(Received for publication, November 17, 1995; and in revised form, March 6, 1996)
From the
Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M.(1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.
It is now well established that the regulation of gene
expression in eukaryotes occurs at the level of chromatin and that
transcription needs changes in chromatin
structure(1, 2) . There is a consensus in the
literature with regard to the nucleosome structure upon transcription
of protein-coding genes. In contrast, the picture that emerges from
studies of ribosomal RNA genes is rather confusing. Electron microscopy
analyses suggest that DNA in transcribing ribosomal gene chromatin is
in an extended unbeaded conformation when spread for
visualization(3, 4, 5) , in some cases
indistinguishable from coprepared naked DNA(3, 5) .
Many biochemical studies, however, demonstrate the presence of
organized histone-containing particles in ribosomal
chromatin(6, 7, 8, 9, 10) .
Such a contradiction is not surprising having in mind that in somatic
cells only a portion of the repeated rRNA genes are transcribed (11, 12) and that electron microscopy is restricted to
transcribed copies, while biochemical analysis assays the entire set of
ribosomal genes. An important contribution in this respect was the
demonstration by psoralen photo-cross-linking that cells in vertebrates (13, 14) and in yeast (15) contain two types
of ribosomal chromatin, one that consists of nucleosomes and represents
the inactive genes and one that lacks a repeating structure and
corresponds to the transcribed copies. If, however, nucleosomes
disappear as distinct entities, it is not clear whether histones are
released from or remain attached to the extended DNA. The existence of
nucleosome-free ribosomal chromatin as revealed by psoralen
cross-linking does not mean absence of histones (13, 16) . Moreover, the same authors have shown that
histone-DNA interactions, different from those in intact nucleosomes,
do exist and allow extensive access of psoralen to histone-complexed
DNA(17) . Studies on Drosophila melanogaster active
ribosomal RNA genes claimed that they are packaged into unstable
nucleosome structure (18) (see, however, (19) ).
Association of histones with transcribed Xenopus laevis rRNA
genes in nucleosome-like structures was demonstrated by the 200-bp
spacing of the cleavage sites of topoisomerase I(20) . In a
study on the chromatin structure of ribosomal genes of the same
organism by UV laser-induced histone-DNA cross-links, we found that
coding sequences and spacer enhancers and promoters were associated
with histones both in actively transcribed embryonic genes and in their
silent counterparts in the erythrocytes (21, 22) .
The presence of histones on transcribed ribosomal genes raises the question about their postsynthetic acetylation. Generally, the level of histone acetylation is higher in transcriptionally active than in silent chromatin(23, 24) . The numerous correlative evidence communicated during the last 30 years was recently fortified by more direct biochemical studies (25, 26, 27, 28, 29) and genetic experiments(30, 31) . Very recently, the problem faced a new development connected with the role of acetylation of individual core histone species as well as the modification of different lysine residues on the same histone molecule (32, 33, 34) . It should be stressed, however, that all of these data come from studies on protein-coding genes. The genes transcribed by RNA polymerase I have not been purposefully studied in this respect merely because it was not clear whether they contain histones at all. Two contradictory results have been reported so far, claiming hyperacetylation of H3 in the active nucleolar chromatin from Physarum polycephalum(9) , and a lack of significant difference between histone acetylation in nuclei, nucleoli, and active ribosomal chromatin from the same organism(35) . This work presents our results on the acetylation of histones bound to ribosomal genes in rat tumor cells, grown in the presence of butyrate to inhibit deacetylation (36) . An antibody capable of recognizing acetylated core histones was used to immunoprecipitate cross-linked protein-DNA complexes generated by irradiation of nuclei with a UV laser. The DNA from the antibody-bound complexes, containing both active and inactive rRNA genes, was enriched in coding rDNA sequences. In a parallel experiment, purified mononucleosomes assumed to contain inactive rRNA gene copies (13) were also immunoprecipitated, but the antibody-bound DNA contained coding rDNA sequences in an amount similar to that in the unfractionated DNA.
To isolate
nuclei, the cells were pelleted, washed twice in 0.14 M NaCl,
once in 10 mM Tris, pH 7.5, 0.14 M NaCl, 3 mM MgCl, 1 mM phenylmethylsulfonyl fluoride, 10
mM sodium butyrate and then suspended in the same buffer
supplemented with 0.5% Triton X-100. After incubation for 10 min on
ice, the suspension was centrifuged, and the pellet was washed twice in
the buffer without Triton X-100. The final nuclear pellet was stored in
0.1 M NaCl, 50 mM Tris, pH 7.5, 10 mM sodium
butyrate.
