Eur. J. Biochem. 75, 545-560 (1977)

Analysis and Reconstruction of Xenopus Ribosomal Chromatin Nucleosomes Raymond REEVES Department of Zoology, University of British Columbia, Vancouver (Received November 22, 1976)

The double-stranded DNA from staphylococcal(micrococca1)-nuclease-derivedchromatin subunits (nucleosomes) from transcriptionally active somatic cells of heterozygous mutant (O/+ nu) embryos of the amphibian Xenopus laevis has been analyzed. T o determine whether the transcriptionally active ribosomal genes of these 0/+ nu embryos are selectively hydrolyzed by micrococcal nuclease cleavage and, more generally to determine whether any particular frequency class of DNA sequences was differentially removed by nuclease treatment of chromatin, DNA reassociation (cot) curves of heterozygote (O/+ nu) mutant subunit (nucleosome) DNA were compared with the cot curves obtained from whole 0/+ nu DNA. The results of these experiments clearly demonstrated that cleavage of the chromatin by micrococcal nuclease, to the point where about 75 of the DNA is present as fragments about 200 base pairs long, does not result in any preferential removal (hydrolysis) of either the ribosomal cistrons or any particular frequency class of gene sequences. However, these same reassociation curves also show that O / + nu DNA has a noticable decrease in sequence complexity in all of its frequency classes relative to that of whole O/ + nu DNA. Further experiments demonstrated that this decreased complexity is probably due to two factors: (a) a change in the nucleotide base composition of the nucleosome DNA relative to that of whole DNA (base composition analyses indicate that the subunit DNA has about 10 ”/, fewer d A f d T residues than whole DNA); and (b) the cleavage by micrococcal nuclease of the base sequences between adjacent chromatin subunits within each of the frequency classes of DNA. Further evidence supporting the similarity of nucleosome and whole cellular DNA comes from DNA-driven cot reassociation experiments involving the renaturation of individually isolated frequency classes of DNA from nucleosomes ( i .e., unique sequences and intermediate repetitive sequences) with an excess of whole (sheared) cellular DNA, which showed that the frequency classes of DNA within nucleosomes are indistinguishable, on kinetic grounds, from those of non-nuclease-treated DNA. Addition experiments involving solution hybridization reactions followed by hydroxyapatite analysis of nucleic acid duplex formation have indicated that nucleosome DNA derived from transcriptionally active cells contains fragments of DNA that anneal to both ribosomal RNAs and poly(A)-containing messenger RNAs. And, finally, the results from chromatin reconstitution experiments using isolated ribosomal DNA (rDNA) are presented that indicate that the association of nucleosomes with DNA in the formation of chromatin subunits is probably not base-sequence specific. All of these experiments are consistent with (but do not prove) the idea that transcriptionally active somatic cell ribosomal and unique-sequence genes can be partially, or perhaps transiently, associated with histones in the form of nucleosomes or chromatin subunits. These results are then discussed in terms of a dynamic model for the association of nucleosomes with transcriptionally active chromatin.

Abbreviations. rRNA, ribosomal ribonucleic acid (including 18-S and 2 8 3 RNA but not 5-S RNA); rDNA, ribosomal deoxyribonucleic acid (the gene sequences coding for rRNA plus the non-transcribed spacer DNA) ; monosome, monomer-sized nu body or nucleosome containing approximately 200 base pairs of DNA derived by short-term micrococcal nuclease digestion of chromatin [1, 11 - 151; cot, product of DNA concentration (in mol/l of nucleotides) and time (in s); equivalent cot,values of cot normaliz-

ed to values expected for 0.12 M phosphate buffer [lo]; phosphate buffer, a buffer prepared from equimolar amounts of mono- and dibasic sodium phosphate (pH = 7.2); rot, product of RNA concentration (in mol/l of nucleotides) and time (in s); standard saline citrate, 0.15 M NaCI, 0.015 M trisodium citrate, pH 7.8. Enzymes. Micrococcal (staphylococcal) nuclease; nucleate 3’-oligonucleotidohydrolase(EC 3.1.4.7).

Chromatin Subunits in Transcriptionally Active Genes

546

The DNA within the ribosomal chromatin (rDNA) of the amphibian Xenopus laevis has recently been shown to be at least partially protected from shortterm digestions with the enzyme micrococcal (staphylococcal) endonuclease by its association with histone aggregates in the form of nucleosomes (or nu bodies) [l - 41. Nucleic acid hybridization studies have indicated that both transcriptionally active and synthetically quiescent ribosomal genes give rise to 200 base-pair fragments of DNA containing both 18-S and 28-S gene sequences when digested with this enzyme [1,2]. Thus, most (85 - 90 %) of the reiterated ribosomal genes in transcriptionally inactive adult red blood cells [l], as well as those within early embryonic blastula cells not yet synthesizing rRNA [2],seem to be packaged into nucleosomes. On the other hand, the cistrons from synthetically active tissue-culture cells have less of their rDNA (70 - 74 %) packaged in this fashion [l]. Furthermore, during early embryogenesis, when the rate of ribosomal RNA synthesis is changing rapidly, there appears to be an inverse correlation between the amount of nuclease protection afforded the ribosomal DNA by nucleosome structures and the rate of rRNA transcription [2]. Experiments involving animals heterozygous for the Xenopus anucleolate ribosomal gene deletion mutation (O/ + nu animals) have clearly demonstrated, however, that a definite limit exists as to the amount of transcriptionally active chromatin that can be attacked by short-term nuclease treatments [2]. For example, after sufficient nuclease hydrolysis to convert virtually all of the mutant animal chromatin DNA into 200 base-pair fragments, nucleic acid saturation hybridization experiments indicate that about 60 o/, of the ribosomal DNA is still present within these gene pieces [2]. Since both genetic and biochemical evidence has indicated that it is very likely that all of the ribosomal genes within these heterozygous mutants must be synthetically active to maintain embryonic viability [S - 71, these results lend strong support to the idea that genomic transcription and DNA/nucleosome (Le., histone) associations need not necessarily be mutually exclusive events. Rather, it seems more probable that chromatin actively engaged in transcription has a dynamic structure and can be, at least partially or perhaps transiently [ 2 ] , arranged with histones into nucleosome structures, although the available experimental results do not rule out the possibility that some maximally active genes that are completely covered with polymerases may be free of such tightly bound structures (unpublished results). The present report extends these earlier investigations into the structure of transcriptionally active Xenopus chromatin. Advantage has again been taken of the heterozygous anucleolate mutation (O/ + nu) to allow the investigation of the nature of the doublestranded DNA fragments found within the nucleo-

somes of transcriptionally active ribosomal chromatin. DNA reassociation ( c o t ) curves of 0/+ nu monomer nucleosome (monosome) DNA have been analyzed and compared to whole (sheared) heterozygote DNA to determine whether any particular frequency class of reiterated sequences is differentially susceptible to micrococcal nuclease digestion. In addition, nucleotide base composition analyses as well as RNA . DNA hybridization experiments have been conducted with monosome DNA fragments to determine whether nucleosomes have an unusual nucleotide base composition or contain fragments of DNA from genes actively engaged in transcription. And, finally, experiments have been performed to determine whether nucleosome histones have any particular DNA base-sequence specificity of association by reassociating purified ribosomal DNA with high-salt-extracted histones to see if apparently normal native chromatin structure could be reconstructed from these genes of highly unusual base composition. The totality of these results indicates that the micrococcal-nuclease-derived monosome DNA is quite similar to whole DNA in most respects and the differences that are observed are probably not due to either selective hydrolysis of any particular frequency class of DNA sequences or to complete enzyme digestion of transcriptionally active DNA sequences. Rather the results are more consistent with a dynamic model of organization of chromatin actively engaged in transcription where nucleosome association with DNA is rather labile in nature and is not related to base-sequence specificity. The implications of these findings are also discussed. MATERIALS A ND METHODS

