ATP binding facilitates target search of SWR1 chromatin remodeler by promoting one-dimensional diffusion on DNA
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Evaluation Summary:
Using biophysical measurements, this work establishes that the SWR1 chromatin remodeling complex, which directs incorporation of the histone variant H2A.Z adjacent to nucleosome depleted regions, preferentially associates with longer DNA fragments. It is proposed that larger stretches of free DNA determine the specificity of the complex in vivo. These findings will be of general interest to researchers interested in understanding how chromatin remodelling enzymes act to influence the localisation of histone variants.
(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, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
One-dimensional (1D) target search is a well-characterized phenomenon for many DNA-binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a conserved yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome-depleted region (NDR) at gene promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single-particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA-binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 μm 2 /s, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cellular chromatin.
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Evaluation Summary:
Using biophysical measurements, this work establishes that the SWR1 chromatin remodeling complex, which directs incorporation of the histone variant H2A.Z adjacent to nucleosome depleted regions, preferentially associates with longer DNA fragments. It is proposed that larger stretches of free DNA determine the specificity of the complex in vivo. These findings will be of general interest to researchers interested in understanding how chromatin remodelling enzymes act to influence the localisation of histone variants.
(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, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This is a nice biophysical study to show that the SWR1 complex can diffuse on DNA in one dimension and is blocked by nucleosomes. Diffusion is stimulated by ATP but does not require ATP hydrolysis distinguishing SWR1 movement from that of other chromatin remodelers.
The work is well done and convincing. It describes a relatively small aspect of SWR1 function, somewhat limiting the broader impact of the study.
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Reviewer #2 (Public Review):
This manuscript provides exciting and timely new insight into the target search mechanism of SWR1, with fundamentally important implications for other remodelers. The authors use extremely elegant single-molecule approaches (TIRF-based fluorescence microscopy and optical trapping in combination with confocal microscopy) to study the interaction of SWR1 with DNA. This study provides the first conclusive demonstration that SWR1 undergoes 1D diffusion along DNA, which plays an important role in finding the correct nucleosomal substrate in vivo by guiding SWR1 molecules that bind to nucleosome-depleted regions towards flanking nucleosomes.
The authors demonstrate that the Swc2 DNA-binding domain of SWR1 alone can diffuse along DNA, indicating an important role in the target search mechanism. Interestingly, a …
Reviewer #2 (Public Review):
This manuscript provides exciting and timely new insight into the target search mechanism of SWR1, with fundamentally important implications for other remodelers. The authors use extremely elegant single-molecule approaches (TIRF-based fluorescence microscopy and optical trapping in combination with confocal microscopy) to study the interaction of SWR1 with DNA. This study provides the first conclusive demonstration that SWR1 undergoes 1D diffusion along DNA, which plays an important role in finding the correct nucleosomal substrate in vivo by guiding SWR1 molecules that bind to nucleosome-depleted regions towards flanking nucleosomes.
The authors demonstrate that the Swc2 DNA-binding domain of SWR1 alone can diffuse along DNA, indicating an important role in the target search mechanism. Interestingly, a significant effect of the nucleotide state on the diffusion of the complete SWR1 complex suggests a key role of the helicase subunit Swr1 in this process, which presents an exciting target for future research.
The authors have also analyzed the effects of Cas9 barriers and nucleosomes on the SWR1 diffusion along DNA. The fact that crossing the Cas9 barrier was observed very rarely argues in favor of the sliding mechanism for SWR1 diffusion. However, the diffusion coefficient of SWR1 significantly increases with increasing monovalent salt concentration, which is a hallmark of hopping-mediated diffusion. For that reason, SWR1 diffusion likely occurs through a combination of hopping and sliding.
SWR1 diffusion seems to be substantially restricted in the presence of nucleosomes, but the interpretation of the data is limited by the fact that (unlike the Cas9 case) the authors used unlabeled nucleosomes. Nevertheless, these data open up exciting new avenues for future research.
The manuscript is very well written, methodologically impressive, and the data are presented in a clear and efficient manner. The conclusions are strongly supported by the data and are of outstanding interest to the field of chromating biology. This manuscript will undoubtedly spark future research into the target search mechanisms of SWR1 and other remodelers and chromatin-associated proteins.
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Reviewer #3 (Public Review):
In this manuscript the authors set out to determine how the SWR1 chromatin remodelling complex interacts with DNA. This was achieved by monitoring the diffusive motion of individual fluorescently labelled complexes along DNA.
It was first observed that the rate of association and dissociation of complexes from DNA was affected by the length of the DNA fragment.
It is concluded from measurements of on and off rates that SWR1 is primarily affected by the rate of association. The authors propose that this could underpin a mechanism that facilitates the enrichment of complexes at regions where large expanses of DNA are exposed.
Diffusive motion of SWR1 complexes is found to be modulated by nucleotide binding, but not to require ongoing ATP hydrolysis. This is an interesting observation that may affect the …
Reviewer #3 (Public Review):
In this manuscript the authors set out to determine how the SWR1 chromatin remodelling complex interacts with DNA. This was achieved by monitoring the diffusive motion of individual fluorescently labelled complexes along DNA.
It was first observed that the rate of association and dissociation of complexes from DNA was affected by the length of the DNA fragment.
It is concluded from measurements of on and off rates that SWR1 is primarily affected by the rate of association. The authors propose that this could underpin a mechanism that facilitates the enrichment of complexes at regions where large expanses of DNA are exposed.
Diffusive motion of SWR1 complexes is found to be modulated by nucleotide binding, but not to require ongoing ATP hydrolysis. This is an interesting observation that may affect the distribution of other chromatin remodelling enzymes.
The diffusive motion of SWR1 complexes is observed to be limited by the presence of bound DNA factors including Cas9 bound to DNA and nucleosomes. The authors propose that the relatively long region of DNA adjacent to nucleosomes at transcriptional start sites contributes to the targeting the activity of SWR1 complexes to +1 nucleosomes.
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