Budding yeast chromatin is dispersed in a crowded nucleoplasm<i>in vivo</i>

Chen, Chen; Shi, Jing; Lim, Hong Hwa; Tamura, Sachiko; Maeshima, Kazuhiro; Surana, Uttam; Gan, Lu

doi:10.1101/056465

Cited by 18 publications

(29 citation statements)

References 78 publications

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“…Picoplankton and yeast chromatin do not show evidence of 30-nm fibers in vivo (Gan et al, 2013; Chen et al, 2016). To test whether the chromatin higher-order structure of these two organisms is conserved in vitro , we imaged yeast chromatin in cell lysate.…”

Section: Resultsmentioning

confidence: 99%

“…(H and I) Histograms of nearestth nearest-neighbor distances, respectively. The in vivo measurements were ing a cryotomogram of sectioned yeast we previously reported (Chen et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

“…Three groups found no evidence of regular 30-nm fibers in mammalian cells (McDowall et al, 1986; Eltsov et al, 2008; Eltsov et al, 2014). We found that in the single-celled organisms Ostreococcus tauri (a eukaryotic picoplankton; herein called picoplankton) and Saccharomyces cerevisiae (a budding yeast; herein called yeast), there was also no evidence of regular 30-nm fibers (Gan et al, 2013; Chen et al, 2016). In contrast, cryo-EM studies have detected regular 30-nm fibers in the isolated nuclei of both starfish sperm (Woodcock, 1994) and chicken erythrocytes (Scheffer et al, 2011).…”

Section: Introductionmentioning

confidence: 89%

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Natural chromatin is heterogeneous and self associatesin vitro

Cai

Song

Chen

et al. 2017

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16The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely 17 found in chromatin within nuclei. To determine how chromatin high-order structure is 18 controlled, we used cryo-ET to study the undigested natural chromatin released from 19 cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the 20 presence of divalent cations, most of the chromatin from both organisms is compacted 21 into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, 22 some of which include face-to-face interactions. In the absence of divalent cations, 23 picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin 24 mostly remains compact with looser nucleosome packing, even after treatment with 25 histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore 26 sensitive to the local environment, but generally nonpermissive of regular motifs, even 27 at the level of oligonucleosomes.28 70 erythrocytes (Scheffer et al., 2011). These and other studies challenge whether 30-nm 71 fibers is the best model of chromatin within somatic cells (Hansen, 2012;; Nishino et al., 72 2012;; van Holde and Zlatanova, 1995).73 5 The different conclusions between in vitro and in vivo chromatin studies raise the 74 question: can the chromatin of cells that do not show evidence of 30-nm fibers in vivo 75 be made to adopt a 30-nm-fiber structure in vitro? To address this question, we used 76 cryo-ET to study the 3-D organization of undigested natural chromatin from 77 picoplankton and yeast. In the presence of even traces of divalent cations, most of the 78 chromatin in both organisms compacts into large masses. Some of the chromatin does 79 form 30-nm fibers, predominantly of the irregular variety. In the absence of divalent 80 cations, picoplankton chromatin decondenses into open zigzags, but yeast chromatin 81 remains either in a mass or folded as irregular 30-nm fibers. To better understand how 82these nucleosomes interact, we classified the chromatin in both 2-D and 3-D, and found 83 that there is no dominant higher-order packing motif, but the orientation of the DNA at 84 the core-particle entry/exit points is not too variable. Therefore, the higher-order 85 structure of natural chromatin is sensitive to environmental conditions, but this 86 sensitivity varies by species. 87 6 RESULTS 88 89Release of natural picoplankton chromatin 90 To study the higher-order structure of natural picoplankton chromatin in vitro, we 91 lysed cells in hypotonic buffer on ice either with or without divalent cations (Fig. 1B). 92This treatment released all cellular contents, including the chromatin, and is expected to 93 produce thinner plunge-frozen samples that generate higher-contrast cryotomograms. 94Compared to intact cells (Fig. 1C), the lysed cells' contents spread over a much larger 95 area and indeed allowed the ice to be thinner, resulting in higher-contrast tomograms 96 (Fig. 1D). The majority of the densities came from remnants of...