Preparation and Micrococcal Nuclease Digestion of Nuclei

Nuclei were isolated from four sources: (a) wildtype (+/ nu) embryos ; (b) embryos heterozygous for the anucleolate ribosomal gene deletion mutation (O/+ nu) [8]; (c) a permanent line of Xenopus embryonic cells growing exponentially in culture; and (d) adult red blood cells. These isolation procedures have been described previously [I, 21 and are a modification of those described by Destree et al. [9] except that they have been modified to minimize any histone rearrangements or protein degradation during the isolations. Cells were homogenized in a buffer containing 10 mM Tris-HC1 (pH 7.8), 1 mM MgC12,0.28 mM phenylmethylsulfonyl fluoride, 0.05 M NaHS03 sucrose and 0.5% Triton-X 100. All isolations were carried out at 4 "C unless otherwise stated. After washing and pelleting, the nuclei were resuspended in the homogenization buffer (without Triton) containing 0.5 mM MgC12 and 1 mM CaC12. Micrococcal nuclease (Sigma) was added to the solution (with concen-

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547

R. Reeves

trations of nuclei of up to 5 x 108/ml) to give a final nuclease concentration of between 100 - 300 units/ml depending on the experiment. The nuclei were digested at 37 “C for the times indicated and then pelleted at 600 x g for 10 min after adding 2 mM EDTA to stop nuclease action. Preparation of’ Nucleosomes Chromatin particles were freed from the pelleted nuclei by homogenization in a medium containing 10 mM Tris-HC1 (pH 7.5), 2 mM EDTA, 0.15 M sucrose and 0.28 mM phenylmethylsulfonyl fluoride. Nuclear membranes and insoluble material were removed by low-speed centrifugation. The chromatincontaining supernatant was concentrated and dialyzed against a buffer solution containing 10 mM TrisHC1 (pH 7.8), 3.5 mM EDTA and 0.28 mM phenylsulfonyl fluoride. The chromatin subunits were then separated on a 10- 30 % sucrose gradient containing the same buffer as described previously [l].Catalase was used as a molecular-weight marker for the 11-S region of the gradient, which corresponds to the apparent sedimentation value of monomer nucleosome chromatin subunits [ll - 151. Protein determinations were by the method of Lowry [16] using appropriate standards. Culture and Isotopic Labelling of Cells A permanent line of embryonic tissue culture cells of Xenopus was grown under standard conditions as described previously [l]. When nucleic acids were labelled with 32P, the cells were grown in phosphatefree nutrient medium containing serum (low-phosphate medium) to which [32P]orthophosphate(New England Nuclear, spec. act. > 500 Ci/mol) was added. Alternatively DNA was labelled with [3H]thymidine and/or [3H]deoxyguanosine (both from New England Nuclear) was added to complete medium. All other chemicals were of either tissue culture or reagent grade purity. Nucleic Acid Isolations and Electrophoresis DNA was isolated either from nucleosomes or from whole nuclei by a modification of the phenol procedures of Brown and Weber [17,18], as previously described [l]. Double-stranded DNA fragments from nuclease digested chromatin nucleosomes or from sheared whole DNA were separated by electrophoresis on 1.4‘j/,agarose slab gels by the methods described by Sharpe [19]. The DNA in agarose gels was stained with ethidium bromide and photographed under ultraviolet light. The relative sizes of the various double-stranded DNA fragments were determined by comparison with the electrophoretic mobilities of fragments of simian virus 40 (SV40) viral DNA of

known size as previously reported [I]. The relative sizes of denatured single-stranded DNA fragments, on the other hand, were determined by sucrose sedimentation velocity centrifugation under alkaline conditions [20] using as standards 3H-labelled fragments of DNA of known molecular weight. Isotopically labelled 18-S and/or 2 8 3 ribosomal RNAs were extracted and separated into purified fractions by polyacrylamide gel electrophoresis as described by Miller and Knowland [6,7]. Segments of gels corresponding to the 18-S and 28-S regions were cut out and the RNAs eluted by electrophoresis. DNA determinations were either by the method of Burton [21] or by absorbance assuming that native DNA has 1.0 A;?: unit = 50 pg ~ m - RNA ~ . determinations were by absorbance assuming 1.0 unit = 40 pg [22]. D N A Reassociation Kinetics For reassociation studies whole Xenopus DNA was sheared to about 250 nucleotides in length by homogenization in a medium consisting of 67 :{ glycerol in 0.05 M phosphate buffer for 60 min in a Virtis homogenizer at -20 “C as described by Davidson et al. [23]. The resulting fragments (with an average sedimentation value of around 4.4-4.5 S on alkaline sucrose gradients) were then dialyzed against 0.3 M sodium acetate and subsequently precipitated with 2 volumes of cold 100% ethanol. The nucleasederived nucleosome DNA (averaging 200 base pairs in length) was not subjected to shearing but otherwise was treated identically to the sheared DNA preparations. Both the precipitated monosome and sheared DNAs were then redissolved and chemically iodinated with 1251as described below. The isotopically labelled DNA was then reprecipitated and finally redissolved in 50 mM Na3P04 and further purified by chromatography on Sephadex G-200 followed by passage over Chelex 100 (Bio-Rad) resin to remove traces of heavy metals. The kinetics of DNA reassociation and duplex formation were monitored by hydroxyapatite chromatography using standard techniques [lo,241 except that double-stranded DNA was eluted from the 60 “C hydroxyapatite columns using 0.5 M phosphate buffer. After hydroxyapatite fractionation, the duplex DNA preparations were digested with S1 single-strandpreferring exonuclease [25] under conditions that selectively removed unmatched base sequences and single-stranded ‘tails’ so that only base-paired structures remained to be analyzed [lo]. The hydroxyapatite columns were operated well below their nucleic acid carrying capacity. Data obtained at various DNA and sodium ion concentrations were corrected to cot values equivalent to those obtained under standard reassociation conditions of 0.12 M

Chromatin Subunits in Transcriptionally Active Genes

548

phosphate buffer (0.18 M N a+ ) and 60 "C [lo]. cot is the product of DNA concentration in mol/l and time in s [lo]. A digital p d p l l computer was used for determining the best fit of reassociation curves using the least-squares method of second-order reaction analysis described by Britten et al. [lo]. For some reassociation experiments purified DNA preparations were chemically labelled with 1251 in vitro using the thallium trichloride reaction method of Commerford [26]. Specific activities of up to about lo6 counts min-' pg-' were obtained without affecting the hybridization results in any noticable way. Isolation of Unique and Middle (Intermediate) Repetitive Sequences The middle repetitive and unique-sequence components of either sheared or monosome DNA fragments from heterozygous O/ nu animals were isolated by selective elution of duplex DNA from hydroxyapatite columns by standard techniques [ 10, 241. For both sheared and monosome DNA, intermediate repetitive sequences were taken to be those fragments of DNA that reassociated to form duplexes between cot values of 0.05 mol I-' s and 50 mol I-' s whereas unique (single copy) sequences were considered to be the single-stranded fragments unbound at a cot of 1500 m o l l - l s . Each of the isolated DNA fractions was subjected to the same selective hydroxyapatite binding procedure for a second time before use for kinetic studies.

