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(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 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
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 (
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
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 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.
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 11852-11857
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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) .
Growing Cells in Butyrate and Isolation of
Nuclei
Guerin ascites tumor cells were inoculated in albino
rats. On day 7 after transplantation the ascites fluid was collected,
and the cells were pelleted by low speed centrifugation and resuspended
in Dulbecco's modification of Eagle medium (Flow Laboratories,
UK) containing 5% fetal serum, 20 mM sodium butyrate, heparin
(1 unit/ml), and [
H]thymidine. The suspension was
incubated 12 h at 37 °C under gentle shaking. The viability of the
cells was monitored by the trypan blue exclusion test.
, 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.Preparation of Nucleosomes
Nuclei were resuspended
in digestion buffer containing 10 mM Tris, pH 7.5, 10 mM NaCl, 10 mM sodium butyrate, 1 mM CaCl, 0.1 mM phenylmethylsulfonyl fluoride at
a concentration of 3 mg/ml DNA and digested with 240 units/ml
micrococcal nuclease (Sigma) for 15 min at 37 °C. The digestion was
stopped with 5 mM EDTA, and the suspension was chilled on ice
and centrifuged for 10 min at 3000 
g. The supernatant
was saved, and the pellet was resuspended in 6 mM sodium
butyrate, 0.25 mM EDTA. Following an incubation on ice for 30
min, the suspension was centrifuged as above, and the supernatant was
added to the first one. The combined supernatants were concentrated
with Centricon microconcentrator (Amicon), loaded on a 5-25%
sucrose gradient containing 10 mM Tris, pH 7.5, 0.1 M NaCl, 6 mM sodium butyrate, 0.25 mM EDTA and
centrifuged in a Beckman SW28 rotor at 24,000 rpm for 19 h at 5 °C.
The mononucleosome fraction was collected and dialyzed overnight
against the buffer for immunoprecipitation of monosomes.Cross-linking of Proteins to DNA in Nuclei by UV Laser
and Isolation of Protein-DNA Complexes
A nanosecond Nd:YAG laser
was used to irradiate nuclei (and, when necessary, the monosomes)
suspended in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10
mM sodium butyrate at a concentration of about 5 A
units/ml. Generally, irradiation at 266 nm was
performed in a rectangular fused silica cuvette under constant stirring
at a pulse energy of 7 mJ, diameter of the beam 0.5 cm, and repetition
rate 0.5 Hz as described elsewhere for the picosecond regime of
irradiation(37) . In some experiments the cross-linking was
performed by single-pulse laser irradiation, using a flow cuvette and
computer-commanded laser pulse (a technology to be published elsewhere)
with essentially the same result. The cross-linked protein-DNA
complexes were sonicated to reduce the size of DNA to 250-300
base pairs, made 1% in Sarkosyl, and centrifuged through a preformed
gradient of CsCl(37) .Antibody Generation and Purification
Chemically
acetylated histone H4 was used as an antigen. Purified calf thymus
histone H4 was dissolved at 1 mg/ml in 50 mM sodium
bicarbonate buffer, pH 8.0, and treated with 5 mM acetic
anhydride for 1 h on ice. The reaction was terminated by adding Tris,
pH 8.0, to a final concentration of 10 mM. Antibodies were
raised by immunization of rabbits with acetylated histone-tRNA
complexes(38) . The antigen (1 mg in phosphate-buffered saline)
was emulsified with an equal volume of Freund's complete adjuvant
and injected at multiple intradermal sites. Stimulations in an
incomplete adjuvant were repeated on the 7th and 14th day, followed by
an intravenous administration of the same quantity of antigen. Sera
were taken starting 1 week after the booster. The antiacetyl antibodies
were immunospecifically purified from sera by affinity chromatography
using chemically acetylated bovine serum albumin conjugated to
CNBr-Sepharose 4B (Pharmacia Biotech Inc.). Bound antibodies were
eluted using 3.5 M KSCN in 20 mM Tris, pH 8.Immunochemical Analysis and
Immunoprecipitation
The reaction of the antiacetyl antibody with
different antigens was carried out by ELISA, (
)inhibition
experiments, and immunoblotting as described previously(38) .
