High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- University of California, San Diego, United States
- Colorado State University, United States
- University of Pittsburgh, United States
Figures
Figure 1 with 1 supplement
Dual-trap Optical Tweezers Single-molecule Unzipping Assay Unwinds Nucleosomal DNA and Maps Histone-DNA Interactions.
(A) Geometry of the single-molecule unzipping assay. Dashed arrows denote directions of trap movement (20 nm/s) during unzipping (red arrow) or rezipping (black arrow). Two DNA handles connect to …
Figure 1—figure supplement 1
Unzipping Traces of Single Human WT, H2A.Z, M3_M7, uH2B Nucleosomes and Tetrasomes.
(A–E) Representative unzipping traces of WT nucleosomes (A), tetrasomes (B), H2A.Z nucleosomes (C), M3_M7 nucleosomes (D) and uH2B nucleosomes (E). Rezipping traces are not shown and they match bare …
Figure 2 with 1 supplement
Topography Maps of the Nucleosome Revealed by Nucleosome Unzipping at Constant Force.
(A) Representative unzipping traces of bare NPS DNA (black), WT (red), H2A.Z (blue) and uH2B (green) nucleosomes at 28 pN constant force. Unzipped bp are normalized to the beginning of the second …
Figure 2—figure supplement 1
H2A.Z Nucleosomes Assemble More Cooperatively than WT nucleosomes.
(A) Sequence swaps between H2A and H2A.Z reveal important regions for hexasome formation. The native PAGE gel shows the propensity to form hexasomes during assembly of H2A, H2A.Z and swapped mutant …
Figure 3 with 1 supplement
Observation of Multiple Nucleosomal States at the Proximal Dimer Region.
(A) Time traces of number of base pairs unzipped (relative to beginning of the second NPS) with hWT nucleosome for fixed trap separations of 1031 nm, 1045 nm, and 1060 nm (top to bottom). Color …
Figure 3—figure supplement 1
Hopping of the Unzipping Fork Near the Proximal Dimer Region of the Nucleosome.
(A, B) Unzipping traces of human WT nucleosome (A) and bare NPS DNA (B). The slow hopping event near the proximal dimer region of WT nucleosome is indicated with a dashed blue square box; no similar …
Figure 4 with 1 supplement
A ‘Molecular Ruler’ Gauges the Positions of an Elongating Pol II with Near-Basepair Accuracy.
(A) Experimental design of an improved single-molecule nucleosomal transcription assay. A single biotinylated Pol II (purple molecular structure) is tethered between two optical traps. Pol II …
Figure 4—figure supplement 1
Biochemical and Single-molecule Characterization of the ‘Molecular Ruler’.
(A) In vitro transcription assay identifies a major pause site within a single repeat sequence (64 bp). The band corresponding to the pause site is highlighted with a dotted red box. The sequence of …
Figure 5 with 1 supplement
High-resolution Trajectories of Individual Pol II Enzymes Transcribing through WT, H2A.Z and uH2B Nucleosomes.
(A, B) Representative traces of single Pol II enzymes transcribing through single human WT nucleosomes. The gray dotted lines are the pause sites within the ‘molecular ruler’. The inset (black) is …
Figure 5—figure supplement 1
Long-lived Pauses of Pol II in the Nucleosome are Associated with Backtracking and Hopping Dynamics.
(A, B) Representative traces of backtracking (A) and hopping (B) dynamics of Pol II during transcription through an hWT nucleosome. The trace is the same as the red trace in Figure 5A and B. The …
Figure 6 with 1 supplement
Transcriptional Maps of the Nucleosome Reveal that H2A.Z Enhances the Width and uH2B the Height of the Barrier.
(A) Median residence time histograms of Pol II transcription through bare NPS DNA (black), xWT (orange), hWT (red), H2A.Z (blue) and uH2B (green) nucleosomes. Bar width is 1 bp and major peak …
Figure 6—figure supplement 1
Crossing Time, Crossing Probability and Pause-free Velocity of Pol II during Transcription through NPS DNA or Nucleosomes.
(A–E) Histograms of crossing time of Pol II transcription through bare NPS DNA (A), xWT (B), hWT (C), H2A.Z (D) and uH2B (E) nucleosomes. (F) Relative percentage of Pol II molecules that are …
Figure 7 with 1 supplement
Mechanical Model for Pol II Transcription Through the Nucleosome.
(A) Schematic of the mechanical model, showing three different lengths of unwrapped DNA for a given polymerase position along the DNA sequence. The steric spheres are shown in purple (polymerase) …
Figure 7—figure supplement 1
Fitting Nucleosome Energy Profiles Based on Pol II Dwell Times.
(A–C) Mean dwell times (colored dotted lines) calculated from best-fit mechanical model (see Figure 7D–E) whose corresponding nucleosome binding energies (insets, colored lines) are shown for (A) …
Videos
Video 1
Unzipping-rezipping of bare NPS DNA.
https://doi.org/10.7554/eLife.48281.004
Video 2
Unzipping-rezipping of hWT nucleosome.
https://doi.org/10.7554/eLife.48281.007
Video 3
Pol II transcription through bare NPS DNA.