+

D N A . R N A Hybridizations The nucleic acid preparations used for all RNA DNA hybridizations were purified and fractionated as described above, passed over Chelex 100 and Sephadex G-200 columns and finally precipitated with ethanol. The nucleic acids were then dissolved in 0.03 M phosphate buffer and mixed in the appropriate RNA: DNA ratios. The salt concentration was then adjusted to 0.12 M phosphate buffer, sample aliquotes sealed in capillary tubes and the samples heat denatured for 10 min at 95 "C. The capillaries were then quickly cooled to 60 "C and the nucleic acids allowed to reanneai for appropriate times at this temperature to reach a desired cot or rot value. rot is the product of RNA concentration (mol/l of nucleotides) and time (s). The reactions were terminated by diluting the samples twentyfold with 0.03 M phosphate buffer at 4 "C. Duplex formation was monitored by hydroxyapatite chromatography as described. The hydroxyapatite columns and elution buffers contained 0.1 % sodium dodecylsulfate to minimize attachment of single-stranded RNA. The nuclease-resistant duplexes were then precipitated with cold 10% trichloroacetic acid and the acid-insoluble radioactivity counted in an Isocap scintillation counter using

standard techniques. Extent of reassociation was expressed as a percentage of the input radioactivity in the denatured nucleic acids. The RNA . DNA hybridization reactions were of two types: (a) hybridization of a vast excess of purified non-radioactive ribosomal RNA to trace quantities of '251-labelled monosome DNA to determine the fraction of DNA sequences complementary to rRNA in the nucleosome DNA population (this is an RNA-driven or rot curve reaction); and (b) hybridization of trace amounts ~ f ~ ~ P - l a b e lpoly(A)led containing messenger RNA sequences derived from tissue-culture-cell polysomes to a vast excess of monosome-derived DNA (in a DNA-driven reaction) to determine whether nuclease-derived monosome DNA contains fragments of unique sequences coding for transcribed mRNAs. Control experiments established that the hydroxyapatite-retained duplexes were indeed RNA . DNA hybrids and that in the RNA-excess experiments that less than 0.2% of the input tracer radioactive DNA reassociated with itself during the annealing reactions. Isolation of Messenger R N A 32P-labelled poly(A)-containing messenger RNA (mRNA) was obtained from the polysome fraction of Xenopus tissue-culture cells grown for 24 h in lowphosphate medium containing 10 pCi/ml of ["PIorthophosphate. The polysomes were isolated by the method of Woodland [27]. The poly(A)-containing mRNA was then isolated from the polysomes by the methods described by Aviv and Leder [28] using oligo(dT)-cellulose chromatography. The purified [32P]poly(A)mRNA obtained has a specific activity of 3.5 x lo5 counts min-' pg-'. Base Composition of D N A Tissue-culture-cell DNA labelled to a constant specific activity by growth for 6 days in 50 pCi/ml of 32Pcontaining low-phosphate medium was enzymatically hydrolyzed to 5'-mononucleotides. Purified DNA derived from either whole undigested nuclei or from nuclease-derived nucleosomes was incubated overnight at 37 "C with electrophoretically pure pancreatic DNase I (Sigma) at 50 pg/ml in a buffer containing 0.1 M sodium acetate (pH 6.8) and 50 mM magnesium acetate. The pH was changed to 9.0 by adding ammonia; phosphodiesterase I (Russel's viper venom ; Calbiochem) was added (20 units/ml) and incubated for 8 h at 37 "C. The released nucleotides were absorbed to charcoal, washed three times with water, and the nucleotides then eluted from the charcoal by three washes with 50 % ethanol and 0.5 %concentrated ammonia. The nucleotides were concentrated in vacuo and separated by two-dimensional thin-layer chro-

R. Reeves

549

matography on poly(ethyleneimine)(PEI)-cellulose F plates (Merck) using the solvent system of Randerath and Randerath [29]. The separated nucleotides were localized by autoradiography using Kodak R P Royal X-Omat Medical X-ray film. The radioactive spots were cut from the chromatographs and counted in an Isocap scintillation counter (Nuclear Chicago). Isolation of Ribosomal Cistron DNA Erythrocyte DNA was prepared as described previously [I] from the blood of 45 adult frogs yielding about 9 0 m g native DNA. The (dG + dC)-rich ribosomal cistrons (dG + d C = 64 %; e = 1.723 g/ cm3) were isolated by a modification of the method of Dawid et al. [30], which involves the selective precipitation of (dA dT)-rich DNA by poly(L1ysine)titration followed by a final cesium chloride fractionation of the purified cistrons. This procedure yielded 40 pg of pure ribosomal DNA, which represents a recovery of about 22% of the expected theoretical maximal yield [30,31].

+

Isolation of’ High-Salt-Extracted Histones All isolations were carried out at 4 ° C . The preparation of chromatin from purified nuclei form Xenopus culture cells was carried out according to the methods of Marushige and Bonner [32] modified in order to minimize histone degradation by the addition of 50 mM sodium bisulfite, 0.28 mM phenylmethylsulfonyl fluoride and 1 ”/, dithiothreitol to all of the solutions. The final pellet was dissolved in 2.0 M NaCI, 50 mM sodium acetate (pH 5.0), 0.28 mM phenylmethylsulfonyl fluoride and 50 mM sodium bisulfite. The deoxyribonucleoprotein was then precipitated by reducing the NaCl concentration to 0.30 M through dilution. The insoluble DNA . histone complex was pelleted by centrifugation leaving the acidic non-histone proteins in solution under these conditions [33]. The chromatin was redissolved in 2 M NaCl to remove the histones from the DNA [34, 351 and the histones separated from the DNA by Sepharose 4B (Pharmacia) gel filtration chromatography [36]. Histones were analyzed by acid urea polyacrylamide gel electrophoresis using 18 % acrylamide gels containing 2.5 M urea as described by Adamson and Woodland [38] based on a modification of the procedures of Panyim and Chalkley [37]. Reconstitution of Chromatin Total high-salt-extracted histones ( F l , F3, F2a1, F2a2 and F2b) and purified ribosomal gene DNA were reconstituted at 4 “C into chromatin by successive dialyses against solutions of decreasing ionic strength. The 40 pg native rDNA was dissolved in 1 ml 2 M NaCl, 20mM sodium bisulfite, 1 0 m M EDTA,

100 mM Tris-HC1 (pH 7.5) and 0.28 mM phenylmethylsulfonyl fluoride. To this solution was added, by slow dropwise addition with rapid mixing, 1 ml of total histones containing 60 pg proteins in the same buffer solution to give a final solution with a histone :D NA ratio of 1.5 : 1. This solution was then dialyzed successively against the following saline solutions (each containing the above buffer and inhibitors) : 2 M NaCl for 2 h ; 1.5 M NaCl for 12 h ; 1 M NaCl for 2 4 h ; 0.75 M NaCl for 1 2 h ; 0.5 M NaCl for 24 h; and finally 0.4 M NaCl for 24 h. The reconstituted chromatin was then digested with nuclease as described and aliquots taken for analysis of DNA and histones. RESULTS The Ribosomal Genes of Xenopus laevis In diploid wild-type somatic cells of Xenopus the 1000 or so genes that code for ribosomal RNA comprise about 0.2 % of the DNA of the genome [30,31]. The ribosomal DNA in such cells is usually divided evenly between two specific nucleolar organizer regions located on homologous chromosomes [39]. Since each nucleolar organizer forms a prominent nucleolus, wild-type cells usually have two and are hence designated as +/+ nu or 2-nu [39]. There exists a certain heterozygous mutation, the anucleolate mutation, involving the total deletion of the rDNA from the nucleolar organizer region of one of the chromosomes [8]. These mutant animals contain only about half the normal wild-type number of genes (about 500), contain only about 0.1 rDNA within their genome and contain only one nucleolus per somatic nucleus [39]. These animals, designated O/ i nu, are quite viable and synthesize ribosomal RNA at about the same rate as wild-type animals even though they have only half their rDNA content, Since a reduction of the number of ribosomal genes (by further deletion mutations) below this heterozygous 0/+ nu level results in embryonic lethality [5-71, it seems likely that all of the ribosomal genes within the 0/+ nu animals must be transcriptionally active to maintain viability. This assumption is further supported by the finding that in these partial deletion mutations (that have less than the heterozygous rDNA content) the rate of rRNA synthesis is directly proportional to the number of ribosomal genes they possess, even though at a later stage of development these mutants die [6,7]. From all of the available evidence it seems reasonable to assume that 0/+ nu animals have a fully functional set of transcriptionally active ribosomal genes. Previous reports from this [1,2] and other laboratories [3,4,40,41] have indicated that micrococcalnuclease-derived monomer nucleosome DNA frag-

Chromatin Subunits in Transcriptionally Active Genes

550 100

i

4

04 10-2

I 10-1

10'

