affinity chromatography of mammalian and yeast nucleosomes-walker-1990.pdf · nucleosomes were...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 10, Issue of April 5. pp. 5736-5746,199O Printed in U.S.A.

Affinity Chromatography of Mammalian and Yeast Nucleosomes TWO MODES OF BINDING OF TRANSCRIPTIONALLY ACTIVE MAMMALIAN NUCLEOSOMES TO ORGANOMERCURIAL-AGAROSE COLUMNS, AND CONTRASTING BEHAVIOR OF THE ACTIVE NUCLEOSOMES OF YEAST*

(Received for publication, November 21, 1989)

Janis Walker, Thelma A. Chen, Richard Sterner, Michael Berger, Fred Winston& and Vincent G. Allfrey From the Laboratory of Cell Biology, Rockefeller University, New York, New York 10021 and the *Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115

The reasons for the selective binding of nucleosomes from transcriptionally active genes to organomercurial-agarose columns have been investigated. At least two modes of binding are identified by a new two-stage elution procedure that discriminates between nucleosomes which are retained by the Hg-column because of their salt-labile associations with SH-reactive nonhistone proteins, and nucleosomes in which a confor- mational change has made the thiol groups of histone H3 accessible to SH-reagents. The first class is released from the column in 0.5 M NaCl; the second class is eluted in 10 mM dithiothreitol which displaces the bound H3-thiols. In mammalian cells, both classes of Hg-bound nucleosomes are enriched in the DNA sequences being transcribed at the time, and their histones H3 and H4 are hyperacetylated. In yeast cells, in which histone H3 lacks cysteinyl residues, only a small fraction of nucleosomes binds to the mercury column, and it has no enrichment of DNA sequences derived from the actively transcribed GAL, HIS4, and ACT1 genes. Since few nucleosomes remain on the column after elution in 0.5 M NaCl, the bound nucleosomes of yeast are retained primarily because of salt-labile associations with thiol-reactive nonhistone proteins. Thus, the presence of histone H3-thiol groups appears to be essential for the mercury binding of the second class of nucleosomes which, in mammalian cells, is derived from the transcriptionally active genes. The results support models of reversible nucleosome unfolding during transcription in mammalian cells to reveal previously inaccessible H3-SH groups, and they also indicate that other thiol-containing proteins, including high mobility group 1 and 2, become closely but transiently associated with the chromatin subunits during their transcription.

It has been shown previously that the chromatography of nucleosomes on organomercurial-agarose columns results in the selective retention of the transcriptionally active DNA sequences of the tissue or cell type examined (l-3). The binding to the Hg-column of nucleosomes containing specific DNA sequences, such as c-fos and c-myc, coincides closely

* This work was supported by Grants GM17383 and CA14908 from the National Institutes of Health and bv Grant NP-228s from the American Cancer Society. The costs of piblication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed.

with the timing and extent of their expression, as determined by run-off transcription assays on the isolated nuclei (3).

In light of the evidence that nucleosomes in transcriptionally active chromatin have an altered configuration in which the previously shielded sulfhydryl groups of histone H3 become accessible to SH-reagents (4, 5), we have assumed that the H3-sulfhydryl groups play an important, if not the pre- dominant, role in the binding of active nucleosomes to the mercury column.

Here we examine the mercury binding of nucleosomes in more detail and show that there are two separable modes of Hg binding of the transcriptionally active chromatin subunits. We present new evidence that histone H3-thiol groups are directly involved in the Hg binding of the transcribed DNA sequences of mammalian cells. In addition, we examine the DNA sequences of nucleosomes which adhere to the column because of their associations with other sulfhydryl-containing proteins and find that they also originate in transcriptionally active chromatin. Finally, we show that although some yeast nucleosomes (in which there are no histone H3-sulf’hydryl groups) may bind to the column by virtue of aggregation or associations with other proteins, they are not enriched in the transcriptionally active DNA sequences of the GAL, HIS4, or

ACT1 genes.

EXPERIMENTAL PROCEDURES

Isolation and Endonuclease Digestion of Mammalian Nuclei-All procedures were done at 4 “C unless otherwise specified. Nuclei from livers of freshly killed Sprague-Dawley rats (150-180 g in weight) were prepared essentially as described by Blobel and Potter (6), with the following modifications; sodium butyrate was present at all times to inhibit histone deacetylase activities, and one more centrifugation step (1000 X g for 10 min in buffer A (0.25 M sucrose, 50 mM Tris- HCl, pH 7.5, 25 mM KCl, 5 mM MgCl,, 5 mM sodium butyrate)) was added before centrifugation of the nuclei through 2.3 M sucrose in buffer A.

The nuclear pellet was resuspended in buffer B (10 mM Tris-HCI, pH 7.4), containing 25 mM KCl, 5 mM MgC12, 0.5 mM CaCl*, 0.35 M sucrose, 5 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride (Sigma) and 0.1 mM 1,2-epoxy-3-(paranitrophenoxy) propane (Ko- dak). Nuclear suspensions containing 20 AZeO units/ml (determined in 0.1 N NaOH) were digested for 5 min at 37 “C with 8 units of micrococcal nuclease/ml (Cooper Biomedical). The reaction was stopped by the addition of ice-cold 30 mM EGTA,’ pH 7, to a final coccentration of 3 mM and the nuclei centrifuged at 10,000 X g for 20 min (7). This procedure avoids lysis of the nuclei (8) and yields a supernatant fraction, S, containing 4-9% of the total nuclear DNA and comprised mainly of monomeric nucleosomes (Fig. 4).

1 The abbreviations used are: EGTA, [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; HMG, high mobility group; kb, kilobase; bp, base pair.

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Fractionation of Transcriptionally Active Nucleosomes 5737

Nucleosomes were prepared from HeLa cells (2.) and 3T3 cells (3) as described. COLO 320 HSR cells (ATCC) were seeded at a density of 2 x 105/m1 in RPMI-1640 medium (GIBCO/Bethesda Research Laboratories) containing 10% denatured fetal bovine serum (Hy- clone) and 10 pg/ml gentamycin sulfate. Cells were collected at a density of 4-6 X 10”/ml by centrifugation at 500 X g for 7 min at 4 ‘C. The cells were washed three times in ice-cold Dulbecco’s phos- phate-buffered saline containing 5 mM butyrate before isolating the nuclei by the procedure employed for 3T3 cell nuclei (3). Nuclear suspensions containing 1 mg/DNA/ml were treated with 10 units/ml micrococcal nuclease for 6 min at 37 “C to release 10% of the total DNA.

Isolation and (Saccharomvces

Endonuclease Digestion of Yeast Nuclei-Yeast cells cereuisiae) were grown at 29 “C in YPGlu (1% yeast

extract, 2% peptone (Difco), 2% glucose), or YPGal (wherein galactose was substituted for glucose as the carbon source), until they reached a density of 1 x 10’ cells/ml. The cells were then brought to the spheroplast stage essentially as described by Jerome and Jaehning (9). The spheroplasts were lysed and nuclei isolated as described by Schultz (10). The nuclei were washed in buffer B without CaC12 and resuspended at a concentration of 20 A 260 units/ml. After adjustment of the CaC12 concentration of 0.5 mM and addition of micrococcal nuclease (18 units/ml), the nuclei were incubated at 35 “C for 5 min, at which time digestion was stopped by the addition of EGTA to a final concentration of 5 mM. The nuclei were centrifuged at 10,000 X g for 20 min, and the supernatant (S), representing 3.9-10.6% of the total nuclear DNA, was employed for the chromatographic fractionation of nucleosomes.

