DNase I sensitivity of ribosomal genes in isolated nucleosome core ...

volume 8 Number 11980 Nucleic Ac ids Research

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

Chandrakant P.Giri and Martin A.Gorovsky

Department of Biology, University of Rochester, Rochester, NY 14627, USA

Received 21 August 1979

ABSTRACT

The level of chromatin structure at which DNase I recognizes conforma-tlonal differences between inert and activated genes has been investigated.Bulk and ribosomal DNA's of Tetrahymena pyriformis were differentiallylabeled in vivo with [1%C]- and [3H]-thymidine, respectively, utilizinga defined starvation-refeeding protocol. The H-labeled ribosomal geneswere shown to be preferentially digested by DNase I in isolated nuclei.Staphylococcal nuclease digested the ribosomal genes more slowly than bulkDNA, probably owing to the higher GC content of rDNA. DNase I and staphylo-coccal nuclease digestions of purified nucleosomes and of nucleosome coreparticles isolated from dual-labeled, starved-refed nuclei were indistin-guishable from those of intact nuclei. We conclude from these studies thatDNase I recognizes an alteration in the internal nucleosome core structureof activated ribosomal genes.

INTRODUCTION

Most of the DNA in eukaryotic nuclei is transcriptionally inert (see 1

for a review) and exhibits both periodic and particulate (nucleosome) struc-

ture. Non-specific nucleuses, particularly staphylococcal nuclease and

pancreatic DNase I, have been especially useful in probing the structure

of chromatin (see 2-4 for reviews). Staphylococcal nuclease digests bulk

chromatin in five stages (5). The first stage is cleavage between groups

of about eight nucleosomes (6-8). The second stage involves preferential

cleavage between nucleosomes to produce an oligomeric series of particles

from which an oligomeric series of DNA molecules can be extracted. This

basic repeating unit contains 160-240 base pairs (bp) of DNA depending on

the cell type. Further digestion with staphylococcal nuclease produces a

third, metastable intermediate containing about 160-168 bp of DNA (5,9)

recently named a chromatosome (5). Continued digestion results in a some-

what more stable intermediate referred to as the nucleosome core or core

particle which invariably contains about 145 bp of DNA. Nucleosomes and

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Nucleic Acids Research

chromatosomes contain 2 molecules each of histone H2A, H2B, H3 and H4 (the

four Inner hlstones) and one molecule of HI. Core particles lack HI.

Finally, extensive digestion leads to the so-called limit digest where

cleavage occurs at specific sites within nucleosome cores to give a series

of DNA fragments 145-40 bp in length differing in size by 10-20 bp.

Pancreatic DNase I shows less specificity than staphylococcal nuclease

for internucleosome regions. This enzyme appears to digest both inter- and

Intranucleosome regions of chromatin to give a series of DNA fragments whose

lengths are multiples of approximately 10 bases when analyzed under denatur-

ing conditions. Since this 10b series can be obtained by digestion of

isolated nucleosome cores (but not from free DNA), DNase I is a sensitive

probe of the internal structure of nucleosome cores.

Staphylococcal nuclease and DNase I have also been used to study the

changes (or lack thereof) in chromatin structure which occur during gene

activation. There is general agreement that presumably active rlbosomal

(10-21) and non-ribosomal (19,22-26) genes show both periodic and partlcu-

late structure after digestion with staphylococcal nuclease. The periodicity

of the presumably active genes is indistinguishable from that of bulk chroma-

tin (10,11,19,26). There is some controversy over whether active genes show

the same susceptibility as bulk chromatin to staphylococcal nuclease.

Earlier studies suggested both types of genes were digested at the same

rates. More recent studies indicate that active genes are more susceptible

to brief staphylococcal nuclease digestion than bulk genes (13,17,18,26-28).

He have found (Vavra, Girl, Bowen and Gorovsky, unpublished observations)

that the relative rates of digestion of presumably active genes In

Tetrahymena macronuclei are extremely sensitive to digestion conditions,

possibly explaining some of these differences.