1 00

102

Equivaient Cot

104

103

(rnol-'s)

+

Fig. 1. Reassociation profilcs of' m e n o p u s 01 + tiu monomer uucleosome D N A , niechanicull~sheared 01 D N A and sheared E. coli D N A . Data were obtained at various D N A and phosphate buffer concentrations; however all data were normalized to cot values expected for 0.12 M phosphate buffer at 62 'C (equivalent cot) [lo]. The mechanically sheared DNAs averaged about 250 base pairs in length before denaturation, whereas the nuclease-derived monomer nucleosome DNA averaged about 200 base pairs in length (see text). All DNA preparations were chemically labelled in vitro with to specific activities of about lo6 counts min-' pg DNA-' as described in the text and reassociation analyxd by means of hydroxyapatite chromatography t o detect duplex formation [lo]. The lines through the data were obtained by computer analysis using the least-squares best-fit method of Britten e l al. [lo]. The root-mean-squares for the computer fits of the various curves are: sheared, A) staphylococcal-nucleaseM) Mechanically sheared O / + nu Xenopus D N A ; (A 1.56%; monomer 2.45:/,; E. coli 1.782,. (W-derived monomer nucleosome 0/+ nu Xenopus D N A ; (0- 0 ) mechanically sheared E. coli DNA

ments from transcriptionally active cells contain pieces of genes coding for ribosomal RNAs. All of these studies, however, have suffered from the limited sensitivity of the biochemical assay methods used for the detection of DNA sequences complementary to rRNA present within the monosome population. It was thus of interest to reinvestigate whether monosome DNA fragments derived from the transcriptionally active heterozygous O/ + nu mutants contained detectable fragments of not only ribosomal DNA but also of unique-sequence DNAs that code for messenger RNAs, by using the much more sensitive and quantitative methods of solution nucleic acid hybridization followed by hydroxyapatite analysis of duplex formation [lo]. As a first step in such an analysis the kinetics of reassociation of DNA from monosomes derived from 0/+ cells were compared to those of sheared 0 / + nu DNA to determine whether any particular frequency class of DNA sequences is differentially sensitive to nuclease cleavage. D N A Reassociat ion Kinetics

+

A comparison of the reassociation curves of O/ nu heterozygote DNAs that have either been sheared by homogenization to fragments of about 250 nucleotides in length or have been cleaved by nuclease to monosome-length fragments (about 200 nucleotides) is shown in Fig. 1. The DNA for both curves was obtained from stage-42 tadpoles [42] and labelled in vitvo with '25iodine to a specific activity of 8.5

x lo5 counts min-' pg-'. Reassociation and analysis of duplex DNA hybrids were as described in the Methods section. From this figure it is evident that the heterozygote monosome DNA fragments (the lower curve) contain the same classes of repetitive DNAs in about the same proportions as does the sheared O/+ nu DNA not subjected to nuclease attack (the upper curve). Thus, the non-repetitive(unique)-sequence DNAs of both populations of fragments correspond to about 55-56% of the total DNA (Table 1). Likewise, the intermediate repetitive sequences of both the monosome and sheared DNAs represent about 29 - 31 % of the total DNA (Table l), with the remainder of the sequences (12- 15 "/,) being made up of the fast and zero-time binding DNAs (Table 1). Micrococcal nuclease digestion of chromatin down into nucleosomes thus does not appear to preferentially hydrolyze any particular class of gene sequences. However, another equally obvious conclusion to be drawn from Fig.1 is that the monosome cot curve is shifted noticably to the left of the cot curve for sheared DNA. That this shift to lower cot values in the monosome curve represents a true decrease in complexity of all of the different frequency classes of DNA relative to the reassociation curve of E. coli DNA (shown in the center of the figure) is inferred from two observations: (a) the close fit of the various cot curve points with the computer-derived leastsquares best-fit curves for the two different populations of fragments; and (b) the minimal overlap of cot

551

R. Reeves

Table 1. Frequency components of reassociated D N A s K is the second-order rate constant for reassociation expressed as I mol-' s-'. Since K is proportional to ln.'/N where I is the average length of single-stranded D N A in nucleotides and N is the complexity of the DNA [43], the difference in rate constants between sheared DNA (average length of about 250 nucleotides) and nuclease-derived subunit D N A (about 200 nucleotides) leads to the expectation that the monomer nucleosome D N A should reassociate about 0.24 times as slowly as the sheared DNA (see text for discussion). Kinetic complexity ( N ) measured as the total length of different D N A sequences expressed as nucleotide pairs relative to the sequence complexity of sheared E. coli (given in the last line of the table). The complexity of B. coli D N A is assumed to be 4.3 x lo6 base pairs and to have an average base dC) [64] composition of 50 "/, (dG

+

Source of DNA

Component

Quantity

cotll, (mixture)

2 7

mol I-' s ~~

cot^/, (pure)

~~

K

Complexity ( N )

6.98 x 10-4 1.84

1.95 x 109" 7.35 x lo's

~~

0 / + nu, sheared

non-repetitive intermediate repetitive fast and zero-time binding

56.2 31.0 12.8

2.55 x 103 1.75 -

-

0 / + nu, monomer

non-repetitive intermediate repetitive fast and zero-time binding

55.0 29.8 15.2

2.28 x 103 1.55

1.25 x 103 0.462

7.98 x 10-4 2.16

1.35~ 4.98 x 10'

-

-

-

-

non-repetitive

90.0

-

4.1

0.24

4.3 x lo6

E. coli, sheared

1.43 x 103 0.54

+ +

The difference in base composition of sheared Xenopus DNA (Table 2; 38.3% d G dC) and E. coli DNA (50% d G + dC) slows the rate of renaturation of Xenopus D N A by a factor of 0.77 [43] relative to E. coli. dC) and E. coli (50 % d G dC) slows the rate The difference in base cornposition of monomer Xenopus DNA (Table 2; 48.6 % d G of renaturation of Xenopus monomer nucleosome D N A by a factor of 0.972 [43] relative to E. coli.

100 90 --

+

+

-

80 -4

70 -60 --

D

F 50.-

m

'Y 40$ 3020 _lo --

C+ 1c-2

100

10' Equivalent c o t

102

I 03

(moi i - ' ~ )

+

Fig. 2. DNA-driven reassociation profiles fbr i.~olated unique-sequence nwtiorner nucleosome O/ nu D N A and .for isolated middle repetitive monomer nucleosome O / + nu D N A in the presence of a vast e ~ c e s sof sheared +/+ tiu D N A . Both the unique and middle repetitive DNAs were isolated individually by differential hydroxyapatite chromatography as described in the text and then chemically labelled with "'I. These radioactive 0/+ nu DNAs were then mixed with unlabelled sheared driver D N A at ratios of between 200- 10000: 1 of driver: labelled DNA, denatured and allowed to reassociate under various phosphate buffer and temperature conditions. The kinetics of duplex formation were monitored by hydroxyapatite chromatography. The abscissa refers t o the cot values of driver DNA corrected for the various DNA and phosphate buffer concentrations to give cot values expected for 0.12 M phosphate buffer at 62 "C (equivalent cot) [lo]. The lines through the data points were computer derived as described [lo]. The solid line is the D N A reassociation curve for mechanically sheared 0 / + nu DNA shown in Fig. 1. The root-mean-square for each computer analysis is: unique, 2.85 %; intermediate repetitive, 3.36%. (O-------*) Unique-sequence monomer nucleosome 0/+ nu Xenopus D N A ; (0-0) middle repetitive-sequence monomer nucleosome 0/ + nu XenopusDNA

points for the two different DNA fragment populations. There are a number of possible explanations for the observed shift to lower cot values of the monosome DNA. Three alternatives will be considered in detail.