Sequential Fractionation of Nucleosomes by Hg Affinity Chromatog- raphy-The recovered S fractions of mammalian nuclear digests, already 3 mM in EGTA, were made 5 mM in total chelator by the addition of Na,EDTA to 2 mM. The nucleosomes were loaded on 1 X 6-cm columns of Affi-Gel501 (Bio-Rad) at a flow rate of 20 ml/h. In large scale preparations (150 ml of more of the S fraction), the nucleosomes were applied to 2.5 x 3-cm columns at flow rates up to 60 ml/h. The columns were washed with buffer C (10 mM Tris-HCl, pH 7.5, 25 mM KCl, 25 mM NaCl, 5 mM sodium butyrate, 5 mM Na2EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM l&epoxy- 3-(paranitrophenoxy) propane to elute the unbound nucleosomes. When this elution was complete, as monitored by the A,,, of the eluate, the retained nucleosomes were then eluted in two stages. The first employed 0.5 M NaCl in buffer C to displace nucleosomes adhering to the Hg column through salt-labile associations with bound nonhistone nuclear proteins. The second stage used 10 mM dithiothreitol (DTT) in buffer C to displace nucleosomes bound to the Hg column through their intrinsic thiol groups. In several experiments, Hg affinity chromatography was preceded by chromatography on 1.5 X 90.cm columns of Sephacryl S-200 (Pharmacia LKB Bio- technology Inc.) in order to separate nucleosomes from smaller chromatin fragments, adventitious proteins, and RNA fragments (1). This step does not diminish the yield of Hg-bound nucleosomes.

DNA and Protein Determinations-The DNA contents of the chromatographically separated nucleosome fractions were determined using the Hoechst 33258 fluorochrome reaction (ll), and measuring fluorescence with a Perkin-Elmer 650-40 fluorescence spectropho- tometer.

Protein contents of the nucleosome fractions were measured using the bicinchoninic acid assay (Pierce Chemical Co.) (12), following dialysis to remove DTT.

DNA Sizing and Hybridization-DNA was prepared from the unbound and Hg-bound nucleosome fractions as described (1, 3) and digested with 50 pg/ml RNase A for 1 h at 37 “C, 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 (13), and dissolved in 10 mM Tris-HCl, pH 7.4, 1 mM Na*EDTA for DNA sizing (14), and slot- blot hybridization experiments.

Nucleosomal DNA from each sample was blotted onto a pre-wetted nylon membrane (Zeta-Probe, Bio-Rad) in a slot-blot apparatus (Schleicher & Schuell) in quantities of 5 or 10 pg, following the alkaline procedure (15). Membrane-bound DNAs were hybridized to “random primed,” ‘* P-labeled probes (16), using the Southern procedure, as described (13). The probes employed for rat liver nucleosomes were plasmids encoding rat serum albumin, a mixture of four non-overlapping subclones (“JB,” C, B, and D) (17), and rat transferrin cDNA (18). The ribosomal DNA probe was the plasmid I-19 (19) containing 4.8 kb of mouse genomic DNA including the gene for 28 S rRNA.

The probe for c-fos was the 1-kb PstI-PuuII restriction fragment

of FBJ osteosarcoma proviral DNA (20). The v-myc probe was the 1.5 kb PstI-AhaIII restriction fragment of the avian myelocytomatosis virus MC-29 (ATCC 41008). The probe for histone H4 DNA was a 710.bp DNA fragment from plasmid pHu4A containing human histone H4 gene and its flanking sequences (2). The probe for the yeast GAL loci was the 2-kb EcoRI fragment excised from plasmid 4812 (21). The probe for yeast HIS4 was the 1.2-kb SalI-BglII fragment excised from plasmid pFw 45 (22). The probe for yeast ACT1 was the 1.6.kb HindIII-BamHI fragment from the ACT1 gene cloned into pBR322 (plasmid pCC69).* The filters were washed (13), dried, and exposed to Kodak X-OMat AR5 film for various times with a DuPont Cronex I-G Plus intensifying screen at -80 “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.

Gel Electrophoretic Analyses-The proteins of the run-through and adsorbed nucleosome fractions from Hg affinity columns were sub- jected to electrophoresis in 15% polyacrylamine gels containing 0.1% SDS (24). For electrophoretic separations of the acetylated forms of the histones, they were prepared by the protamine displacement technique (25), purified by ion-exchange chromatography on BioRex- 70 (Bio-Rad) and analyzed in gels containing 15% polyacrylamide, 5.5% (v/v) acetic acid, 8 M urea, 0.3% Triton X-100, as described (1, 26). The gels were stained with 0.25% Coomassie Brilliant Blue-R, or with Procion blue MX-R (Fluka), or with silver (27). Gels stained with Procion blue were used for quantitation of the histones by laser- scanning densitometry.

Carborymethylation of Nucleosomal Proteins-To test whether histone HS-thiol groups are directly involved in the Hg-binding reaction, the reactivity of HS-thiols with iodo[“H]acetate was compared in nucleosome fractions on and off the column. On-column derivatization were carried out on the total Hg-bound nucleosome fraction and on the nucleosomes remaining following elution with buffer C containing 0.5 M NaCl. In either case, the columns were first washed at 23 “C with buffer C (adjusted to pH 8) to remove unbound nucleosomes. When the Alw returned to base line, one column volume of buffer C containing 1.5 mM iodo[3H]acetic acid (188 mCi/mmol, Du Pont-New England Nuclear) was pumped into the column, which was allowed to stand without further movement of buffer at 23 “C in the dark for 45 min. The column was then washed with buffer C at 4 “C until the A960 returned to base line, at which time the nucleosomes were eluted in buffer C containing 10 mM DTT.

Off-column derivatizations were carried out on the unbound nucleosome fraction and on the two nucleosome fractions eluted successively in buffer C containing 0.5 M NaCl, followed by buffer C containing 10 mM DTT. After dialysis to remove DTT, all fractions were reacted with 1.5 mM iodo[“H]acetic acid for 45 min at pH 8 and 23 “C in the dark. In all cases, the histones were extracted in 5% (w/ v) guanidine HCl, pH 6.8, and purified by ion-exchange chromatography on Bio-Rex 70, prior to electrophoretic analysis and fluorography of the gels, or scintillation spectroscopy of the excised 3H- labeled H3 bands.

To determine the extent to which nucleosomal H3-thiol groups are accessible to SH-reagents in the mercury-bound nucleosomes, equal aliquots of the S fraction were applied to parallel Hg columns. After washing to remove the unbound nucleosomes, both columns were eluted with buffer C containing 0.5 M NaCl to remove nucleosomes retained by salt-labile associations with nonhistone proteins. One column was then eluted with 10 mM DTT, while the second column was treated with 15 mM nonradioactive iodoacetate at 23 “C for 45 min in the dark to block any accessible SH groups. The nucleosomes were then eluted in buffer C containing 10 mM DTT. The histones of both samples were reacted with iodo[RH]acetate to carboxymethylate the accessible H3-thiols. The histones of each sample were separated electrophoretically and the “H activities of the histone H3 bands were compared by fluorography. Control experiments using purified histone H3 show that H3, once bound to the mercury column, cannot be displaced by iodoacetate treatment under these conditions, and that its thiol groups do not react with iodo[“H]acetate. After elution in 10 mM DTT, the thiol groups of the released histone H3 are fully reactive.

RESULTS AND DISCUSSION

Binding of Transcriptionally Active Nucleosomes to Mercury Columns Is Completely Thiol-dependent

In earlier studies of the ribosomal genes of Physarum polycephalum, it was found that the nucleosomes containing

’ C. Clark-Adams and F. Winston, unpublished results.

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5738 Fractionation of Transcriptionally Active Nucleosomes

the rRNA coding sequences “unfold” during transcription to reveal the previously shielded SH groups of histone H3 and that the reactivity of the H3-thiols was lost when rDNA transcription ceased (4). A similar “unfolding” of the nucleosomes during the transcription of other genes is indicated by H3-thiol reactivity in the non-nucleolar chromatin of Physa- rum (28), as well as in the active chromatin of mammalian (2) and avian cells (5).

