Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer

Chromatin remodelers are sophisticated nano-machines that are able to alter histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the histone octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique “one pot assays” for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.

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Generation of Remosomes by the SWI/SNF Chromatin Remodeler Family

Scientific Reports ◽

10.1038/s41598-019-50572-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Manu Shubhdarshan Shukla ◽

Sajad Hussain Syed ◽

Ramachandran Boopathi ◽

Elsa Ben Simon ◽

Sunil Nahata ◽

...

Keyword(s):

Dna Repair ◽

High Resolution ◽

Restriction Enzymes ◽

Dna Interactions ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Dna Accessibility ◽

Nucleosomal Dna ◽

Dnase I Footprinting ◽

High Resolution Microscopy

Abstract Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed “remosomes”. Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.

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Nucleosome Remodeling by the Human SWI/SNF Complex Requires Transient Global Disruption of Histone-DNA Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3653-3662.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3653-3662 ◽

Cited By ~ 36

Author(s):

Sayura Aoyagi ◽

Geeta Narlikar ◽

Chunyang Zheng ◽

Saïd Sif ◽

Robert E. Kingston ◽

...

Keyword(s):

Restriction Enzyme ◽

Atp Hydrolysis ◽

Dnase I ◽

Cross Linking ◽

Dna Interactions ◽

Nucleosome Remodeling ◽

Histone Octamer ◽

Nucleosomal Dna ◽

Single Cross ◽

A Site

ABSTRACT We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.

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Dinucleosome specificity and allosteric switch of the ISW1a ATP-dependent chromatin remodeler in transcription regulation

Nature Communications ◽

10.1038/s41467-020-19700-1 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Saurabh K. Bhardwaj ◽

Solomon G. Hailu ◽

Lola Olufemi ◽

Sandipan Brahma ◽

Soumyadipta Kundu ◽

...

Keyword(s):

Transcription Regulation ◽

Histone Acetyltransferase ◽

Mutational Analysis ◽

Higher Order ◽

Chromatin Organization ◽

Atpase Domain ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Nucleosomal Dna ◽

Accessory Subunit

AbstractOver the last 3 decades ATP-dependent chromatin remodelers have been thought to recognize chromatin at the level of single nucleosomes rather than higher-order organization of more than one nucleosome. We show the yeast ISW1a remodeler has such higher-order structural specificity, as manifested by large allosteric changes that activate the nucleosome remodeling and spacing activities of ISW1a when bound to dinucleosomes. Although the ATPase domain of Isw1 docks at the SHL2 position when ISW1a is bound to either mono- or di-nucleosomes, there are major differences in the interactions of the catalytic subunit Isw1 with the acidic pocket of nucleosomes and the accessory subunit Ioc3 with nucleosomal DNA. By mutational analysis and uncoupling of ISW1a’s dinucleosome specificity, we find that dinucleosome recognition is required by ISW1a for proper chromatin organization at promoters; as well as transcription regulation in combination with the histone acetyltransferase NuA4 and histone H2A.Z exchanger SWR1.

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Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

10.1101/376806 ◽

2018 ◽

Author(s):

Jaewon Kirk ◽

Ju Yeon Lee ◽

Yejin Lee ◽

Chanshin Kang ◽

Soochul Shin ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Chromatin Remodeler ◽

Base Pairs ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Histone Octamer ◽

Single Molecule Fret ◽

Single Molecule Studies ◽

Dna Unwrapping

AbstractChromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. Here, we employ a single-molecule fluorescence approach to characterize nucleosome remodeling by yeast CHD1 (Chd1p). We show that Chd1p translocates nucleosomes in steps of multiple base pairs per ATP. ATP binding to Chd1p induces a transient unwrapping of the exit-side DNA, and facilitates nucleosome translocation. ATP hydrolysis induces nucleosome translocation, which is followed by the rewrapping upon the release of the hydrolyzed nucleotide. Multiple Chd1ps binding to a single nucleosome sequentially moves a histone octamer with a preference to the center of DNA fragments, suggesting a new mechanism for regularly spaced nucleosome generation by Chd1p. Our results reveal the unique mechanism by which Chd1p remodels nucleosomes.Significance StatementThere are four major ATP-dependent chromatin remodeler families: SWI/SNF, ISWI, CHD, and INO80/SWR1. The remodeling mechanisms of SWI/SNF and ISWI chromatin remodelers have been elucidated through extensive single-molecule studies, but it remains poorly understood how CHD chromatin remodeler operate. We use single-molecule FRET techniques, and show that Yeast CHD1 uses unique mechanisms to remodel a nucleosome.

