(Received for publication, May 22, 1995; and in revised form, June 1, 1995)
From the
A homogeneous oligonucleosome complex was prepared by
reconstitution of highly hyperacetylated histone octamers onto a linear
DNA template consisting of 12 tandemly arranged 208-base pair fragments
of the 5 S rRNA gene from the sea urchin Lytechinus
variegatus. The ionic strength-dependent folding of this
oligonucleosome assembly was monitored by sedimentation velocity and
electron microscopy. Both types of analysis indicate that under ionic
conditions resembling those found in the physiological range and in the
absence of histone H1, the acetylated oligonucleosome complexes remain
in an extended conformation in contrast to their nonacetylated
counterparts. The implications of this finding in the context of a
multistate model of chromatin folding (Hansen, J. C., and Ausio,
J.(1992) TIBS 197, 187-191) as well as its biological
relevance are discussed. In our opinion, one of the important landmarks in the chromatin
field during the last two decades has been the realization that
histones are not merely passive structural players but have an
important functional role in the modulation of genomic expression (2, 3, 4) . In other words, they have both a
structural and a functional role. At the structural level, histones
provide the protein blocks that allow for the hierarchical folding of
DNA in nucleosomes, higher order, and chromosomal structures. At the
highest level of compaction the structures arising from such
organization (chromosomes) may be essential to prevent the shearing of
long eukaryotic DNA molecules during the transmission of the genetic
material from one cell to another in the course of cell division (5, 6) in eukaryotes. At the lower levels, chromatin
structure and nucleosomes may have also been selected during the
evolutionary transition from prokaryotes to eukaryotes, as the scaffold
that provides support and modulates the fine tuning of the more complex
functional mechanisms of transcription and replication. All these
levels of folding must be reversible in order to accommodate the
different functional needs during the cell cycle. The dynamic aspects
of this chromatin folding and its implications both at the structural
and functional level are still poorly understood. During the cell
cycle, histones undergo several chemical post-translational
modifications that could presumably be involved in the modulation of
chromatin folding. One of the more extensively characterized of such
modifications has been histone acetylation. Histone acetylation is a
dynamic post-translational metabolic modification (7) that has
been strongly correlated with transcriptional activity and with the
processes of histone deposition (such as during replication) or histone
displacement (such as during spermatogenesis) (for detailed reviews see (8, 9, 10, 11) ). At the
structural level, histone acetylation is a lysine amidation reaction
catalyzed by acetyltransferases. By virtue of its chemical nature, it
alters the net positive charge balance of the N-terminal regions
(tails) of the core histones (histones H2A, H2B, H3, and H4), and as
such, it has been long hypothesized to weaken the histone-DNA
interactions involved at the different levels of chromatin folding. A
disruption of the protein-DNA interactions would be expected to lead to
the loss of stability (folding) of both the chromatin fiber and its
constitutive subunits, the nucleosomes. Nevertheless, the experimental
effort to provide support to this hypothesis has led in the past, quite
often, to conflicting results. At its best it has provided evidence for
only minor changes in chromatin folding. The structural effect of
histone acetylation on the conformation of the nucleosome core particle (12) (consisting of a double set of each H2A, H2B, H3, and H4
histones and 146-base pair DNA) has been found to be
small(12, 13, 14, 15) . A larger
effect was observed in the case of nucleosomes consisting of longer
DNA(16) . Very small charges were also reported at the higher
order structure level of chromatin folding(17, 18) . Despite all this, there is increasing evidence that the major effect
of the histone tails (and hence acetylation) on chromatin folding (19) arises from their interactions with the linker DNA region
connecting adjacent nucleosomes in the chromatin fiber. We have
recently proposed a coupled multistate model of chromatin dynamics to
account for the contribution of chromatin folding in the modulation of
genetic activity (1) . The results that follow, while providing
experimental support for this model, also point to the importance of
acetylation in the mechanisms of chromatin folding and dynamics.
1.2% agarose gels were prepared in TBE buffer (25 mM Tris borate, 0.5 mM EDTA, pH 8.0), according to Maniatis et al.(23) .