The accessibility of H3-thiol groups in transcribing nucleosomes, as contrasted to their nonreactivity in nontranscribing nucleosomes, suggested that the nucleosomes of active genes could be isolated by mercury affinity chromatography, and a procedure for the separation of transcriptionally active and inactive nucleosomes has been described (1). It is based on the selective retention of the thiol-reactive nucleosomes by organomercurial-agarose columns when mixtures of active and inactive nucleosomes are passed through the column. In the original method (l), nucleosomes released from isolated nuclei during a limited digestion with micrococcal nuclease were applied to the Hg 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.

This one-step elution technique 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 (3).

Here we explore the binding reaction in more detail. To determine whether the binding of nucleosomes to the Hg column is entirely due to interaction between the organomercurial column and accessible thiol-groups of the nucleosomal proteins, we first tested the effects of thiol-blocking agents. The nucleosomes released by a limited micrococcal nuclease digestion of isolated HeLa cell nuclei were reacted with sodium iodoacetate to carboxymethylate the accessible SH groups. When the thiol-blocked nucleosomes were applied to the Hg column under the same conditions used for the chromatographic isolation of transcriptionally active chromatin subunits (l-3), over 99% of the total DNA appeared in the unbound nucleosome fraction (Fig. 2). Blocking of the thiol groups also prevents the binding of nucleosomes from other cell types (data not shown). (The small amount of residual binding after treatment with 5 mM iodoacetate can be attributed to incomplete derivatization of the protein thiol group.) In contrast, control experiments, using HeLa nucleosomes in which the thiol groups had not been reacted with iodoacetate, showed that 12.0% of the applied nucleosomal DNA was retained by the Hg column (average of 14 experiments), and in 3T3 cells, a range of nucleosomal DNA binding between 11.3 and 25.7% was observed.

We conclude that all chromatin subunits retained by the Hg column under these conditions are bound through their constituent thiol groups. The results rule out the participation of DNA (in any form in which it appears in the Hg-bound nucleosomes) as a factor in the mercury binding of transcriptionally active DNA sequences.

This conclusion is supported by experiments showing that the Hg column retains no more than 0.3% of purified HeLa DNA labeled with [3H]thymidine to high specific activity (2.24 x 10’ dpm/mg). Earlier observations on the ability of this organomercurial column to retain thio-phosphorylated DNA while rejecting normal DNA (29) confirm the view that bind-

ing of active nucleosomes to the Hg column is entirely due to the accessible thiols of the DNA-associated proteins.

Chromatographic Separation of Two Classes of Nucleosomes Containing Reactive Thiol Groups

In the original procedure (l), the compactly beaded nucleosomes of inactive chromatin pass directly through the mercury column, while the retained nucleosomes of transcriptionally active DNA sequences are eluted in buffered 10 mM dithiothreitol. Although there is compelling evidence that the nucleosomes of transcriptionally active genes contain histone H3 molecules with accessible thiol-groups (2, 4, 5, 28), no direct evidence for the Hg binding of nucleosomal H3 had been obtained. Indeed, electrophoretic analyses of the protein complements of the DTT-eluted nucleosome fraction clearly indicate the presence of many nonhistone proteins, some of which appear to be present in stoichiometric proportions to the histones, and also contain reactive SH groups (1, 2). Because many nonhistone proteins of molecular mass below 200 kDa could not be removed by size exclusion chromatography on Sephacryl S-200prior to Hg affinity chromatography (2), we decided to test for their possible involvement in the Hg binding of the associated nucleosomes.

In considering which of the thiol-reactive nonhistone proteins might be closely associated with transcriptionally active DNA sequences, attention was first directed to the thiol- containing high mobility group proteins, HMG-1 and HMG- 2. Their presence in active chromatin is indicated by their selective release along with transcribed DNA sequences during limited digestions with DNase I (30) or micrococcal nuclease (5, 31). HMG-1 and HMG-2 have also been shown to markedly stimulate RNA synthesis in chromatin (32) and in reconstituted transcription systems using purified RNA po- lymerases II and III (33).

The ease of extraction of the high mobility group proteins when chromatin is treated with 0.35 M NaCl (34) suggested that nucleosomes which bind to the mercury column through the thiol groups of HMG-1 and HMG-2 could be released by raising the ionic strength of the eluting buffer. The subfrac- tionation achieved when the Hg-bound nucleosomes of HeLa cells are exposed successively to buffers containing 0.35 M

NaCl, 0.5 M NaCl, and 10 mM DTT, is shown in Fig. 1. DNA analyses of the eluates show that salt-labile associations with thiol-reactive proteins are responsible for the retention of about 60% of the total Hg-bound nucleosomal DNA (Table I). All of the salt refractory nucleosomes are released from the column when 10 mM DTT is added to the eluting buffer. Similar experiments on nucleosomes from rat liver, COLO 320 cells, and 3T3 cells confirm that a substantial fraction is released by 0.5 M NaCl and that all these cells also contain a nucleosome fraction requiring DTT for its displacement from the column (Fig. 2 and Table I). In every case, DNA analyses show that all of the DNA originally bound to the Hg column is recovered in the combined salt-labile and DTT-eluted nucleosomes. Treatment of the column with 0.5 or 2 M NaCl after DTT elution releases no detectable DNA or protein.

A two-step chromatographic procedure which separates the Hg-bound nucleosomes according to their mode of binding employs 0.5 M NaCl to release nucleosomes retained through salt-labile associations with thiol-reactive nonhistone proteins. Subsequent elution of the column with 10 mM DTT displaces nucleosomes containing histone H3 molecules with accessible thiol groups. Typical elution diagrams are shown for four different cell types in Fig. 2. If the S fraction is adjusted to 0.5 M NaCl before application to the Hg column, there is no appreciable fraction of the Hg-bound nucleosomes

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which elutes in 0.5 M NaCl; all of the bound nucleosomes are released in 10 mM DTT (Fig. 20). Similar chromatographic separations of nucleosomes released from 3T3 cell nuclei (after digestion of 10.2% of the total nuclear DNA by micrococcal nuclease) show that 18.5 -+ 7.2% of the released DNA was retained by the mercury column. Of the bound DNA, 62 + 4.8% was recovered in the nucleosomes eluted in 0.5 M

FIG. 1. Fractionation of nucleosomes by mercury affinity chromatography. The mixture of nucleosomes released by a limited micrococcal nuclease digestion of HeLa cell nuclei was applied to an organomercurial-agarose column. After elution of the unbound nucleosome fraction, the Hg-bound nucleosomes were fractionated by stepwise increments in the salt concentration of the elution buffer to 0.35 and 0.5 M NaCl. This releases nucleosomes that are bound to the column through salt-labile associations with SH-reactive nonhistone proteins. The remaining nucleosomes, containing reactive histone H3-thiol groups, were displaced from the column by the addition of 10 mM DTT to the elution buffer. This step removes all nucleosomes from the column, and a final wash with 0.5 M NaCl shows no residual DNA binding. DNA distribution in the eluates was monitored by absorbance at 260 nm. Arrows indicate the successive changes in eluting buffer to 0.35 M NaCl, 0.5 M NaCl, and 10 mM DTT, in that order.

NaCl, while 38 f 4.6% was recovered in the nucleosomes released by 10 mM DTT (average of five experiments).

