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

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0904497107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 1936-1941 ◽

Cited By ~ 35

Author(s):

Manu Shubhdarshan Shukla ◽

Sajad Hussain Syed ◽

Fabien Montel ◽

Cendrine Faivre-Moskalenko ◽

Jan Bednar ◽

...

Keyword(s):

Restriction Enzymes ◽

Dna Interactions ◽

One Pot ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Force Microscopy ◽

Histone Octamer ◽

Distinct Variable ◽

Nucleosomal Dna ◽

Dnase I Footprinting

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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SWI-SNF-Mediated Nucleosome Remodeling: Role of Histone Octamer Mobility in the Persistence of the Remodeled State

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3058-3068.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3058-3068 ◽

Cited By ~ 73

Author(s):

Mariela Jaskelioff ◽

Igor M. Gavin ◽

Craig L. Peterson ◽

Colin Logie

Keyword(s):

Restriction Enzyme ◽

Enzyme Assay ◽

Atp Hydrolysis ◽

Dynamic State ◽

Nucleosome Remodeling ◽

Histone Octamer ◽

Nucleosomal Array ◽

Histone Octamers ◽

Dna Ends

ABSTRACT SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling–restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.

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G-actin conformational change and polymerization induced by paraquat

Biochemistry and Cell Biology ◽

10.1139/o98-019 ◽

1998 ◽

Vol 76 (4) ◽

pp. 583-591 ◽

Cited By ~ 3

Author(s):

Isabella DalleDonne ◽

Aldo Milzani ◽

Roberto Colombo

Keyword(s):

Conformational Changes ◽

Mammalian Cells ◽

Structural Changes ◽

Cell Injury ◽

Atp Hydrolysis ◽

Protein Composition ◽

Dnase I ◽

Cross Linking ◽

Cytoskeletal Protein ◽

Actin Assembly

Paraquat (1,1´-dimethyl-4,4´-bipyridilium dichloride) is a broad-spectrum herbicide that is highly toxic to animals (including man), the major lesion being in the lung. In mammalian cells, paraquat causes deep alterations in the organization of the cytoskeleton, marked decreases in cytoskeletal protein synthesis, and alterations in cytoskeletal protein composition; therefore, the involvement of the cytoskeleton in cell injury by paraquat was suggested. We previously demonstrated that monomeric actin binds paraquat; moreover, prolonged actin exposure to paraquat, in depolymerizing medium, induces the formation of actin aggregates, which are built up by F-actin. In this work we have shown that the addition of paraquat to monomeric actin results in a strong quenching of Trp-79 and Trp-86 fluorescence. Trypsin digestion experiments demonstrated that the sequence 61-69 on actin subdomain 2 undergoes paraquat-dependent conformational changes. These paraquat-induced structural changes render actin unable to completely inhibit DNase I. By using intermolecular cross-linking to characterize oligomeric species formed during paraquat-induced actin assembly, we found that the herbicide causes the formation of actin oligomers characterized by subunit-subunit contacts like those occurring in oligomers induced by polymerizing salts (i.e., between subdomain 1 on one actin subunit and subdomain 4 on the adjacent subunit). Furthermore, the oligomerization of G-actin induced by paraquat is paralleled by ATP hydrolysis.Key words: actin, paraquat, subdomain 2, DNase I, ATP hydrolysis.

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Architecture of the SWI/SNF-Nucleosome Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00693-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6010-6021 ◽

Cited By ~ 104

Author(s):

Mekonnen Lemma Dechassa ◽

Bei Zhang ◽

Rachel Horowitz-Scherer ◽

Jim Persinger ◽

Christopher L. Woodcock ◽

...

Keyword(s):

Transcription Factor ◽

Catalytic Subunit ◽

Cross Linking ◽

Dependent Manner ◽

Transcription Activator ◽

Entry Site ◽

Dna Footprinting ◽

Histone Octamer ◽

Nucleosomal Dna ◽

Protein Cross Linking

ABSTRACT The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. SWI/SNF interactions with nucleosomes were mapped by DNA footprinting and site-directed DNA and protein cross-linking when SWI/SNF was recruited by a transcription activator. SWI/SNF was found by DNA footprinting to contact tightly around one gyre of DNA spanning ∼50 bp from the nucleosomal entry site to near the dyad axis. The DNA footprint is consistent with nucleosomes binding to an asymmetric trough of SWI/SNF that was revealed by the improved imaging of free SWI/SNF. The DNA site-directed cross-linking revealed that the catalytic subunit Swi2/Snf2 is associated with nucleosomes two helical turns from the dyad axis and that the Snf6 subunit is proximal to the transcription factor recruiting SWI/SNF. The highly conserved Snf5 subunit associates with the histone octamer and not with nucleosomal DNA. The model of the binding trough of SWI/SNF illustrates how nucleosomal DNA can be mobilized while SWI/SNF remains bound.

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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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High-Resolution Mapping of Changes in Histone-DNA Contacts of Nucleosomes Remodeled by ISW2

Molecular and Cellular Biology ◽

10.1128/mcb.22.21.7524-7534.2002 ◽

2002 ◽

Vol 22 (21) ◽

pp. 7524-7534 ◽

Cited By ~ 84

Author(s):

Stefan R. Kassabov ◽

Nathalia M. Henry ◽

Martin Zofall ◽

Toshio Tsukiyama ◽

Blaine Bartholomew

Keyword(s):

The Novel ◽

Histone Fold ◽

Histone Octamer ◽

Nucleosome Structure ◽

High Resolution Mapping ◽

Nucleosomal Dna ◽

Before And After ◽

Resolution Mapping ◽

A Site ◽

Dna Template

ABSTRACT The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.

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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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CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179993 ◽

1990 ◽

Vol 48 (1) ◽

pp. 248-249

Author(s):

John Trinickt ◽

Howard White

Keyword(s):

Electron Microscopy ◽

Atp Hydrolysis ◽

Myosin Head ◽

Bound State ◽

Cross Linking ◽

Weakly Bound ◽

Cryo Electron Microscopy ◽

Myosin Subfragment 1 ◽

Attached State ◽

High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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