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(Received for publication, May 15, 1995; and in revised form, June 30, 1995) From the
We show that nondenaturing agarose gels can be used for the
study of the structure and dynamic properties of native
(uncross-linked) chromatin. In gels containing 1.7 mM Mg
In chromatin an octamer of histones H2A, H2B, H3, and H4
associated with 146 bp ( The mechanism of chromatin folding and the structure of the 30-nm
fiber have been studied by sedimentation
methods(7, 8, 9) , enzymatic and chemical
digestions(10, 11, 12) , electric, flow, and
photochemical dichroism(13, 14, 15) , x-ray
diffraction(16, 17) , small angle x-ray
scattering(18, 19, 20) , neutron
scattering(21, 22) , transmission electron
microscopy(23, 24, 25, 26, 27, 28, 29, 30, 31, 32) ,
and scanning force microscopy(33) . The results obtained in
these studies have suggested different models for the folding and
structural organization of the 30-nm chromatin fiber. These models
differ essentially in the location of the linker DNA. In the one-start
helix models, the linker DNA is folded and connects laterally
consecutive
nucleosomes(8, 13, 17, 23, 32) .
In the twisted-ribbon models, a two-start helix is formed by pairs of
nucleosomes with the linker DNA parallel to the fiber
axis(24, 26) . Finally, in several continuous (11, 18, 28) and discontinuous (27) crossed-linker models, and in the variable zigzag
nucleosomal ribbon model(30, 31) , the linker DNA is
extended in the fiber interior. In this work we show that
nondenaturing agarose gel electrophoresis can be used for the study of
the folding of chromatin. We have found that, when electrophoresis is
performed in the presence of Mg
Figure 1:
Electrophoresis of chromatin fragments
on a low ionic strength nondenaturing gel. a, chromatin
fragments of different length (lanes with +) and the DNA
isolated from these fragments (lanes with -) were
electrophoresed on a 0.1
Figure 2:
Electrophoresis of chromatin fragments on
nondenaturing gels containing Mg
With chromatin samples containing more than 6
nucleosomes, the changes in electrophoretic mobility produced by the
increase of the molecular weight of the chromatin are very small. Note
that in gels containing Mg
In gels
of higher ionic strength in absence or in presence of
Mg Furthermore, as
can be seen in Fig. 3a, when chromatin is treated with
increasing NaCl concentrations and then electrophoresed on agarose gels
with Mg
Figure 3:
Effect of NaCl concentration on the
electrophoretic mobility of chromatin fragments on nondenaturing gels
containing Mg
The
slow and rapid bands of each lane shown in Fig. 2b contain the same number of nucleosomes. For instance, the analysis
of a sample similar to that of lane 4 of Fig. 2b on a second-dimension denaturing gel shows that both the slow and
rapid bands contain the DNA corresponding to 3-4 nucleosomes (see Fig. 4, lanes 2 and 3). One possible
interpretation of this surprising observation is that the slow band is
due to the association of the oligonucleosome fragments that produce
the rapid band. This possibility is demonstrated in the cross-linking
experiment shown in Fig. 4. It can be seen that whereas the slow
band produces cross-linked structures of low mobility in
second-dimension denaturing gels (lane 4), the rapid band does
not give rise to cross-linked material (lane 5). This result
indicates that small fragments of chromatin containing 3-4
nucleosomes can produce associated structures having the same
electrophoretic properties as chromatin fragments of higher molecular
weight. The association of small chromatin fragments is confirmed in
the electron microscopy studies presented below. We have assumed that
two small fragments with few nucleosomes associate to form a structure
containing about 6 nucleosomes, according to the equation f + f &cjs0633; F, where f and F correspond, respectively, to the small and associated fragments.
The densitometric analysis of gels loaded with samples containing
fragments with about 4 nucleosomes has allowed us to estimate that the
apparent association constant for this assembly reaction determined at
four different concentrations is 1.4 (± 0.7)
Figure 4:
Chemical cross-linking of different
chromatin bands. The slow and rapid bands of a sample similar to that
shown in lane 4 of Fig. 2b were cut out of the
gel, washed twice with TEAB buffer containing 1.7 mM
Mg
The histone composition of the
chromatin samples containing more than 6 nucleosomes is the same as
that of whole nuclei samples, but the chromatin samples containing few
nucleosomes have a low H1-H5 content (see Fig. 5b). Nevertheless, second-dimension
electrophoresis shows that the slow band detected in chromatin samples
containing less than 6 nucleosomes has approximately the same
H1-H5 content as normal chromatin (see Fig. 5a, lane 2 and legend), indicating that these histones are
necessary for the association of small chromatin fragments to form the
band that has the same mobility as the fragments of higher molecular
weight.