We have characterized in the past (19, 20) the folding behavior of reconstituted
oligonucleosome complexes using a DNA template consisting of 12
tandemly arranged copies of a 208-base pair fragment of 5 S rRNA gene
from the sea urchin L. variegatus and chicken erythrocyte core
histones. In the present work we have reconstituted 208-12
oligonucleosome complexes with the same DNA template but using highly
acetylated core histones obtained from HeLa cells grown in the presence
of sodium butyrate (to inhibit the cell deacetylases) and core histones
obtained from HeLa cells grown in the absence of butyrate (used as a
control). To this purpose, the chromatin from the butyrate-treated
cells was fractionated based on its differential solubility in the
presence of divalent cations to yield fractions highly enriched in
highly hyperacetylated core histones(14) . Upon depletion of
histone H1 the core histones from both butyrate (acetylated) and
non-butyrate (control) treated cells were purified under nondenaturing
conditions using hydroxylapatite as described under ``Experimental
Procedures.'' Fig. 1shows the electrophoretic analysis
of the core histones (panels A and B) as well as the
DNA template and the resulting reconstituted oligonucleosome complexes (panel C).
Figure 1:
A, acetic
acid-urea-Triton-polyacrylamide gel electrophoresis of hyperacetylated
histone octamers (lanes1), native histone octamers
(control) (lanes2), histone composition of the
chromatin isolated from HeLa cells grown in the presence of 5 mM sodium butyrate (lane3), histone composition of
the chromatin isolated from HeLa cells grown in the absence of sodium
butyrate (lane4). The smallnumbers on the side of the histone labels (H2A, H2, H2B, H3, and H4) point to the
different extents of acetylation of the histone fractions. B,
SDS-polyacrylamide gel electrophoresis of the samples shown in A. C, agarose (1.2%) native gel electrophoresis of
208-12 DNA template (lane D), oligonucleosome 12-mer
reconstituted with native histone octamers (lane N),
oligonucleosome 12-mer reconstituted with hyperacetylated histones (lane A),
The acetylated histones used in this work (Fig. 1A, lanes1) correspond to
fraction a of the chromatin fractionation procedure (14) and
contain an average of In
agreement with previous observations(19, 25) , the
electrophoretic mobility of the 208-12 oligonucleosome complexes
reconstituted with nonacetylated core histones is very similar to that
of their 208-12 DNA template. Under the electrophoretic
conditions used here (see ``Experimental Procedures'') the
acetylated oligonucleosome 12-mers also exhibit an almost identical
mobility (Fig. 1C, laneA).
Sedimentation equilibrium analysis carried out with both samples (see
also Fig. 2) provided linear plots of log (absorbance) versus the square of the radial distance for the concentration
gradient at equilibrium. The molecular weight established from these
plots M
Figure 2:
Plot of log Yi-Yo versus r
The
salt-dependent hydrodynamic behavior of the control and hyperacetylated
oligonucleosome complexes is shown in Fig. 3in comparison with
the hydrodynamic data obtained from oligonucleosomes reconstituted with
chicken erythrocyte histones.
Figure 3:
Effect of the ionic strength (milimolar
NaCl concentration) on the average sedimentation velocity coefficient
of 208-12 oligonucleosome complexes reconstituted with HeLa cell
native octamers (solidline) or with chicken
erythrocyte histone octamers (brokenline) (19) (A) and 208-12 oligonucleosome complexes
reconstituted with highly hyperacetylated HeLa cell histones (B). The buffer was 10 mM Tris-HCl, 0.1 mM EDTA, 3 mM sodium butyrate (pH
7.5).
As can be seen in Fig. 3A, both the HeLa control oligonucleosomes
(reconstituted with histones from non-butyrate-treated HeLa cells) and
the oligonucleosomes reconstituted with chicken erythrocyte histones
behave very similarly. As has already been exhaustively
discussed(19) , this dependence of the sedimentation
coefficient within this ionic strength range (0-100 mM NaCl) corresponds to a folding pattern of the oligonucleosome
structure such as that shown in Fig. 5B. Unfortunately,
our results do not allow us to establish whether this folding is due to
the bending of the internucleosomal DNA linker regions(26) , to
an additional wrapping of these DNA regions about the histone octamer,
or to a combination of both effects. However, it is obvious that the
increase in the salt within the 0-100 mM range provides
charge screening of the phosphates in the DNA backbone that allows for
adjacent nucleosomes to come together.