Purified mononucleosomes were immunoprecipitated following a protocol described elsewhere (25) except that IgG Sorb was used to bind antibodies (see above), and all incubations were carried out overnight at 4 °C.
The experimental approach we followed is outlined in Fig. 1. Cross-linking was used to assay the acetylation of histones, bound to the rRNA genes regardless of whether they were wrapped in nucleosomes or existed in an extended anucleosomal conformation. The experiments with the purified mononucleosomes addressed the same question solely for the nucleosome-organized gene copies.
Figure 1: Strategy for studying acetylation of histones associated with the ribosomal genes independently of their chromatin structure (using UV laser-irradiated nuclei) and, alternatively, with those that are organized in nucleosomes (using isolated monosomes).
Figure 2:
Characterization of the affinity-purified
antiacetyl antibody. a, ELISA of the reaction of the antibody
with chemically acetylated (-
) and nonmodified
(
-
) histone H4; (
- - - -
), response
of the nonimmune IgG to chemically acetylated H4. b, ELISA of
the reaction of the antibody to chemically acetylated
(
-
) and nonmodified (
-
)
BSA; (
- - - -
), response of the nonimmune IgG to
chemically acetylated BSA. c, inhibition study of the binding
of the antibody (
) and nonimmune IgG (
) to physiologically
acetylated H4, extracted from rat tumor cells grown in the presence of
butyrate. The antibody was mixed with H4 in free solution, and the
residual unbound antibody was back-titrated with chemically acetylated
H4. d, immunoblotting of histones from butyrate-treated Guerin
ascites tumor cells after electrophoresis in 15% polyacrylamide/acetic
acid/urea/Triton gel. Proteins were blotted and stained with Amido
Black (left lane) or reacted with the antibody (right
lane). The zone of H4 is shown. The number of acetylated groups is
indicated by numbers 0-4.
Another characteristic is the reaction of the antibody with histones as a function of the number of acetylated lysines. To test this, acetylated histones from tumor cells grown in the presence of butyrate were separated on a polyacrylamide gel, blotted on filters, and revealed with the antibody. Acetylation of H4, which is best separated in this gel, is presented in Fig. 2d. Again, the antibody showed no reaction with nonacetylated H4. The immune reaction increases upon increasing the level of acetylation; the most intensive bands on the immunoblot are the tri- and tetraacetylated forms of H4, while the protein pattern of the histone is dominated by its mono- and diacetylated molecules. The antibody, therefore, recognizes any acetylated H4 molecule, but the reaction is much stronger with hyperacetylated forms.
The precipitation ability of the antiacetyl antibody is demonstrated in Fig. 3. The electrophoretic analysis of the antibody-bound fraction shows the presence of acetylated molecules of histones H4, H3, and H2B. This is well illustrated with histone H4, which is best resolved in the electrophoretic system used; the antibody-bound fraction contains mainly tetra- and triacetylated molecules, while the unbound fraction is enriched for unmodified and low acetylated forms of H4.
Figure 3: Antibody-precipitated proteins. Electrophoresis in 15% polyacrylamide acetic acid/urea/Triton gel of proteins extracted from monosomes (obtained from butyrate-treated cells) before immunoprecipitation (a) and after fractionation into unbound (b) and antibody-bound (c) material.
Figure 4:
Dot hybridization analysis of DNA
immunoprecipitated with antiacetyl antibody from cross-linked
protein-DNA complexes. Total rat DNA is DNA purified from the
cross-linked complexes, which were fractionated by the antibody into
bound and unbound DNA. Three identical filters were prepared for
hybridization, each one containing tree dots of immobilized total rat
DNA (1.0, 0.5, and 0.25 µg) and three dots of p20 DNA, containing BamHI-EcoRI fragment of rat rDNA, coding for 28 S RNA
(corresponding to a 1.0-, 0.5-, and 0.25-µg insert) and were
hybridized to P-labeled total rat DNA (A), bound
DNA (B), and unbound DNA (C), respectively. The three
hybridization mixtures were prepared in such a way as to contain an
equal quantity of DNA (based on [
H]thymidine
incorporation) and an equal amount of
P-radioactivity in
equal final volumes. The content of ribosomal DNA sequences in the
tested DNA preparations is illustrated by their hybridization to p20
DNA (bottom lane), the enrichment of the bound DNA for coding
rDNA and, respectively, the depletion of the unbound DNA of these
sequences can be seen as B to A and C to A signal ratios, respectively. Hybridization to total rat DNA (upper lanes of this figure and in Fig. 5) is shown to
demonstrate that the antibody-bound DNA is a subset of the total rat
DNA (see ``Discussion'').