The relative proportions of the salt-labile and DTT-eluted fractions has been found to vary, depending upon the extent of endonuclease digestion. The more extensive the digestion, the lower the proportion of DTT-eluted nucleosomes. This relationship is shown for HeLa nucleosomes in Fig. 3. The greater susceptibility to endonuclease attack of the unfolded nucleosomes in the DTT-eluted fraction is in accord with earlier observations on the rapid degradation by micrococcal nuclease of the SH-reactive nucleosomes of ribosomal genes (4). Therefore, to minimize variability in experiments involv- ing comparisons of nucleosome structures in cells exposed to growth factors or inhibitors (e.g. 3, 35), it is important to select conditions that provide a reproducible release of DNA from all nuclear preparations tested.

Properties of the NaCl-eluted and DTT-eluted Nucleosomes

DNA Site Differences-Fig. 4 displays the DNA sizes of the unbound nucleosomes and each of the mercury-bound nucleosome fractions of rat liver, HeLa, COLO 320, 3T3 cells, as determined by ethidium bromide staining of the electrophoretically separated DNA fragments. Densitometric analysis of these patterns shows that the monomeric nucleosomes pre- dominate in all fractions (Table I). For example, in 3T3 cells, monomers comprise 70-80% of the total DNA in the run- through, 0.5 M NaCl-eluted, and DTT-eluted nucleosome fractions. Dimers make up most of the remainder (Table I). Faint bands corresponding to trimers and higher order nucleosomal arrays are also visible. The spacing of those bands indicates that the nucleosomal repeat in the unbound fraction is -195 bp.

Although the repeat length does not differ appreciably from 195 bp in the mercury-bound nucleosome fractions, there is a significant difference in the amount of DNA present in the monomeric nucleosomes of the various fractions. In 3T3 cells,

TABLE I

Proportions and monomeric DNA lengths of nucleosome fractions separated by two-stage Hg affinity chromatography

Nucleosomes present aso Cell type Nucleosome Distribution Monomeric DNA

fraction of DNA Mono- Di- Tri- Oligo- length& mers mers mers men

% of total’ % % % % b

Rat liver Peak 1 88.0 88 12 Trace 170 Rat liver Peak 2 5.5 76 24 Trace 195 Rat liver Peak 3 6.5 82 16 2 175

BALB/c 3T3 Peak 1 81.5 80 20 170 BALB/c 3T3 Peak 2 11.5 79 21 Trace 195 BALB/c 3T3 Peak 3 7.0 72 20 8 170

HeLa Peak 1 88 75 15 3 7 155 HeLa Peak 2 7 37 33 30 Trace 170 HeLa Peak 3 5 93 6 1 155

COLO 320 Peak 1 75 75 24 1 Trace 175 CCL0 320 Peak 2 19 84 13 3 180 COLO 320 Peak 3 6 75 20 3 Trace 170

Yeast Peak 1 84 48 39 13 Trace 160 Yeast Peak 2 12 26 30 21 23 180 Yeast Peak 3 4 Trace Trace Trace 100 170?

” Proportion of peak DNA present as mono-, di-, tri- and oligo-nucleosomes, as determined by laser-scanning densitometry of the ethidium-stained DNA bands in DNA-sizing gels (14).

’ Size, as determined by the position of the absorption maximum of each DNA band relative to the migration distances of HaeIII fragments of +X174 DNA.

’ Proportion of total DNA applied to Hg column recovered in each peak.

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FIG. 2. Two-stage chromatographic separations of mercury- hound nucleosomes from mammalian cells. Nucleosomes released by limited micrococcal nuclease digestions of nuclei from rat liver (A), COLO 320 cells (B), 3T3 cells (C and D), and HeLa S-3 cells (E and F) were applied to organomercurial agarose columns. After elution of the unbound nucleosomes (peak l), nucleosomes retained on the column because of salt-labile associations with SH-reactive nonhistone proteins were released in buffered 0.5 M NaCl (peak 2). The remaining nucleosomes, containing reactive histone HS-SH groups, were eluted in 10 mM DTT (peak 3). If the nucleosome mixture is adjusted to 0.5 M NaCl before application to the Hg column, no peak 2 is observed, as shown for 3T3 cell nucleosomes in panel D. Nu- cleosomes bound through their intrinsic H3-thiol groups are released as usual in 10 mM DTT. If the S fraction of HeLa nuclei is treated with iodoacetate to block reactive thiol groups prior to chromatography, 99.3% of the DNA (as determined by the Hoechst assay) appears in the run-through peak, and less than 0.7% is bound to the column (F).

the average DNA size of the monomer peak eluted in 0.5 M NaCl is -25 bp longer than that of the unbound nucleosomes or in the DTT-eluted nucleosomes (Table I). Similar differences are noted in the nucleosome fractions from rat liver, COLO 320, and HeLa cells; all show longer DNA lengths in the 0.5 M NaCl-eluted nucleosome fraction (Fig. 4 and Table I). The longer DNA lengths indicate the presence of “linker” DNA sequences in nucleosomes that are retained on the Hg column because of salt-labile associations with DNA-binding proteins containing reactive thiol groups. Since we will show that those nucleosomes contain transcriptionally active DNA sequences, the results suggest that the proteins responsible for the Hg-binding are associated with the linker DNA in transcriptionally active chromatin and that they provide some protection from endonuclease attack.

Nonhistone Proteins of the Hg-bound Nucleosome Frac- tions-The known association of HMG-1 and HMG-2 with linker DNA (36, 37) supports the view that those thiol- containing HMG proteins may play a role in the Hg binding of the salt-labile nucleosome fraction. Treatment of the S fraction with 0.5 M NaCl to release the HMG proteins (which can then be separated from the nucleosomes by chromatography on Sephacryl S-200) effectively eliminates the 0.5 M

NaCl-eluted nucleosome peak during subsequent fractiona- tions on the Hg column (Fig. 20). Alternatively, the HMG proteins can be displaced from nucleosomes that are already bound to the Hg column by treatment with 0.5 M NaCl. This releases any nucleosomes held by salt-labile associations with HMGs (or other proteins), and leaves those proteins covalently attached to the Hg column through their reactive thiol groups. The proteins can then be eluted along with the remaining nucleosomes in 10 mM DTT.

Electrophoretic analyses of the proteins in the DTT eluates consistently show the presence of bands corresponding in mobility to HMG-1 and HMG-2 (Fig. 5A). In the Hg-bound nucleosomes of COLO 320 cells, HMG-1 and HMG-2 occur in stoichiometric proportions to the histones (Table II). The identity of the HMG-1 band was confirmed by its reactivity in Western blots with a polyclonal antibody specific for HMG- 1 (data not shown).

The presence of HMG-1 and HMG-2 in the Hg-bound nucleosomes does not preclude the possibility that other nonhistone proteins play a role in the retention of the salt-labile nucleosome fraction, since many other SH-reactive proteins are recovered in the DTT eluates of all the cell types we have examined. In order to determine which of those proteins are

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Percent of nuclear DNA digested

FIG. 3. DNA distribution in mercury-bound nucleosome fractions varies with the extent of endonuclease digestion. HeLa cell nuclei were treated with micrococcal nuclease to degrade increasing amounts of DNA, and the released nucleosomes were fractionated by two-stage mercury affinity chromatography, as shown in Fig. 2. The DNA contents of the 0.5 M NaCl-eluted and DTT- eluted nucleosome fractions were measured by the Hoechst assay, and the oronortion of DTT-eluted nucleosomes (neak 3) in the total Hg-bouid &cleosomes (peak 2 + peak 3) was’calcul&ed for each experiment. Linear regression analysis of the data (Pearson product- moment correlation; P < 0.05) shows a progressive drop in recovery of the DTT-eluted nucleosomes as endonuclease digestion proceeds. This indicates that the nucleosomes of the DTT-eluted fraction are highly susceptible to further endonuclease digestion, possibly as a consequence of increased DNA accessibility due to higher levels of acetylation of the component histones.

displaced by 0.5 M NaCl, comparisons were made of the electrophoretic patterns of the DTT-eluted nucleosome fractions prepared by two different procedures. In the first method, the S fraction was passed through Sephacryl S-200 to remove proteins below 200 kDa that were not associated with the nucleosomes. Then the nucleosomes were applied to the mercury column and eluted successively in 0.5 M NaCl and 10 mM DTT. (Passage of the S fraction through Sephacryl S-200 does not diminish the recovery of nucleosomes in either fraction.) In the second method, the S fraction was immediately adjusted to 0.5 M in NaCl and passed through Sephacryl S-200 to remove the salt-released proteins before mercury affinity chromatography. The Hg-bound nucleosomes were then eluted in 10 mM DTT. After both procedures, the proteins present in the DTT-eluted nucleosome fractions were separated electrophoretically and stained. Comparisons of the banding patterns show which proteins were removed by prior treatment of the S fraction with 0.5 M NaCl. Among the more prominent salt-extractable proteins are polypeptides of M, 39,000, 37,000, and 35,000. All three proteins are completely removed in 0.5 M NaCl (Fig. 5B).

