(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, 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.
In chromatin an octamer of histones H2A, H2B, H3, and H4
associated with 146 bp ()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) ).
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, 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.
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 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.
Figure 2:
Electrophoresis of chromatin fragments on
nondenaturing gels containing Mg. 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.
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, 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).
In gels
of higher ionic strength in absence or in presence of
Mg, 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
.
Furthermore, as
can be seen in Fig. 3a, when chromatin is treated with
increasing NaCl concentrations and then electrophoresed on agarose gels
with 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.
Figure 3:
Effect of NaCl concentration on the
electrophoretic mobility of chromatin fragments on nondenaturing gels
containing Mg. 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.
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) 10
M
.
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 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.
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 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 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, 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).
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. 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.
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. 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) ).
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 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).
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(32) .
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 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.
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 (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) .