The first of these possibilities is that the shift has somehow been caused by the difference in length between the sheared DNA fragments (which average about 250 nucleotides in length) and the monosome fragments (which average about 200 nucleotides in

552

Fig. 3 . Autoradiagraphs of 3 2 P-labelled j'-monod~oxynucIeoiides derived from nuclease hydrolysates of X. laevis tissue-culture cell (line X j X ) D N A and separated by two-dimensioncrl thin-layer chromatography as described in the text. (A) 32P-labelled nucleotides from 11-S monomer nucleosome D N A : (Bj "P-labelled nucleotides from whole, bulk DNA that has been sheared. m'dC, 5-methyldeoxycytidine

length). Upon close inspection, however, this seems to be rather unlikely, since the smaller monosome DNA fragments should, if anything, reassociate at a slower rate than the longer sheared fragments and the monosome curve in this case would be shifted to the right of the sheared cot curve rather than to the left [43]. The extent of this expected shift due to length can be calculated from the finding that the reassociation constant K is proportional to 1°.5/N (where I is the average length in nucleotides of the DNA fragments and N is the kinetic complexity [43]. When this calculation is made for the two given populations of DNA it is found that on length considerations alone the monosome population of DNA fragments would

Chromatin Subunits in Transcriptionally Active Genes

be expected to reassociate (as an average of both the unique and intermediate sequence rates) about 0.24 times as slowly as the sheared DNA and the monosome curve should be shifted by about this amount to the right of the sheared DNA cot curve. From Fig. 1 it is quite evident that this is not the case. This point will be returned to below. The second possibility to be considered is that the nucleosome fragments released by nuclease treatment represent a subset or partial complement of the total cellular DNA sequences present in the native chromatin of the 0/+ nu embryos [44,45]. To investigate this possibility further, intermediate repetitive and non-repetitive sequences of monosome 0/+ nu DNA were isolated individually by hydroxyapatite chromatography and then chemically labelled with 125i0dine. These isotopically labelled DNA fractions were then individually annealed with a great excess of sheared wild-type (+/+ nu) DNA obtained from adult red blood cells. The results of these DNAdriven hybridization reactions are shown in Fig. 2. The solid line represents the cot reassociation curve for sheared 0/+ nu DNA (from Fig. 1). The upper curve (with a computer-derived root-mean-square of 2.85 %) is the reassociation curve of monosome 0/+ nu unique-sequence DNA driven in its reassociation by up to a lo4-fold excess of unlabelled sheared erythrocyte DNA. The for reassociation of this nonrepetitive DNA is 1.47 x lo3 mol 1-l s with a rate 1 mol-' s-'. These values constant ( K ) of 6.8 x are very similar to those of sheared unique-sequence DNA annealed to itself (Table 1) and probably indicate that the nuclease-derived monosome DNA contains unique sequences that are quite similar to those of undigested Xenopus DNA. In a similar series of experiments to those just described '2sI-labelled monosome O/ + nu intermediate repetitive DNA was annealed to a vast excess of unlabelled sheared driver DNA and the cot curve determined. From Fig.2 it is seen that this middle repetitive DNA reassociates with a cotli2 value of 0.61 m o l l - ' s and with a rate constant ( K ) of 1.64 1 mol-' s-l (the lower curve with a root-meansquare of 3.36%). Again, these results are very close to the values obtained for middle repetitive sheared Xenopus DNA (Table 1). The most reasonable conclusion to be drawn from these experiments is that micrococcal nuclease digestion of native chromatin probably does not release a specific subset or partial complement of the gene sequences present in the total Xenopus genome. The third possible explanation to be considered for the shift of the monosome cot curve to lower complexity is that the nucleotide base composition of the nuclease-derived nucleosomes is different from that of sheared whole DNA [43]. To explore this possibility a deoxynucleotide base composition analy-

R. Reeves

553

Table 2. Deoxynucleoside 5 '-monophosphate base composition of undigested and monosome [" P I D N A Results are expressed k standard error. msdC = 5-methyldeoxycytidine Nucleoside

Bulk DNA

Monosome DNA

mol "/, total nucleotides ~~~~

dA dT dG dC msdC Number of determinations

~~~~

~~~

31.0 k 0.5 30.7 k 0.5 19.1 f 0.7 18.3 k 0.7 0.9 k 0.03

25.5 25.9 24.4 20.9 3.3 4

3 ~~~~

d G + dC" dA + dT dAjdT dGjdC" a

38.3 k 0.8 61.7 k 0.7 1.01 0 99

f 0.6 f 0.6 0.5 f 0.5 k 0.3

~~

48.6 f 0.7 51.4 f 0.7 1.01 1.01

Includes msdC.

sis of the two types of DNA was performed. Exponentially growing Xenopus culture cells in lowphosphate medium were allowed to incorporate [""porthophosphate for 6 days until their DNA had reached a constant specific activity. Nuclei were then isolated and the sample split and one half treated with nuclease to give nucleosomes and the other half used for whole DNA extraction. Since micrococcal nuclease gives rise to deoxynucleotide 3'-monophosphate residues it was decided that enzyme-derived 5'-monophosphate residues obtained by combined pancreatic DNase I and phosphodiesterase I (snake venom) digests should be analyzed. Base composition analysis of 5'-monophosphate nucleotides has also been shown to be more reliable and quantitative than is analysis of 3'-residues [30]. Fig. 3 shows autoradiographs of the separated deoxyribonucleotides in whole and nucleosome DNA. In addition to the four major nucleotides, both samples are seen to contain a small amount of 5-methyldeoxycytidylic acid. This finding is in agreement with those of others that somatic chromosomal DNA in X.luevis contains this modified base [31]. Table 2 gives a quantitative estimate of the base composition of bulk and nuclease-derived monosome DNA. From both the autoradiographs and the data in Table 2 it is evident that the monosome DNA contains about 10% less combined dA and dT residues than does the undigested whole DNA. These results cannot be explained on the basis of non-equilibrium of precursor nucleotide pools, since the ratio of the base pairs (dA/dT and dG/dC) are near unity in the undigested bulk DNA. On the other hand, these results are most easily interpreted in terms

+

of an increased sensitivity of (dA dT)-rich DNA within native chromatin to be attacked by shortterm exposure to micrococcal nuclease, a conclusion in agreement with the findings of Bernardi [46] for pure DNA. However, it should be noted that these quantitative base-composition results for monosomes of 200 base pairs released from chromatin by shortterm nuclease treatment stand in contrast to the early findings of Clark and Felsenfeld [47], who reported that the much shorter fragments of doublestranded DNA released from long-term nuclease limit digestions of chromatin did not seem to differ in mean base composition from whole DNA. These early studies, however, analyzed base composition by the less sensitive method of hyperchromic spectrumshift measurements and no data were presented for their conclusions [47]. Nevertheless, it would be of some interest to analyze the base composition of these limit-digest DNA fragments by the quantitative method described here. Could this increase in d G + dC content of monosome DNA revealed by the base composition analysis account for the decrease in sequence complexity (and hence in K values) seen in the nucleosome cot curve of Fig.l? The answer appears to be 'not entirely'. For example, the difference in average base composition of sheared Xenopus DNA (38.3%) and monosome DNA (48.6%) would be expected to increase the rate constant ( K ) of renaturation of monosome DNA by a factor of about 0.22 relative to that of whole DNA [43]. On the other hand, the monosome cot curve of Fig.1 shows that the nucleosome unique sequence DNA has increased its rate constant by about a factor of 0.125 relative to the sheared DNA and that the intermediate repetitive rate constant has increased by a factor of 0.33 relative to sheared DNA. This gives a rough average of about 0.23 as the total relative rate increase in the reassociation of monosome DNA relative to sheared DNA. However, to get a more accurate estimate of how much faster monosome DNA actually reassociates than does sheared DNA, it is also necessary to add to the observed average rate increase seen in Fig.1 the expected rate decrease of monosome reassociation due to the difference in fragment lengths of the two populations as discussed above. When this value is added (0.24) the total cumulative reassociation rate difference between monosome and sheared DNA fragments can be calculated to be about 0.47. Of this total about 0.22 of the rate difference can be attributed to the increase in d G + dC content of monosome DNA relative to whole DNA. This leaves a factor of about a 0.25-fold increase in rate of monosome DNA to be accounted for. As will be discussed later, this finding is compatible with the idea that about 25% of each of the DNA frequency classes of chromatin (and hence probably of each gene sequence) is sus-

Chromatin Subunits in Transcriptionally Active Genes

554 I

1

Fig.4. Hybridization profilesfor excess ribosomal R N A annealed to trace quantities qf'either '2'1-labelled wwnomer nucleosome O / + nu DNA or 1Z51-labelledsheared O / + nu D N A . The hybridization was carried out in solution and duplex formation monitored by hydroxyapatite chromatography as described in the text. The specific activity of both preparations of DNA was about 8 x lo5 counts min-' kg D N A - ' . The ribosomal RNA ( 1 8 3 + 28-S) was electrophoretically purified before hybridizations. The abscissa represents the logarithm of the ribonucleotide concentration (in mol nucleotide/l) multiplied by time (s) (i.e., rot values) [24]. (0) Hybridization curve of nuclease-derived monomer nucleosome 0/+ nu DNA annealed to ribosomal R N A ; (W) hybridization curve of mechanically sheared 0/+ nu DNA annealed to ribosomal R N A

ceptible to complete digestion during nuclease release of nucleosomes.