A prominent band of molecular mass 40 kDa is consistently observed in the DTT-eluted nucleosomes of all the mammalian cell types we have examined. Its properties and those of several other proteins in the active nucleosome fractions will be reported elsewhere.

The results suggest, but do not prove, that some nucleosomes are retained on the Hg column because of salt-labile associations with proteins other than HMG-1 and HMG-2.

Histone Stoichiometry in Active and Inactive Nucleosome Fractions-The histone composition of the nucleosomes in the unbound, 0.5 M NaCl-eluted, and 10 mM DTT-eluted fractions was analyzed by densitometry of the stained histone bands separated by SDS-polyacrylamide gel electrophoresis.

A 3T3 B COLO-320

std I 2 3

FIG. 4. DNA complements of nucleosome fractions of 3T3 cells (A), COLO 320 cells (B), rat liver (C) and HeLa cells (D), as separated by two-stage mercury affinity chromatography. The DNA of all fractions was compared by DNA-sizing experiments. A: left lane, Hue111 restriction fragments of 0X 174 DNA employed as size markers; lanes Z-3, DNA of nucleosome peaks 1, 2, and 3 of BALB/c 3T3 cells; B: left lane, DNA size markers; lanes 1-3, DNA of nucleosome peaks 1, 2, and 3 from COLO 320 cells; C: lanes l-3, DNA of nucleosome peaks 1, 2, and 3 from rat liver; right lane, HaeIII restriction fragments of pBR322; D: left lane, pBR322 DNA size markers; lanes 1-3, DNA of nucleosome peaks 1, 2. and 3 from HeLa cells. Note that, in all cases, DNA lengths of the monomeric nucleosomes eluted in 0.5 M NaCl (peak 2) exceed those of the corresponding unbound (peak 1) and DTT-eluted (peak 3) nucleosome fractions (see Table I).

Typical histone patterns are evident in all nucleosome fractions from rat liver, COLO 320, (Fig. 5A) HeLa, and 3T3 cells. The proportions of the individual core histones in the unbound and each of the Hg-bound nucleosome fractions are summarized in Table II.

The occurrence of all four core histones, H2A, H2B, H3, and H4, in stoichiometric proportions in each of the fractions, together with the DNA sizing results, confirms the presence of intact nucleosomes in both the active and inactive DNA sequences of all the cell types examined. This agrees with earlier observations on the stoichiometry of the core histones in nucleosomes derived from the transcribing domains of the ribosomal genes of Physarum (28). Such evidence for histone stoichiometry argues against the view that transcription is accompanied by a selective loss of any histones from the nucleosome core.

Histone Hyperacetylation in the Hg-bound Nucleosome Fractions-Although all of the nucleosome fractions contain stoichiometric amounts of histones H2A, H2B, H3, and H4, the histones of the Hg-bound nucleosome fractions differ from those of the unbound nucleosomes in their postsynthetic modifications.

The acetylation of l-4 lysine residues in the NH,-terminal regions of histones H3 and H4 results in a stepwise reduction in positive charge which permits electrophoretic separation and densitometric quantitation of each modified form. The proportions of the various acetylated isoforms of histone H4 are shown for the unbound, 0.5 M NaCl-eluted, and DTT- eluted nucleosomes in Table III. In HeLa cells, the two Hg- bound nucleosome fractions are hyperacetylated, whereas the unbound nucleosomes are not. In the DTT-eluted fraction

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Fractionation of Transcriptionally Active Nucleosomes B kd

- 200

- 92.5

- 69

H4 - . .r.e-.r. d -14

1 2 3 4 5 6 7 8 9 10 22:: r

111, - 21.5

123 4

FIG. 5. Protein complements of nucleosome fractions separated by Hg affinity chromatography. A, proteins of the unbound, 0.5 M NaCl-eluted, and 10 mM DTT-eluted nucleosome fractions of rat liver and COLO 320 cells were separated electrophoreti- tally in SDS-polyacrylamide gels. Lane I, proteins of the unbound nucleosome fraction of rat liver; lane 2, proteins of the Hg-bound nucleosomes released in one step with 10 mM DTT; lane 3, proteins of the Hg-bound nucleosomes released in 0.5 M NaCl; lane 4, proteins of the remaining nucleosomes released with 10 mM DTT; lane 5, mixture of calf thymus histones and duck erythrocyte HMG proteins. Lanes 6-9, proteins of COLO 320 nucleosome fractions, in the order described for rat liver nucleosomes (lanes 1-4); lane 10, molecular weight standards. Note the prominent bands corresponding to HMG- 1 and HMG-2 in the DTT eluate, and the presence of all four core histones in every nucleosome fraction. Histone stoichiometry is shown for rat liver and COLO 320 cells in Table II. B, comparison of the nonhistone proteins of DTT-elutable nucleosome fractions isolated with and without prior removal of proteins extractable in 0.5 M NaCl. Lane I, electrophoretic banding pattern of DTT-eluted nucleosomal proteins. These nucleosomes had been passed through Sepha- cryl S-200 (to remove proteins of M, below 200,000) before Hg affinity chromatography. Note the three prominent bands at 35, 37, and 39 kDa. Lane 2, banding pattern of proteins in the DTT-eluted nucleosome fraction after exposure of the S fraction to 0.5 M NaCl and removal of the released proteins on Sephacryl S-200 before Hg affinity chromatography. Note the absence of the protein bands of M, 35,000, 37,000, and 39,000. Lane 3, electrophoretic pattern of the proteins released by 0.5 M NaCl and separated from the nucleosomes by Sephacryl S-200 chromatography. Note the presence of the 35-, 37-, and 39-kDa proteins.

