Yeast Chd1p remodels nucleosomes with unique DNA unwrapping and translocation dynamics

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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CHD7 interacts with the nucleosome acidic patch for its efficient activity via its N-terminal region

10.1101/2020.11.19.389429 ◽

2020 ◽

Author(s):

Eunhye Lee ◽

Chanshin Kang ◽

Pasi Purhonen ◽

Hans Hebert ◽

Karim Bouazoune ◽

...

Keyword(s):

Single Molecule ◽

Developmental Disorders ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Charge Syndrome ◽

Terminal Region ◽

Open Chromatin ◽

Chromatin Remodeler ◽

Nucleosome Remodeling ◽

Chromatin Remodelers

AbstractChromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed integrative biophysical analysis on CHD7 chromatin remodeler using crosslinking-mass spectrometry (XL-MS), cryo-Electron Microscopy (cryo-EM) and single-molecule Förster Resonance Energy Transfer (smFRET). We uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. The cryo-EM analysis on the N-CRD_Chromodomain bound to nucleosome reveals that the N-CRD interacts with the acidic patch of nucleosome. Furthermore, smFRET analysis shows the mutations in the N-CRD result in slow or highly-fluctuating remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

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Quantitative structural information from single-molecule FRET

Faraday Discussions ◽

10.1039/c5fd00110b ◽

2015 ◽

Vol 184 ◽

pp. 117-129 ◽

Cited By ~ 15

Author(s):

M. Beckers ◽

F. Drechsler ◽

T. Eilert ◽

J. Nagy ◽

J. Michaelis

Keyword(s):

Single Molecule ◽

Structural Information ◽

Monte Carlo Sampling ◽

Dye Molecules ◽

Distance Information ◽

Single Molecule Fret ◽

Single Molecule Studies ◽

Archaeal Transcription ◽

Short Time ◽

Fluorescence Energy

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

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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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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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Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

eLife ◽

10.7554/elife.69387 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jee Min Kim ◽

Pat Visanpattanasin ◽

Vivian Jou ◽

Sheng Liu ◽

Xiaona Tang ◽

...

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Dissociation Kinetics ◽

Additional Function ◽

Fast Kinetics ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Genome Wide ◽

Regulatory Dna ◽

Temporal Interactions

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending ‘tug-of-war’ that controls a temporally shifting window of accessibility for the transcription initiation machinery.

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Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

10.1101/207886 ◽

2017 ◽

Cited By ~ 1

Author(s):

Luuk Loeff ◽

Stan J.J. Brouns ◽

Chirlmin Joo

Keyword(s):

Single Molecule ◽

Adaptive Immune System ◽

Dna Unwinding ◽

Type I ◽

Base Pairs ◽

Spatiotemporal Resolution ◽

Adaptive Immune ◽

Single Molecule Fret ◽

Target Dna ◽

Loaded Mechanism

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

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High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation

Physical Chemistry Chemical Physics ◽

10.1039/d0cp01921f ◽

2020 ◽

Vol 22 (28) ◽

pp. 15853-15866 ◽

Cited By ~ 1

Author(s):

Hsuan-Lei Sung ◽

David J. Nesbitt

Keyword(s):

High Pressure ◽

Single Molecule ◽

Pressure Effects ◽

Protective Mechanisms ◽

Single Molecule Fret ◽

Single Molecule Studies

Protective mechanisms of the piezolyte trimethylamine N-oxide counteracting the pressure effects are revealed by single molecule studies at extreme pressures.

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Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

Chemical Science ◽

10.1039/d0sc06134d ◽

2021 ◽

Author(s):

Steffen Wolf ◽

Benedikt Sohmen ◽

Björn Hellenkamp ◽

Johann Thurn ◽

Gerhard Stock ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Single Molecule ◽

Atp Hydrolysis ◽

Md Simulations ◽

Large Protein ◽

Single Molecule Fret ◽

Fluorescence Methods ◽

Dynamics Simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.

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Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

10.1101/2021.04.21.440742 ◽

2021 ◽

Author(s):

Jee Min Kim ◽

Pat Visanpattanasin ◽

Vivian Jou ◽

Sheng Liu ◽

Xiaona Tang ◽

...

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Dissociation Kinetics ◽

Additional Function ◽

Fast Kinetics ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Genome Wide ◽

Regulatory Dna ◽

Temporal Interactions

ABSTRACTConserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending ‘tug-of-war’ that controls a temporally shifting window of accessibility for the transcription initiation machinery.

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Stepwise nucleosome translocation by RSC remodeling complexes

eLife ◽

10.7554/elife.10051 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 36

Author(s):

Bryan T Harada ◽

William L Hwang ◽

Sebastian Deindl ◽

Nilanjana Chatterjee ◽

Blaine Bartholomew ◽

...

Keyword(s):

Free Energy ◽

Atpase Activity ◽

Binding Site ◽

Single Molecule ◽

Chromatin Structure ◽

Atp Hydrolysis ◽

Size Distributions ◽

Dna Translocation ◽

Step Size ◽

Single Molecule Fret

The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1–2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

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