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Published in final edited form as: Curr Opin Cell Biol. 2012 Mar 27;24(3):359–366. doi: 10.1016/j.ceb.2012.03.003

Revisiting Higher-order and Large-scale Chromatin Organization

Qian Bian a,1, Andrew S Belmont b,✉
PMCID: PMC3372662  NIHMSID: NIHMS368905  PMID: 22459407

Abstract

The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct “30 nm” chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30 nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding.

Introduction

For several decades, the path towards understanding higher order organization of chromatin appeared clear: solve the 30 nm chromatin fiber structure and then determine its path within interphase and mitotic chromosomes. Underlying these two goals, however, were two major assumptions- first, that a unique structure exists for “the” 30 nm chromatin fiber and second, that the 30 nm chromatin fiber as it exists in vitro is the basic structural motif underlying further chromatin folding in vivo.

These two long-standing assumptions increasingly have been challenged in recent years. A family of 30 nm chromatin fiber structures has emerged together with speculation concerning alternative pathways for chromatin compaction in vivo that bypass the 30 nm fiber. 3C (chromosome conformation capture) molecular methodologies and new microscopy approaches suggest a surprising plasticity of chromatin folding challenging conventional ideas for how chromatin folds into chromosomes.

Higher order folding of chromatin into 30 nm fibers in vitro and in situ

Salt-dependent folding in vitro of “beads on a string” oligonucleosomes into a roughly 30 nm diameter fiber led to the natural assumption of a unique “30 nm”, higher-order chromatin folding motif. The solenoid model, the first proposed for its structure, postulated a one-start, helical folding of the nucleofilament with the linker DNA bent between adjacent nucleosomes. At low ionic strength, unfolded oligonucleosomes typically show a zig-zag appearance, leading to two alternative model classes, the twisted ribbon model in which the zig-zag folds to form the fiber such that the zig-zag is along the fiber trajectory versus the crossed-linker model in which a two-start helical folding is created through extension of the straight linker DNA across the fiber cross-section (reviewed in [1]). Interestingly, inter-nucleosome cross-linking patterns suggest both solenoid and zig-zag model features can co-exist within a single 30 nm fiber [2*].

Electron microscopy (EM) of oligonucleosomes reconstituted with linker histone on tandem arrays of nucleosome positioning DNA sequences with variable linker lengths now reveals a family of higher-order chromatin fibers. A nucleosome repeat length (NRL) of 167 bp produces a 21 nm diameter ladder-like crossed-linker pattern [3] consistent with previous X-ray crystallography of a nucleosome tetramer (167 NRL) [4] and predictions from cross-linking experiments (177 NRL) [5]. NRLs of 177, 187, 197, and 207 bp produced 34 nm diameter chromatin fibers, but a jump to a 44 nm diameter was seen for NRLs of 217, 227, and 237 bp [6]. The invariance of fiber diameter over a 177-207 NRL range suggests an important role for specific inter-nucleosome contacts, consistent with the strong disruption of compaction observed for 202 NRL arrays reconstituted with histone H4 acetylated at K16 [7]. H4K16 is predicted to contact the acidic patch on histone H2A on adjacent nucleosomes to stabilize higher-order folding [8]. While linker histone plays an important role in modulating higher order folding, the exact location of the linker histone relative to the nucleosome and linker DNA remains unknown.

Surprisingly, whereas the 21 nm fiber has a density of 6.1 nucleosomes/11 nm [3], the density of the compacted 34 nm fiber is nearly double at 11.2 nucleosomes/11 nm [6], suggesting inter-digitation of adjacent nucleosome gyres and a much higher compaction than previous estimates.

