1. Structural Biology and Molecular Biophysics
  2. Chromosomes and Gene Expression

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

  1. Chao Yu Zhen
  2. Roubina Tatavosian
  3. Thao Ngoc Huynh
  4. Huy Nguyen Duc
  5. Raibatak Das
  6. Marko Kokotovic
  7. Jonathan B Grimm
  8. Luke D Lavis
  9. Jun Lee
  10. Frances J Mejia
  11. Yang Li
  12. Tingting Yao
  13. Xiaojun Ren  Is a corresponding author
  1. University of Colorado Denver, United States
  2. Janelia Research Campus, Howard Hughes Medical Institute, United States
  3. Colorado State University, United States
https://doi.org/10.7554/eLife.17667
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Cite this article
  1. Chao Yu Zhen
  2. Roubina Tatavosian
  3. Thao Ngoc Huynh
  4. Huy Nguyen Duc
  5. Raibatak Das
  6. Marko Kokotovic
  7. Jonathan B Grimm
  8. Luke D Lavis
  9. Jun Lee
  10. Frances J Mejia
  11. Yang Li
  12. Tingting Yao
  13. Xiaojun Ren
(2016)
Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin
eLife 5:e17667.
https://doi.org/10.7554/eLife.17667
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  3. Download .RIS
8 figures, 39 videos and 3 additional files

Figures

Figure 1 with 3 supplements
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The Cbx family members exhibit distinct dynamics in living mES cells.

(A) The sequences encoding the five Cbx proteins were fused with HaloTag to generate the HaloTag-Cbx fusions that were stably expressed in wild-type (PGK12.1) mES cells. The expression level of …

https://doi.org/10.7554/eLife.17667.002
Figure 1—source data 1

Source data for Figure 1D–Eand Figure 1—figure supplement 1B and 2A.

https://doi.org/10.7554/eLife.17667.003
Figure 1—figure supplement 1
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Comparison of the Dm and Df1 analysis.

(A) Distributions of the track (trajectory) length (in frames) for H2A-HaloTag, HaloTag-NLS, and HaloTag-Cbx7. (B) Normalized histograms of the log diffusion coefficient Df1 calculated from the …

https://doi.org/10.7554/eLife.17667.004
Figure 1—figure supplement 2
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Control experiments for testing the effects of the endogenous Cbx7 protein on the kinetic fractions of the exogenous HaloTag-Cbx7 fusion.

(A) Normalized histograms of the log maximum likelihood diffusion coefficient Dm for HaloTag-Cbx7 in Cbx7−/− mES cells (N = 25 cells, n = 5119 trajectories). (B) Fraction of the CB (red), ID (cyan), …

https://doi.org/10.7554/eLife.17667.005
Figure 1—figure supplement 3
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Control experiments for analyzing the protein level of HaloTag-Cbx7 and for testing whether HaloTag-Cbx7 occupies Polycomb target promoters.

(A) Western blotting of nuclear extracts from wild-type mES cells expressing HaloTag-Cbx7 in the presence or absence of Dox indicated above lanes. The endogenous and fusion proteins are marked at …

https://doi.org/10.7554/eLife.17667.006
Figure 2 with 1 supplement
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H3K27me3 is important for the targeting of Cbx7 and Cbx8 to chromatin, but plays a less important role for Cbx2, Cbx4, and Cbx6.

(A) Normalized histograms of the log maximum likelihood diffusion coefficient logDm for HaloTag-Cbx2 (N = 27 cells, n = 2471 trajectories), HaloTag-Cbx4 (N = 21 cells, n = 3254 trajectories), …

https://doi.org/10.7554/eLife.17667.015
Figure 2—source data 1

Source data for Figure 2A–C and Figure 2—figure supplementary 1B–C.

https://doi.org/10.7554/eLife.17667.016
Figure 2—figure supplement 1
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Additional experiments for HaloTag-Cbx in Eed−/−and Ezh2−/−mES cells.

(A) Schematic representation of the hypothetical model for the targeting of Cbx-PRC1 to chromatin via the Cbx CD interaction with H3K27me3 mediated by PRC2. The current research tests whether …

https://doi.org/10.7554/eLife.17667.017
Figure 3 with 1 supplement
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The Cbx7 CD is not efficient for the targeting of Cbx7 to chromatin.