Figure 5:
Schematic representation of the dynamic
transitions of chromatin. A, ``textbook''
representation of the histone H1 modulation of chromatin structure.
This represents the currently accepted model for the transition of the
higher order structure (HOS, 30-nm fiber) to the low order
structure (LOS) of chromatin. According to this model,
depletion (displacement) of histone H1 in vivo (by phenomena
still not known), leads to an unfolding from HOS to LOS. This extended
LOS conformation of chromatin would allow for the mechanisms of
recognition of specific DNA sequences by activating factors and would
eventually allow for chromatin activation (transcription, replication). B, multistate folding of chromatin structure(1) . This
is our model based on the experimental results presented in this paper
and in (19) . According to this model, upon
removal/displacement of histone H1, under physiological
``native'' conditions, the chromatin fiber only partially
unfolds, thus remaining in intermediate folded conformation
(intermediate order structure, IOS). Complete unfolding of this IOS
chromatin fiber into a completely unfolded conformation (LOS) is only
achieved by lowering the concentration of DNA counterions, either by
decreasing the ionic strength or by removing the highly cationic
domains (histone tails) of the core histones by trypsinization. We have
recently provided thorough and detailed evidence for this
model(19) . C, based on the model of panelB, a hypothesis is made, and experimentally supported in
this paper, that a HOS
In contrast, oligonucleosomes
reconstituted with histones from butyrate-treated HeLa cells exhibit a
completely different behavior. At low salt (10 mM Tris-HCl,
0.1 mM EDTA, 3 mM sodium butyrate, pH 7.5) the
sedimentation coefficient of these complexes ( As the salt concentration
increases, the sedimentation coefficient of the hyperacetylated
oligonucleosome complexes also increases, reaching a plateau at about
100 mM NaCl with an s A similar
change in conformation would also explain the slight shift observed in Fig. 3A for the curves corresponding to chicken
erythrocyte oligonucleosomes and to hyperacetylated histone
oligonucleosomes due to the differences in their basal levels of
acetylation (0.6 acetyl residues/histone H4 for chicken erythrocytes
and 1.2 acetyl residues/histone H4 in butyrate-untreated HeLa cells (32) ). Further experimental evidence in support of this
comes from the electron microscopy analysis shown in Fig. 4. In
contrast to what happens to nonacetylated oligonucleosome
fibers(19) , hyperacetylated oligonucleosomes remain in an
unfolded conformation within the range of salt concentrations analyzed
here. At low ionic strength (see Fig. 4, 0 mM)
the fibers exhibit an extended conformation that is reminiscent of that
exhibited by trypsinized oligonucleosomes (19) and agrees well
with the hydrodynamic data.
Figure 4:
Effect
of the ionic strength on hyperacetylated 208-12 oligonucleosome
complexes as visualized by electron microscopy. The bar is 0.2
µm. The numbers correspond to the milimolar NaCl
concentration.