In contrast, recent cryo-electron microscopy tomography of avian erythrocyte nuclei [9**] (212 bp NRL) demonstrates an ~32 nm diameter fiber formed by a left-handed two start helical folding of nucleosomes with ~6.5 nucleosomes/11 nm density, closer to previous estimates of nucleosome density. Density within the fiber interior suggests linker DNA crossing the fiber cross-section. Docking of nucleosome structure within the EM reconstruction and molecular modeling leads to a geometry similar to the crossed-linker model. There is a prominent face-to-face orientation of nucleosomes and a more homogenous radial distribution of nucleosomes, not unlike that predicted in a solenoid model, yet nucleosomes remain separated rather than close packed and no inter-digitation of nucleosomes is observed. Chromatin within intact erythrocyte nuclei is compacted into dense chromocenters. This study used isolated nuclei and buffer conditions that presumably decondensed these chromocenters into dispersed 30 nm chromatin fibers. The compaction of these 30 nm chromatin fibers relative to their conformation in vivo therefore remains unclear.

Thus we already are now at four distinct “30 nm” structures (Fig. 1). Interestingly, imposing just a few, simple geometrical constraints was shown to produce a family of possible “30 nm” chromatin fiber structures [10], as now more recently demonstrated by molecular dynamics simulations [11*,12*].

Figure 1. Four different higher-order chromatin structures and counting.

Figure 1

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(A) 21 nm diameter zig-zag structure formed after reconstitution with both core and linker histones using a 167 bp nucleosome repeat length. Reprinted from Fig. 4, reference [3], Copyright 2008 National Academy of Sciences, USA. (B-C) 33 nm diameter (B) versus 44 nm diameter (C) higher-order fibers formed after reconstitution with both core and linker histones but with nucleosome repeat lengths of 197 bp (B) or 207 (C). Reprinted from Fig. 1, reference [6], Copyright 2006 National Academy of Sciences, USA. (D-E) Transverse (left) or longitudinal (right) sections through individual (D) or averaged (E) tomograms of 32 nm diameter higher-order fibers visualized within cryo-sections of avian erythrocyte nuclei [9]. These fibers contain ~6.5 nucleosomes per 11 nm length of fiber. Reprinted from Figs. 1&2, reference [9]. (F) Molecular model [6] of one start helical structure proposed for 33 nm diameter reconstituted fiber (B). Inter-digitation of neighboring gyres of helix results in high nucleosome fiber density of ~ 11 nucleosomes per 11 nm fiber length. Reprinted from Fig. 3, reference [6], Copyright 2006 National Academy of Sciences, USA. (G) Molecular model for two-start, cross-linker type structure proposed for 32 nm fiber visualized by cryo-EM (D-E). The approximately 6.5 nucleosomes per 11 nm of fiber length corresponds more closely to previous estimates of fiber compaction. Reprinted from Fig. 3, reference [9], Copyright 2011 National Academy of Sciences, USA. Scale bars = 50 nm (A), 100 nm (B-C), 30 nm (D), or 15 nm (E).

Chromatin array oligomerization- an in vitro model for in vivo compaction

FISH analysis reveals most genome regions in higher metazoan somatic nuclei are condensed well above the 30 nm fiber. Interestingly, polycation concentrations which preserve large-scale chromatin folding within isolated nuclei [13] are similar to those causing inter-fiber associations and chromatin aggregation in vitro [14], suggesting oligomerization of chromatin arrays as an in vitro model for chromatin compaction in vivo [15]. Oligomerization is cooperative and, like higher order chromatin folding, dependent on linker histone and histone tails, particularly H4. However, its dependence on histone tails appears to be largely through nonspecific, charge interactions [16*]. This contrasts with specific interactions, in particular between H4K16 and an acidic patch on H2A [7,17], implicated in higher order folding. Whereas H2A variant H2ABbd has a reduced acidic patch and reduced higher order folding but increased oligomerization, histone variant H2A.Z with an increased acidic patch has the opposite behavior, suggesting competition between chromatin inter-fiber and intra-fiber interactions [18]. Moreover a histone mutation mimicking acetylation at H3K56 has no effect on higher order chromatin compaction but increases oligomerization of chromatin arrays with nucleosome free regions, which simulates sites of nucleosome assembly where this mark is enriched [19*]. In contrast, p300 mediated histone acetylation at the HIV promoter leads to both higher order chromatin decondensation and inhibition of array oligomerization [20**]. Which effect is most closely related to the accompanying in vitro transcriptional activation remains unclear.