There are few studies on the effects of more extensive digestion of

active genes by staphylococcal nuclease. Mathis and Gorovsky (29) have

reported that the pattern of DNA fragments derived from Tetrahymena ribo-

somal genes after extensive digestion with staphylococcal nuclease differs

slightly from that of bulk chromatin, suggesting that the structure of the

nucleosome cores containing this presumably active gene sequence may be

altered.

In contrast to staphylococcal nuclease, DNase I clearly distinguishes

between chromatin containing active or inactive genes. Welntraub and

Groudlne (24) demonstrated that globin genes in chick erythrocyte nuclei

were preferentially digested by DNase I, but not by staphylococcal nuclease.

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They also showed that both actively transcribed genes (globin sequences •In

embryonic cells) and genes which had stopped transcribing (globin sequences

in adult erythrocytes) were preferentially digested by DNase I. Miller et al.

(30) have shown that globin genes in Friend erythroleukemia cells are prefer-

entially digested by DNase I prior to induction of globin mRNA synthesis.

These results, and related studies on ovalbumin gene expression in chick

oviduct (31,32), suggest that the DNase I sensitive state is not simply

related to the process of transcription. Rather, DNase I probably recognizes

a conformational state that is necessary, but not sufficient, for transcrip-

tion to occur. We shall therefore refer to genes which are preferentially

digested by DNase I as transcriptionally activated rather than active. These

initial observations on the preferential digestion of activated genes have

since been extended to include both ribosomal (29,33,34), and non-ribosomal

(35—40) genes in other species.

The molecular basis by which DNase I distinguishes activated genes is

not known. The altered conformation of activated chromatin could occur at

either of three levels of chromatin structure. DNase I could recognize a

change in the higher order packing of nucleosomes which occurs in regions of

activated chromatin. In this case, the internal structure of nucleosomes

(or nucleosome cores) containing activated sequences would not differ from

that of bulk chromatin, and DNase I would not preferentially digest activated

genes in isolated nucleosomes. Alternatively, since DNase I is a sensitive

intranucleosome probe, it could recognize an alteration in the Internal core

structure of activated nucleosomes. In this case, DNase I should preferen-

tially digest DNA sequences in isolated core particles which are derived

from activated genes. Finally, it is possible that the DNase I - sensitive

conformation of activated genes requires some or all of the linker region

or the 20 bp of DNA (and/or HI) that distinguishes chromatosomes from core

particles. In this last case, activated sequences in isolated nucleosomes

(or chromatosomes) would be DNase I sensitive while those in nucleosome

cores would not.

Conflicting results have been published regarding the DNase I sensitivity

of activated genes in isolated nucleosomes. Weintraub and his associates

(24,39) have maintained that preferential DNase I digestion of both globin

and Integrated viral genes can be demonstrated in isolated nucleosomes. It

should be pointed out, however, that their preparations of isolated nucleo-

somes were not, in fact, purified nucleosome monomers. Rather, they were

solubilized chromatin from staphylococcal nuclease-digested nuclei which,

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when analyzed on sucrose gradients, contained about 80% nucleosome monomers.

Garel and Axel (23), on the other hand, were unable to preserve the DNase I -

sensitive state of ovalbumin gene sequences either in isolated nucleosomes

of the type studied by Weintraub and his colleagues, or in sucrose gradient

purified nucleosome monomers.

In an attempt to determine the level of chromatin structure at which

DNase I distinguishes between activated and bulk chromatin, we have compared

the DNase I sensitivity of Tetrahymena ribosomal genes and bulk chromatin

in isolated (macro-) nuclei, sucrose gradient purified mononucleosomes and

purified nucleosome cores. Our data demonstrate that the structural features

which DNase I recognizes in activated ribosomal genes occur at the level of

nucleosome core part icles.

MATERIALS AND METHODS

Cel l c u l t u r e and l a b e l i n g

Stock c u l t u r e s of Tetrahymena pyriformis (formerly amlcronucleate s t r a i n

GL) were grown in enriched proteose peptone medium as descr ibed previous ly

(41). Cells were differentially labeled using a previously described modi-

fication (10) of the defined starvation-refeeding protocol of Engberg et^ a l .

