From the
The question as to how the strands of chromatin are packed into
the eukaryotic nucleus has intrigued biologists and biochemists for
many years. Although there is ample space in the nucleus for the
eukaryotic genome, the enormous length of the DNA molecules requires
that they be extensively folded. In addition, the folding must be such
as to allow regulated access to certain regions of the genome. The
discovery of the nucleosome as a basic organizing unit cast the problem
in more defined focus (see Refs. 1 and 2). Even so, it was realized
that the condensation afforded to the DNA by nucleosomes (about a 5:1
compaction ratio) could not account for the many thousand-fold
compaction that must exist in vivo. Some kind of higher order
folding of the nucleosomal fiber must therefore exist. Many models have
been proposed for this folding, most of which predicate a regular
helical folding of the fiber. It is the purpose of this review to
critically re-examine the evidence concerning this condensed structure
of chromatin.
We must begin, however, with two caveats.
First, most of our evidence concerning chromatin fiber structure has
been based on in vitro studies of isolated fragments of
chromatin. There is some reason for concern that these may not closely resemble the fibers as they exist in the nucleus (see, for
example, Ref. 3). Only a few experiments have been conducted in which
chromatin fibers have been examined in situ, with minimum
perturbation of the nuclear structure. Second, past experiments have
all too often utilized cells that are relatively inactive, for example
chicken erythrocytes and invertebrate sperm. The folding of chromatin
in such cells may not be typical of eukaryotic cells in general. Such
concerns must be kept in mind in evaluating existing data. From Beads-on-a-string to Solenoids
The first electron microscope observations of chromatin fibers to
reveal their nucleosomal structure utilized the low salt spreading
technique developed by Miller and Bakken
(4) . Both Woodcock
(5) and Olins and Olins
(6, 7) reported
regularly spaced beadlike objects along extended DNA filaments. It has
since been realized that this method, while ideal for visualizing
nucleosomes, seriously distorts the chromatin fiber itself.
The
first evidence that the fiber might be more compact under some
circumstances appears to be found in a study of SV40 minichromosomes by
Griffith
(8) . At low salt Griffith observed a zigzag circular
chain of nucleosomes, but at 150 m
M NaCl the structure had
condensed to yield a smooth fiber of close packed nucleosomes about 10
nm in thickness. Similar structures were reported under a variety of
circumstances (see Refs. 9 and 10, for example). However, conditions
reported by various laboratories for production of the ``10-nm
filament'' were contradictory, and it is now believed that such a
structure is an artifact produced by EM
A major advance was made when Finch and
Klug
(10) demonstrated that even low concentrations of
Mg
Several major electron microscopic studies appeared
in the succeeding years; especially important are the careful analyses
by Rattner and Hamkalo
(12) and Thoma et al. (13) . Thoma et al. described a progressive
folding from a zigzag structure at very low ionic strength to the
solenoid at NaCl concentration above about 60 m
M or at
Mg
During the years immediately following, a considerable number of very
specific models for the condensed fiber were put forward. Some of
these, such as the models proposed by McGhee et al. (14) and Butler
(15) , were essentially refinements of
the Finch and Klug
(10) solenoid, making specific disposition
of the linker DNA, a question that Finch and Klug had left open.
Others, like those of Worcel et al. (16) , Staynov
(17) , Woodcock et al. (18) , and Williams
et al. (19) seem to have been inspired by the zigzag
chain presumed to exist at lower ionic strength. The first of these
represents a twisting of the zigzag chain, Staynov's model has a
crossed linker pattern, and the latter two are two-start helices built
on the zigzag motif. Illustrations of most of these models are to be
found in Refs. 1 and 2. We shall not discuss them in greater detail
here for a simple reason: in our opinion (see below) it is unlikely
that any regular helical model describes significant portions
of the chromatin fiber. In certainty, there exists no convincing
evidence for any one of the specific models that have been proposed.