Nucleosomes are Present in Transcriptionally Active Chromatin The cot reassociation curves of Fig. 1 indicate that nuclease-derived nucleosome are not missing any detectable frequency classes of DNA sequences. This finding is consistent with the previously reported evidence that the transcriptionally active ribosomal cistrons of these mutant animals are probably at least partially associated with histones in the form of nucleosomes or nu bodies [2]. However, since these earlier experiments were of limited sensitivity, owing to the use of the filter hybridization technique [lo], it was of considerable importance to analyse the monosome DNA of 0/+ nu animals by the more sensitive technique of solution hybridization followed by hydroxyapatite analysis. The results of such an analysis of '251-labelled heterozygote monosome DNA hybridized to a great excess of unlabelled electrophoretically purified, ribosomal RNA (18-S + 28-S) is shown in Fig.4. The sheared heterozygote 0/+ nu DNA reached a plateau of hybridization at about 0.038% of the DNA hybridized (as determined by a double-reciprocal plot method [48]) whereas the monosome DNA from the same embryos reached a plateau of saturation of about 0.024%. These results indicate that about 63 '%, of the ribosomal sequences of these transcriptionally active embryo cells are protected from micrococcal nuclease attack by being associated with histones in the nucleosome form.

1hese results are consistent with previous findings, although the level of protection detected by the solution hybridization method of assay leads to a slightly higher value for nucleosome protection than previously observed [2]. In addition to containing fragments of synthetically active ribosomal genes, Fig. 5 indicates that monomeric nucleosomes may also contain fragments of genes coding for embryonic messenger RNAs. In these experiments 32P-labelled poly(A)-containing messenger RNA obtained from embryonic tissue-culture cells was annealed (under conditions of vast DNA excess) to monomer nucleosome nu DNA with the results shown in Fig. 5. The mRNA reassociates with DNA that anneals with a cotli2 of about 1.6 x lo3 mol I-' s, which is similar to the nonrepetitive cotli2values of Xenopus DNA seen in Table 1. Although these results are consistent with the suggestion that fragments of transcriptionally active mRNA genes are present in nucleosome DNA fragments, they cannot be taken as direct evident supporting this contention, however. The reason for this uncertainty is that one is not sure that all of the mRNA genes are transcriptionally active in the starting population of cells from which the nucleosomes were released. This is in marked contrast to the situation with the ribosomal RNA hybridizations just mentioned, where the mutant heterozygote O/+ nu embryos probably did have all of their ribosomal genes active and the hybridization results can be interpreted with much more confidence. Nevertheless, all of these experimental results are entirely consistent with the hypothesis that histone association with DNA is not always of

+/+

555

R. Reeves

_______;

70

1 .?O

I ,68

-

." ?

60 50

Q

>-

I40 4

30 n N 0

-~2 0 10

0

3

2

1

4

log c o t

Fig. 5. Hyhridization profile oJ 32P-lubelled poly(A)-containing messenger R N A s annealed to monomer nucleosome -ti+ nu D N A under cotidifions of vust D N A C . Y ~ L ' S SThe . 3ZP-labelledm R N A preparation was obtained from tissue-culture cell (line X58) polysomcs by oligo(dT)-cellulose chromatography as described in the text. ~ counts min-' pg The RNA had a specific activity of 2 . 3 lo5 RNA-'. The logarithm of the driver DNA concentration (nucleotide/l) multiplied by time (s) (log c o t ) is given on the abscissa. Hybridization was in solution and duplex analysis was by hydroxyapatide chromatography Table 3. Steps in the purijkation o f rihosomul cistron D N A Densities given for each of the multiple peaks of DNA when they are present Step

Purified ribosomal cistron D N A First polylysine precipitation

Amount of D N A

DNA recovered

Mean density of D N A

m&

x

gicm3

90.1

100

10.1

Second polylysine precipitation

Isolated rDNA from c s c l gradient Theoretical maximum recovery rDNA recovered ( 5,, of maximum)

~

11.2

0.950

1.05

0.0401

0.045

0.1802

0.20" 22.25

1.698 (a) 1.700 (b) 1.720 (a) 1.704 (b) 1.712 (c) 1.723 1.723 ~

-

See [30] and [31].

an inhibitory nature and that transcriptionally active genes can contain nucleosomes, finding consistent with previous reports from this [1,2] and other laboratories [40,41,49 - 531. Reconstitution of Ribosomal Chromatin The results presented so far seem to indicate that micrococcal nuclease is non-specific in its attack of

,

1

68

I

!

1.70

,

*

1

.72

.

,

1.74

aensity ( g / c m 3 )

Fig. 6. Isolation ($,DNA hj, poly( ~-1ysIni~) prrcipiruriori und cesium clzloride gradient sepurutions. This figure depicts the cesium chloride gradient buoyant density profiles of aliquots of DNA after various purification steps: (a) bulk native DNA (sheared); (b) supernatant DNA after the first polylysine precipitation; (c) supernatant after the second polylysine precipitation ; (d) purified ribosomal cistron D N A isolated from the peak of density p = 1.723 g/cm3 as shown in line (c), Centrifugation was at 30000 rev./min for 70 h at 20 ' C in a 50.1 rotor of the Beckman ultracentrifuge. (0 -0) CsCl density gradient

various frequency classes of DNA sequences and is non-discriminating between transcriptionally active and inactive chromatin when it liberates nucleosomes. However, the enzyme does seem to have a greater propensity to degrade chromatin rich in dA + dT residues relative to that enriched for dG + dC residues. To investigate this aspect of the nuclease activity further and to determine experimentally whether nucleosomes may have any base-sequence specificity in their association with DNA, the ribosomal genes of Xenopus (which are composed of about 64 dG dC residues [30]) were isolated and then reassociated by decreasing salt dialysis with isolated histones. It was the aim of these experiments to determine whether native chromatin (as judged by the nuclease release of nucleosomes) could be reconstructed from this purified gene DNA with its abnormally high dG dC content or whether a significant portion of the reconstituted chromatin would have an unusual nuclease sensitivity due to its unusual base composition.

