactivated will be presented in a subsequent paper. This work was supported by research grants from the United States Public Health Service (CA-14908 and GM-17383) (VGA), by travel grant 1-3570 from the National Cancer Institute (LCB), and by Associazione Ztaliana Ricerea su Cancro, 1990 ‘Oncology’ Grant 8800797-44 (LCB).

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