For many years there have been those who were skeptical of helical
models. The skeptics fell into two classes. First were the proponents
of the ``superbead'' hypothesis, which held that the
condensed fiber represented a linear array of ``beads,'' each
containing some roughly defined number of nucleosomes (see Refs. 1 and
2 for a description of this model and criticism of it). Second, there
were a few who, although not wedded to the superbead hypothesis,
remained skeptical of any regular ordering in the chromatin fiber (see,
for example, Refs. 20-25). However, such skeptics have been a
decided minority, with most scientists who are concerned about
chromatin higher order structure arguing the merits of one or another
of the helical models. Perhaps it is time to reopen the whole question
by asking: ``how substantial is the evidence for a
regular, helical structure in chromatin fibers?'' What Is the Evidence?
The transmission electron
microscope, using negative staining or metal shadowing, provided much
of the earlier data concerning chromatin structure. It was realized by
many workers in the field that these treatments, in addition to the
chemical fixation and extreme dehydration usually employed, could well
produce artifacts. In fact, the possibility of distortion during sample
preparation has been put forward as an explanation for the remarkable
paucity of EM images showing regular helical structure in condensed
fibers
(25) . Paper after paper presents multiple (presumably
selected) images of fibers in which tiny bits are pointed out by arrows
as indicating whatever model the authors champion. The vast majority of
the fibers in such images show no evidence at all for regular
structure. The argument that it was there in the nucleus but was
destroyed in extraction is hardly supported by recent studies of
sectioned or frozen nuclei, which also show little evidence for regular
structure
(26, 27, 28) .
Scanning
transmission electron microscopy has been used by Gerchman and
Ramakrishnan
(29) in a very careful study of chicken
erythrocyte chromatin. Again, fibers of very non-uniform diameters were
observed, a result that correlates with the large variation in mass per
unit length derived from these studies. Both factors argue against the
idea of regular helices.
There exist two electron microscope studies
that do provide some evidence for helical structures. Williams et
al. (19) made optical transforms of selected regions of EM
images; these show cross-like patterns that constitute evidence for
local helical structure. However, these experiments must be evaluated
with the understanding that the regions selected were of very limited
extent, and the chromatin utilized had unusually uniform linker
lengths. It is quite possible (even likely) that small local regions of
regular structure exist, perhaps in regions of unusual regularity in
nucleosome spacing. In a more recent study of end-on views of short
chromatin fibers, Bartolomé et al. (30) show
images depicting what appears to be a helical coiling of the fiber
periphery. It is interesting to note that in this as well as in many
other studies (see, for example, Ref. 13) both left and right helices
have been reported, sometimes in the same chromatin preparation.
Interpretation of such observations, in terms of models, is complicated
by the fact that some models demand left helices, some right, and some
are ambiguous.
Advances in electron microscopic techniques over the
past two decades have not changed the picture materially. Careful
attempts, using such modern techniques as EM tomography, to map the
location of individual nucleosomes in a fiber produced no evidence for
regular helices
(20, 27) .
In summary,
despite two decades of careful observation, it cannot be said that
electron microscopy has provided convincing evidence for more than
fragments of regular helical structure in the chromatin fiber.
A wide variety
of scattering experiments, using x-rays and neutrons on chromatin
fibers in solution, in swollen gels, and in partially oriented
quasi-crystalline arrays, have been carried out in attempts to deduce
structural parameters. Because it is difficult to obtain highly
oriented samples, the scattering patterns observed in most of these
studies are almost or completely radially symmetric. This led to
misinterpretation of early experiments; the scattering maxima at 5.5,
3.7, and 2.7 nm were thought to represent successive higher orders of
the 11-nm reflection originating from a solenoid of 11-nm pitch. This
is now believed not to be the case, since the latter three reflections
are observed from chromatin solutions at low ionic strength in which
the condensed fiber no longer exists; indeed all maxima other than that
at 11 nm can be generated by scattering from individual nucleosomes
(22) . Furthermore, the preferential orientation of the latter
reflections is at right angles to that of the 11-nm reflection (see,
for example, Ref. 31).
Nevertheless, the fact that the 11-nm maximum
in the scattering curves is observed only under conditions in which the
condensed fiber is stable is widely held to argue for a helical
structure with 11-nm pitch. There are, we believe, good reasons to be
skeptical even of this inference. Theoretical calculations by Koch
(22) demonstrate that nucleosome-size objects, packed tightly
but randomly to give a cylinder with 30-nm outer diameter,
produce a scattering curve remarkably similar to that observed
experimentally, including a maximum near 11 nm. Thus, a regular helical
structure is not required to produce the observed pattern of scattering
maxima.