The purified protein-DNA complexes were immunoprecipitated with the
antiacetyl antibody as reported elsewhere(21) . Briefly, 100
µl of IgG Sorb (The Enzyme Center, Malden, MA) were suspended in
0.5 ml of 1% bovine serum albumin (BSA) in phosphate-buffered saline
and agitated 30 min at 20 °C to block the sites for nonspecific
absorption. After centrifugation, the pellet was suspended in a 0.5-ml
mixture of cross-linked material and antibody in 50 mM HEPES,
pH 7.5, 2 M NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium
deoxycholate, 5 mM EDTA, 0.1% BSA, and incubated overnight at
4 °C under constant shaking. Following centrifugation, the pellet
was washed five times in the same solution, three times in 50 mM HEPES, pH 7.5, 0.15 M NaCl, 5 mM EDTA, and the
remaining material was eluted with 3.5 M KSCN in 20 mM Tris, pH 8.2.Hybridization
All DNA preparations were purified
by treatment with RNase (50 µg/ml, 20 min, 37 °C), followed by
Proteinase K digestion (100 µg/ml for at least 4 h at 37 °C),
extraction with phenol-chloroform, and precipitation with ethanol. The
DNA samples to be immobilized on membranes were denatured in 0.5 M NaOH, 1.5 M NaCl for 10 min at 37 °C and 1 min in
boiling water and dotted on Hybond N filters (Amersham Corp.). The
filters were immersed in 0.5 M NaOH, 1.5 M NaCl for 5
min and then in 0.5 M Tris, pH 7.2, 1.5 M NaCl for 30
s, blotted dry, and exposed to UV light for 2 min to cross-link DNA to
the filter. The filters were prehybridized in 5 SSC (1
SSC = 0.15 M NaCl, 0.015 M sodium citrate), 5
Denhardt's solution, 0.5% SDS, 50 mM phosphate
buffer, pH 7.0, 200 µg/ml denatured salmon sperm DNA for 2 h at 62
°C. Hybridization was carried out under the same conditions for 16
h at 62 °C using DNA labeled by random priming to specific
activities of 2-9 10
cpm/µg at a final
concentration of 100 µg/ml. Following hybridization, filters were
washed twice in 2 SSC, 1% SDS for 15 min at 65 °C and twice
in 1 SSC, 0.1% SDS for 15 min at 65 °C. Filters were
blotted dry and autoradiographed at -80 °C using intensifying
screen. The hybridization signals were quantified by densitometry of
the exposed film.Electrophoresis
Histones were extracted with 0.25 N HCl, precipitated with 20% trichloroacetic acid, and
separated by electrophoresis in 15% polyacrylamide/acetic
acid/urea/Triton gels(39) .
Antibody Characterization
The polyclonal antibody we
raised should meet certain criteria in order to serve our purpose. It
should be specific for the 
-N-acetyl group of lysine and
must not recognize the nonmodified parental molecules. This is
illustrated in Fig. 2. Using ELISA, a clear reaction of the
antibody with chemically acetylated H4 is observed while the reaction
with nonacetylated H4 is similar to that observed with the nonimmune
IgG (Fig. 2a). The ability of the antibody to react
with the -N-acetyl group of proteins other than H4, the
histone that was used to raise the antibody, is demonstrated by
chemically acetylated bovine serum albumin (Fig. 2b).
The antibody was further characterized by inhibition of the immune
reaction (Fig. 2c). We show this test not only to
confirm the specificity of the antibody but rather to demonstrate its
ability to react with the antigen in solution. Such an ability is conditio sine qua non if the antibody is to be used for
immunoprecipitation. Besides, as far as the inhibition experiments have
been carried out with physiologically acetylated histones, this test
justifies the application of the antibody in studying histone
acetylation in nuclei. The same result was obtained when the inhibition
was carried out with cross-linked protein-DNA complexes. This means
that linking of the antigene to DNA by UV laser does not affect the
binding ability of the antibody.
-) 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.