The results presented in the previous section conclusively
show that histone acetylation has a positive role in the folding
dynamics of the chromatin fiber. In the absence of linker histones
under ionic strength conditions, similar to those of the physiological
environment, acetylated polynucleosomal fibers remain in an extended
conformation in contrast to their nonacetylated counterpart. These
results suggest that, at the chromatin level, the major structural
effect of the acetylation of the N-terminal regions of the histones is
on the linker DNA connecting adjacent nucleosome particle. This is
fully consistent with the finding that histone acetylation reduces the
nucleosome core particle linking number change previously reported by
Norton et al.(31) . It is also in good agreement with
our earlier observation that the linker region is attacked more readily
by micrococcal nuclease in hyperacetylated stripped
chromatin(33) . Nevertheless, all these data are in apparent
contradiction with the results recently obtained with SV40
minichromosomes from viral particles isolated from butyrate-treated
cells(34) . Although the histones of the SV40 minichromosomes
exhibited a high extent of acetylation, no reduction in the level of
constrained supercoiling could be detected(34) . In an
attempt to explain these experimental discrepancies it has been argued (34) that whereas in the in vivo systems histone
acetylation occurs in nucleosomes that are already assembled, in the in vitro oligonucleosome systems used by Norton et al.(31) oligonucleosomes were assembled (reconstituted) from
histones that were already acetylated. However, this explanation seems
very unlikely for the following reasons. 1) Our previously reported
enhanced nuclease accessibility of the linker regions (33) was
observed in a native (nonreconstituted) chromatin. 2) As will be
discussed next, some of the structural effects of histone acetylation
on chromatin folding can be mimicked ``in vitro''
(to a certain extent) by histone trypsinization (removal of the histone
N-terminal domains by trypsin). It was shown, in this case, that the
same structural effects can be observed regardless of whether in
situ trypsinized oligonucleosomes or oligonucleosomes
reconstituted from trypsinized core histones were used(19) . 3)
It was also shown that the reconstituted 208-12 oligonucleosome
system has a folding behavior identical to that of ``native''
oligonucleosomes isolated from chicken erythrocyte
chromatin(19) . This also argues against any DNA sequence
specificity as being responsible (34) for the results retained
with the hyperacetylated oligonucleosome construct used by Norton et al.(31) and which is very similar to that used in
this paper. The predominant effect of histone acetylation on the
linker DNA region also explains the larger conformation effect observed
at the nucleosome level (16, 35) when compared with
the core particle(14) . The results obtained here with the
hyperacetylated oligonucleosome complexes together with our earlier
studies with native and reconstituted trypsinized oligonucleosomes
provide experimental support to the multistate model of chromatin
folding (1) (see Fig. 5). Although the role of the
core histone tails in chromatin folding, has been long
recognized(36, 37) , these regions seem to play a much
more important role in the dynamic modulation of this folding than was
initially envisaged. In the absence of H1, the interactions of the core
histone tails with the linker DNA regions provide the charge
neutralization necessary to allow, under physiological conditions, the
bending (or additional wrapping about the histone octamer) of this DNA
region. This brings adjacent nucleosome particles together in a
partially folded intermediate order state (IOS). This IOS structure
is not easily amenable to transcription(39) . Transcription may
be favored upon transition to LOS modulated by histone acetylation. In
fact both transcription initiation and elongation by RNA polymerase III
have been shown to be significantly inhibited by the LOS In the presence of linker histones the
extent of compaction of the chromatin fiber increases dramatically
giving rise to the higher order structure (HOS) organization. Early
studies on the effect of histone acetylation on the HOS of the
chromatin fiber showed an almost negligible
effect(17, 18) . Therefore, the biological relevance
of our finding has to do with the still unsolved issue of the linker
histone stoichiometry in transcriptionally active chromatin (40) (i.e. with whether or not the transcriptionally
active domains are deficient or not in this particular kind of
histone). Although the problem remains largely unsettled, there is
increasing evidence in support of an altered interaction between
histones of the linker family and acetylated
nucleosomes(41, 42) . Upon displacement of histone H1
from its native binding position and/or removal of histone H1 from the
linker DNA region (by mechanisms yet not quite understood), the major
role of histone acetylation could be that of maintaining the
transcriptionally active chromatin fiber in a transitionally unfolded
conformation. Such an unfolding would allow the RNA polymerase to move
along the coding regions of the gene during transcriptional elongation.