A better case for a pervasive helical structure could be
made if there were strong evidence in oriented samples for splitting of
the 11-nm reflection about the axis, providing at least a hint of a
``cross'' pattern in the reflections. However, the results of
the most careful x-ray diffraction studies of oriented fibers
(31) show no such effect. Very weak splitting of the 11-nm
reflection has been observed in neutron scattering from fibers
(32) . However, interpretation is complicated by the fact that
similar results were observed with both intact chromatin and samples
from which histone H1 had been removed
(33) ; the latter should
not exist as condensed fibers (see Refs. 10 and 13).
In
conclusion, scattering data provide no evidence for a pervasive,
regular helical structure in the condensed fiber. Indeed, they seem to
be more consistent with the picture that has emerged from EM studies, a
fiber that is generally irregular though tightly packed, with at best a
roughly helical structure interspersed by occasional short regular
regions.
It is
often argued that evidence for preferential orientation of nucleosomes,
with their short axes more or less perpendicular to the fiber axis,
supports helical models. Such evidence comes from two kinds of
experiments, and neither has provided a quantitative result.
X-ray
and neutron scattering studies of oriented chromatin fibers do exhibit
some anisotropy. It appears clear that the 11-nm reflection has
preferential meridional orientation, whereas the remaining reflections
are more equatorial (see, for example, Refs. 31-33). However, to
our knowledge no attempt has been made to quantitate the degree of
orientation from such data.
Numerous attempts have been made to
employ linear electric dichroism and flow dichroism measurements to
provide such quantitative information (see Refs. 1 and 22 for reviews
of published results). The maximum dichroism values observed are
invariably low, with both positive and negative values being reported,
but most results clustering around
However, to interpret the
data in this way assumes that all of the nucleosomes make
approximately the same angle with respect to the fiber axis. There is,
in fact, an alternative explanation for a very low value for the
dichroism, that the nucleosomes are nearly randomly oriented with
respect to the fiber axis, so that even complete alignment of the
fibers still results in nearly random alignment of DNA chromophores.
This possibility does not seem to have been considered.
A further
complication in interpreting linear dichroism data arises from the fact
that the orientation of the linker DNA (which comprises about 25% of
the whole) is unknown. An attempt has been made to correct for this,
using photochemical dichroism
(34) ; the results were similar to
those described above.
The most reasonable conclusion from all of
the above studies is that there is some evidence for orientation.
However, the degree of orientation is unknown, and a weak preferential
orientation does not in itself argue for a regular structure.
Again, the results seem most simply explained by the existence of
no more than a very irregular helix, with only limited regions of
regularity.
Clearly, attempts to observe details of structure in the
condensed fiber have not been very successful. In part, this results
from the extremely tight packing of nucleosomes. Another way to
approach the problem is to ask how the more readily observable
structure present at lower ionic strength might be expected to
fold as salt is added.
The conformation adapted by chromatin fibers
in the absence of divalent cations and at monovalent salt
concentrations of only a few millimolar has been the subject of some
controversy. The flattened ``zigzag'' structures first
reported by Thoma et al. (13) have been accepted by
many as truly representing the extended conformation and have in fact
influenced the development of several models for the condensed helix.
However, Thoma et al. (13) pointed out very clearly
that the zigzag structure they observed could well be the consequence
of flattening of an open helix by absorption to the EM grid. Indeed,
the results of numerous physical studies of chromatin fibers in dilute
salt solution are best explained in terms of some kind of an open
helical conformation
(21, 29, 35, 36, 37, 38) .
Further evidence that the fiber at low salt is not a flattened zigzag
comes from recent scanning force microscopy experiments
(39, 40, 41) (Fig. 1). Neither do these
experiments give evidence for a regular helical structure; rather, an
irregular, quasi-helical conformation is observed.
If such an irregular
structure is to condense further, it would seem likely that the primary
process might be an axial contraction, leading not to a helix of any
defined sense or regularity but to an irregular packing of nucleosomes.
How such a condensation might proceed would depend on the behavior of
linker DNA as the salt concentration is increased. The conventional
view, supported by data of Yao et al. (43) , is that
the linker bends, thereby allowing close contacts of nucleosomes.
