Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction
- Department of Biology, Johns Hopkins University, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Department of Biochemistry and Cell Biology, Stony Brook University, United States
- Institute of Systems Genetics, Langone Medical Center, New York University, United States
- Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, United States
Figures
Figure 1 with 3 supplements
Diffusive behaviors of protein fusions to HaloTag (Halo) reveal chromatin-bound and free populations in live yeast.
(A, B) Normalized histograms and two-component Gaussian fits for H2A.Z-Halo (A) and Halo-H2B (B) show the log diffusion coefficient distributions. The Gaussian fit for HaloTag is shown for reference …
Figure 1—figure supplement 1
Cell growth, labeling and SPT analysis of Halo-tagged proteins.
(A) Growth of strains bearing SWR1, H2A.Z (HTZ1) and H2B (HTB1) fusions to HaloTag. Saturated cultures at optical density 1.0 were spotted (1:5 serial dilutions) on CSM plates with or without 1% …
Figure 1—figure supplement 2
Spot-On kinetic modeling analysis.
(A) Histogram of displacements over time intervals of 10, 20, 30, 40 and 50 ms (dt 1–5) for H2A.Z. First four displacements were included for each track. Kinetic fitting shown as dashed line and …
Figure 1—figure supplement 3
H2A.Z-Halo distribution in cell division cycle.
(A) FACS analysis shows DNA content of the synchronized cell population upon time of release from α-factor arrest, from 0’ – 120.’ (B, C) Normalized histogram and two-component Gaussian fit for …
Figure 2 with 2 supplements
H2A.Z chromatin binding is substantially reduced upon abrogation of the deposition pathway by SWR1 inactivation.
(A) Time course of H2A.Z-Halo labeling, rapamycin treatment and image acquisition in Swc5-AA cells. Rapamycin treatment for an hour before SPT, and imaging performed in continued presence of …
Figure 2—figure supplement 1
Swc5 is required post-recruitment for SWR1 activity.
(A,B) Normalized histograms and two-component Gaussian fits for Swr1-Halo imaged in Swc5-AA cells in the absence (A) or presence (B) of rapamycin. (C) Spot-On analysis of the same imaging data. …
Figure 2—figure supplement 2
Reduction in chromatin-bound H2A.Z in a time course of Swc5-AA.
(A) Schematic of cell staining and imaging conditions. (B,C) SPT data from different time points after rapamycin treatment were pooled for normalized diffusion histogram (B) and Spot-On analysis (C).…
Figure 3 with 2 supplements
RNA polymerase II is critical for H2A.Z eviction.
(A) Normalized histograms and two-component Gaussian fits for H2A.Z-Halo imaged in cells co-depleted for Rpb1 and Swc5. (B) H2A.Z-Halo distributions in cells co-depleted for Ino80 and Swc5. (C) …
Figure 3—figure supplement 1
Efficient nuclear depletion of Swc5 in double anchor-away (SWC5-FRB; RPB1-FRB) strain.
(A, B) Deconvolution fluorescence microscopy shows nuclear depletion of Swc5-FRB-GFP in rapamycin-treated, single anchor-away cells, and in Swc5-FRB-GFP; Rpb1-FRBdouble anchor-away cells. Images of …
Figure 3—figure supplement 2
H2A.Z diffusion histograms in cells for single AA of Rpb1 and Ino80.
(A, B) H2A.Z-Halo distributions in Rpb1-AA strain without (A) and with (B) depletion of Rpb1. (C) Spot-On results for chromatin bound H2A.Z upon Rpb1 depletion. Single AA of Rpb1 causes only a …
Figure 4 with 1 supplement
Kin28 phosphorylation of RNA polymerase II CTD is critical for H2A.Z eviction.
(A) Schematic representation shows the three Pol II kinases Kin28, Bur1 and Ctk1 recruited at initiation, early-elongation and elongation phases respectively of Pol II and corresponding …
Figure 4—figure supplement 1
H2A.Z diffusion histograms in cells after single depletion of Kin28.
(A, B) H2A.Z-Halo distributions in Kin28-AA strain without (A) and with (B) depletion of Kin28. (C) Spot-On results for chromatin bound H2A.Z upon Kin28 depletion. (D) Reproduction of Gaussian fits …
Figure 5
Cycle of H2A.Z eviction and deposition.
RNA polymerase II assembled genome-wide in the PIC and Rpb1 CTD Ser5 phosphorylated by Kin28 constitutively transcribes short noncoding RNAs (with m7G cap) and evicts H2A.Z-H2B dimers from the +1 …
Videos
Video 1
H2A.Z-Halo single molecules imaged in wild type cells.
Molecules were tracked with JF552 dye. Time is indicated on top in seconds and outline of nuclear regions are marked in yellow.
Video 2
Swr1-Halo single molecules imaged in wild type cells.
Molecules were tracked with JF646 dye. Time is indicated on top in seconds and outline of nuclear regions are marked in yellow.
Video 3
H2A.Z-Halo single molecules imaged after anchor-away of swc5.
Molecules were tracked with JF552 dye. Time is indicated on top in seconds and outline of nuclear regions are marked in yellow.
Additional files
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Supplementary file 1
List of strains used in this study.
- https://cdn.elifesciences.org/articles/55667/elife-55667-supp1-v2.docx
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Supplementary file 2
List of results from MSD and Spot-On analysis.
- https://cdn.elifesciences.org/articles/55667/elife-55667-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55667/elife-55667-transrepform-v2.docx