Figure 5:
Histone composition of different chromatin
samples. The samples used for the nondenaturing gel shown in Fig. 2b (lanes 1-8) were analyzed on a
SDS-polyacrylamide gel (Panel b, lanes 2-9). Lanes 1 and 2 of the second-dimension SDS gel
presented in Panel a correspond, respectively, to the analysis
of the histones of the rapid and slow (indicated by a star)
bands of a sample similar to that shown in lane 4 of Fig. 2b. Lane 1 in Panel b corresponds to
histones of a whole nuclei sample. The H1-H5 content (estimated
from densitometric analysis) relative to the amount of these histones
found in whole nuclei is, respectively, 0.1 (lane 1) and 0.8 (lane 2) in Panel a, and 0.03 (lane 2), 0.2 (lane 3), 0.3 (lanes 4 and 5), 0.5 (lanes 6 and 7), 0.6 (lane 8), and 0.8 (lane 9) in Panel b.
Figure 6:
Electron micrographs of rotary-shadowed
chromatin fragments. Chromatin fragments containing 3-4
nucleosomes in 0.1
The measurements
presented in Fig. 7show that the diameter of the structures
extracted from the slow bands corresponding to samples containing from
2-3 to 5-6 nucleosomes (Panels b-d) is about 33
nm, i.e. the same diameter found for chromatin samples
containing a larger number of nucleosomes (see Panel a). In
contrast, the rapid band of samples containing few nucleosomes does not
produce the typical circular structure (see Fig. 6d).
Before electrophoresis, in presence of 1.7 mM Mg
Figure 7:
Diameter of chromatin fragments.
Histograms of diameter measurements (corrected from metal deposition)
corresponding to rotary-shadowed preparations of chromatin extracted
from different bands of gels containing 1.7 mM
Mg
As can be seen in Fig. 8, the highly magnified images obtained from the associated
small chromatin fragments present in the slow electrophoretic bands
(indicated by a star in Fig. 2) are equivalent to the
typical images of larger chromatin fragments that we have described
previously(32) . The structures assembled from small chromatin
fragments, and the fragments of higher molecular weight have
approximately the same electrophoretic mobility because they become
very compact in presence of Mg
Figure 8:
High magnification of folded chromatin
fragments produced by spontaneous association of small fragments
containing 3-4 nucleosomes. Micrographs are printed in negative
contrast. The bar represents 20
nm.
The remarkable changes in the electrophoretic behavior of
chromatin fragments observed when electrophoresis is performed in the
presence of Mg The main conclusions of this work can be
summarized in the scheme presented in Fig. 9. Our results show
that about 6 nucleosomes are enough to produce the compact structures
having approximately the same electrophoretic mobility as the folded
chromatin fragments of higher molecular weights. Early sedimentation
studies (8) showed a marked structural transition when the
chromatin fragments analyzed contained 6 nucleosomes. We have found
that even small fragments (containing 3-4 nucleosomes) can
associate giving rise to compact particles with the same mobility as
the folded fragments of higher molecular weight. When observed in the
electron microscope all these structures are apparently equivalent.
