Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

Eltsov, Mikhail; MacLellan, Kirsty; Maeshima, Kazuhiro; Frangakis, Achilleas S.; Dubochet, Jacques

doi:10.1073/pnas.0810057105

Cited by 376 publications

(398 citation statements)

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“…We isolated chicken erythrocyte nuclei in such buffer and vitrified them in buffer containing 20 mM NaCl with 20% 40 kDa dextran. The osmotic effect of this particular dextran maintains fiber compaction that is equivalent to that of 2 mM MgCl 2 ionic strength (17) (Fig. S1).…”

Section: Resultsmentioning

confidence: 90%

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Scheffer

Eltsov

Frangakis

2011

Proc. Natl. Acad. Sci. U.S.A.

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140

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Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

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Section: Resultsmentioning

confidence: 90%

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Scheffer

Eltsov

Frangakis

2011

Proc. Natl. Acad. Sci. U.S.A.

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140

122

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“…S7 suggests how the bent linkers may destabilize i Ϯ 1 interactions in cis (within fibers) and promote nucleosome interactions in trans (between fibers). Internucleosomal interactions in trans may account for partially interdigitated 30-nm fibers in the interphase nucleus (11) and for global chromatin condensation in metaphase chromosomes where divalent cation levels are sharply increased (53) and no distinct 30 nm fibers are observed (54).…”

Section: Discussionmentioning

confidence: 99%

Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions

Grigoryev

Arya

Correll

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

245

358

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The architecture of the chromatin fiber, which determines DNA accessibility for transcription and other template-directed biological processes, remains unknown. Here we investigate the internal organization of the 30-nm chromatin fiber, combining Monte Carlo simulations of nucleosome chain folding with EM-assisted nucleosome interaction capture (EMANIC). We show that at physiological concentrations of monovalent ions, linker histones lead to a tight 2-start zigzag dominated by interactions between alternate nucleosomes (i ؎ 2) and sealed by histone N-tails. Divalent ions further compact the fiber by promoting bending in some linker DNAs and hence raising sequential nucleosome interactions (i ؎ 1). Remarkably, both straight and bent linker DNA conformations are retained in the fully compact chromatin fiber as inferred from both EMANIC and modeling. This conformational variability is energetically favorable as it helps accommodate DNA crossings within the fiber axis. Our results thus show that the 2-start zigzag topology and the type of linker DNA bending that defines solenoid models may be simultaneously present in a structurally heteromorphic chromatin fiber with uniform 30 nm diameter. Our data also suggest that dynamic linker DNA bending by linker histones and divalent cations in vivo may mediate the transition between tight nucleosome packing within discrete 30-nm fibers and self-associated higher-order chromosomal forms.chromatin structure ͉ electron microscopy ͉ mesoscopic modeling ͉ Monte Carlo simulations ͉ linker histone T he DNA in eukaryotic chromatin is packed and functionally regulated by histones and nonhistone architectural proteins (1). The primary packing level is represented by an array of repeating units, the nucleosomes, where the DNA is wound around histone octamers (2). Further compaction is achieved through a hierarchy of folding levels, including the 30-nm chromatin fiber (secondary level) and more compact and self-associating tertiary and quaternary forms whose structures are unknown (3-6). Because DNA conformation in chromatin and nucleosome packing are intimately connected to DNA/protein recognition and gene regulation, there has been intense interest in understanding chromatin structure, energetics, and dynamics. Some experimental studies have suggested that nucleosomal arrays fold in a zigzag arrangement with relatively straight linkers and a 2-start nucleosome interaction pattern that brings each nucleosome in proximity to its second nearest neighbor (7-10) consistent with chromatin fibers observed in situ (11). Other evidence suggests that chromatin condensed with linker histones and divalent cations such as Mg 2ϩ can form either zigzag structures with various nucleosome topologies (12, 13) or solenoid-like arrangements. The latter class of structures has bent DNA linkers and predominant interactions between either nearest neighbor nucleosomes (14, 15) and/or between every fifth or sixth nucleosome along the chain (16,17).The picture has became more complex recently with our heighte...

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“…Nevertheless, although the fiber is well characterized in vitro, the question of its in vivo significance is still much debated (Maeshima et al 2010;Fussner et al 2011;Hansen 2012;Ghirlando and Felsenfeld 2013). In particular, no higher order structures, including the 30-nm fiber, are observed by cryo-electron microscopy in native mitotic chromosomes (Eltsov et al 2009;Nishino et al 2012), nor are they apparent in nuclei of tissue-culture cells analyzed by correlative electron spectroscopic imaging (Ahmed et al 2010). As originally described, the 30-nm fibrr contains one or more helical stacks of nucleosomes with an eponymous diameter of approximately 30 nm (Finch and Klug 1976) (Fig.…”

Section: -Nm Fibermentioning

confidence: 99%

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

Muskhelishvili

Travers

2016

Biophys Rev

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We argue that dynamic changes in DNA supercoiling in vivo determine both how DNA is packaged and how it is accessed for transcription and for other manipulations such as recombination. In both bacteria and eukaryotes, the principal generators of DNA superhelicity are DNA translocases, supplemented in bacteria by DNA gyrase. By generating gradients of superhelicity upstream and downstream of their site of activity, translocases enable the differential binding of proteins which preferentially interact with respectively more untwisted or more writhed DNA. Such preferences enable, in principle, the sequential binding of different classes of protein and so constitute an essential driver of chromatin organization.Keywords DNA supercoiling . DNA translocases . Chromatin organization . Superhelicity gradients . DNA untwisting . DNAwrithing Dynamic changes of DNA supercoiling act as a driving force behind the alterations of genetic activity and DNA compaction both in eukaryotes and prokaryotes. Both these processes involve formation of spatially organized nucleoprotein structures by DNA architectural proteins. Whereas in eukaryotes the paradigmal example is the histone octamer, in prokaryotes a similar function is attributed to a small class of highly abundant nucleoid-associated proteins. We argue that, depending on the primary sequence organization, the changes of superhelicity elicit various alterations of local DNA geometry, while these, in turn, facilitate the assembly of distinct nucleoprotein structures pertinent to genetic function. Genome-wide conversion of the superhelical energy into various three-dimensional DNA structures thus acts as an analogue code coordinating the energy supply with the genetic expression of the chromosome.Recent studies make it increasingly clear that the DNA double helix carries at least two types of encoded information and that these include the well-known genetic code and the structural information determining the form and physical properties of the double helix itself. Since both these information types are inscribed in the same DNA sequence, and yet one is discrete whereas the other is continuous, they have been respectively dubbed the digital and analog DNA codes (Marr et al. 2008;Travers et al. 2012;Muskhelishvili and Travers 2013). The potential of the DNA to adopt distinct configurations is strongly modulated by DNA supercoiling, which is increasingly recognized as one of the major forces coordinating the cellular DNA transactions in both prokaryotes (including Archaea) and eukaryotes.DNA supercoiling can be generated by the simple application of torque to the double helix (Strick et al. 1998) but, importantly, because the DNA molecule itself possesses chirality-it is, after all, a right-handed double helix-the local structures stabilized by changes in superhelical density depend on both the sign and strength of the applied torque. For example, both positive and negative torque (respectively overand underwinding of the double helix) can change the intrinsic coiling of ...

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Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

Cited by 376 publications

References 37 publications

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

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