Consistent with this model are the experimental observations that core
histone hyperacetylation co-maps with the whole DNase I-sensitive
domain in the chicken Recently, a positive structural role for
histone acetylation that allows transcription factor access to
nucleosomal DNA has been postulated(45) ; yet, the distribution
of acetylated histone, which co-maps with the entire DNase I
sensitivity of the chicken Despite the early
correlation between transcription and histone
acetylation(46, 47) , the causative structural
connection between both has remained elusive for many years. As the
pieces of this complicated puzzle seem to start coming together, the
results of the structural studies, such as those reported here, call
for the imperative need to use hyperacetylated chromatin complexes in
any of the in vitro studies designed to understand the
molecular mechanisms involved in the eukaryotic transcription of pol II genes (48) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
Chicken blood was obtained from
commercial slaughterhouses in Victoria and processed immediately after
collection. The 208-12 DNA template consisting of 12 tandem
repeats of a 208-base pair fragment of the 5 S rRNA gene from the sea
urchin Lytechinus variegatus(25) was a generous gift
of Dr. Robert Simpson. HeLa (S3 strain) cells were purchased from ATCC
(American Type Culture Collection; Rockville, MD). Sephacryl S-1000 was
obtained from Pharmacia Biotech Inc., and Hydroxylapatite BioGel HTP
was from Bio-Rad.HeLa Cells
HeLa cells (S3 strain) were grown in
spinner culture at 37 °C in the presence or absence of sodium
butyrate as described elsewhere(14) .Preparation of Histone Octamers
Chicken
erythrocyte histone octamers were obtained as described
elsewhere(20) . Control and highly hyperacetylated histone
octamers from HeLa cells were prepared from chromatin fractions
isolated as described previously(14) . Only fraction a (14) was used as a source of hyperacetylated histones. The
chromatin fractions (6 mg) were then dialyzed against 0.633 M NaCl, 0.1 M potassium phosphate, 1 mM dithiothreitol (pH 6.7) with or without 5 mM sodium
butyrate and loaded onto a hydroxylapatite column (1.5
15 cm at
a flow rate of 16 ml/8 fractions/h) previously equilibrated with the
same buffer. After elution of the linker histones under these
conditions (about 100 ml) the eluting buffer was changed to 1 M NaCl in the same buffer to elute the histone octamers. The histone
octamers obtained in this way were concentrated in a centricon 10 and
used immediately thereafter or kept frozen at -60 °C.
Preparation of the 208-12 DNA Template
The
208-12 DNA template used in the oligonucleosome reconstitutions
was prepared as described in (19) .Oligonucleosome Reconstitution
This procedure was
carried out as described previously(19) .Gel Electrophoresis
SDS-15% polyacrylamide gel
electrophoresis was carried out according to Laemmli(21) .
Acetic acid, 8 M urea, 0.5% Triton X-100, polyacrylamide gel
electrophoresis was carried out according to (22) with a few
minor modifications. The acrylamide:bisacrylamide ratio was 30:1
(separating gel) and 20:1 (stacking gel). The sample buffer was 10 M urea, 5% acetic acid, 5% -mercaptoethanol, and 0.3%
Pyronine Y.
Electron Microscopy
Preparation of the samples for
electron microscopy was carried out according to Labhart and Koller (24) as described in (19) . Micrographs were taken with
a Philips 301 electron microscope.Analytical Ultracentrifuge
Characterization
Sedimentation velocity and sedimentation
equilibrium analysis were performed on a Beckman model E analytical
ultracentrifuge with a computer-interfaced UV scanner (Ultrascan
Interface and Data Analysis Program version 1.70; Borries Demeler
(Missoula, MT). Experimental conditions and analysis of the runs were
as described previously (19) or as indicated in the figure
legends.
DNA BstE II digest used as a DNA
marker (lane M).
17 acetyl groups/histone
octamer(14) . The core histones obtained from
non-butyrate-treated HeLa cells are shown in Fig. 1A, lanes2, and in Fig. 1B, lane2. Fig. 1also shows a native agarose gel of the
oligonucleosome complexes reconstituted with hyperacetylated histones (panelC, laneA) and histones of
low level acetylation (Fig. 1C, laneN) in comparison with the 208-12 DNA used as a
template (Fig. 1C, laneD).