(42). Bulk cellular DNA was labeled during exponential growth with

[methyl-^Cj-thymidine (0.17 yCi/ml of culture). rDNA was preferentially

labeled with [methyl-3H]-thymidine (6.25 yCi/ml) during a 2-hour refeeding

following 24-hours of starvation in 10 mM tris-HCl, pH 7.5. Nuclei were

isolated from these double-labeled cells as previously described (41) and

were stored frozen at -20°C. Except where stated, a l l buffers contained

0.2 mM phenylmethyl-sufonyl fluoride (PMSF) to inhibit proteolysis.

Isolation of mononucleosomes

Nuclei were washed twice with RSB (10 mM tris-HCl, pH 7.5, 3 mM HgCl,,

10 mM NaCl) containing 0.5Z NP-40 and once with RSB alone. Washed nucleiQ

were resuspended in RSB at a concentration of approximately 10 nuclei/ml.

CaCl_ was added to a final concentration of 0.1 mM. The nuclear suspension

was allowed to equilibrate at room temperature for about 5 minutes and was

then digested with staphylococcal nuclease (Worthington, 400 units/ml) for

15 minutes. The reaction mixture was then chilled and spun immediately at

1500 x g for 10 minutes. The nuclear pellet was washed twice with RSB and

then lysed with 10 mM Na-EDTA, pH 7.5, followed by centrifugation as above.

The RSB and EDTA supernatant fractions were then fractionated on linear

5-20Z sucrose gradients containing tris-EDTA (10 mM tris-HCl, pH 7.5, 1 mM

200



Na2EDTA) or RSB. Centrifugation was at 35,000 rpm for 10 hours In a Spinco

50.1 rotor at 4°C. Five-drop fractions were collected utilizing a Buchler

Auto Densiflow. Aliquots of each fraction were counted in ACS (Amersham-

Searle) or in Liquiscint (National Diagnostics). Appropriate fractions

containing mononucleosomes were pooled and concentrated by dialysis (see

below) .

Isolation of nucleosome cores8

Double labeled nuclei (2 x 10 /ml RSB) were digested with staphylococcal

nuclease (400 units/ml, 30 minutes at room temperature). Digestion was ter-

minated by addition of 200 mM Na2EDTA, pH 7.5, to a final concentration of

10 mM, and the reaction mixture was chilled on ice for about 10 minutes.

The digest was spun (1500 x g for 10 minutes) and the pellet washed with

10 mM tris-HCl, pH 7.5, 10 mM EDTA. The combined supernatants were adjusted

to 0.1 M KC1 by addition of 1.0 M KC1 and stirred for 30 minutes at 0°C. The

KCl-treated chromatin was spun at 16,000 x g for 10 minutes and the super-

natant was purified by centrifugation on sucrose-tris-EDTA gradients as

described above except that centrifugation was for 12 hours at 37,000 rpm

in an SW 41 rotor. This KCl-solubilization procedure has been shown to

result in a relatively homogeneous preparation of nucleosome core particles

(5,43-46) .

CsCl-Hoechst gradients

DNA was Isolated from undigested and from slightly DNase I digested

dual-labeled nuclei and analyzed on CsCl-Hoechst gradients as previously

described (10,29). The percentage of H-radioactivity attributable to rDNA3 14in each gradient was calculated by determining the H/ C ratio at points

3 14

along the lighter edge of the H peak, multiplying the total C counts by

this ratio and subtracting this value from the total H counts in the

gradient. This analysis Is based on two reasonable assumptions: 1) the

lighter edge of the H-peak does not contain rDNA, and 2) the density dis-

tribution of 3H-labeled and ^C-labeled bulk DNAs are identical.

