Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin
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Evaluation Summary:
In this study, Kim and co-workers track the dynamics of a large set of different ATP-dependent chromatin remodelers in living cells by utilizing state-of-the-art single-molecule imaging. They report that the remodelers exhibit very high turnover rates at target loci/nucleosomes, find evidence for cooperativity among the remodelers, and reveal the role of ATP hydrolysis in those interactions. These observations allow the authors to put forward a model for tug-of-war activities that modulate the accessibility of promoter regions for transcriptional activity. This manuscript brings important new information to the remodeler and chromatin dynamics field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
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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Evaluation Summary:
In this study, Kim and co-workers track the dynamics of a large set of different ATP-dependent chromatin remodelers in living cells by utilizing state-of-the-art single-molecule imaging. They report that the remodelers exhibit very high turnover rates at target loci/nucleosomes, find evidence for cooperativity among the remodelers, and reveal the role of ATP hydrolysis in those interactions. These observations allow the authors to put forward a model for tug-of-war activities that modulate the accessibility of promoter regions for transcriptional activity. This manuscript brings important new information to the remodeler and chromatin dynamics field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
In this study, Kim and co-workers track the dynamics of a large set of different ATP-dependent chromatin remodelers in living cells by utilizing state-of-the-art single-molecule imaging. They report that the remodelers exhibit very high turnover rates at target loci/nucleosomes, find evidence for cooperativity among the remodelers, and reveal the role of ATP hydrolysis in those interactions. These observations allow the authors to put forward a model for tug-of-war activities that modulate the accessibility of promoter regions for transcriptional activity. This manuscript brings important new information to the remodeler and chromatin dynamics field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Strength: Chromatin remodelers regulate chromatin-templated functions through mobilizing and positioning nucleosomes; however, the molecular mechanisms underlying the binding and target-search remain obscure. Kim et al. utilize the powerful live-cell single-molecule tracking technique to investigate the binding and target-search kinetics of a comprehensive set of ATP-dependent chromatin remodelers. They endogenously tag the catalytic subunits of 6 major chromatin remodeling complexes and find that these remodelers reside at chromatin with 4-7 s mean residence times. Their results indicate these chromatin remodelers frequently transition between bound and free states. By using the remodeler mutants that are defective in binding or hydrolyzing ATP, they uncover that the ATPase activity is critical for …
Reviewer #1 (Public Review):
Strength: Chromatin remodelers regulate chromatin-templated functions through mobilizing and positioning nucleosomes; however, the molecular mechanisms underlying the binding and target-search remain obscure. Kim et al. utilize the powerful live-cell single-molecule tracking technique to investigate the binding and target-search kinetics of a comprehensive set of ATP-dependent chromatin remodelers. They endogenously tag the catalytic subunits of 6 major chromatin remodeling complexes and find that these remodelers reside at chromatin with 4-7 s mean residence times. Their results indicate these chromatin remodelers frequently transition between bound and free states. By using the remodeler mutants that are defective in binding or hydrolyzing ATP, they uncover that the ATPase activity is critical for dissociation of remodelers from chromatin. They reveal that ATP binding rather than ATP hydrolysis facilitates mobility of chromatin-bound fraction of remodelers at chromatin. Finally, they calculate the temporal occupancy of remodelers. Based upon these novel results, they provide a 'tug-of-war' model that explain how remodelers temporally control accessibility of promoters for transcription initiation. These results well support the authors; claims and conclusions and provide novel insights into the dynamic process underlying how remodelers search, locate, and bind target sites.
Weakness: There is no major weaknesses of the manuscript. Re-analysis of some data will strength this manuscript but will not change the claims and conclusions.
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Reviewer #2 (Public Review):
Much progress has been made in the understanding of the mechanisms involved in chromatin remodeling, and recent advances in cryogenic electron-microscopy applied to the field have revealed the structural organization of many remodelers, and the way they engage their nucleosomal substrate. Currently, one of the least explored and documented aspect -and one of the most challenging to address- is the dynamic and the kinetics governing the interaction of remodelers with nucleosomes, as well as the concomitant dynamics of different remodelers at a specific nucleosomal location in vivo. These are interesting questions of broad interest as the vast majority of chromatin remodelers are involved in the regulation of DNA accessibility, particularly in the context of allowing or preventing gene expression and DNA repair.