= 2.92
10
(nonacetylated oligonucleosome complexes) and M
= 2.93
10
(acetylated oligonucleosome
complexes) is fully consistent with the presence of 12
nucleosomes/molecule of DNA template(19) .
obtained from the sedimentation equilibrium analysis of the
reconstituted hyperacetylated oligonucleosome 12-mers. High speed,
meniscus depletion sedimentation equilibrium was carried out at 2400
rpm and 17 °C. r is the radial distance in cm. y is the direct absorbance (265 nm) reading in the recording chart,
at a given i radial position. y is the absorbance
reading in the sample-depleted region close to the meniscus. The insets show electron microscopy images obtained from the same
sample. The number of nucleosomes observed in these micrographs (12) coincides with the number of nucleosomes estimated from
the molecular mass determined from the slope of the plot shown in this
figure.
LOS transition upon histone H1 depletion
is possibly modulated in vivo by acetylation of the histone
octamers through a physical counterion mechanism virtually identical to
that described in panelB. Acetylated chromatin in
this LOS conformation would thus become available for specific DNA
sequence recognition and binding of activating factors (45) and/or to allow transcriptional elongation by
polymerases.
22 S) is much lower
than that of the control counterparts (
27 S) for HeLa histone
oligonucleosomes or 29 S for chicken erythrocyte-histone
oligonucleosomes). Simple modeling calculations using the Kirkwood
formalism (27) (see also (19) for a detailed analysis)
suggest that such a decrease in S value can be accounted for by an
unfolding of the oligonucleosome fiber as a result of a partial
unraveling of the nucleosomal DNA coil at the flanking sides of the
acetylated nucleosome particle. The model is fully consistent with
earlier data on the melting properties of hyperacetylated nucleosome
core particles(14, 28) . It was shown that
hyperacetylated nucleosome core particles exhibited a significant
increase in the first melting transitions (14) corresponding to
the DNA regions at the ends of the DNA
supercoil(29, 30) . Indeed, a release of the DNA
affecting about 18.5 base pairs within these regions has been more
recently proposed (31) .
= 29 S (Fig. 3B). This value is very similar to the
sedimentation coefficient of the control oligonucleosomes at low salt
(see Fig. 3A), in which the linker DNA regions are in a
rather extended conformation. These hydrodynamic data clearly indicate
that under ionic strength conditions similar to those found under
physiological conditions and in the absence of histone H1 acetylated
oligonucleosome fibers retain an extended conformation.
(
)It is clear from all our studies that under ionic
strength conditions similar to those found in the cell nucleus,
chromatin depleted of linker histones does not exhibit an extended
conformation unless the histones are hyperacetylated. For simplicity,
we have represented this intermediate state of folding as one in which
nucleosomes are arranged in a contacting bidimensional zigzag
organization, which is consistent with simple hydrodynamic modeling
calculation(19, 20) . Nevertheless the possibility of
more complex three-dimensional arrangements arising from some kind of
helical folding cannot be excluded(38) .
IOS
transition(39) .
-globin gene(43) . It has also been
shown that transcriptionally active chromatin has a loosened linker
folding(44) .
-globin domain, argues against an
exclusively ``recognition'' role of histone acetylation. A
widespread distribution of histone acetylation may indeed play an
important role in the elongation stage through the dynamic equilibrium
mechanisms described above (see Fig. 5).
We are very grateful to Dr. R. T. Simpson at the
National Institutes of Health for generously providing the p5S,
208-12 plasmid construct. We are very indebted to Prof. J. A.
Subirana and to Dr. M. T. Casas from the Departament
d'Enginyera
Qumica (Universidad
Politcnica de Catalunya, Barcelona) for allowing
us to use their electron microscope facilities. We are also thankful to
Dr. Crisanto Gutierrez for allowing us to use the electron microscopy
facility of the Centro de Biologia Molecular Severo Ochoa (Consejo
Superior de Investigaciones Cientficas,
Universidad Autonoma, Madrid). We thank Dr. A. Fabra from the Institut
de Recerca Oncolologica (Barcelona) for generous financial support for
some of the expenses incurred by M. Garcia-Ramirez during this work.
Finally we thank Maree Roome for careful typing of the manuscript and
Phil Rice for observant reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.