Figure 5: Dot hybridization analysis of DNA from immunoprecipitated mononucleosomes. Total rat DNA was isolated from nuclei and sonicated to about 300 base pairs; input DNA is isolated from purified mononucleosomes before their fractionation into unbound and antibody-bound DNA. Filters and hybridization mixtures were prepared as described in the legend to Fig. 4. The content of rDNA in bound and unbound DNA can be seen as C to B and D to B signal ratios, respectively (bottom lane). Upon immunofractionation of monosomes, the input DNA showed somewhat reduced content of rDNA as compared to total DNA (see ``Discussion''). The inset represents a digest of nuclei with micrococcal nuclease. Left three lanes are stained with ethidium bromide, the right three lanes are Southern analyses of DNA with p20. The asterisk marks the position of mononucleosomal DNA.
The observed enrichment of bound DNA in ribosomal genes might eventually be due to a specific affinity of rDNA to IgG Sorb, unrelated to the presence of the antibody. This possibility was tested by two experiments in which the antibody was either omitted from the solution with which the cross-linked complexes were incubated prior to addition to IgG Sorb, or replaced by nonimmune IgG. The amount of material absorbed under these conditions did not exceed 5% of that absorbed in the presence of the antiacetyl antibody. What is the nature of this DNA, an average probe DNA or selectively attached rDNA? To determine this, equal amounts of genomic rat DNA and DNA from nonspecifically absorbed complexes were analyzed for the presence of rDNA by hybridization to p20 DNA as described above. The signals obtained with the two DNA preparations did not significantly differ (not shown), i.e. nonspecifically absorbed DNA is bulk DNA. This holds true upon immunoprecipitation of noncross-linked chromatin.
Since the presence of histones on transcribed rDNA has been demonstrated, it was reasonable to examine their level of acetylation. The evidence so far reported that links histone acetylation to transcription was based on studies with transcribed and repressed protein-coding genes. Therefore, a study designed to assay the acetylation of histones bound to rDNA is justified only if it addresses the problem active/inactive genes. The interpretation of any biochemical study of ribosomal genes in chromatin should take into account the fact that in the somatic cell only a part of repeated rRNA genes is transcribed(11, 12) . To solve the problem we exploited the evidence that, in vertebrates and in yeast, half of these genes were packaged in nucleosomes and were inactive, while the other half were nucleosome-free and contained the active gene copies(13, 14, 15) . To analyze directly the active genes is a difficult task, one has to isolate a chromatin fraction that is not organized in nucleosomes. Another approach is to compare mammalian somatic cells with markedly different levels of rRNA synthesis. This does not solve the problem, however, because according to the same studies(13, 14) , the 1:1 ratio of active versus inactive ribosomal gene copies remained constant, independent of the transcriptional activity of these genes. However, one can easily isolate mononucleosomes, shown to represent the inactive ribosomal chromatin(13) . Accordingly, our experimental approach consisted of two parallel procedures. The first one assays the acetylation of histones bound to both active and inactive rRNA genes. This was accomplished by cross-linking proteins to DNA in the nuclei by irradiation with UV laser. An important property of the laser is that it cross-links in nanosecond time intervals, thus ``freezing'' in vivo existing protein-DNA interactions(37, 41) . It must be mentioned that the reversible acetylation of histones does not affect their cross-linking to DNA, although the covalent link between histones and DNA was shown to proceed via the N-terminal tails(43) , where the acetylatable lysines had been located. After cross-linking, an antibody that specifically recognizes acetylated histones but not their nonmodified parental molecules was used to select DNA fragments linked to acetylated histones. DNA of these fragments was then analyzed for the presence of sequences coding for mature rat 28 S RNA. The second procedure aimed to assay the acetylation of histones bound to the inactive nucleosome-organized rRNA genes. To this end, mononucleosomes from micrococcal nuclease-digested nuclei were immunofractionated, and DNA of the antibody-bound nucleosomes was analyzed as above.