49% of the H4 molecules occur in their tri- and tetra-acetylated forms; this is 53% higher than the tri- and tetra-acetyl- H4 content of nucleosomes eluted in 0.5 M NaCl. In COLO 320 cells, the tri- and tetra-acetyl H4 content of the DTT- eluted nucleosomes is 65% higher than that of the nucleosomes eluted in 0.5 M NaCl. Given that both of the Hg-bound fractions contain the transcribed DNA sequences of rat liver, HeLa, and 3T3 cells (Fig. 7), these results place the hyperacetylated forms of histone H4 in both classes of transcriptionally active chromatin subunits. This confirms earlier observations on the high levels of histone acetylation in the total Hg-bound nucleosome fractions of liver (1) and HeLa cells (2). The link between hyperacetylation and transcription is further strengthened by the observation that nucleosomes containing the transcribed oc-D-globin DNA sequences of avian red cells are immunoprecipitated by antibodies specific for hyperacetylated histone H4 (38). The very high levels of histone tri- and tetra-acetylation in the DTT-eluted nucleosomes which are bound to the column through their histone H3-thiol groups (see below), are also in accord with the finding that hyperacetylation of the nucleosomes uncovers histone HS-thiol groups (40). All these findings are consistent with

TABLE II Histone and HMG protein content of mercury-bound

nucleosome fractions

Protein Proportion” in nucleosomes eluted in

0.5 M NaCl 10 mM dithiothreitol

Rat liver Histone H3 Histone H4 Histone H2A Histone H2B

COLO 320 cells Histone H3 Histone H4 Histone H2A Histone H2B HMG-1 HMG-2

2.0 + 0.03 2.18 + 0.04 1.89 + 0.14 1.97 + 0.06 2.09 f 0.08 2.07 k 0.02 1.85 r 0.14

2.0 zk 0.04 2.05 f 0.15 1.86 f 0.07 2.00 -c 0.02

2.0 f 0.11

2.0 + 0.03 1.89 k 0.14 1.66 -c 0.07 1.94 + 0.06 1.14 + 0.02 1.21 + 0.02

a Determined by densitometry of Procion Blue-stained proteins following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densities were corrected for differences in dye binding of each histone fraction and are expressed relative to those observed in the corresponding proteins of the unbound nucleosome fraction which contain two molecules of each histone in stoichiometric proportions. Average of three determinations rtr S.D.

TABLE III Histone H4 acetylation levels in nucleosome fractions separated by

two-stage Hg affinity chromatography Prooortion of histone containine”

Cell type Nucleosome fraction 0 1 2 3

4 N-Ace- tyl groups

HeLa HeLa HeLa

% % % % % Peak 1 61 38 1 ND* ND Peak 2 20 31 16 10 22 Peak 3 11 21 15 18 31

COLO 320 Peak 1 20 40 3 30 8 COLO 320 Peak 2 26 21 15 34 3 COLO 320 Peak 3 14 13 11 15 46

’ As determined by laser-scanning densitometry of the Coomassie- stained histone H4 isoforms separated by electrophoresis on acid- urea-Triton gels (26).

* ND, not detectable.

the hypothesis that acetylation of the histones facilitates transcription by increasing DNA accessibility to RNA polym- erases (39).

Role of Histone H3-Thiols in Nucleosome Binding to the Mercury Column-The isolation of transcriptionally active nucleosomes by Hg affinity chromatography was originally suggested by observations on ribosomal gene chromatin showing that the thiol groups of histone H3 react with SH reagents only when the rDNA is being transcribed (4). The presence of SH reactive H3 molecules in the active chromatin has been amply confirmed in a variety of cell types (1, 2, 5, 28) and also demonstrated in the isolated nucleosomes of active ribosomal genes (4).

We have now compared the reactivity of the H3-thiols in the different nucleosome fractions separated by Hg affinity chromatography. Iodo[“H]acetate was used to label the histone H3-thiols of each nucleosome fraction following its elution from the mercury column. Comparisons of the electrophoretically purified H3 bands show that the accessibility of the histone HS-thiol groups differs greatly in the Hg-bound and unbound nucleosomes. In rat liver, for example, the 3H activity of the carboxymethylated H3 in the DTT-eluted nucleosomes (847 dpm/pg) was seven times greater than that of histone H3 in nucleosomes of the salt-eluted fraction (120

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dpm/pg), and no H3-thiol reactivity was detected in nucleosomes that did not bind to the mercury column. The 7-fold difference in H3 reactivity between the salt-eluted and DTT- eluted nucleosome fractions indicates a much greater confor- mational change in the latter. The great disparity in H3-thiol reactivity of the salt-eluted and DTT-eluted nucleosomes was also evident in HeLa nucleosome fractions, as shown by fluorography and densitometry of the “H-carboxymethylated H3 band separated by SDS-polyacrylamide gel electrophoresis (Fig. 6A).

Although the reactivity of H3-thiols in the DTT-eluted nucleosomes is high, there has been no direct chemical evidence that those accessible HS-thiols become covalently linked to the mercury column. (Clearly, Hg binding to histone HS-SH groups cannot account for the nucleosomes which are eluted from the column in 0.5 M NaCl and whose retention

FIG. 6. Comparison of histone H3-thiol reactivity in nucleosome fractions separated by Hg affinity chromatography. A, nucleosomes from HeLa cell nuclei were applied to the organomer- curia1 column, eluting the Hg-bound nucleosomes successively in 0.5 M NaCl and 10 mM DTT, as shown in Fig. 2. Each of the Hg-bound nucleosome fractions was reacted with iodo[“H]acetate under identi- cal conditions in order to carboxymethylate the accessible thiol groups of the constituent proteins. The proteins were extracted, separated by SDS-polyacrylamide gel electrophoresis, and their “H activities detected by fluorography. The densitometric tracing of the histone region of the fluorogram shows little ‘H-carboxymethylation of histone H3 in the 0.5 M NaCl-eluted nucleosomes (a). In contrast, the H3-thiols of the DTT-eluted nucleosomes react with iodo[3H]acetate (b), indicating unfolding of the nucleosome cores in that fraction. B, inaccessibility of histone HB-thiols when the DTT-elutable nucleosomes are still attached to the mercury column. The Hg-bound nucleosomes of HeLa cells were reacted on the column with iodo[RH] acetate and then eluted in one step with 10 mM DTT (lane I), or in two steps as shown in Fig. 2E, analyzing the proteins of the DTT- eluted fraction. Fluorography of the electrophoretically separated protein bands shows negligible ‘H-carboxymethylation of the histone H3 bands when nucleosomes are bound to the Hg column (lanes 1 and 2). In contrast, nucleosomes eluted in DTT and then reacted with iodo[YH]acetate show reactivity of their H3-thiols (lane 3). C, H3-thiol groups of Hg-bound nucleosomes cannot be blocked by nonradioactive iodoacetate. Equal aliquots of COLO 320 nucleosomes were applied to parallel mercury columns, and the unbound and 0.5 M NaCl-elutable nucleosome fractions were removed. One column was then eluted with 10 mM DTT in the usual way, and the released nucleosomes were reacted with iodo[“H]acetate. The 3H-carboxymethylated H3 molecules were detected by-fluorography of the protein bands in SDS-oolvacrvlamide gel electroohoresis gels (lane I). The second columnwas treated with non-radibactive iodoacetate prior to elution of the nucleosomes, which were then displaced by DTT and reacted with iodo[“H]acetate. Subsequent fluorography of the electrophoretically separated H3 band shows that the H3-thiols were reactive (lane 2), and therefore, could not he blocked by nonradioactive acetate when the nucleosomes were bound to the organomercurial.

depends on salt-labile interactions with thiol-reactive nonhistone proteins.)

If the SH groups of histone H3 participate directly in the Hg binding of the DTT elutable nucleosomes, those groups will not be accessible to SH reagents, such as iodoacetate, as long as the nucleosomes remain linked to the Hg column; but they would become accessible after the nucleosomes were displaced from the column. Therefore, to test for the covalent linkage of histone HS-thiols to the organomercurial, we compared the reactivity of H3-thiols with iodo[“H]acetate when HeLa nucleosomes were bound to the Hg column (on-column labeling), with that of histone H3 in nucleosomes which were reacted with iodo[“H]acetate after their elution (off-column labeling). The mercury-bound nucleosomes showed very little “H-carboxymethylation of their constituent histone H3 molecules as compared with the extent of “H-carboxymethylation of histone H3 in nucleosomes after their release from the column (Fig. 6B).