Higher order chromatin folding within nuclei and chromosomes

The assumption of 30 nm chromatin fibers as the basic building block of further chromosome folding is derived from a relatively small number of observations. These include visualization of 30 nm chromatin fibers in isolated mitotic chromosomes swollen by low salt isolation buffers [23,24], the clearly distinct and regular packing of well spaced 30 nm chromatin fibers within intact nuclei of a few specialized cell types such as starfish sperm nuclei [25], and the clear presence of 30 nm chromatin fibers at specific, less compacted chromosomal loci, for example in polytene chromosome puffs after inhibition of transcription [26]. Given these in situ observations, more condensed chromosome regions whose substructure cannot be easily visualized were assumed to be formed by these same fibers.

New experimental approaches now question this assumption [21*,22*]. First, cryo-EM of mitotic chromosomes fails to reveal any substructure above 11 nm packing of nucleosomes, leading to a “melt” chromosome model in which strong inter-fiber nucleosome interactions of comparable strength to intra-fiber interactions cause mixing of nucleosomes into a uniform mass [27]. Second, using conventional chemical fixation and embedding but energy spectroscopic imaging (ESI) for improved chromatin contrast, Bazzet-Jones and colleagues fail to observe any hint of 30 nm chromatin structure in a range of cell types, with no 30 nm type fiber structures visualized within more compact chromatin regions [28*,29*].

Reconciling the absence of any apparent substructure above 11 nm in cryo-EM images of mitotic chromosomes with the observation of distinct mitotic chromosome substructure above 30 nm by conventional light microscopy in fixed and live cells [30] and now by super-resolution light microscopy [31**] remains difficult. Fixation by high pressure freezing and other cryo-methods producing vitreous ice is generally considered ideal, yet estimated millisecond freezing rates are still slow relative to FRET FCS measurements of microsecond trinucleosome array dynamics and sub-millisecond trinucleosome decompaction [32**]. Possible effects of temperature dependent changes in DNA pitch during freezing are unknown. Meanwhile, the ESI studies described above used formaldehyde fixation and immunostaining procedures previously shown to significantly perturb chromatin ultrastructure [33]. Considerable cell extraction is observed in these ESI images, particularly in the ES cells where unfolded nucleosomal filaments (defined here as “10 nm chromatin fibers”) were predominately observed. Conventional TEM of ES cells using standard fixation conditions which do not cause cell extraction show extensive regions of condensed chromatin, including at the nuclear periphery [34]. Repeating this work using stronger fixation conditions and eliminating the pre-embedding immunostaining would be desirable.

Still, reconciling the ~240 nm (~25-50 kb) persistence length estimated from molecular simulations of 30 nm fiber folding [1] with the highly compacted chromatin prevalent in most nuclei and mitotic chromosomes remains difficult. The possibility that a major fraction of the genome is compacted in a non-canonical 30 nm fiber structure is significant, while EM tomography of chromosomes assembled in a Xenopus oocyte extract demonstrates the ability to achieve high compaction without canonical 30 nm fibers in such a reconstituted system [35].

Lessons from 3C

A similar challenge to the concept of further chromosome compaction through folding of 30 nm chromatin fibers now comes indirectly from molecular biology based 3C methods. In 3C experiments, intact nuclei are fixed with formaldehyde to preserve physical interactions between chromatin loci. Cross-linked chromatin is then cut with restriction enzymes and this is followed by inter-molecular DNA ligation of neighboring cross-linked fragments. The capability of 3C to map chromatin interactions has been expanded by combination with high-throughput sequencing approaches and newer derivatives of 3C, including 4C [36,37], 5C [38,39*], Hi-C [40**], e4C [41**] and ChIA-PET [42**,43**]. These derivatives allow mapping the interactions between one locus to the whole genome [36,37], mapping interactions between many foci within a chromatin domain [38,39*], identifying, at lower resolution, genome-wide interactions [40**], and/or detecting interactions mediated by specific proteins [41**,42**,43**].