The fraction of rDNA digested was then calculated from the CsCl

gradient patterns of partial digests according to the following formulae./Fraction of \ /Fraction of \

(1) Fraction rDNA [starting rDNAI (rDNA at zero]remaining - yemalnlng / \ timeafter digestion/Fraction of \/Fraction of \ /Fraction oft/Fraction of\

(starting rDNAHrDNA at zerol-Hbulk DNA Ibulk DNA a t l\remaining /Vtime / \remaining .'vero time /

201



(2) Fraction rDNA digested » 1 - (Fraction of starting rDNA remaining)

Dialysis o£ nucleosomes and core particles

Sucrose gradient purified mononucleosomes or core particles were

dialyzed againat RSB at 4°C for 16 hours using a Spectrapore No. 6 dialysis

membrane (MW cutoff - 2000). In some experiments the particles were diacon-

centrated by vacuum dialysis using a collodion bag (25,000 MW cutoff;

Schleicher and Schuell, Inc., No. 100). Similar results were obtained in

either case.

Kinetic analyses

Isolated nuclei, RSB soluble nucleosomes, dialyzed nucleosomes and core

particles were digested with DNase I at room temperature in RSB. At indi-

cated time intervals, the percentages of radioactivity soluble in 5Z TCA-1

M NaCl-5 mM inorganic pyrophosphate were determined by counting aliquots of

the reaction mixture on triplicate filters as described previously (47).

Staphylococcal nucleate digestions were performed in RSB containing 0.1 mM

CaCl2.

RESULTS

Tetrahymena ribosomal genes (rDNA) can be preferentially labeled u t i l i z -

ing a defined starvation-refeeding scheme modified slightly from that origin-

ally developed by Engberg et_ al. (42). This procedure yields nuclei in14

which the bulk. UNA has been labeled with C-thymidine during exponential

growth while rDNA is preferentially labeled with H-thymidine during a 2-hour

refeeding period. When DNA from these double-labeled nuclei is analyzed on

CsCl-Uoechst equilibrium density gradients (Fig. 1A) , we routinely find that

50-601 of the tritium label bands as rDNA although the rDNA is only about

2Z of the total genome. Thus, by comparing the nuclease digestion properties3 14

of H and C labeled chromatin, we are mainly comparing the properties of

a specific activated gene (see below) with that of bulk., largely inactive,

chromatin. I t should be noted, however, that neither isotope exclusively14

labels a single type of chromatin: C-labeled bulk DNA contains activated

sequences (48) while approximately 50Z of the H-label is in bulk DNA

Fig. 1A).

Nuclease digestion of intact nuclei

A clear picture of the kinetics of digestion of rDNA and bulk DNA in

intact nuclei is an essential prerequisite to studies on isolated nucleo-

somes and nucleosome cores. He have previously presented brief comparisons

of the kinetics of ataphylococcal nuclease and DNase I digestion of rDNA and

202



-o

FRACTION

Figure 1_. Equilibrium centriguation of nuclear DNA from starved-refedcells in CsCl-Hoechst density gradients. CsCl (A.5 ml, startingdensity D 1.638) containing approximately 1 UR Hoechst 33258 perpg DNA was centrifuged in a Beckman Type 50 rotor for 24 hr at42,000 rpm followed by 48 hr at 33,000 rpm. Fractions were col-lected from the top of the gradient using a Buchler Auto-Densiflow.(• • [1*C]-thymidine labeled bulk DNA; • - • 3H-thymidine labeledstarved-refed, DNA). (J) Undigested;® 1"C digested to 6Z TCAsoluble, rDNA 31Z digested;© "*C digested to 9Z TCA soluble, rDNA55Z digested.

bulk DNA in isolated nuclei (10,29). These studies have been extended and

have been performed under conditions (digestion buffer, method for determin-

ing TCA solubility, etc.) which parallel those used for purified chromatin

subcomponents (see below) .