However, sedimentation data of Butler and Thomas
(45) indicate
no contraction of dinucleosomes over a wide salt range,
implying that the linker remains stiff. This is in accord with the
biochemical study of thymine dimer formation in core particles and
linker DNA in situ (46) and with recent EM
observations by Horowitz et al. (27) . This would imply
an ``accordion-like'' contraction, which, if non-uniform,
might well lead to the superbeads appearance of the fiber reported by
several researchers (see Refs. 1 and 2 for discussion). Furthermore, an
axial condensation of this kind would avert a potential problem that
has been largely overlooked by many of the proponents of helical
models: how can constrained helical chromatin regions condense or
decondense without invoking severe superhelical stress on the DNA? Of
course, depending on the sense of the helix imposed, either the
condensation or the decondensation could be accommodated by
topoisomerases; however, the lack of convincing evidence for a
eukaryotic gyrase poses a serious problem for whichever process
requires the introduction of negative supercoiling. The only kind of
helical chromatin fiber that could readily undergo a
condensation/decondensation cycle is one in which both senses of the
helix are mixed in equal proportions. Interestingly, Bartolomé
et al. (30) report just this from their EM studies of
end views of chromatin fragments. Summary and Directions for Future Research
Clearly, chromatin fibers condense to irregular, rodlike structures
at ionic conditions approaching those in the nucleus. Evidence for some
degree of helical coiling can be found. Yet, despite years of
experimental study, it seems to us that there is very little reason to
believe that the condensed chromatin fiber contains substantial amounts of any regular helical structure. Nor are there
substantive reasons to believe that the fibers observed and studied
in vitro represent artifactually distorted remnants of more
regular in vivo structures. Furthermore, what we now know of
the irregularity of nucleosome arrangements in fibers at low ionic
strength argues for a corresponding irregularity under physiological
conditions. For these reasons, to continue arguments about the virtues
of particular higher order structures seems pointless.
Careful
perusal of the chromatin literature shows that many authors have
expressed skepticism concerning the existence of regular helical
structure in the fiber (see especially Refs. 20-22). Very
recently, evidence from EM tomography
(27) has provided the
most direct experimental challenge to regular helix models for the
condensed fiber. As the authors state: ``The reconstitutions show
no single, symmetrical arrangement of nucleosomes within the fibers,
but rather a continuum of structures . . . ''.
What are needed
now are more investigations, by the most non-destructive means
possible, of chromatin fibers from a variety of sources, with special
attempts made to focus on structure in nuclei. Equally important would
be the study of chromatin fibers with exactly equally spaced
nucleosomes. Such fibers might be obtained by isolation of satellite
chromatin or by reconstitution of both histone cores and linker
histones onto long tandem repeats of a very strong and specific
nucleosome positioning signal. Such structures would be predicted to
form regular helices at low ionic strength, which might then fold into
equally regular condensed structures. The behavior of such model
structures in microscopy, scattering, and dichroism studies would
provide a benchmark against which the data on native fibers could be
meaningfully evaluated.
We thank Dr. M. Koch (EMBL, Hamburg) and C. L.
Woodcock (University of Massachusetts) for critical reading of the
manuscript and useful suggestions and Drs. S. H. Leuba and G. Yang
(University of Oregon, Eugene) for supplying the images in
Fig. 1
.
ABSTRACT
INTRODUCTION
Electron Microscopy
Low Angle Scattering Studies
Evidence for Nucleosome Orientation
Structure at Low Ionic Strength; Folding of the
Fiber
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
staining procedures (see Ref. 11 for a recent evaluation).
Nevertheless, the term 10-nm filament still persists in the literature
and should be discarded.
(0.2 m
M) produced condensation of the
filaments of nucleosomes into an irregular fiber about 30 nm in
diameter. The term ``30-nm fiber'' has since become
associated with the salt-compacted structures. However, we believe that
there are now good reasons to substitute for this designation the term
``condensed fiber'' (see below). The fibers observed by Finch
and Klug
(10) showed occasional diagonal striations,
interpreted as evidence for helical structure. Earlier x-ray
diffraction studies on chromatin fibers had indicated maxima
corresponding to spacings of about 11, 5.5, 3.7, and 2.7 nm.