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Chromatin remodelers couple inchworm motion with twist-defect formation to slide nucleosomal DNA

10.1101/297762 ◽

2018 ◽

Author(s):

Giovanni B. Brandani ◽

Shoji Takada

Keyword(s):

Genome Organization ◽

Electrostatic Interactions ◽

Defect Formation ◽

Energy Landscape ◽

Molecular Machines ◽

Biological Processes ◽

Complete Understanding ◽

Chromatin Remodelers ◽

Nucleosomal Dna ◽

Dynamics Simulations

ABSTRACTATP-dependent chromatin remodelers are molecular machines that control genome organization by repositioning, ejecting, or editing nucleosomes, activities that confer them essential regulatory roles on gene expression and DNA replication. Here, we investigate the molecular mechanism of active nucleosome sliding by means of molecular dynamics simulations of the Snf2 remodeler in complex with a nucleosome. During its inchworm motion driven by ATP consumption, the remodeler overwrites the original nucleosome energy landscape via steric and electrostatic interactions to induce sliding of nucleosomal DNA unidirectionally. The sliding is initiated at the remodeler binding location via the generation of twist defects, which then spontaneously propagate to complete sliding throughout the entire nucleosome. We also reveal how remodeler mutations and DNA sequence control active nucleosome repositioning, explaining several past experimental observations. These results offer a detailed mechanistic picture of remodeling important for the complete understanding of these important biological processes.

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5-Phenyl-10,15,20-Tris(4-sulfonatophenyl)porphyrin: Synthesis, Catalysis, and Structural Studies

Molecules ◽

10.3390/molecules23123363 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3363 ◽

Cited By ~ 3

Author(s):

Aitor Arlegui ◽

Zoubir El-Hachemi ◽

Joaquim Crusats ◽

Albert Moyano

Keyword(s):

Alder Reaction ◽

Water Soluble ◽

Experimental Conditions ◽

One Pot ◽

Force Microscopy ◽

Diels Alder Reaction ◽

Transmission Electron ◽

Porphyrin Synthesis ◽

The One ◽

Water Soluble Porphyrin

A convenient protocol for the preparation of 5-phenyl-10,15,20-tris(4-sulfonatophenyl)porphyrin, a water-soluble porphyrin with unique aggregation properties, is described. The procedure relies on the one-pot reductive deamination of 5-(4-aminophenyl)-10,15,20-tris(4-sulfonatophenyl)porphyrin, that can be in turn easily obtained from 5,10,15,20-tetraphenylporphyrin by a known three-step sequence involving mononitration, nitro to amine reduction and sulfonation of the phenyl groups. This method provides the title porphyrin in gram scale, and compares very favorably with the up to now only described procedure based on the partial sulfonation of TPP, that involves a long and tedious chromatographic enrichment of the final compound. This has allowed us to study for the first time both the use of its zwitterionic aggregate as a supramolecular catalyst of the aqueous Diels–Alder reaction, and the morphology of the aggregates obtained under optimized experimental conditions by atomic force microscopy and also by transmission electron cryomicroscopy.

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Poly(dA-dT) Promoter Elements Increase the Equilibrium Accessibility of Nucleosomal DNA Target Sites

Molecular and Cellular Biology ◽

10.1128/mcb.21.11.3830-3839.2001 ◽

2001 ◽

Vol 21 (11) ◽

pp. 3830-3839 ◽

Cited By ~ 102

Author(s):

J. D. Anderson ◽

J. Widom

Keyword(s):

Free Energy ◽

Promoter Region ◽

Physiological Role ◽

Dna Interactions ◽

Promoter Elements ◽

Polypurine Tract ◽

Nucleosomal Dna ◽

Target Sites ◽

The Stability

ABSTRACT Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A16 element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 ± 0.03 and 0.35 ± 0.04 kcal mol−1, respectively, and is accompanied by 1.5- ± 0.1-fold and 1.7- ± 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A16 elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.

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Models for chromatin remodeling: a critical comparison

Biochemistry and Cell Biology ◽

10.1139/o03-038 ◽

2003 ◽

Vol 81 (3) ◽

pp. 169-172 ◽

Cited By ~ 24

Author(s):

K van Holde ◽

T Yager

Keyword(s):

Diffusion Model ◽

Chromatin Remodeling ◽

Experimental Studies ◽

Nucleosome Remodeling ◽

Defect Generation ◽

Critical Comparison ◽

Nucleosomal Dna ◽

Reptation Model ◽

Cast Doubt ◽

Random Diffusion

Nucleosome remodeling has been shown, in many cases, to involve cis displacement of nucleosomes on the DNA. This process seems similar to the long-recognized random diffusion of nucleosomes along DNA, but the remodeling process is unidirectional and ATP dependent. Several years ago, we developed a model for nucleosome migration, based on the diffusion of "twist-defects" within the nucleosomal DNA. This has been modified into a model that incorporates ATP-dependent defect generation, and can account for many observations concerning remodeling. However, certain experimental studies in recent years have cast doubt on the applicability of the twist-diffusion model for remodeling, and seem to favor instead a "reptation" model. We discuss herein these problems and propose a resolution.Key words: nucleosome, remodeling, chromatin.

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