Previous studies Indicated that DSase I preferentially digests rlbo-

somal genes in intact nuclei (29) . Figure 2A clearly i l lustrates that in

RSB, DNase I digestion releases tritium labeled DNA more rapidly than bulk14

C-DNA from intact, dual-labeled, starved-refed nuclei. The difference in3 14

rates of release of H and C observed in Fig. 2A is likely to be a minimum

203



10 10 30 40 60 80

MINUTES

10 JO 30 40 SO

MINUTES

."I

) ?0 30 4O W 60 70

PERCENT MC DIGESTED

Figure 2. Nuclease digestion of intact nuclei.( " o - o - o, 3H) . ®

A typical DNase IA typical Staphylococcaldigestion (•-•-•, ""C:

nuclease digestion. ^J Summary of all digestions on intact nuclei(>, DNase I; Q , Staphylococcal nuclease).

estimate of the actual differences in rates of digestion between the ribo-

somal genes and inert genes owing to the lack of complete specificity of

the tritium labeling and to the presence of (about 20-401) activated non-

ribosomal genes in the bulk DNA. Detailed kinetic analyses of the release3 14

of H and C from double-labeled starved-refed nuclei (48, and unpublished

observations) indicate that 30-40Z of the tritium digests about five times

faster than the bulk of macronuclear chromatin. Since only 50-602 of the

total tritium is in rDNA, it is necessary to demonstrate that the rapidly

digesting tritium-labeled component in nuclei is, in fact, rDNA. This is

clearly demonstrated in Fig. 1. DNA was isolated from nuclei both before

and after partial digestion with DNase I and analyzed on CsCl-Hoechst

gradients. Analyses of seven such gradients in which the partially digested14 T

DNA (Range of C digestion - 2-22Z; Range of H digestion - 4-40X) banded

sharply enough to allow quantitation, demonstrated that the rDNA digested

approximately 6 times faster than the bulk DNA (Vavra, Bowen and Gorovsky,

unpublished observations). Thus, the conclusion is inescapable that the

rapidly (DNase I) digested tritium-labeled component is the ribosomal gene-

containing chromatin (rChr).

When nuclei are digested with staphylococcal nuclease, the tritium-14

labeled DNA consistently digests slightly slower than C-labeled bulk

(Fig. 2B). In studies to be reported elsewhere (Vavra and Gorovsky, in

preparation) we show that kinetics similar to those in Fig. 2B are also

obtained with purified double-labeled DNA, suggesting thar staphylococcal

204



nuclease Is recognizing the difference (10-15Z) in GC content which exists

between rDNA and bulk DNA (49,50). Staphylococcal nuclease is known to

show a distinct preference for AT sequences (51-53). These results differ

slightly from previous studies which indicated that, in a different buffer,

rDNA and bulk DNA in isolated nuclei were digested at the same rate by

staphylococcal nuclease (10). This discrepancy illustrates the fact that

the relative rates of digestion of active and inactive genes by staphylo-

coccal nuclease are extremely dependent on the digestion conditions (Vavra,

Girl, Bowen and Gorovsky, unpublished observations).

Fig. 2C summarizes our results on dual-labeled, starved-refed nuclei

digested In RSB and presents the data in a useful form to be described In

detail elsewhere (Vavra and Gorovsky, in preparation). In brief, the per-

centage of H which is solubilized is plotted against the percentage of14

C solubilized at each point in a digestion. Such an analysis proves to

be relatively independent of the kinetic order of the reaction (48, in

preparation). More importantly for the experiments that follow, i t allows

a number of independent experiments to be graphed together even if the

actual rates of reaction vary. Such a graphical representation has the

added virtue that preferential digestion is readily demonstrated by devia-

tion from a slope of one. Figure 2C clearly Illustrates that, under the

conditions used here, staphylococcal nuclease preferentially digests bulk

chromatln while DNase I preferentially digests rChr.