Antibody-bound DNA from Cross-linked Protein-DNA
Complexes Containing Both Nucleosome-organized and Anucleosomal rRNA
Genes Is Enriched in Sequences Coding for rRNA
Following
irradiation, nuclei were sonicated to reduce the size of DNA to about
300 base pairs and passed through CsCl to separate the covalently
linked protein-DNA complexes from free DNA and proteins. Such a
fragment size was used to avoid a situation when tandemly arranged rRNA
genes could be a part of a long stretch of nonribosomal DNA, carrying
covalently linked acetylated histone. After immunoprecipitation, equal
quantities of precipitated DNA (``bound'' DNA) and
nonprecipitated DNA (``unbound'' DNA) were screened for the
presence of coding rDNA sequences, using as a probe the plasmid p20
containing a 1.6-kilobase pairs BamHI-EcoRI fragment
coding for mature 28 S rat rRNA. This fragment has been subcloned from
RrIV into pBR322 (40) . Bound, unbound, and total rat DNA
(DNA purified from the cross-linked protein-DNA complexes before
immunoprecipitation) were labeled with [
P]ATP
and hybridized to p20 DNA immobilized on filters. The signal ratio
bound DNA/total DNA gives the enrichment (or, alternatively, the
depletion) of antibody-bound DNA in coding rDNA sequences. Several
independent experiments showed 5-20-fold enrichment of bound DNA
in these sequences, while their amount in the unbound DNA was
respectively reduced. An experiment with about 20-fold enrichment of
bound DNA in rDNA is demonstrated in Fig. 4. In parallel dots,
made on the same membrane, the three labeled DNA preparations were
hybridized to immobilized total rat DNA in order to illustrate that
immunofractionation of protein-DNA complexes on the basis of acetylated
core histones resulted also in immunofractionation of DNA. In this way
a subset of sequences was selected (bound DNA) which differed in
sequence complexity from the total DNA (see ``Discussion'').
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'').
Antibody-bound DNA upon Immunoprecipitation of Monosomes
Is Not Enriched in Coding rDNA
Sucrose gradient-purified
mononucleosomes were directly immunoprecipitated with the antiacetyl
antibody and were further processed as the cross-linked material. A
typical autoradiography of a dot hybridization analysis is shown in Fig. 5. In this case the hybridization of antibody-bound and
unbound DNA to p20 were compared to the hybridization of
``input'' DNA (DNA isolated from the purified mononucleosomes
before precipitation) to p20. In the above experiments using the
cross-linked protein-DNA complexes from irradiated nuclei, the input
DNA is in fact a total DNA since the laser cross-linking is a random
process with respect to the DNA sequence(41) . In contrast,
nucleases preferentially cleave transcriptionally active chromatin, so
that the resulting mononucleosomes might be deficient in certain DNA
sequences. In our experiments the input DNA showed somewhat reduced
content of rDNA compared with total DNA (Fig. 5, A and B). One explanation of this finding suggests that micrococcal
nuclease treatment has preferentially eliminated nuclease-sensitive
active (anucleosomal) ribosomal gene copies. The results of Fig. 5could also be obtained if rDNA has not been cleaved to
monosomes upon digestion of nuclei and hence could not have been in
this fraction. An efficient protection of rDNA from nuclease attack in
comparison to bulk chromatin was reported but, nevertheless, rDNA of
monomeric size has been obtained(42) . In our digestion
experiments the presence of rDNA in the ladder of DNA fragments,
including monomeric ones, was demonstrated by Southern blot analysis
using p20 DNA (Fig. 5, inset). Different independent
experiments with precipitated mononucleosomal DNA showed a signal ratio
bound DNA/p20 DNA to input DNA/p20 DNA of 1 or less (Fig. 5). It
follows, therefore, that the histone molecules associated with
nucleosome-organized rRNA gene copies are not acetylated. Essentially
the same results were obtained upon immunoprecipitation of
laser-irradiated monosomes.
)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.
))
We thank D. Angelov for the help with the laser
irradiation and I. Stancheva for generous gifts of plasmid p20.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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