Dynamic 1D search and processive nucleosome translocations by RSC and ISW2 chromatin remodelers
- Department of Biology, Johns Hopkins University, United States
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Howard Hughes Medical Institute, United States
- Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, United States
Figures
Figure 1 with 2 supplements
RSC and ISW2 diffusion on lambda DNA and impacts of ionic strength.
(A) Schematic: RSC and ISW2 bind nucleosome-free DNA at yeast promoters. (B) Aligned trajectories for RSC, ISW2, and dCas9. (C) Rolling-window analysis assigns diffusion coefficients (Dcoef) and …
Figure 1—figure supplement 1
RSC and ISW2 diffusion analysis under varied conditions.
(A) Schematic of the microfluidics chamber used, with four solution compartments (1–3 separated by laminar flow, 4 as reservoir). (B) Construct map displaying RSC and ISW2 labeling via 3x-FLAG tag …
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Figure 1—figure supplement 1—source data 1
Original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified RSC complex.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data1-v1.pdf
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Figure 1—figure supplement 1—source data 2
Original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified RSC complex with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data2-v1.pdf
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Figure 1—figure supplement 1—source data 3
Red channel fluorescence scan of original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified JFX650-labeled RSC complex.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data3-v1.pdf
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Figure 1—figure supplement 1—source data 4
Red channel fluorescence scan of original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified JFX650-labeled RSC complex with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data4-v1.pdf
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Figure 1—figure supplement 1—source data 5
Original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified ISW2 complex.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data5-v1.pdf
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Figure 1—figure supplement 1—source data 6
Original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified ISW2 complex with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data6-v1.pdf
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Figure 1—figure supplement 1—source data 7
Red channel fluorescence scan of original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified JFX650-labeled ISW2 complex.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data7-v1.pdf
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Figure 1—figure supplement 1—source data 8
Red channel fluorescence scan of original sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel showing purified JFX650-labeled ISW2 complex with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp1-data8-v1.pdf
Figure 1—figure supplement 2
Halo-tagged remodeler functional validation, labeling, and purification.
Native PAGE analysis of nucleosome sliding by HaloTagged RSC (A) and ISW2 (B). RSC shifts centered nucleosomes (43N43; Cy5-DNA) to end positions, while ISW2 moves end-positioned nucleosomes (80N3; …
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Figure 1—figure supplement 2—source data 1
Original native PAGE analysis of nucleosome sliding by HaloTagged RSC.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original native PAGE analysis of nucleosome sliding by HaloTagged RSC with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
Original native PAGE analysis of nucleosome sliding by HaloTagged ISW2.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data3-v1.zip
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Figure 1—figure supplement 2—source data 4
Original native PAGE analysis of nucleosome sliding by HaloTagged ISW2 with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data4-v1.zip
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Figure 1—figure supplement 2—source data 5
Original red channel fluorescence scan of a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX650-Halo-RSC used for quantifying Halo-RSC labeling efficiency.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data5-v1.pdf
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Figure 1—figure supplement 2—source data 6
Original red channel fluorescence scan of a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX650-Halo-RSC used for quantifying Halo-RSC labeling efficiency with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data6-v1.pdf
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Figure 1—figure supplement 2—source data 7
Original green channel fluorescence scan of a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX554-Halo-RSC used for quantifying Halo-RSC labeling efficiency.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data7-v1.pdf
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Figure 1—figure supplement 2—source data 8
Original green channel fluorescence scan of a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX554-Halo-RSC used for quantifying Halo-RSC labeling efficiency with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data8-v1.pdf
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Figure 1—figure supplement 2—source data 9
Original image of protein staining of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX 650 and JFX 554-Halo-RSC used for quantifying Halo-RSC labeling efficiency.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data9-v1.pdf
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Figure 1—figure supplement 2—source data 10
Original image of protein staining of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of JFX 650 and JFX 554-Halo-RSC used for quantifying Halo-RSC labeling efficiency with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data10-v1.pdf
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Figure 1—figure supplement 2—source data 11
Original image of flamingo stained SDS–PAGE gel of fractions from glycerol gradient purification for the RSC-JFX650 preparation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data11-v1.pdf
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Figure 1—figure supplement 2—source data 12
Original image of flamingo stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the RSC-JFX650 preparation with annotations.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data12-v1.pdf
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Figure 1—figure supplement 2—source data 13
Original red channel image of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the RSC-JFX650 preparation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data13-v1.pdf
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Figure 1—figure supplement 2—source data 14
Original red channel image of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the RSC-JFX650 preparation with annotations.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data14-v1.pdf
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Figure 1—figure supplement 2—source data 15
Original image of flamingo stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the ISW2-JFX650 preparation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data15-v1.pdf
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Figure 1—figure supplement 2—source data 16
Original image of flamingo stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the ISW2-JFX650 preparation with annotations.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data16-v1.pdf
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Figure 1—figure supplement 2—source data 17
Original red channel image of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the ISW2-JFX650 preparation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data17-v1.pdf
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Figure 1—figure supplement 2—source data 18
Original red channel image of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel of fractions from glycerol gradient purification for the ISW2-JFX650 preparation with annotations.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig1-figsupp2-data18-v1.pdf
Figure 2 with 1 supplement
Bimolecular remodeler–remodeler interactions during one-dimensional (1D) encounters on DNA.