Nuclease digestion of isolated nucleosomes

Double-labeled nuclei from starved-refed cells were briefly digested

with staphylococcal nuclease (4Z H and 11Z C rendered TCA soluble). The

nuclei were washed with RSB and chromatin was solubilized with EDTA as

described in Experimental Procedures. Both the RSB-soluble and EDTA-soluble

fractions were then fractionated in sucrose gradients containing RSB or

EDTA, respectively. As shown in Fig. 3A, the RSB-soluble fraction contained

predominantly nucleosome monomers, a few dimers and some uncharacterized,3 14

slow sedimenting material. The H and C radioactivity profiles were

almost superimposable across the entire gradient. The EDTA-soluble fraction

(Fig. 3B) contained nucleosome monomers, dimers and higher oligomers. Care-

ful analyses of this and numerous similar gradients indicate that H-contain-14

Ing monomers and oligomers sedlmented slightly faster than C-containing

particles in EDTA-contalning gradients. These results are consistent with

the observations of Gottesfeld and co-workers (38,54) who showed that DNase II

generated monomers enriched in active genes sediment at 14S rather than at

206



Q_O

10 20 30

FRACTION

10

NUMBER

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Figure 3̂ Sucrose gradient analyses of isolated nucleosomes and coreparticles. Sedimentation in all cases is from left to right.Bracketed regions indicate fractions vhich were pooled for subsequentnuclease digestions. IN and 2N Indicate mono- and di-nucleosomesrespectively (•-•-•, ""C-bulk DNA; o - o - o, 3H-starved-refed DNA) .

Q RSB-soluble fraction in an RSB-containing gradient. ® RSB in-soluble, EDTA-soluble fraction. ^ ) 0 . 1 M KC1 soluble fraction.

(5) 0.1 M KC1 insoluble, EDTA soluble fraction. Samples in B-D wereal l analyzed in EDTA-containing gradients; see text for experimentaldetails.

IIS like bulk mononucleosomes. I t should be noted that we have not observed

a significant accumulation of tritium in a rapidly appearing, slowly sedi-

menting form such as has been described by Johnson, et̂ a^. (17,18) for

rDNA-containing chromatin of Fhysarum.

Fractions corresponding to mononucleosomes from the EDTA-solubilized

chronatin were pooled, dialyzed against RSB and incubated with or without

DNase I . The kinetics of digestion, shown in Fig. 4A, clearly indicate that

208



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Figure k_. Nuclease digestion of isolated, sucrose gradient purified,mononucleosomes. d A typical DNase I digestion (•-•-•, C;o - o - o; 3H). (J/ A typical staphylococcal nuclease digestion.

f) Sunmiary of all digestions on isolated nucleosomesQ , Staphylococcal nuclease digestion of purified total mono-

nucleosomes; B , DNase I digestion of purified, total moncmucleo-somes; , DNase I digestion of RSB-soluble, mononucleosomes; I,DNase I digestion of RSB insoluble, EDTA soluble mononucleosomes;t, DNase I digestion of 0.1 M KC1 insoluble mononucleosoroes; seetext for experimental details).

the H-labeled, rDNA-enriched chromatin was digested more rapidly than the14

C-labeled bulk chromatin. No detectable release of TCA-soluble radio-

activity was observed when DNase I was omitted. Preferential digestion of

tritium was not observed if nucleosomes were dialyzed against 1 mM MgCl2

(Instead of 3 mM), or if MgCl- was added directly to a final concentration

of 3 mM instead of by dialysis. Repeated freezing and thawing also elimin-

ated preferential digestion (data not shown).

Isolated nucleosomes which have been dialyzed against RSB are partially

precipitated. Although Garel and Axel (23) have shown that the characteris-

tic 10 baae ladder generated by DNase I is similar when digestion is performed

on intact nuclei or on nucleosomes prepared as we have described, the pos-

sibility still remained that the DNase I sensitivity we observed was due to

differential solubility of activated and inert nucleosomes. Gottesfeld and

his co-workers (38,54) have, in fact, demonstrated that transcrlptionally

active chromatin solubilized by brief digestion with DNase II or staphylo-

coccal nuclease is more soluble than bulk chromatin in 2 mM Mg -containing

buffers.

Two lines of evidence argue that the preferential release of tritium

from dual-labeled, starved-refed nucleosomes is not due simply to trivial

207



differences In the solubility of activated and inert nucleosomes. 1) Soluble

mononucleosomes which were released by the RSB-wash and sedimented in RSB

gradients (Fig. 3A) also show preferential release of tritium by DNase X

(Fig. 4C). 2) If the tritium-labeled activated nucleosomes were more

accessible to DNase I simply because they were more soluble, we might also

expect them to be more accessible to staphylococcal nuclease; this is not

the case. Mien EDTA-released, RSB-dialyzed mononucleosomes are re-digested

with staphylococcal nuclease, H-labeled DMA becomes TCA soluble more slowly14

than C-DNA, just as in intact nuclei (Fig. 4B) .