+

+

Chromatin Subunits in Transcriptionally Active Genes

556

11s

0.3

0

I

0.2

T

0.1

30 50 F r a c t i o n number

10

70

F2a2

0

1

2

. . . . . 3

Distance

4

5

migrated

6

7

I

I

8

(cm)

Table 3 gives data on the efficiency of the isolation steps involved in the polylysine/CsCl purification of ribosomal DNA from adult red blood cell native DNA. Fig. 6 depicts the cesium chloride gradient buoyantdensity profiles of aliquots of DNA from the various purification steps: (a) bulk native D N A ; (b) supernatant DNA after the first polylysine precipitation; (c) supernatant DNA after the second polylysine precipitation ; and (d) purified ribosomal gene DNA. This purified erythrocyte rDNA was then reassociated with purified histone from embryonic tissue culture cells as described in the materials and methods section. The reconstituted ribosomal chromatin was digested with nuclease, nucleosomes isolated by sucrose gradient centrifugation, and the resulting monosome DNA and histones analysed. The results of these experiments are shown in Fig. 7. Inserts (a) and (b) in Fig.7A show the electrophoretic patterns of DNA isolated from nuclease treated reconstituted chromatin after 4 min (a) and 8 min (b) of digestion. The lower curve in Fig.7A

Fig. 7. ( A ) Nuclease sensitivity of reconstructed chromatin reconstituted by dialysis of purified DNA (either bulk or isolated r D N A ) with high-salt-extracted histones as explained in the text. ( B ) Densitometer tracings of Xenopus laevis tissue-culture cell (line X58) high-salt-extracted histones electrophoresed on acidjurea/ 18% polyacrylamide gels as described in te.xt (see also (381). (A) The lower absorbance curve (AZ60)shows the profile obtained when the supernatant from an 8-min nuclease digest (at 200 units of nuclease/ml) of reconstituted ribosomal gene chromatin is run on a 10-300/, sucrose gradient as explained in the text. Note that the reconstituted ribosomal chromatin gives rise to a single 11-S peak corresponding t o the monomer chromatin subunit after this length of nuclease digestion. (a) Aliquots of a 4-min digest of reconstructed whole ( i . e . ,sheared but not fractionated) DNA chromatin from which the D N A has been extracted and then electrophoresed on a slab gel of 1.4 % agarose. Note the multiple bands of D N A present in this digest and their relative sizes as determined by comparison with restriction endonuclease fragments of simian virus 40 (SV 40) D N A of known molecular weights electrophoresed on the same gel. This slot contained 15 Fg DNA. Molecular weights of SV 40 D N A indicated above figure. (b) Aliquot of the 8-min nuclease digest of the reconstituted ribosomal D N A chromatin isolated from the 11-S sucrose gradient peak shown in the lower part of the figure. The D N A was isolated and electrophoresed as described. Note that by this length of digestion the only major band is the monomer D N A of about 200 base pairs in size. The monomer-sized D N A fragments were recovered from this gel and combined with the remaining 11-S DNA for use in the hybridization studies. This slot contained 10 pg RNA. (B) (a) Profile of the five histones present in the initial reconstitution mixture. (b) Profile of the histones recovered from nuclease-derived monomer nucleosomes of the reconstituted ribosomal chromatin. The densitometer tracings were made o n stained gels in a Gilford 2400 recording spectrophotometer

depicts the absorption profile of 8-min-digest nucleosomes run on a sucrose gradient before DNA isolation. It is seen that the reconstituted ribosomal chromatin responds to nuclease treatment in much the same way as native chromatin [l, 11- 151, giving rise to a chromatin subunit with a sedimentation value of around 11-S from which DNA of about 200 base pairs in length can be extracted. Fig.7B shows the absorption profiles of stained 18% polyacrylamide gels of histones used in the reconstitution dialysis step (a) and of histones recovered from the nuclease-derived chromatin subunits (b). These results indicate that the purified ribosomal gene DNA, in spite of its very high content of d G d C residues, can be reconstituted with high-salt-extracted histones to give a chromatin product that is at least superficially similar to native chromatin. Thus, it appears to be very unlikely that nucleosome association with DNA has any basesequence specificity (at a gross level). Therefore the finding that short-term nuclease treatment has a slight

+

557

R. Reeves

+

preference to attack (dA dT)-rich DNA may reflect a somewhat looser association of nucleosomes with this type of DNA, or perhaps it may indicate that native chromatin contains stretches or regions of (dA + dT)-rich DNA that are relatively free of histones or nucleosomes. An equally likely alternative explanation for these findings, however, is that monosoma1 DNA contains less dA dT because of the dA + dT preference of micrococcal nuclease [46]. The main observable difference between whole chromatin and the nucleosomes derived from the reconstructed ribosomal chromatin is the absence of the most lysine-rich of the histones in the latter. This finding of an absence of histone F1 in isolated nucleosomes is in agreement with the findings of others [ l l , 12, 14,15,54,55]. However, the results do not indicate whether the F1 histone was at first associated with the (dG + dC)-rich DNA and then subsequently removed as nuclease digestion sequencially proceeds as has been suggested [56] or was simply never associated with the chromatin in the first place. In any case, it is known from a number of studies that the lysine-rich histone F1 is not essential for normal chromatin reconstruction [57- 601.

+

DISCUSSION Abundant evidence has recently been reported from several laboratories indicating that micrococcal nuclease digestion of chromatin from many different organisms cleaves both synthetically active and inactive gene sequences into nucleosome fragments containing about 200 or so base pairs of double-stranded DNA [l-4,40,41,44,45,49- 531. Moreover, it has also been reported that transcriptionally active gene sequences seem to be more accessible to nuclease attack (within limits) than inactive sequences [1,2] and that this susceptibility appears to be inversely related to the rate of gene transcription [2]. Based on these results it has been suggested that chromatin nuclease sensitivity and the association of nucleosomes with DNA may be of a dynamic rather than static nature [2]. The findings of the present report are generally in agreement with this earlier work but extend considerably our knowledge of the nature of the DNA fragments from transcriptionally active chromatin that have been protected from nuclease cleavage by nucleosome association. If indeed both active and inactive genes contain nucleosomes, then nuclease-derived monosome DNA fragments would be expected to be very heterogeneous in nature and generally to reflect the overall repetitive sequence composition of whole DNA. The detailed analysis of the cot reassociation curves of heterozygous 0/+ nu nucleosome DNA reported on here (Fig. 1 and Table 1) reveals that these fragments are indeed

heterogeneous and furthermore contain all of the repetitive sequence classes of whole DNA in about the same relative proportions. By the hybridization of specifically isolated frequency classes of O/ nu monosome DNA to excess whole 0/+ nu DNA (Fig.2) it has also been demonstrated that various repetitive sequences present within the nuclease-derived fragments are very similar, if not identical, to those of whole DNA. Furthermore, RNA-driven solution hybridization reactions have revealed that fragments of transcriptionally active ribosomal genes (coding for 18-S and 28-S rRNA) are present within 0/+ nu monosome DNA and can account for about 63% of the total rDNA present within whole chromatin (Fig.4). Since the mutant animals, from which these nucleosomes were derived, probably had to have all of their ribosomal genes active for survival [5-71 these results provide strong support for the idea that histones can be associated with transcriptionally active DNA [2]. Another line of evidence that is consistent with this interpretation is the finding that purified ribosomal gene DNA can, despite its unusual base composition, be reconstituted with isolated histones to give chromatin with apparently native nucleosome structure, as judged by nuclease digestions (Fig. 7). These chromatin reconstruction experiments also argue against any base-sequence specificity for nucleosomes within native chromatin. The DNA-driven solution hybridization experiments, in which messenger RNAs were annealed to a vast excess of 0/+ nu monosome DNA (Fig.5), cannot be as easily interpreted as those for the rRNA hybridization experiments just mentioned. On the surface these experiments would suggest that uniquesequence genes actively synthesizing messenger RNAs contain nucleosomes. However, this statement cannot be advanced with confidence, since one is not at all certain that the starting population of cells had all of their messenger-coding genes active at the same time. Thus an alternative interpretation of these results could be that the nucleosomes within the unique sequence genes were derived from inactive messagecoding genes within the starting population. This same argument can also be raised against all of the experiments reported so far [44,45,47,49 - 531, that purport to demonstrate nucleosomes within transcriptionally active unique-sequence genes. To answer this question convincingly it will be necessary to develop a system experimentally, analogous to the use of the heterozygous 0/+ nu mutation, for the study of ribosomal gene structure, in which one is fairly certain that all of the cells in the starting population have a particular mRNA gene active. Nevertheless, these results are not inconsistent with active mRNA genes containing nucleosomes. In addition to the above, two further points of interest are evident from the data presented here.