The horizontal gray dashed lines indicate predicted pause sites in the molecular ruler, the three horizontal black dashed lines represent NPS entry, dyad, and NPS exit, respectively. This applies to …
Video 4
Pol II transcription through bare NPS DNA, NPS zoom.
https://doi.org/10.7554/eLife.48281.013
Video 5
Pol II transcription through xWT nucleosome.
https://doi.org/10.7554/eLife.48281.018
Video 6
Pol II transcription through xWT nucleosome, NPS zoom.
https://doi.org/10.7554/eLife.48281.019
Video 7
Pol II transcription through hWT nucleosome.
https://doi.org/10.7554/eLife.48281.020
Video 8
Pol II transcription through hWT nucleosome, NPS zoom.
https://doi.org/10.7554/eLife.48281.021
Video 9
Pol II transcription through H2A.Z nucleosome.
https://doi.org/10.7554/eLife.48281.022
Video 10
Pol II transcription through H2A.Z nucleosome, NPS zoom.
https://doi.org/10.7554/eLife.48281.023
Video 11
Pol II transcription through uH2B nucleosome.
https://doi.org/10.7554/eLife.48281.024
Video 12
Pol II transcription through uH2B nucleosome, NPS zoom.
https://doi.org/10.7554/eLife.48281.025Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | NeutrAvidin (deglycosylated native avidin from egg whites) | Thermo Fisher | Cat# PI31000 | powder dissolved in PBS, 0.5 µM |
| Antibody | Anti-Digoxigenin from sheep | Sigma-Aldrich | Cat# 11333089001, RRID:AB_514496 | powder dissolved in PBS, 0.2 mg/ml |
| Strain | DH5α competent cells | Fisher Scientific | Cat# 18-265-017 | |
| Strain | BL21(DE3) competent cells | NEB | Cat# c2527H | |
| Strain | Agilent SURE 2 Supercompetent Cells | Fisher Scientific | Cat# 200152 | |
| Chemical compound | dNTP set 100 mM Solutions | Fisher Scientific | Cat# R0181 | |
| Chemical compound | NTP set 100 mM Solutions | Fisher Scientific | Cat# R0481 | |
| Chemical compound | 3'-Deoxyadenosine-5'-Triphosphate | TriLink Biotechnologies | Cat# N3001 | |
| Chemical compound | 3'-Deoxyguanosine- 5'-Triphosphate | TriLink Biotechnologies | Cat# N3002 | |
| Chemical compound | 3'-Deoxycytidine-5'-Triphosphate | TriLink Biotechnologies | Cat# N3003 | |
| Chemical compound | 3'-Deoxyuridine-5'-Triphosphate | TriLink Biotechnologies | Cat# N3005 | |
| Chemical compound | Trioxsalen | Sigma-Aldirch | Cat# T6137 | |
| Chemical compound | [α−32P]-ATP | Perkin Elmer | Cat# BLU003H250UC | |
| Commercial assay or kit | T4 DNA ligase | NEB | Cat# 0202L | |
| Commercial assay or kit | E. coli DNA ligase | NEB | Cat# 0205L | |
| Commercial assay or kit | Phusion high-fidelity DNA polymerase | NEB | Cat# M0530S | |
| Commercial assay or kit | DraIII-HF | NEB | Cat# R3510S | |
| Commercial assay or kit | BsaI-HF | NEB | Cat# R3535L | |
| Commercial assay or kit | BglI | NEB | Cat# R0143S | |
| Commercial assay or kit | EagI-HF | NEB | Cat# R3505S | |
| Commercial assay or kit | SapI | NEB | Cat# R0569S | |
| Sequence-based reagent | Lambda DNA | NEB | Cat# N3011S | |
| Recombinant DNA reagent | Primers for making constructs and DNA templates | This paper | IDT custom order | Supplementary file 1 |
| Recombinant DNA reagent | pGEM-3z/601 | Addgene | Cat# 26656 | |
| Recombinant DNA reagent | pGM3z-8×repeat-2×BsaI | This paper | N/A | |
| Software, algorithm | LabVIEW VIs | Comstock et al. (2011) | RRID:SCR_014325 | |
| Software, algorithm | Matlab scripts for data analysis | This paper | RRID:SCR_001622 | |
| Software, algorithm | Python scripts for data analysis | This paper | RRID:SCR_008394 | |
| Software, algorithm | ImageJ | NIH (open source) | RRID:SCR_003070 | |
| Other | Pierce Streptavidin Magnetic Beads | Thermo Fisher | Cat# 88816 | |
| Other | BD 1 mL Insulin Syringe with Slip Tip | Fisher Scientific | Cat# 14-823-434 | |
| Other | Dual-trap time-shared high resolution optical tweezers | (Comstock et al., 2011) | N/A | |
| Other | Multi-channel optical tweezers chamber | This study | N/A | |
| Other | 1 µm carboxylated polystyrene beads | Bang Laboratories, Inc. | Cat# SVP-10–5 | |
| Other | 1.26 µm streptavidin polystyrene beads | Spherotech | Cat# PC04001-PC04N | |
| Other | Streptavidin Coated Magnetic Particles | Spherotech | Cat# SVM-08–10 | |
| Other | Hi-Trap Q HP columns 5 × 1 mL | Genesee Scientific | Cat# 17-1153-01 | |
| Other | Ni-NTA Agarose | Qiagen | Cat# 30210 | |
| Other | HisTrap HP 1 × 5 mL | Genesee Scientific | Cat# 84–208 | |
| Other | Amicon Ultra-0.5 Centrifugal Filter Unit, 100K | Millipore Sigma | Cat# UFC500324 | |
| Other | Amicon Ultra-0.5 Centrifugal Filter Unit, 3K | Millipore Sigma | Cat# UFC510024 | |
| Other | Zyppy Plasmid Maxiprep Kit | Zymo Research | Cat# D4028 | |
| Other | Typhoon imager | GE Healthcare | TRIO Variable Mode |
Additional files
-
Supplementary file 1
Oligos used in this study.
- https://doi.org/10.7554/eLife.48281.028
-
Supplementary file 2
Pol II backtrack analysis.
- https://doi.org/10.7554/eLife.48281.029
-
Transparent reporting form
- https://doi.org/10.7554/eLife.48281.030