An emerging theme from these experiments has been the high frequency of observed interactions between distal regulatory regions (promoters, enhancers, insulators, and DNase I HS sites) separated by several to thousands of kb, leading to a chromatin “hub” model of organization [44*]. While attention has focused on the high frequency of long-range interactions, multiple interactions between regulatory elements separated less than the estimated ~25-50 kb 30 nm fiber persistence length are equally surprising. 30 nm chromatin fiber looping over such short DNA stretches would be unlikely without local kinking or fiber discontinuities, perhaps related to local chromatin structure, and/or buckling of 30 nm fibers induced by strong inter-nucleosome interactions. In vitro studies have revealed disruption of both higher order folding and oligomerization of nucleosome arrays over several kb surrounding local recruitment of the p300 HAT [20**], raising the possibility of stretches of 10 nm structure, encompassing regulatory regions, acting as flexible connectors facilitating looping of adjacent 30 nm chromatin fiber segments.

Recent studies have employed computational modeling to extrapolate the comprehensive datasets generated from 3C-based assays into estimated 3D structures of large-scale chromatin folding [39*,40**,45*,46*]. 3C interaction frequencies are used as distance constraints for model reconstruction. A model generated for a 500kb region surrounding the alpha-globin locus predicts its folding into 1 or 2 highly compact chromatin domains, consisting of rosettes of 50- 60kb chromatin loops, in its transcriptionally inactive state. A less compact structure is predicted in cells where the alpha-globin locus is expressed, with active genes clustered towards the globule center.

On a still larger scale, Hi-C methods have pointed to spatial segregation of Mbp domains separating active and inactive genomic regions in a functional compartmentalization of the genome [40**]. Interestingly, these Hi-C identified domains match closely with comparable size genomic domains with distinctive early versus late replication timing [48**].

Interpretation of these 3C-based results is tempered by uncertainty regarding key assumptions regarding this methodology. First, is the question of whether rare cross-linking events represent temporal fluctuations occurring within a large fraction of the cell population versus rare but stable conformation heterogeneity within the cell population. Second, is the question of what is the actual nature of the cross-linking events. Whereas the 3C ligation event typically is depicted as between two naked DNA elements cross-linked at a single molecular complex, even at 20-fold reduced formaldehyde fixation levels than commonly used for 3C-based methods internucleosome cross-linking events are frequent within nucleosome arrays [2*]. UV irradiation, a similar short range cross-linking method, produces up to 20% inter-fiber nucleosome cross-linking events using oligomerized nucleosome arrays as a substrate [47*]. Extensive cross-linked protein/DNA networks therefore may be the actual template for 3C ligation events. This might explain why many loci connected by long-distance 3C interactions show microscopic versus molecular separation distances as measured by FISH [41**]. Taking this idea one step further, these cross-linked networks could include protein/RNA complexes from nuclear bodies, linking DNA fragments from different chromosomes attached to the same nuclear body. Understanding which of these possibilities occur in 3C experiments will be critical for deriving correct structural information from 3C data.

Direct visualization of large-scale chromatin organization and dynamics

FISH reveals interphase chromatin compaction for most higher metazoan species is significantly higher than that predicted for 30 nm chromatin folding in most tissue types and for most genomic loci, transcriptionally active or inactive. One exception is the Hox B locus which in its active form has a compaction on the order expected for an extended 30 nm chromatin fiber [49]. Both FISH [50,51] and DNA replication pulse-chase studies [52] have shown spatially distinct, localized chromatin domains, or “globules”, 100’s to 1000’s of kb in size, as predicted by 3C methodologies. At the EM level, compacted domains and fibers with diameters well above 30 nm have been observed, with locally decondensed segments showing suggestions of loose coiling of ~30 nm fibers [53]. Live cell microscopy shows similar compacted, fiber-like conformation for lac operator tagged engineered regions comprised of BAC trangenes with high transcriptional activity [54**], while in vivo immuno-gold labeling which avoids detergent extraction and prolonged staining procedures also reveals large-scale chromatin fibers for gene amplified chromosome regions [55].

