©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chromatin Higher Order Structure: Chasing a Mirage?(*)

Kensal van Holde (1)(§), Jordanka Zlatanova (1) (2)

From the (1) Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305 and the (2) Institute of Genetics, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

ABSTRACT
INTRODUCTION
Electron Microscopy
Low Angle Scattering Studies
Evidence for Nucleosome Orientation
Structure at Low Ionic Strength; Folding of the Fiber
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

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() staining procedures (see Ref. 11 for a recent evaluation). Nevertheless, the term 10-nm filament still persists in the literature and should be discarded.

A major advance was made when Finch and Klug (10) demonstrated that even low concentrations of Mg(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.

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 Mgconcentration 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

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?


Electron Microscopy

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.


Low Angle Scattering Studies

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.


Evidence for Nucleosome Orientation

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 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.

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.


Structure at Low Ionic Strength; Folding of the Fiber

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.


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.

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.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995.

§
To whom correspondence should be addressed. Tel.: 503-737-4155; Fax: 503-737-0481.

The abbreviations used are: EM, electron microscopy, SFM, scanning force microscopy, bp, base pairs.


ACKNOWLEDGEMENTS

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 .


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