According to our previous observations(32) , this is due to the
fact that the images obtained correspond to the top view of the folded
chromatin fragments (see above). These findings suggest that the
structure of folded chromatin fibers must be very compact and simple
enough to be started and stabilized with a basic element containing
about 6 nucleosomes. Such a structure is compatible with the compact
one-start helix model proposed from the detailed analysis of the
electron microscope images of small chromatin fragments in presence of
1.7 mM Mg
Figure 9:
Scheme
for the self-organization of chromatin fibers based on the
physicochemical and structural properties of the chromatin fragments
analyzed in this study. Mg
In agreement with
our findings about the self-assembly of small chromatin fragments,
other laboratories using different techniques have reported results
about the association of oligonucleosomes to form higher order
structures(35, 40, 41, 42) . Taken
together, these observations indicate that chromatin fibers are able to
self-organize even when there is no covalent continuity in the DNA of
the fiber. This stresses the fundamental role of histones in organizing
the chromatin structure. Histones H1-H5 are necessary to produce
the folding of chromatin fragments. The characteristic electrophoretic
behavior of the chromatin fragments analyzed in this study is not
observed in samples depleted of these histones. Moreover, the
association of small fragments does not take place in the
oligonucleosome fraction that has a low H1-H5 content; only the
fraction having the normal content of these histones can form
self-assembled structures. These results are consistent with previous
findings indicating the essential role of histones H1-H5 for
chromatin assembly (43) and folding(44) . Recent
results (45) have shown that histones H1 and H5 suppress the
mobility of the histone octamers positioned on constructs of 5 S rRNA.
Furthermore, neutron scattering analysis of chromatin containing
deuterated histone H1 has indicated that this histone is located in the
interior of the folded chromatin at a distance from the fiber axis of
about 6 nm(46) . This internal location of H1 (and presumably
H5) is probably responsible for the stabilization of the folded
structure. It is very likely that the observed association of small
chromatin fragments is originated by the same interactions that cause
the spontaneous folding of the chromatin fiber. Since the association
constant corresponding to this assembly (
Volume 270,
Number 38,
Issue of September 22, pp. 22514-22521, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
SELF-ASSEMBLY OF SMALL CHROMATIN FRAGMENTS AND FOLDING OF THE
30-nm FIBER (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, chicken erythrocyte chromatin fragments
having from about 6 to 50 nucleosomes produce well defined bands. These
bands have an electrophoretic mobility that decreases only slightly
with molecular weight. This surprising behavior is not observed in low
ionic strength gels. Fragments with less than 6 nucleosomes and low
content of histones H1-H5 give rise to broad bands in gels with
Mg
. In contrast, fragments containing only 3-4
nucleosomes but with the normal H1-H5 content are able to form
associated structures with a mobility similar to that observed for high
molecular weight chromatin. Electron microscopy results indicate that
the associated fragments and the fragments of higher molecular weight
show similar electrophoretic properties because they become very
compact in the presence of Mg
and form cylindrical
structures with a diameter of
33 nm. Our results suggest that the
interactions involved in the self-assembly of small fragments are the
same that direct the folding of larger fragments; in both cases, the
resulting compact chromatin structure is formed from a basic element
containing 5-7 nucleosomes.
)of DNA forms the core of the
nucleosome(1, 2) . The two turns of DNA (about 165 bp)
around these core histones are sealed by histone H1 (H5 in avian
erythrocytes)(3, 4, 5) . Nucleosomes
connected by linker DNA form a filament that can fold into a condensed
chromatin fiber of about 30 nm in diameter (reviewed in (6) ). , chicken erythrocyte
chromatin fragments of a relatively high molecular weight change
dramatically their mobility due to the formation of very compact
structures. Furthermore, we have found that under these conditions
small oligonucleosomes are able to associate forming structures with
the same structural characteristics as larger chromatin fragments. The
results obtained using gel electrophoresis have been interpreted taking
into account the analysis by electron microscopy of the same samples.
Our electrophoretic method does not require the cross-linking of the
chromatin samples, but the structures produced by the association of
uncross-linked oligonucleosome fragments are stable enough to form the
well defined bands detected in nondenaturing gels at the end of the
electrophoresis. This suggests that the forces that allow the
self-assembly of oligonucleosomes are the same that direct the folding
of chromatin of higher molecular weight. We have also investigated the
role of histones H1-H5 in these structural transitions.
Preparation of Chromatin Fragments
Chicken
erythrocyte nuclei were obtained in 90 mM KCl, 30 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 0.4
mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM
triethanolamine-HCl (pH 7.4) and digested with micrococcal nuclease
(Sigma, 1 unit/mg of DNA) for 30-90 min in the presence of 1
mM CaCl
at 37 °C(32, 34) .