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Section: Resultsmentioning

confidence: 99%

“…(H and I) Histograms of nearestth nearest-neighbor distances, respectively. The in vivo measurements were ing a cryotomogram of sectioned yeast we previously reported (Chen et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 89%

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Natural chromatin is heterogeneous and self associatesin vitro

Cai

Song

Chen

et al. 2017

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“…Internal friction is associated to an onset in the effective drag coefficient due to intramolecular conformational barriers (43). Intrachromosomal contacts associated to chromatin loops, such as those documented by 3C techniques (44) or by cryo-EM technologies (8,45), could represent such internal barriers. The RIF model was tested in silico using our kinetic Monte Carlo simulations of coarse-grained homopolymer chains with internal association (46).…”

Section: Transient Chromatin Contacts Play An Essential Role In Chrommentioning

confidence: 99%

In vivo, chromatin is a fluctuating polymer chain at equilibrium constrained by internal friction

Socol

Wang

Jost

et al. 2017

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Chromosome mechanical properties determine DNA folding and dynamics, and underlie all major nuclear functions. Here we combine modeling and real-time motion tracking experiments to infer the physical parameters describing chromatin fibers. In vitro, motion of nucleosome arrays can be accurately modeled by assuming a Kuhn length of 35-55 nm. In vivo, the amplitude of chromosome fluctuations is drastically reduced, and depends on transcription. Transcription activation increases chromatin dynamics only if it involves gene relocalization, while global transcriptional inhibition augments the fluctuations, yet without relocalization. Chromatin fiber motion is accounted for by a model of equilibrium fluctuations of a polymer chain, in which random contacts along the chromosome contour induce an excess of internal friction. Simulations that reproduce chromosome conformation capture and imaging data corroborate this hypothesis. This model unravels the transient nature of chromosome contacts, characterized by a life time of ~2 seconds and a free energy of formation of ~1 k B T.. CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/192765 doi: bioRxiv preprint first posted online Sep. 24, 2017; Physics of chromosome folding drives and responds to all genomic transactions. In cycling budding yeast cells, large-scale organization of chromosomes in a Rabl-like conformation has been established by imaging and molecular biology approaches (1-4). Yet, the structure of the chromatin fiber at smaller length scales remains more controversial (5). For instance, the recurrent detection of irregular 10-nm fibers by cryo-TEM of thin nuclear sections is questioning the relevance of solenoid or helicoid models of nucleosome arrays (6)(7)(8). This problem has not been clarified by probing the motion of chromosomes in vivo, although dynamic measurements offer a unique opportunity to infer structural properties of genome organization (9). Indeed, we and others have shown that chromosome dynamics in yeast is characterized by sub-diffusive behavior detected by a non-linear temporal variation of the mean square displacement (MSD) of chromosome loci (10-16):( ) = Γwith , , and t the anomaly exponent, amplitude, and time interval, respectively. A subdiffusive response was detected over a broad temporal time scale covering four time decades with an anomaly exponent in the range 0.4-0.6. This response appeared to be consistent with the Rouse model, a generic polymer model that describes chromosomes as a series of beads connected by elastic springs. The length of the springs is related to the mechanical properties of chromatin fiber, namely equal to twice its persistence length (hereafter denoted as the Kuhn length b). The link between the flexibility and the amplitude of the MSD (17) suggested that chromosomes are highly flexible in yeast with b of 1-5 nm (10). Inconsistent with structural and mechanical mode...

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“…Additionally, a further series of experiments suggests that nucleosomes are highly interdigitated and do not form regular 30 nm fibres but irregular folded structures. These findings are best described by a polymer melt model (Joti et al 2012;Chen et al 2016a;Eltsov et al 2008;Fussner et al 2012;Hsieh et al 2015;Nozaki et al 2017;Ou et al 2017). However, alternative higher-order structures, which are incompatible with the polymer melt model, have been described for metaphase and interphase chromosomes.…”

Section: Introductionmentioning

confidence: 98%

Characterizing higher order structures of chromatin in human cells

Schwartz

Németh

Diermeier

et al. 2018

Preprint

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Packaging of DNA into chromatin regulates DNA accessibility and, consequently, all DNA-dependent processes, such as transcription, recombination, repair, and replication. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This defined organization of chromatin has been recently questioned using microscopy techniques, proposing a rather irregular structure. To gain more insight into the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. Thus, we assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions by utilizing statistical data analyses and computer modelling. We found no difference in the degree of compaction between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially compacted domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

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Budding yeast chromatin is dispersed in a crowded nucleoplasmin vivo

Cited by 18 publications

References 78 publications

Natural chromatin is heterogeneous and self associatesin vitro

Natural chromatin is heterogeneous and self associatesin vitro

In vivo, chromatin is a fluctuating polymer chain at equilibrium constrained by internal friction

Characterizing higher order structures of chromatin in human cells

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