We conclude from these studies that the conformational features which

DNase I recognizes in activated genes are retained in isolated, sucrose-

gradient purified nucleosomes.

Nuclease digestion of isolated nucleosome cores14

Nuclei in which the bulk ( C-labeled) chromatin had been digested to

varying extents (MOZ to V30Z TCA solubility) by staphylococcal nuclease

a l l yielded purified mononucleosomes in which the tritium-labeled DNA was

preferentially digested by DNase I . Nucleosomes prepared from more exten-

sively digested preparations might be expected to contain increased percent-

ages of particles which had been trimmed to produce chromatosomes or nucleo-

some cores. These results suggested that the activated conformation might

be maintained in purified nucleosome cores. Core particles were isolated

from double-labeled, starved-refed nuclei after staphylococcal nuclease

digestion (7-30Z TCA solubility of bulk chromatin) by 0.1 M KC1 precipita-

tion of contaminating nucleosomes. These core particles sediment exclusively

as monomers (Fig. 3C). As is the case for unfractionated monomers (Fig 3B)

as well as 0.1 M KCl-precipitable nucleosomes (Fig. 3D), H-labeled core14

particles sediment slightly faster than bulk, C-labeled particles.

Gradient purified nucleosome cores contained about 143 bp of DNA, and approx-

imately equal amounts of histones H2A, H2B, H3 and H4 (data not shown).

Histone HI was not detectable in purified nucleosome cores, attesting to

the absence of contamination by chromatosomes or intact nucleosomes.

Purified core particles were dialyzed against RSB and then digested

either with DNase I or staphylococcal nuclease. In purified nucleosome

cores, as in intact nuclei and nucleosome monomers, H-labeled DNA was

preferentially digested by DNase I (Fig. 5A), but was digested more slowly

than bulk DNA by staphylococcal nuclease (Fig. 5B). Similar preferential

digestion of H-labeled chromatin in 0.1 M KCl-insoluble nucleosome monomers

and dimers was also observed (Fig. 4C).

208



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PERCENT MC DKJESTED

Figure 5̂. Nuclease digestion of isolated, sucrose gradient purifiednucleosome core particles. ^ A typical DNase I digestion (•-•-•,1>>C; o - o - o, 'H) . @ A typical staphylococcal nuclease digestion.Q Summary of all digestions on isolated nucleosome cores (•,DNase I; CX, Staphylococcal nuclease).

Figure 6 summarizes our results to date on DNase I and staphylococcal

nuclease digestion of intact nuclei, sucrose gradient purified mononucleo-

somes and purified nucleosome cores isolated from dual-labeled, starved-

refed cells. Considering the variability expected of experiments using

small amounts of material, dual isotope counting and extensive manipulation

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PERCENT "C DIGESTED

Figure b_. Comparison of nuclease digestion of intact nuclei (•, o)purified mononucleosomes (•I.D) and purified nucleosome coreparticles (A, A). ^ J DNase I. @ Staphylococcal nuclease.

209



of nuclear fractions, the kinetics of digestion of ribosomal genes in

nucleosomes and core particles are remarkably similar to kinetics of diges-

tion in intact nuclei.

DISCUSSION

The present study was undertaken primarily to determine the level of

chromatin structure at which DNase I distinguishes between the conformations

of activated and inert genes. Our results clearly indicate that the prefer-

ential digestion by DNase I of DNA which has been labeled during refeeding

of starved Tetrahymena is similar in intact nuclei, isolated nuclesomes and

purified nucleosome cores. It has been demonstrated that this defined

starvation-refeeding scheme preferentially labels the ribosomal genes (10,

11,29,42; see Fig. 1). Using CsCl-density gradients, we have demonstrated

that the ribosomal gene accounts for most, if not all, of the tritium-

labeled DNA that is preferentially digested by DNase I in intact nuclei.