To prove that hyperacetylated histones were responsible for the immunoprecipitation, proteins of the antibody-bound fraction were analyzed by polyacrylamide gel electrophoresis (Fig. 3). The preferential precipitation of the hyperacetylated histone molecules is well illustrated with H4. The bands of tri- and tetraacetylated molecules dominated the picture. H2B and H3 were also acetylated. Such a result is to be expected, because histones are the best known acceptors of acetyl groups(23) . Beside them, the only nuclear proteins shown to undergo acetylation are HMG proteins(23) . The lack of hyperacetylated forms of these proteins as well as their much lower quantity, compared to histones, makes their contribution to precipitation negligible, if any. Nevertheless, the antibody-bound fraction was dotted on filters and reacted with a biotinylated anti-HMG1 antibody, which cross-reacted also with HMG2. No reaction was observed (data not shown). As for HMG14/17, their amount is much lower than that of HMG1/2.
The antibody-bound DNA obtained upon immunofractionation of the cross-linked protein-DNA complexes was 5-20-fold enriched in coding rDNA sequences. This means that rRNA gene copies have been associated with acetylated histones. The question is which of them, the active genes, the inactive ones, or both? By analogy with protein-coding genes one may assume that at least the transcribed copies should be acetylated. However, the possibility that all rRNA genes were associated with acetylated histones could not be ruled out. The question was answered by the alternative approach, immunoprecipitation of monosomes, shown to contain the inactive rDNA(13) . The antibody-bound monosomal DNA contained coding rDNA sequences in an amount that did not significantly differ from that of the input DNA. The lack of enrichment is interpreted to mean that nucleosome-organized rRNA genes have been associated with nonacetylated histones. It follows, therefore, that the acetylated histones that enriched the antibody-bound fraction of the cross-linked protein-DNA complexes in coding rDNA have been associated with the rest of the gene copies, the anucleosomal ones, claimed to be transcriptionally active(13) .
To check the reliability of both the
experimental approach and the antibody in selecting defined DNA
sequences, in a control experiment we assayed the distribution of DNA
from centromeric heterochromatin, shown to be associated with
underacetylated histones(34, 44) . To this end, UV
laser cross-linked histone-DNA complexes from mouse cells were
immunoprecipitated with either the antiacetyl antibody used in this
study or an antibody against histone H2A. The two antibody-bound
fractions were analyzed for the presence of mouse satellite DNA. The
content of satellite sequences in the anti-H2A-precipitated DNA was
similar to that in bulk mouse DNA, while in the antiacetyl
antibody-bound DNA the amount of satellite sequences was dramatically
reduced. ()
Another conclusion from the experiments with
the ribosomal genes is that immunofractionation on the basis of
acetylated histones resulted also in fractionation of DNA, separating a
subset of it (antibody-bound DNA) which differs in sequence complexity
from total DNA. This can be seen upon comparing the hybridization
signals total DNA/total DNA and bound DNA/total DNA (upper lines of Fig. 4, A and B, and 5, A and C). Under the conditions of the experiment, when equal amounts
of total rat DNA were dotted on the membranes and hybridized to equal
amounts of P-labeled total DNA and bound DNA, the signal
bound DNA/total DNA has been repeatedly found lower than the signal
total DNA/total DNA. The dependence of the filter hybridization on the
amount of reiterated DNA on one hand and, on the other, the
experimental evidence that the fractionation of chromatin fragments on
the basis of acetylated histones results in selection of transcribed
protein coding DNA(25, 26, 45) , which
generally represents single copy genes, suggest an explanation of this
finding.
An intriguing observation in the above cited studies of ribosomal chromatin by the psoralen strategy (13, 14, 15) was that in vertebrates the changes in the rate of rRNA synthesis did not result in changes in the ratio of active/inactive ribosomal chromatin structures(13, 14) , while yeast can rapidly change the portion of active genes in response to altered growth conditions(15) . A conclusion was made that the regulation of rRNA synthesis in vertebrates is achieved at the level of transcription initiation of the available anucleosomal genes rather than by activation/inactivation of gene copies(15) . It was recently reported that genes which are not transcribed at a moment but are ``poised'' to transcription have been associated with acetylated histones(26, 45, 46) . In the light of this evidence, our data that histones associated with anucleosomal (transcribed) ribosomal genes are acetylated, while those bound to nucleosome-organized (inactive) gene copies are not, support such a view for the modulation of rRNA synthesis.