Digested nuclei were extracted overnight at 4 °C with 40 mM NaCl, 1 mM EDTA, 0.4 mM PMSF, and 10 mM
triethanolamine-HCl (pH 7.4), and finally centrifuged at 2,500 
g for 10 min. The soluble chromatin in the supernatant was
concentrated (Diaflo PM1O, Amicon) to 5-6 mg of DNA/ml and
fractionated by sedimentation on a 5-20% linear sucrose gradient
containing 5 mM EDTA, 0.4 mM PMSF, and 10 mM
triethanolamine-HCl (pH 7.4). When necessary, prior to density gradient
centrifugation, the digested chromatin was depleted of histones
H1-H5 following the method of Ruiz-Carrillo et
al.(35) . Selected fractions from the sucrose gradients
were dialyzed against the indicated buffers (see
``Results''); other fractions were concentrated (Centricon
10, Amicon) to 2-5 mg/ml and further fractionated by
nondenaturing gel electrophoresis (see below). Chromatin of high
molecular weight was obtained as described elsewhere (32) and
further purified by sucrose gradient sedimentation followed by
nondenaturing gel electrophoresis. The length of the isolated DNA
corresponding to the different chromatin fragments was analyzed on
agarose gels stained with ethidium bromide. The histone composition of
the different samples was analyzed on SDS-polyacrylamide gels stained
with the fluorescent dye Nile red(36) . Photographic negatives
were scanned with a Shimadzu CS-9000 densitometer.Nondenaturing Gel Electrophoresis
Electrophoresis
of chromatin fragments on gels containing 0.1 TB (9 mM Tris borate, pH 8.3) was performed following essentially the
procedure previously described for the analysis of core particle
DNA-histone complexes (37) but using 0.5% agarose gels instead
of polyacrylamide gels. These gels were pre-electrophoresed, and the
buffer was recirculated between the electrode compartments.
Nondenaturing 0.5% agarose gels (10 cm long) of higher ionic strength
contained TB and the concentrations of MgCl indicated in
the figure legends. In this case, pre-electrophoresis and buffer
recirculation were omitted. Chromatin samples directly obtained from
the sucrose gradients or in the buffers indicated in the figure legends
(in all cases without Mg) were mixed with 0.25 volume
of 5
TB containing 12.5% Ficoll and loaded onto the
TB-Mg gel. Generally, electrophoresis was carried out
at 100 V for about 1.7 h. The apparent equilibrium constant for the
reaction of association of small chromatin fragments (see
``Results'') was calculated from the densitometric analyses
of the electrophoretic bands corresponding to the small and associated
fragments of chromatin samples having different concentrations (ranging
from 0.19 to 1.7 mg of DNA/ml).
Chemical Cross-linking
After electrophoresis the
selected bands were cut out of the gel, washed twice with 1.5 ml of
TEAB buffer (90 mM triethanolamine-borate, pH 8.6) containing
1.7 mM MgCl for 2 h, and treated with 1.5 ml of
0.1% glutaraldehyde for about 15 h at 4 °C in the same buffer.
Finally, the agarose block containing fixed chromatin fragments was
loaded onto an Ultrafree-CL tube (0.45 µm Durapore membrane,
Millipore), and centrifuged at 2,500 g for 30 min. The
chromatin eluted from each band was used in electron microscopy
experiments. The chromatin samples prepared without gel electrophoresis
were fixed with 0.1% glutaraldehyde for about 15 h at 4 °C in the
indicated buffers before electron microscopy. The specific
cross-linking conditions of the chromatin fragments used for
second-dimension electrophoretic analyses are described (see
``Results'').Electron Microscopy
Spread preparations of the
resulting samples (final concentration 2-10 µg of DNA/ml)
were obtained as described previously in carbon-coated copper grids
pretreated with Alcian blue(38) . The grids were
rotary-shadowed with platinum-carbon at an angle of about 7°.
Micrographs were obtained at a magnification of 30,000 using a
Hitachi H7000 transmission electron microscope. Images from the
original negatives were acquired either directly using a
transilluminator and a Hamamatsu C2400 camera or at a higher
magnification with the same camera attached to a modified Zeiss
Axiotron microscope equipped with a 5 objective. Additional
experimental details have been described elsewhere(32) .