Accordingly, Finch and Klug
(10) postulated a helical
``solenoid'' model with a pitch of about 11 nm and suggested
that the series of maxima represented successive orders of diffraction
from such a helix. The model was simple and seemed to accommodate much
of the data available at the time. It took root in the textbooks and
continues to be regarded by many as the appropriate description of the
condensed fiber.
concentration above 0.3 m
M. The
beads-on-a-string form was considered by these authors to be an
artifact of fiber stretching and/or H1 depletion, whereas the 10-nm
nucleofilament was viewed as an artifactual consequence of staining
conditions. The model of Thoma et al. (13) for
chromatin condensation (folding with increasing ion concentrations from
an open zigzag through a closed zigzag to a helical condensed fiber)
has dominated thinking in this field for many years. Indeed, it is this
picture that helped to generate the rash of alternative models for the
condensed fiber that appeared in the 1980s. The solenoid model had
fixed the idea that some kind of regular structure must
describe the condensed fiber, but there was widespread controversy
concerning the details of that structure. 1980-1986: An Orgy of Model Building
A/ A =
-0.1. This is a very small value when compared, for example, to
the maximum electric dichroism of naked DNA, which approximates
-1.3. This has been interpreted as indicating that the average
angle between the DNA base planes and the fiber axis is around 60°,
close to the magic angle of 54.7° at which the dichroism passes
through zero and changes sign. This would correspond to a tilt of about
30° of the long axis of the nucleosome away from the fiber axis, a
result consistent with a number of models.
Figure 1:
Model of the chromatin fiber at low
salt, its simulated SFM image, and an actual SFM image. A,
model of a chromatin fiber used in these simulations. The DNA wraps in
a left-handed fashion 1.75 turns around a histone octamer. The
nucleosome is simulated by a disc, 5.5 nm high and 11 nm in diameter.
The radius of curvature of the DNA wrapped around the core is 55
Å, and the pitch of the DNA is 28.6 Å. The DNA has an
average of 10.15 bp/turn around the histone octamer and 10.4 bp/turn in
the linker portion. The exit angle of the DNA is determined by the
tangent at the point it leaves the nucleosome. The length of the linker
DNA is determined using a uniform deviate random number algorithm,
which generates linker lengths between 51 and 73 bp. The linker DNA is
assumed to adopt a straight configuration between nucleosomes. This
model generates three-dimensional, randomly organized fibers with an
average diameter of 30 nm. B, a projection of the fiber onto a
plane without changing the orientation of the nucleosomes. C,
simulated SFM image of the model in A, obtained by convoluting
the plane projection in B with a parabolic tip with a radius
of curvature of 10 nm. D, an experimental SFM image of
glutaraldehyde-fixed chromatin fibers deposited on mica from 5
m
M triethanolamine HCl, pH 7.0. Image sizes are 400
400 nm ( B, C, and D). Heights are encoded by
color, with low regions depicted in dark red and
higher regions in increasingly lighter tones of
red, in a height scale from 0 to 50 nm. (Courtesy of S. H.
Leuba and G. Yang, University of Oregon.)
The significance
of these observations can now be understood in terms of a simple
modeling of chromatin structure
(40, 41, 42) .
At low ionic strength, we expect the linker DNA between nucleosomes to
be rigid, an assumption supported by electron microscopic studies on
dinucleosomes
(43) . Then the rotation of each nucleosome with
respect to the preceding one will be determined by the number of base
pairs in the intervening linker (see, for example, Ref. 20). If linkers
were absolutely uniform, a regular, open helix would result
(21, 22, 42, 44) . However, linkers are
not of uniform length in real chromatin fibers; consequently,
open, irregular helix-like structures like that shown in
Fig. 1
should be generated. As the figure shows, predicted and
actual SFM images are very similar. The open ``helices'' are
predicted and observed to have a diameter of the order of 30 nm and a
mass/length ratio of about 1-2 nucleosomes/11 nm. It is the fact
that the low ionic strength structure also has a diameter of
about 30 nm that makes us wish to abandon the term 30-nm fiber as a
designator for the condensed structure.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.