While it is tempting to model the internal folding of these large-scale chromatin domains as compaction of 30 nm fibers, their structure remains an open question. The close-packing of chromatin and the inability to visualize linker DNA paths, makes extraction of fiber trajectories or distinguishing a short 30 nm fiber segment from two interdigitated 10 nm fibers or a single, supercoiled 10 nm fiber loop difficult.

Just as 3C interactions between sites several to tens of kb apart are difficult to reconcile with canonical 30 nm chromatin fiber folding, 3C interactions between loci at variable distances tens to several hundred kb apart are difficult to reconcile with hierarchical models of chromatin folding. For example, it is difficult to imagine interactions between loci separated by several hundred kb within extended large-scale chromatin fibers with a 1-3 Mbp / micron compaction level [56*]. Yet FISH analysis of several Mbp genomic regions in fact have suggested spatial segregation of transcriptionally active versus inactive regions within these genomic regions [51], and, on a larger scale, segregation of unique from repetitive sequences in the inactive X chromosome [57], suggesting that sequence distances along the DNA are not packed linearly along the interphase chromosome. Indeed, pairing of lac operator repeats over distances of several hundred kb in BAC transgene arrays suggests complex topological looping of DNA can coexist within linear large-scale chromatin fibers [58*].

Conclusions

The last several years has seen an important evolution of how we conceptualize higher order chromatin folding. A family of different “30 nm” fibers has emerged together with an increasing appreciation for higher-level compaction of chromatin above the 30 nm fiber and an increasing willingness to consider alternative models (summarized in Fig. 2) for this compaction. 3C-based methodologies may present outstanding opportunities for genomic analysis of higher levels of chromatin folding through molecular rather than microscopy approaches. However, major uncertainty still exists regarding experimental assumptions at the heart of deriving structural models from these 3C data sets. Moving forward, an important step would be to directly test some of these assumptions through a coordinated approach combining microscopy with 3C-derived and other genomic methodologies.

Figure 2. Models for interphase chromosome large-scale chromatin folding.

Figure 2

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(A) Conventional model showing 30 nm higher order fibers forming clusters of loops. In older models these loop interactions are driven by poorly defined nuclear matrix interactions. This model is derived to a significant extent from extrapolation of radial loop models for mitotic chromosomes rather than direct visualization of interphase chromosomes. (B) Based on cryo-EM and ESI contrast for conventional EM, new models have suggested in vivo interphase chromosome structure consists nearly entirely of 10 nm fibers, locally dispersed or concentrated in compact local domains. (C) Chromonema fiber model, derived from a combination of light and electron microscopy, proposes existence of large-scale chromatin fibers on the order of 100 nm diameter. Irregular folding of 10 and/or 30 nm chromatin fibers underlies these large-scale fibers. Tight packing within these large-scale chromatin fibers has made it difficult to determine the substructure of these fibers and the relative ratio of 10 or 30 nm chromatin fibers. Discrete large-scale fiber segments are connected by less tightly coiled 10 and 30 nm fibers. (D) Chromatin hub model suggested by 3C experiments, in which looping interactions are formed through close interactions between various regulatory DNA elements. These loops are usually illustrated as composed of 30 nm fibers but could involve any combination of 10 and 30 nm fibers. (E) Hybrid chromonema / chromatin hub model in which complex topological looping interactions exist within large-scale chromatin fibers.

Acknowledgments

This work was supported by National Institutes of Health grant GM58460 to A.S.B.

Footnotes

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