Behavior of Chromatin Fragments of Different Molecular
Weights in Electrophoretic Gels of Low Ionic Strength
The
electrophoretic mobility of chromatin samples in 0.5% agarose gels
containing 0.1 TB is lower than that found for the DNA
extracted from the same samples (Fig. 1a). The relative
retardation of the mononucleosome band produced by this nondenaturing
agarose gel is lower than that observed in low ionic strength
polyacrylamide gels(37) . Nevertheless, whereas
oligonucleosomes cannot enter the polyacrylamide gels(38) , the
agarose gels described in this work can be used even with chromatin
samples of high molecular weight. The results presented in Fig. 1b show that in 0.1 TB agarose gels the
electrophoretic mobility of native chromatin samples having a wide
range of molecular weights is in all cases lower than that of the
corresponding free DNA obtained from these samples.
TB, 0.5% agarose gel. The size of some
DNA markers (in kb) is indicated. b, plot of log bp versus electrophoretic mobility (relative to the migration of
free DNA of 1 kb) for native chromatin () and chromatin depleted
of H1-H5 (
). The mobility of free DNA (
) is shown
as reference.
Chromatin Electrophoresis on Nondenaturing Gels
Containing Mg
In contrast with the results
presented in the preceding section, when the electrophoresis on 0.5%
agarose gels is performed using buffers of higher ionic strength (see Fig. 2), the mobility of chromatin fragments of increasing
molecular weight shows a complex behavior. In gels containing 1
TB, the plot of the log bp (of the DNA corresponding to a
given chromatin sample) versus electrophoretic mobility (Fig. 2d) has two regions: one with a small
negative slope (corresponding to low molecular weight chromatin
samples) and the other with a high negative slope (corresponding to
chromatin of higher molecular weight). These differences in
electrophoretic behavior are more pronounced using electrophoretic
buffers containing Mg (Fig. 2d). We
have obtained essentially the same results with gels containing 1.7 and
2.5 mM MgCl
. The transition between the two
regions of these plots is observed when the chromatin fragments contain
5-6 nucleosomes.
. a and b, chromatin fragments of different length were
electrophoresed on a 0.5% agarose gel containing TB and 1.7 mM MgCl
. The average length of the DNA of the chromatin
fragments analyzed in a is indicated in the bottom of
this panel. c, the DNA isolated from the chromatin fragments
shown in Panel b was analyzed on TB, 1.7 mM MgCl gels. The size of some DNA markers (in kb) is
indicated. d and e, relationship between the length
of DNA in chromatin fragments and mobility (relative to the migration
of free DNA of 1 kb) in gels containing TB and 0 (
), 1.7
(), and 2.5 (
) mM MgCl
. In Panel
d the analyzed chromatin fragments contained histones H1-H5;
in Panel e chromatin fragments were depleted of H1-H5.
Some chromatin samples produce additional bands (indicated by stars in Panels a and b) with a relatively low
mobility; in Panel d these slow bands are indicated by small stars (gels with 1.7 mM MgCl) and large stars (gels with 2.5 mM MgCl). The
mobility of free DNA in TB gels containing 0 (), 1.7 (
),
and 2.5 () mM MgCl
is shown as
reference.
, the negative slope of the
plots shown in Fig. 2d corresponding to samples with
more than 6 nucleosomes has a very high value. This surprising behavior
of native chromatin produces an apparent increase in the
electrophoretic mobility of the high molecular weight chromatin
fragments relative to the mobility of free DNA. In fact, whereas the
mobility of naked DNA corresponding to chromatin fragments containing 6
nucleosomes is roughly 2-fold higher than that observed for the native
fragments, chromatin samples containing about 50 nucleosomes (i.e. DNA of
10 kb) show approximately the same mobility than the
naked DNA extracted from these samples (see the intersection of the
chromatin and DNA curves in Fig. 2d).
Electrophoretic Mobility of Chromatin Fragments Depleted
of Histones H1-H5
In agarose gels of low ionic strength,
the electrophoretic bands corresponding to chromatin without
H1-H5 migrate faster than the equivalent bands of chromatin
containing all the histones, but slower than the corresponding free DNA
obtained from these samples (see Fig. 1b)., the slope of the plots of log bp versus
relative mobility of chromatin depleted of histones H1-H5
(see Fig. 2e) are different from that obtained in gels
of low ionic strength. However, in contrast with the results obtained
in the case of native chromatin fragments with all the histones, the
plots of log bp versus mobility of samples without H1-H5
in gels of relatively high ionic strength cannot be divided in two
regions (compare the plots presented in Fig. 2, d and e). This indicates that histones H1-H5 are presumably
responsible for the complex electrophoretic behavior of native
chromatin in gels containing Mg
.