(A) Schematic representation of Cbx7 variants. (B) Normalized histograms of the log maximum likelihood diffusion coefficient Dm for HaloTag-Cbx7 replicated from Figure 1E and for HaloTag-CDCbx7 (N = …

https://doi.org/10.7554/eLife.17667.033
Figure 3—source data 1

Source data for Figure 3B

https://doi.org/10.7554/eLife.17667.034
Figure 3—figure supplement 1
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Control experiments for analyzing the protein levels of the HaloTag-Cbx7 variants.

To determine whether the Cbx7 variants are correctly expressed in wild-type mES cells, we performed immunoblotting of nuclear extracts from wild-type mES cells stably expressing HaloTag-Cbx7 and its …

https://doi.org/10.7554/eLife.17667.035
Figure 4 with 1 supplement
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Effects of the Cbx7 CD on the residence time of Cbx7 at chromatin.

(A) Time-lapse imaging of HaloTag-Cbx7 at constant integration (τint = 30 ms) and dark (τd = 170 ms) time in wild-type mES cells. The red arrow indicates a molecule that binds to chromatin. The …

https://doi.org/10.7554/eLife.17667.038
Figure 4—source data 1

Source data for Figure 4B and Figure 4—figure supplementary 1

https://doi.org/10.7554/eLife.17667.039
Figure 4—figure supplement 1
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Control experiments for determine photobleaching constant of JF549.

We performed photobleaching of H2A-HaloTag labelled with JF549 dye. Wild-type mES cells stably expressing H2A-HaloTag were incubated with 500 nM of JF549 dye. Live-cell image stacks were taken using …

https://doi.org/10.7554/eLife.17667.040
Figure 5 with 1 supplement
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Disruption of the complex formation of Cbx7-PRC1 facilitates the targeting of Cbx7 to chromatin.

(A) Normalized histograms of the log maximum likelihood diffusion coefficient Dm for HaloTag-Cbx7 in wild-type mES cells replicated from Figure 1E and for HaloTag-Cbx7 in Ring1a−/−/Ring1b−/− (N = 29 …

https://doi.org/10.7554/eLife.17667.045
Figure 5—source data 1

Source data for Figure 5A and Figure 5—figure supplementary 1

https://doi.org/10.7554/eLife.17667.046
Figure 5—figure supplement 1
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Cumulative frequency distribution of the dwell times for determining the residence times of HaloTag-Cbx7 in Ring1a−/−/Ring1b−/− and Bmi1−/−/Mel18−/− mES cells.

Cumulative frequency distribution of the dwell times for HaloTag-Cbx7 replicated from Figure 4B and for HaloTag-Cbx7 in Ring1a−/−/Ring1b−/− (N = 38 cells, n = 7825 trajectories) and Bmi1−/−/Mel18−/− …

https://doi.org/10.7554/eLife.17667.047
Figure 6 with 1 supplement
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CDCbx7 and ATLCbx7 together constitute a DNA-binding entity.

(A) Schematic representation of Cbx7. The sequence of amino acids of ATL motif is shown. The basic amino acids are underlined and mutated to alanine to generate ATLm. (B) EMSA for the determination …

https://doi.org/10.7554/eLife.17667.052
Figure 6—figure supplement 1
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Control experiments for analyzing the Cbx7 variants purified from BL21 cells by SDS-PAGE.
https://doi.org/10.7554/eLife.17667.053
Figure 7 with 1 supplement
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CDCbx7 and ATLCbx7 together control the targeting of Cbx7 to chromatin.

(A) Schematic representation of Cbx7 variants. The underlined ATL amino acids were mutated into alanine or glycine. (B) Normalized histograms of the log maximum likelihood diffusion coefficient Dm …

https://doi.org/10.7554/eLife.17667.054
Figure 7—source data 1

Source data for Figure 7B and Figure 7—figure supplementary 1.

https://doi.org/10.7554/eLife.17667.055
Figure 7—figure supplement 1
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Cumulative frequency distribution of the dwell times for determining the residence times of HaloTag-Cbx7△ATL, HaloTag-Cbx7ATLm, and HaloTag-Cbx7△CD-ATL in wild-type mES cells.