Further evidence that the H3-thiols of the Hg-bound nucleosomes are linked covalently to the organomercurial was provided by “SH-blocking” experiments. Equal aliquots of COLO 320 nucleosomes were applied to parallel Hg columns, and the unbound and salt-labile nucleosome fractions were removed. One column was eluted with 10 mM DTT in the usual way, while the second column was treated.with 15 mM nonradioactiue iodoacetate to block any accessible H3-thiol groups, before eluting the nucleosomes in 10 mM DTT. The histones of both samples were reacted with iodo[“H]acetate to carboxymethylate the accessible H3-thiols. The radioactivity of the electrophoretically separated H3 bands was compared by fluorography. The results (Fig. 6C), showing the equivalent in “H-carboxymethylation of the two H3 samples, despite the prior exposure of one set of Hg-bound nucleosomes to excess nonradioactive iodoacetate, indicate that the H3- thiols could not be blocked by the SH reagent as long as the nucleosomes were bound to the Hg column.

We conclude that the H3-thiols of the DTT-elutable nucleosomes are covalently linked to the mercury column. The validity of this conclusion was confirmed by control experiments showing that (a) pure histone H3 is not displaced from the mercury column by iodoacetate, and (b) on-column labeling of bound histone H3 by iodo[“H]acetate is negligible.

Transcribed DNA Sequences in the Mercury-bound Nucleo- some Fractions-We have shown that nucleosomes retained by the mercury column and eluted in one step by dithiothreitol contain the transcriptionally active DNA sequences of rat liver, HeLa, and 3T3 cells (l-3). Here we examine whether the nucleosome fractions that differ in their mode of Hg binding both originate in the active genes of the cell type examined.

We tested the DNA sequences of the salt-eluted and DTT- eluted nucleosomes of BALB/c 3T3 cells with probes for c- fos and c-myc genes. The response of quiescent 3T3 cells to fresh serum involves a transient activation of c-fos and c-myc as the cells reenter the growth cycle. Run-off transcription assays show that c-fos is activated almost immediately; it peaks at 15 min and then declines abruptly, returning to the initial low level of expression by 30 min. The transcription of c-myc begins at about 30 min, is maximal at about 60 min, and declines to preactivation levels in 3-4 h (3, 41). This reproducible program of sequential gene activation and repression allows a comparison of the nucleosome structures of the same gene in its active and inactive states. The distribution of the c-fos DNA sequences in the chromatographically separated nucleosome fractions changes dramatically when the oncogene is activated. At zero time, virtually all of the c-

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fos DNA appears in the unbound nucleosome fraction, but at 15 min the c-fos sequences are recovered in the Hg-bound nucleosomes. The transcribed c-fos DNA appears in both the salt-eluted and DTT-eluted nucleosome fractions (Fig. 7A).

nucleosome fraction (82.5 density units/pg DNA) and a 4- fold enrichment in the DTT-eluted nucleosomes (21.3 density

A similar shift of c-myc DNA from the unbound to the mercury-bound nucleosome fractions is seen at 60 min (Fig. 7R). By that time, c-fos transcription has been repressed. It has been shown that c-fos DNA is not present after 30 min in the Hg-bound nucleosome fraction eluted in one-step with 10 mM DTT (3). Because that fraction includes both of the nucleosome classes separated by the two-step elution procedure, it follows that binding of nucleosomes to the mercury column depends upon the transcription of the associated DNA sequences, and is rapidly reversible for both classes of Hg- bound nucleosomes. This implies that, when transcription ceases, two modifications of chromatin structure must take place: 1) whatever proteins are responsible for Hg binding of the salt-labile nucleosome fraction are released, and 2) the nucleosomes with accessible H3-thiol groups revert to a compact configuration in which the SH groups are shielded. In the c-fos gene, these changes can occur within 15 min (3).

The recovery of transcribed DNA sequences in both the salt-eluted and DTT-eluted nucleosomes has been observed in other cell types and is illustrated for the albumin and transferrin genes of rat liver in Fig. 7C. Densitometric analyses of the slot-blot hybridizations for albumin gene DNA sequences show a 13-fold enrichment in the 0.5 M NaCl-eluted

Time (min) 1 2 3

A O- 15 c g&

C D

1

z

::

2 c & :.: i'y& '@A@ .*. :&+;

3e w

*Ar _. +W

Alb Tran rDNA c-myc H4

FIG. 7. Enrichment of transcriptionally active DNA sequences in the mercury-bound nucleosome fractions of mammalian cells. DNAs prepared from the unbound (peah 1), 0.5 M NaCl-eluted (per& 2), and 10 mM DTT-eluted (peak 3) nucleosomes of three different cell types (mouse 3T3 cells, rat liver, and human (HeLa) cells) were probed for their DNA sequence content by slot- blot hybridizations to the indicated “‘P-labeled DNA probes. A, comparison of C-/OS DNA distribution in nucleosome fractions from quiescent 3T3 cells when c-fos is repressed (0 time) and from cells at the peak of c-fos transcription (15 min). Lane 1, nucleosomes of peak 1; lane 2, peak 2; lane 3, peak 3. Note the shift in c-fos DNA from the unbound to the Hg-bound nucleosome fractions when c-fos is activated and that both the NaCl-eluted and DTT-eluted nucleosome fractions contain the transcriptionally active DNA sequences. The c- fos DNA reappears in the unbound nucleosomes when transcription is terminated (3). B, comparisons of c-myc DNA distribution in nucleosome fractions from quiescent 3T3 cells when c-myc is repressed (0 time) and when it is fully active (60 min). Lanes l-3, as described in panel A. Note that the c-myc signal appears in both the NaCl-eluted and DTT-eluted nucleosome fractions at t = 60 when c- myc is actively transcribed, but not at 1= 0. C, enrichment of albumin and transferrin gene DNA sequences in the Hg-bound nucleosome fractions of rat liver. DNA of peaks l-3 in descending order. D, distribution of c-myc and histone H4 gene DNA in the Hg-hound nucleosome fractions of HeLa cells. DNA of peaks 1-3 in descending order.

units/pg DNA), as compared with the unbound nucleosome fraction (6.31 density units/pg DNA). Transferrin DNA is also enriched in both of the Hg-bound nucleosome classes; 34-fold enrichment in the 0.5 M eluted fraction (45.7 density units/pg DNA), and g-fold in the DTT-eluted nucleosomes (12.1 density units/pg DNA), as compared with the unbound nucleosome fraction (1.3 density units/pg DNA). When genes are not fully expressed, as in the case of the multiple ribosomal genes of hepatic cells, the enrichment of rDNA sequences in the Hg-bound nucleosomes is not as pronounced (Fig. 7C). This is also the case for the c-myc genes of HeLa cells (Fig. 70).

In synchronized HeLa cell cultures, the histone H4 gene DNA is greatly enriched in the Hg-bound nucleosomes during its period of maximal transcription in the S-phase of the cell cycle (2), but this enrichment of H4 DNA is not as evident in a randomly dividing cell population with fewer cells in the S- phase (Fig. 70).

We conclude that two-stage Hg affinity chromatography

A a

GAL HISI AFT?