We think it likely that the rapidly digested H-labeled DNA in isolated

nucleosomes and nucleosome cores Is also rDNA. rDNA represents the major-

ity of the H-labeled DNA, and is rapidly digested by DNase I in nuclei.

The kinetics of solubilization of H and C by both DNase I and staphylo-

coccal nuclease are virtually identical in intact nuclei, nucleosomes and

core particles. We think it unlikely that this correspondence is fortuitous.

Our finding that DNase I preferentially digests ribosomal genes in

purified nucleosome cores provides the first direct evidence that this

enzyme detects an alteration in the Internal core structure of nucleosomes.

This conclusion is supported by previous observations that the staphylo-

coccal limit digest pattern and the pattern of fragments produced by DNase I

are altered in rChr (29) and by electron microscopic observations which

indicate that ribosomal genes may adopt a nucleosome-free, extended conforma-

tion prior to initiation of transcription (55,56). Since purified core

particles do not contain either histone HI or linker DNA, it is clear that

these nucleosome components do not play a positive role in determining the

DNase I sensitivity of activated ribosomal genes. Villeponteaux et al.

(57) have reached similar conclusions for activated avian globin genes by

demonstrating that selective removal of histone HI and H5 does not alter

the selective digestion of globin sequences by DNase I in erythrocyte nuclei.

The precise structural changes which occur in activated nucleosomes

are not known. Simpson (5) has recently suggested that removal of HI and

an accompanying loosening of nucleosome structure might be responsible for

210



conversion of a repressed gene to a transcriptionally active DNase I sensi-

tive state. While our results do not preclude such a mechanism from playing

a role in gene activation in̂ vivo, they do suggest that additional altera-

tions in the structure of nucleosome cores must also occur. Moreover, it

should be noted that tritium-labeled activated nucleosomes and core par-

ticles are not highly extended structures; they have sedimentation pro-

perties which are very similar to bulk nucleosomes and core particles. We

have not observed an extended slowly sedimenting particle containing M.40 bp

of DNA such as that derived by staphylococcal nuclease digestion of ribosomal

chromatin in Physarum (17,18).

We also wish to point out an apparent paradox between the electron

microscopic and the biochemical observations on the structure of activated

ribosomal transcription units. When viewed in the electron microscope,

active ribosomal genes are invariably extended to an extent which precludes

typical nucleosome structure (55,56,58-61). In Oncopeltus, the extended

state appears before the onset of transcription (56). Nonetheless, we and

others (12,13,17,18) have been able to demonstrate that ribosomal genes

are contained in typical nucleosome particles. One possible way to recon-

cile these observations is to suggest that active genes retain a periodic

structure which reflects the periodic distribution of proteins (probably

histones) along them, but that, depending on the methods used to study

them, they may be unfolded or extended under conditions where bulk, in-

active nucleosomes are not. It has been shown that nucleosomes can be folded

and unfolded reversibly in̂ vitro while maintaining their periodic structure

(62-64) . Similar changes could occur in_ vivo or during preparation for

electron microscopic or biochemical investigation. At present, the actual

state of active genes in vivo (folded, unfolded, or in dynamic equilibrium

between the two states) is unknown. Similarly, the relationship between

DNase I sensitivity and the degree of chromatin folding is not clear.

Finally, it is of interest to consider the molecular basis for gene

activation. Recent evidence suggests that both histone acetylation (65-

68) and specific non-histone proteins (69) may play important roles in

maintaining the DNase I sensitivity of activated genes. While our results

do not bear directly on the quest'ion of which molecules are involved, they

do predict that the responsible agent(s) will be associated with conforma-

tionally altered nucleosome core particles derived from Tetrahymena ribo-

somal genes.

211



ACKHOWLEDGEMENTS

We wish to thank Dr. C. David Allis for analyzing the histone composi-

tions of nucleosomes and core particles and Ms. Josephine Bowen for

technical assistance.

This work was supported by a research grant from the NIH, a National

Research Service Award to CPG and a Research Career Development Award to

MAG.

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