+

558

Chromatin Subunits in Transcriptionally Active Genes

These concern the two detectable biochemical differences between whole and nucleosome DNA. The first difference, as seen in Fig. 1 and Table 1, is that monosome 0/+ nu DNA reassociates faster, and at lower cot values, than whole O/+ nu DNA. Since, as explained earlier, the increase in rate of reassociation of monosome DNA by about a factor of 0.25 relative to that ofwhole DNAcannot beexplained by suchobvious factors as fragment length, base composition, differential sensitivity of particular sequence frequency classes to nuclease digestions, etc., another explanation must be looked for. The most likely one that is consistent with all of the data is that about 25% of each gene (or of each frequency class of DNA), on the average, is completely hydrolyzed during the enzymatic production of nucleosomes. The reasons for stressing this alternative are: (a) although all of the sequence frequency classes of whole DNA are present in monosome DNA, the entire cot curve for nucleosome DNA is noticably shifted to the left of the cot curve for whole DNA; (b) the unique and intermediate repetitive sequence fragments that remain in monosome DNA are very similar (if not identical) in hybridization properties to the same sequences in whole DNA; (c) no particular gene class (active or inactive) is differentially attacked by micrococcal nuclease under the conditions used here; (d) under the conditions used to isolate nucleosomes, 25 - 30 of the chromatin DNA is completely hydrolyzed leaving the remaining 70-75X of the DNA as nucleosome protected fragments; and (e) a reduction of the bases within each gene (or sequence class) by about 25 % during enzymatic release of nucleosomes could easily account for the reduced complexity and increased reassociation rates seen for monosome DNA. The second observable difference between whole and nucleosome DNA is the apparent increase by about 10% in the number of d G + dC base residues in the nucleosome DNA released by short-time nuclease digestions. In the light of the chromatin reconstruction experiments mentioned earlier, which seem to indicate that nucleosomes do not have any base-sequence specificity in their association with DNA, this finding needs clarification. If, indeed, this promiscuous association of nucleosomes with DNA is reflective of the state of chromatin in vivo, there are several interpretations for the apparent sensitivity of DNA rich in deoxyadenylic and thymidylic acid residues to nuclease digestion. The simplest, and most trivial, explanation is that these results reflect the known preferential cleavage of (dA dT)-rich regions of naked DNA by micrococcal nuclease [46]. More interesting interpretations are also possible, however. For example, the results might reflect the condition that nucleosomes found in predominately (dA + dT)rich regions of DNA are somewhat less firmly associ-

+

ated with the DNA than they are in other regions. Or, alternatively, the results might signify that in vivo there may be regions or stretches of DNA not associated with histones as suggested by Georgiev [61-631. Available data d o not allow one to distinguish between these possibilities at the present time. In evaluating the present study a number of possible alternative explanations for the observed results, other than the occurrence of nucleosomes or subunits composed of histones within chromatin, must be considered. For example it is conceivable that DNA fragments obtained by nuclease digestions were protected by proteins other than nucleosome histones. However, considering the results of a number of mixing experiments reported previously [2] this explanation seems unlikely and this conclusion is further supported by the results of the chromatin-reconstitution experiments reported on here. Another possibility that must also be considered is that RNA polymerase molecules, rather than nucleosomes, protect actively transcribed chromatin from nuclease digestion. This explanation also seems rather unlikely to explain the present results for the following reason. Although accurate molecular size and subunit composition estimates have yet to be reported for Xenopus RNA polymerase I (the nucleolar polymerase) [66, 671, this enzyme(s) appears to be similar to other type I polymerases which have an apparent sedimentation value of 14-15 S on sucrose gradients and whose molecular weight ranges between 450000 and 500 000 [67]. If such a polymerase molecule is about 10 nm in diameter [68], one molecule would be expected to cover about 2 x lo4 daltons of B-form DNA. This would correspond to about 30- 35 base pairs of DNA being protected by one polymerase molecule, a value that is much smaller than the nucleosome-protected DNA fragments (of about 200 base pairs) used in this study. Thus, the most likely explanation for the origin of the double-stranded DNA fragments used in the present study is the protection of these fragments from micrococcal nuclease attack by association of the DNA with histones in the form or chromatin subunits or nucleosomes in vivo. The findings presented in this report are consistent with the previous biochemical evidence that suggests that the association of histones in the form of nucleosomes with chromatin may be of a rather dynamic and labile nature [ 2 ] (unpublished results). This earlier evidence suggested that transcriptionally inactive ribosomal genes are almost completely associated with nucleosomes [l,21 (unpublished results) and, furthermore, that as the genes became increasingly active in transcription the amount of protection from micrococcal nuclease digestion decreased as a function of the rate of gene transcription [2] (unpublished results). However, it was also found that it was highly probably that at least some transcriptionally active

559

R. Reeves

ribosomal genes were still partially, or perhaps transiently, associated with nucleosomes [2], a finding further supported by evidence reported here. However, it should be noted that this evidence of the labile nature of the association of nucleosomes with DNA is not inconsistent with the suggestion that at one extreme of the transcription range (where maximally active ribosomal genes may be completely covered with RNA polymerase molecules) some genes may be generally void of the tightly bound globular form of the nucleosomes [2] (unpublished results). The biochemical data thus suggest heterogeneity within the arrangement of nucleosomes on transcriptionally active ribosomal genes and it is of some interest that recent electron-microscopic studies by Scheer et al. [69] imply that during different stages of amphibian oogenesis the structural morphology of the transcriptionally active amplified ribosomal genes of these animals may vary in a characteristic way as a function of their rates of initiation of ribosomal RNA synthesis. Furthermore, these authors also found that different ribosomal genes, within the same nucleolus and often even those adjacent to each other on the same DNA strand, could differ markedly in their structural morphology and apparent rates of transcription. In addition, other electron-microscopic evidence has been advanced purporting to show that amplified ribosomal genes within amphibian oocytes that are maximally active in transcription may be devoid of globular structures recognizable as nucleosomes [70,71]. However, it was also concluded that histones or other proteins might still be associated with this ribosomal DNA in some other arrangement or configuration, since the chromatin strand present in these genes was still much too thick to be naked DNA [70]. Thus, the available biochemical and morphological data concerning the structure of transcriptionally active ribosomal genes in amphibians may not be as divergent as it initially appeared. In conclusion, the use of micrococcal nuclease as a non-specific endonuclease to cleave chromatin into nucleosomes has allowed the detailed biochemical analysis of the histone-protected fragments of DNA derived from both transcriptionally active and inactive genes. In future experiments the use of micrococcal nuclease to release nucleosomes from all gene sequences in conjunction with the use of pancreatic DNase I, which may preferentially digest transcriptionally active gene sequences [53], should allow for further detailed analysis of both synthetically active and quiescent chromatin.

REFERENCES 1 2 3 4 5 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24

25. 26. 27 28 29 30 31 32 33 34

~

35 36 37. 38 39 40

This research was conducted under a grant from the National Research Council of Canada. I would also like to thank D r John B. Gurdon for the generous gift of the heterozygous 0 / + nu Xenopus.

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R . Reeves, Department of Zoology, University of British Columbia, 2075 Wesbrook Place, Vancouver, British Columbia, Canada, V6T 1W5

Note Added in Proof. Recently, Hagashinakagawa et al. [Dev. Biol. 55, 375- 386 (1977)] have reported isolating amplified ribosomal gene chromatin from transcriptionally active Xenopus oocytes and have detected in these the presence of proteins which comigrate with histones H2a, H2b, H3 and H4. In addition, they have found that some fraction of the Xenopus rDNA (both gene and spacer regions) can be cut into subunit fragments by micrococcal nuclease digestion of nuclei or nucleoli. Furthermore, Matsui and Busch [Exp. Cell Res., in press (1977)l have reported that “euchromatin” isolated by DNase I1 digestion of purified Novikoff hepatoma nucleoli contains all histone fractions and has a structural heterogeneity similar to that of euchromatin containing active single copy sequences.