Reviewer #2 (Public Review):
Much progress has been made in the understanding of the mechanisms involved in chromatin remodeling, and recent advances in cryogenic electron-microscopy applied to the field have revealed the structural organization of many remodelers, and the way they engage their nucleosomal substrate. Currently, one of the least explored and documented aspect -and one of the most challenging to address- is the dynamic and the kinetics governing the interaction of remodelers with nucleosomes, as well as the concomitant dynamics of different remodelers at a specific nucleosomal location in vivo. These are interesting questions of broad interest as the vast majority of chromatin remodelers are involved in the regulation of DNA accessibility, particularly in the context of allowing or preventing gene expression and DNA repair.
In this work by Kim et al., (Dr. Carl Wu lab, and colleagues), the authors address these questions using an elegant and powerful approach, single-molecule tracking (SMT), that allows them to directly visualize and characterize the kinetics of various remodelers in vivo. Overall, the work is well designed, executed and presented. Here, the use of C-terminal Halo tags integrated into the genome at their endogenous locations controls for expression levels and helps ensure that full/normal complexes are being assessed. The technical merit and analysis modes of the work are strong. Another strength is the comparison of the in vivo dynamics and kinetics of a broad set of six remodelers (RSC, SWI/SNF, CHD1, ISW1, ISW2, INO80) representing all families of remodelers that act concomitantly at gene promoters or gene bodies. This allows the authors to provide strong evidence and notable comparisons and reach several significant conclusions:
• All remodelers engage their chromatin target very transiently and at high frequency (~4-7 second time scale).
• All remodelers use the ATPase activity to increase their dissociation rates (as revealed by characterizing the kinetics of Walker B mutants) while ATP binding itself enhances their browsing of nucleosomes (as revealed by characterizing the kinetics of Walker A mutants).
• Multiple, including functionally antagonistic, remodelers repeatedly engage and compete in a tug of war in order to maintain or change nucleosome positioning at promoters.
All these conclusions are well supported by the data presented.
Conceptually, it is noticeable that the in vivo dynamics characterized in this work are conserved between remodelers from different families, which widely vary in their composition, structure and function. This work further consolidates the ongoing theme of unification of all remodelers which engage nucleosomes in a very similar manner to perform highly regulated DNA translocation, and now, as highlighted by this work, while following similar dynamics and kinetics in vivo and capitalizing similarly on the ATP binding and hydrolysis.
The manuscript is well written, references to the context and related work are well chosen and cited, and the data are compelling.
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Reviewer #3 (Public Review):
The paper by Kim et al uses elegant Halo-tag imaging techniques to study the dynamics of a set of 6 nucleosome remodelers in yeast. They model their data to show two populations of slower and faster turnover or movement, and find that remodeler mobility sits appropriately between that of histones and free Halo tag. The authors find that ATP binding (and not necessarily hydrolysis) regulates the rapid movement, as mutations in the Walker A (and to a lesser extent, Walker B motif in CHD1 and ISW2) reduce movement. They suggest that ATP hydrolysis facilitates a rapid movement along the chromatid fiber. Transcriptional elongation is not a source of remodeler movement, on the other hand.
Complementing other data that supports a model of a "tug-of-war" between various types of remodelers at promoters, the authors …
Reviewer #3 (Public Review):
The paper by Kim et al uses elegant Halo-tag imaging techniques to study the dynamics of a set of 6 nucleosome remodelers in yeast. They model their data to show two populations of slower and faster turnover or movement, and find that remodeler mobility sits appropriately between that of histones and free Halo tag. The authors find that ATP binding (and not necessarily hydrolysis) regulates the rapid movement, as mutations in the Walker A (and to a lesser extent, Walker B motif in CHD1 and ISW2) reduce movement. They suggest that ATP hydrolysis facilitates a rapid movement along the chromatid fiber. Transcriptional elongation is not a source of remodeler movement, on the other hand.
Complementing other data that supports a model of a "tug-of-war" between various types of remodelers at promoters, the authors argue for nucleosomal "pushing and pulling" (split among pushers and puller enzyme complexes that turn over rapidly) as a mode for balancing promoter accessibility.
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