Two-color kymographs of RSC-JFX554 and ISW2-JFX650 (A) or RSC-JFX554 and RSC-JFX650 (B) diffusing together on naked DNA. Three types of encounter events are observed: short colocalizations (C), long …
Figure 2—figure supplement 1
Detection of remodeler–remodeler interactions and dwell-time estimation.
(A) Simulated images illustrating diffusive molecules and camera noise, with distinct molecule types shown in different colors. (B) Single-molecule trajectories obtained from the analysis of images …
Figure 3 with 2 supplements
Nucleosomes constrain one-dimensional (1D) diffusion of RSC and ISW2.
(A) Optical tweezers and confocal microscopy assay for measuring remodeler diffusion on lambda DNA nucleosome arrays. (B) Force distance plots are used to assess the number of nucleosomes on arrays, …
Figure 3—figure supplement 1
Remodeler–nucleosome interactions and nucleosome sliding assays.
(A) Electrophoretic mobility shift assay (EMSA) of lambda nucleosome arrays with increasing octamer concentration. The asterisk indicates a condition that produced ~10 nucleosomes/array. (B) …
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Figure 3—figure supplement 1—source data 1
Original DNA scan of electrophoretic mobility shift assay of lambda nucleosome arrays with increasing octamer concentration.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Original DNA scan of electrophoretic mobility shift assay of lambda nucleosome arrays with increasing octamer concentration with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig3-figsupp1-data2-v1.pdf
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Figure 3—figure supplement 1—source data 3
Original green channel scan of electrophoretic mobility shift assay of lambda nucleosome arrays with increasing octamer concentration.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig3-figsupp1-data3-v1.zip
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Figure 3—figure supplement 1—source data 4
Original green channel scan of electrophoretic mobility shift assay of lambda nucleosome arrays with increasing octamer concentration with annotation.
- https://cdn.elifesciences.org/articles/91433/elife-91433-fig3-figsupp1-data4-v1.zip
Figure 3—figure supplement 2
Nucleosome translocation durations and half-lives.
1-Cummulative distribution function (CDF) survival curves of nucleosome translocation events for (A) RSC and (B) ISW2 in the presence of 1 mM ATP. Half-lives from single-exponential fitting printed …
Figure 4
ATP-dependent processive nucleosome translocation by RSC and ISW2.
Kymographs of RSC (A) and ISW2 (B) showing static colocalization in 1 mM ATPγS. Kymographs of RSC (C) and ISW2 (D) moving directionally with nucleosome signals in 1 mM ATP. The green laser is either …
Figure 5 with 1 supplement
Translocation direction relative to one-dimensional (1D) diffusion on linker DNA supports RSC and ISW2 ‘push–pull’ models.
Trace fragmentation plots illustrate translocating regardless of changes in speed or changes in direction (blue), non-translocating (red), and free diffusion (green) segments for RSC (A) and ISW2 (B)…
Figure 5—figure supplement 1
Various translocation scenarios support the push–pull model for RSC–ISW2.
Pie charts of ‘pushing’ and ‘pulling’ counts for RSC (A) and ISW2 (B); various translocation scenarios are displayed separately within the pie chart. The identities of the wedges are indicated with …
Tables
Table 1
crRNA sequences for dCas9-binding oligonucleotide sequences used for lambda DNA preparation.
| Identity | Sequence |
|---|---|
| Cas9 crRNA sequence ‘lambda 1’ | 5′-/ AltR 1/rGrUrG rArUrA rArGrU rGrGrA rArUrG rCrCrA rUrGrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / AltR2/-3′ |
| Cas9 crRNA sequence ‘lambda 2’ | 5′-/ AltR 1/rCrUrG rGrUrG rArArC rUrUrC rCrGrA rUrArG rUrGrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / AltR2/-3′ |
| Cas9 crRNA sequence ‘lambda 3’ | 5′-/AltRl /rCrArG rArUrA rUrArG rCrCrU rGrGrU rGrGrU rUrCrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / AltR2/-3′ |
| 3x-biotin-cos1 oligo | 5′-/5Phos/ AGG TCG CCG CCC TT/iBiodT/TT/iBiodT/TT/3BiodT/-3′ |
| 3x-digoxigenin-cos2 oligo | 5′-/5Phos/ GGG CGG CGA CCT TT/iDigN/TT/iDigN/TT/3DigN/-3′ |
| Adapter oligo for lambda DNA dual end biotin labeling | 5′-/5Phos/ GGG CGG CGA CCT TGC A-3′ |