, a remarkable structural transition is
observed at about 0.6 M NaCl. This transition is not observed
with chromatin depleted of histones H1-H5 (Fig. 3b). Taking into account that histones
H1-H5 are dissociated from chromatin when the NaCl concentration
is 0.6 M(34) , these results indicate that the
presence of histones H1-H5 is necessary in order to maintain the
integrity of the structures that produce the typical electrophoretic
bands of native chromatin.
. a, native chromatin; b, chromatin depleted of H1-H5. The different samples in
TB plus 2.5 mM EDTA were treated with the indicated
concentrations of NaCl, incubated for about 30 min at room temperature,
and loaded onto the gel. Lane D corresponds to the DNA
isolated from the chromatin fragments analyzed in Panels a and b. The size of some DNA markers (in kb) is
indicated.
Well Defined Chromatin Bands in Gels Containing
Mg
Native chromatin fragments containing the
four core histones and histones H1-H5 produce broad bands in low
ionic strength gels (Fig. 1a). In contrast, fragments
with 6 or more nucleosomes form well defined bands in gels with
Mg: Self-assembly of Chromatin Fragments with
3-4 Nucleosomes
(Fig. 2, a and b). This
observation is probably related with the almost invariable mobility in
gels with Mg
observed for native chromatin samples of
increasing molecular weight (see above). Nevertheless, note that, even
in gels containing Mg
, chromatin fragments with less
than 6 nucleosomes produce broad bands (Fig. 2b, lanes 1-5). Furthermore, in samples corresponding to
fragments containing from 3-4 (Fig. 2, b and c, lane 4) to 5-7 (Fig. 2, b and c, lane 7) nucleosomes, these broad bands
are accompanied with a narrow slower band (indicated by a star) that shows the same mobility as the fragments of higher
molecular weight. The relative mobility of the additional slower bands
is also indicated by stars in Fig. 2d. These
bands are not observed in gels without Mg
.
10
M.
for 1 h, cross-linked with 0.2% glutaraldehyde (30
min at 0 °C), and analyzed on a second-dimension TB gel containing
0.05% SDS (stained with ethidium bromide). The results obtained with
the slow and rapid bands are shown in lanes 4 and 5,
respectively. Part of the samples treated with glutaraldehyde shown in lanes 4 and 5 were incubated (30 min at 37 °C)
with proteinase K (200 µg/ml in presence of 1% SDS) and analyzed in
lanes 6 and 7, respectively; the same samples
uncross-linked (lanes 2 and 3) and the original
chromatin sample used in this experiment (lane 1) are shown as
reference. The size of some DNA markers (in kb) is
indicated.
Cross-linking of Chromatin Bands in the Gel and Electron
Microscopy Analysis: The Folding of Chromatin Fragments Is Responsible
for the Changes of Electrophoretic Mobility Observed in Gels Containing
Mg
The similar mobility, in gels with
Mg, observed for chromatin structures produced by the
assembly of small fragments and by native chromatin fragments having a
wide range of molecular weights (see above) suggests that all these
chromatin samples adopt a compact conformation that make them
indistinguishable in the gel. In order to test this possibility,
chromatin samples containing 2-3, 3-4, and 5-6
nucleosomes were electrophoresed under nondenaturing conditions in
presence of Mg
and, after electrophoresis, the slow
band was cross-linked in the gel with glutaraldehyde; then the
cross-linked material was eluted from the gel and finally analyzed in
the electron microscope. We have observed that, in all cases, in the
presence of 1.7 mM Mg
, the slow band
produces circular structures (see Fig. 6e) similar to
those obtained with fragments containing from about 6 to 35 nucleosomes
(see Fig. 6g and (32) ).
TEAB (a), TEAB (b), and
TEAB plus 1.7 mM MgCl (c). The same
sample after electrophoresis on a nondenaturing gel containing 1.7
mM MgCl produces two bands similar to those of lane 4 of Fig. 2b; micrographs of the
chromatin extracted from the rapid and slow (indicated by a star in Fig. 2b) bands in TEAB plus 1.7 mM
MgCl are shown in Panels d and e,
respectively. Chromatin fragments of higher molecular weight
(24-34 nucleosomes) in TEAB (f); the same sample was
electrophoresed under nondenaturing conditions in the presence of
Mg, extracted from the gel in TEAB plus 1.7 mM MgCl
and analyzed in the electron microscope (g). Micrographs are printed in negative contrast. The bar
represents 300 nm.