Cumulative frequency distribution of the dwell times for HaloTag-Cbx7 replicated from Figure 4B and for HaloTag-Cbx7△ATL (N = 17 cells, n = 3956 trajectories), HaloTag-Cbx7ATLm (N = 24 cells, n = …

https://doi.org/10.7554/eLife.17667.056
Figure 8
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Proposed models for the targeting of Cbx-PRC1 to chromatin.

(A) The Cbx7-PRC1 and Cbx8-PRC1 complexes are targeted to chromatin by co-recognition of H3K27me3 and DNA. The Cbx7- and Cbx8-PRC1 complexes are guided to genomic loci by the CD interaction with …

https://doi.org/10.7554/eLife.17667.063

Videos

Video 1
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H2A-HaloTag in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.007
Video 2
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HaloTag-NLS in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.008
Video 3
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HaloTag-Cbx2 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.009
Video 4
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HaloTag-Cbx4 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.010
Video 5
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HaloTag-Cbx6 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.011
Video 6
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HaloTag-Cbx7 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.012
Video 7
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HaloTag-Cbx8 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.013
Video 8
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HaloTag-Cbx7 in Cbx7 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.014
Video 9
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HaloTag-Cbx2 in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.018
Video 10
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HaloTag-Cbx4 in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.019
Video 11
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HaloTag-Cbx6 in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.020
Video 12
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HaloTag-Cbx7 in Eed KO mES cells (Fractional studies)
https://doi.org/10.7554/eLife.17667.021
Video 13
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HaloTag-Cbx8 in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.022
Video 14
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HaloTag-Cbx2 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.023
Video 15
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HaloTag-Cbx4 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.024
Video 16
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HaloTag-Cbx6 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.025
Video 17
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HaloTag-Cbx7 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.026
Video 18
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HaloTag-Cbx8 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.027
Video 19
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HaloTag-Cbx7/Y-Eed in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.028
Video 20
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HaloTag-Cbx8/Y-Eed in Eed KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.029
Video 21
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HaloTag-Cbx7/Y-Ezh2 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.030
Video 22
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HaloTag-Cbx8/Y-Ezh2 in Ezh2 KO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.031
Video 23
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HaloTag-CDCbx7 in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.032
Video 24
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HaloTag-Cbx7F11A in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.036
Video 25
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HaloTag-Cbx7△CD in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.037
Video 26
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HaloTag-Cbx7 in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.041
Video 27
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HaloTag-CDCbx7 in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.042
Video 28
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HaloTag-Cbx7F11A in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.043
Video 29
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HaloTag-Cbx7△CD in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.044
Video 30
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HaloTag-Cbx7 in Ring1a/Ring1b dKO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.048
Video 31
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HaloTag-Cbx7 in Bmi1/Mel18 dKO mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.049
Video 32
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HaloTag-Cbx7 in Ring1a/Ring1b dKO mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.050
Video 33
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HaloTag-Cbx7 in Bmi1/Mel18 dKO mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.051
Video 34
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HaloTag-Cbx7△ATL in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.057
Video 35
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HaloTag-Cbx7ATLm in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.058
Video 36
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HaloTag-Cbx7△CD-ATL in wild-type mES cells (Fractional studies).
https://doi.org/10.7554/eLife.17667.059
Video 37
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HaloTag-Cbx7△ATL in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.060
Video 38
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HaloTag-Cbx7ATLm in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.061
Video 39
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HaloTag-Cbx7△CD-ATL in wild-type mES cells (Residence time studies).
https://doi.org/10.7554/eLife.17667.062

Additional files

Supplementary file 1

Fractional sizes and diffusion constants of the CB, ID, and FD populations obtained from live-cell SMT analysis of the Cbx family proteins and their variants.

https://doi.org/10.7554/eLife.17667.064
Supplementary file 2

Residence times, transient (F1tb) and stable (F1sb) chromatin-binding fractions of Cbx7 and its variants.

https://doi.org/10.7554/eLife.17667.065
Supplementary file 3

U-track parameters used in this research.

https://doi.org/10.7554/eLife.17667.066

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