FIG. 8. Hg affinity chromatography of yeast nucleosomes. Nucleosomes released by a limited micrococcal nuclease digestion of spheroplast nuclei were applied to the organomercurial column. After washing to remove the unbound nucleosomes, the column was eluted successively with buffered 0.5 M NaCl and 10 mM DTT. Elution was monitored by absorbance at 260 nm, and Hoechst DNA assays. DNA sizing experiments (insets) show the presence of nucleosomal arrays in both the unbound and salt-eluted fractions, but no nucleosomes are detectable in the DTT-eluted material. A, nucleosomes of cells grown in glucose-containing medium. B, nucleosomes of cells grown in galactose-containing medium. C, distribution of specific yeast DNA sequences in nucleosomes separated by Hg affinity chromatography, as determined by slot-blot hybridizations to ‘“P-labeled DNA probes for the GAL, HIS4, and ACT1 genes. a, comparisons of the GAL DNA sequence contents of unbound (peak 1), 0.5 M NaCl-eluted (peak 2), and DTT-eluted (peak 3) fractions of yeast cells grown in glucose- containing medium in which the GAL loci are repressed. b, comparisons of the GAL DNA sequence contents of unbound (peak I), 0.5 M NaCl-eluted (peak 2), and DTT-eluted (peak 3) fractions of yeast cells grown in galactose-containing medium when the GAL loci are active. No enrichment of the GAL signal is seen in either of the Hg- bound nucleosome fractions; and there is no enhancement of the GAL signal in the Hg-bound nucleosomes during the shift from glucose to galactose-containing media (see Table IV). c and d, comparisons of the HIS4 DNA sequence contents of the unbound (peak l), 0.5 M NaCl-eluted (peak 2), and DTT-eluted (peak 3) fractions. No enrichment of this active gene is seen in the Hg-bound fractions of cells grown in glucose (c) or galactose (d) (see Table IV). e and f, comparisons of the ACT1 DNA sequences contents of the unbound (peak 1) 0.5 M NaCl-eluted (peak 2), and DTT-eluted (peak 3) fractions. No enrichment of this active gene is seen in the Hg-bound fractions of cells grown in glucose (e) or galactose (f) (see Table IV).

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Nucleosome fraction

TABLE IV Distribution of GAI, HIS4, and ACT1 DNA in yeast nucleosome fractions

Growth medium Gene probe DNA/fraction Gene distribution

Pee gene/g DNA” gene/fraction* 7% of total Unbound nucleosomes Gal + GAL 8,475 2.42 20,480 90.2

0.5 M NaCl-eluted fraction Gal + GAL 1,199 1.29 1,540 6.8 DTT-eluted fraction Gal + GAL ‘413 1.66 .687 3.0

Unbound nucleosomes Gal - GAL 6,664 1.16 7,722 82.3 0.5 M NaCl-eluted fraction Gal - GAL 2,538 0.642 1,630 17.4 DTT-eluted-fraction Gal - GAL 373 0.071 26 0.3

Unbound nucleosomes Gal + HIS4 8.475 3.44 2.913 96.3 0.5 M NaCl-eluted fraction Gal + HIS4 11199 0.330 40 1.3 DTT-eluted fraction Gal + HIS4 413 1.78 73 2.4

Unbound nucleosomes Gal + ACT1 8.475 1.19 10.106 90.8 0.5 M NaCl-eluted fraction Gal + ACT1 1;199 0.492 ‘590 5.3 DTT-eluted fraction Gal + ACT1 413 1.07 440 3.9

‘As determined by hybridization of 10 pg of DNA to the 32P-labeled DNA probe and densitometry of the autoradiogram. Radioactivity expressed as density/slot-blot x 10-6/pg DNA (average of three determinations.

’ Product of total DNA/fraction x radioactivitylpg DNA.

provides direct access to two classes of isolatable nucleosomes containing transcribing DNA sequences, and that the thiol reactivity of active nucleosomes is a complex phenomenon which can be attributed to proteins associated with the transcribed linker DNA, as well as to the histone H3 molecules of the unfolded nucleosome cores.

Hg Affinity Chromatography of Nucleosomes Fails to Select for Transcriptionally Active DNA Sequences of Yeast

Histone H3 of yeast lack cysteinyl residues (42) and therefore cannot bind to the column by covalent linkage of thiols to the organomercurial. This fundamental difference between the H3 histones of yeast and mammalian cells suggested a direct test of the hypothesis that the accessibility of HS-thiols is a key factor in the binding of transcriptionally active nucleosomes to the mercury column.

The great difference in expression of the GAL genes when S. cereuisiae is cultured in galactose-containing media, as compared with glucose-containing media (21), provided the necessary base for comparing the nucleosome structures of the same gene in its active and inactive states. Nucleosomes were prepared by micrococcal nuclease digestion of spheroplast nuclei from both cultures and applied to the mercury columns in the usual way. After elution of the unbound nucleosome fraction, the columns were washed successively with 0.5 M NaCl and 10 mM DTT, following the protocols developed for the separation of active and inactive mammalian nucleosomes.

Although a detectable amount of Hg binding was observed, and arrays of nucleosome-sized DNAs were evident in the 0.5 M NaCl-eluted fraction, no nucleosomes were detected in the DTT-eluted fraction (Fig. 8). This is in accord with the absence of histone H3-thiols in yeast. We conclude that the small amount of DNA in the DTT-eluted fraction is retained because of aggregation on the column matrix or its association with other proteins. The nucleosomes released by 0.5 M NaCl must have been retained through salt-labile interactions with thiol-reactive nonhistone proteins, but the latter could not have been HMG proteins which, in yeast, lack cysteinyl residues (23,43).

The DNA recovered from each fraction was analyzed for its content of GAL DNA sequences by quantitative slot-blot hybridizations to the 32P-labeled GAL probe. The results (Fig. 8C and Table IV) show that there is no enrichment of the GAL loci in the Hg-bound nucleosomes of yeast cells grown in galactose-containing media. It is also evident that the great

difference in GAL transcription between cells grown in glucose or galactose (21) cannot be detected in the Hg-bound fractions of the different cultures.

Similar tests using probes for the actively transcribed HIS4 and ACT1 genes confirm that there is no enrichment of the active genes in the Hg-bound fractions (Fig. 8C and Table IV).

This striking difference in the chromatographic behavior of yeast nucleosomes, as contrasted to mammalian nucleosomes in which histone H3 contains a cysteinyl residue, lends support to the original hypothesis that unfolding of the nucleosomes during transcription to reveal the previously shielded thiol groups of histone H3 is responsible for the Hg binding of transcribed DNA sequences (1). The chemical evidence for the covalent attachment of histone HS-thiols to the mercury column supports this view; but it is equally clear that other modes of binding exist which do not involve a direct participation of histone H3. Other proteins are responsible for the Hg binding of a variable, but always significant fraction of the transcribed DNA sequences of mammalian cells.

Those proteins do not seem to be present or enriched in the transcribed nucleosomes of yeast cells, or, if present on the isolated nucleosomes, they do not contain SH groups that are accessible to the organomercurial functions of the column.

Conclusion

We have shown that the nucleosomes of active genes in mammalian cells are altered in two ways that permit their isolation by mercury affinity chromatography: 1) a change in conformation of the nucleosome core makes the histone H3- thiol groups accessible for mercury binding, and 2) a transient association with other SH-reactive proteins, possibly HMG- 1 and HMG-2 on the DNA linkers, accounts for the retention on the Hg column of other transcriptionally active nucleosomes. The latter are released from the column when the Hg- binding proteins are detached at high ionic strengths (0.5 M NaCl). After removal of the salt-labile nucleosomes, the nucleosomes with reactive HB-thiol groups are displaced from the column by dithiothreitol. Both classes of nucleosomes contain transcriptionally active DNA sequences, but their chromatographic behavior is dynamically coupled to transcription, as shown for the c-fos and c-myc genes of 3T3 cells.

There are other differences between the Hg-bound nucleosome fractions, such as the lower resistance to endonuclease digestion of the DTT-eluted nucleosomes that suggest a higher content of single-stranded DNA. This is consistent

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with recent observations on the presence of topoisomerase I/ DNA adducts in the DTT-eluted nucleosome fraction.3

The contrasting chromatographic behavior of the transcriptionally active nucleosomes of yeast rules out their isolation by this technique. But it also raises interesting and important questions about the essential need for HS-thiols in the separation of active from inactive nucleosomes of mammalian cells. New techniques that are independent on mercury binding are now being applied to the problem.

Acknowledgments-We are grateful to Dr. Ann Ho for her advice and assistance in computer-assisted plotting and statistical analysis of the data. The polyclonal antibody to high mobility group protein HMG-1 was generously provided by Drs. C. S. Teng and C. T. Teng.

1.

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4.

5.

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