, the samples containing few nucleosomes are
mixtures of the circular structures and small particles (Fig. 6c). Electrophoresis causes the separation of
these two components. Control experiments show that at low ionic
strength (Fig. 6a) or in absence of Mg
(Fig. 6b) small fragments cannot associate.
Without Mg
, folding of very large fragments cannot
take place (Fig. 6f).
. N represents the number of nucleosomes
of each sample; the structures analyzed in Panels b-d were extracted from the slow bands (indicated by stars in Fig. 2) produced by samples containing few nucleosomes. The
mononucleosome sample (Panel e) is included as reference. The
curves correspond to Gaussian fittings. The mean (±1 S.D.)
diameters are 33 ± 4 (a), 34 ± 5 (b),
32 ± 4 (c), 34 ± 4 (d), and 12 ±
2 (e) nm.
. In agreement with
this interpretation, we have found previously (32) that
fragments containing from about 6 to 35 nucleosomes show approximately
the same diameter (33 nm) in the electron microscope because in
presence of Mg
they are highly packed, favoring the
vertical placement of the resulting short cylindrical structures on the
grid. This gives rise to images corresponding to the top view of folded
chromatin (see Fig. 8and Fig. 4and Fig. 5of (32) ).
has allowed us to detect the assembly
of oligonucleosomes and the folding of larger fragments. Note that the
experiments with the nondenaturing agarose gels have been performed
using uncross-linked chromatin samples. Thus, our findings derived from
the electrophoretic studies presented in this study correspond to
native chromatin samples. While this investigation was in progress,
Krajewski et al.(39) reported that current agarose
gels without Mg
can be used for the structural
analysis of chromatin cross-linked with glutaraldehyde under different
conditions before electrophoresis. We have used glutaraldehyde
cross-linking exclusively after electrophoresis in order to
stabilize samples for electron microscopy analysis. In this case,
before spreading, it is necessary to make a large dilution of the
sample that causes nucleosome dissociation in the uncross-linked
chromatin (not shown).
(32) .
induces the folding of
chromatin fragments containing 6 or more nucleosomes and the
self-assembly of fragments with about 3 nucleosomes, provided that they
have the normal H1-H5 content. Folded chromatin (top view in the center of the figure) is represented by the model
of the internal structure of chromatin fibers previously proposed by
Bartoloméet al.(32) . The
nucleosomes (
11-nm diameter) in the extended chromatin fragments
and the folded fiber (
33-nm diameter) are drown roughly to the
same scale; in both structures the DNA (double helix diameter
2
nm) is represented by a solid
line.
10
M, see ``Results'') is
about 10
-fold lower than the association constant of
histone octamers with DNA (10
M as estimated from the dissociation analysis of core particles in
0.2 M NaCl)(38) , it can be suggested that DNA in
chromatin is packaged following a sequence of compaction steps (core
particle formation
H1-H5 binding
folding of the 30
nm fiber) involving lower amounts of free energy release in each
consecutive level. Although the binding of histone H1 and similar
proteins to naked DNA is very cooperative and the corresponding
association constant is apparently very high (47, 48, 49) , the binding energy of H1 to
chromatin must be relatively low in order to favor the remarkable
dynamic and functional properties of this
histone(50, 51, 52, 53, 54, 55) .
Furthermore, it has to be taken into account that even compact
chromatin containing histones H1-H5 is not necessarily inert from
a functional point of view. It has been found recently that chromatin
with the normal H1-H5 content in the presence of 1.7 mM Mg
can bound reversibly excess core histones,
suggesting that folded chromatin may be involved in the transient
association of the core histones released during
transcription(56) .
)
We thank Onofre Castell and Francesc Bohils (Servei de
Microscòpia Electrònica,
Universitat Autònoma de Barcelona) and Joan
Masoliver and Ramon Baldrich (Centre de Tractament d'Imatges,
Universitat Autònoma de Barcelona) for excellent
technical help with the electron microscope and image analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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