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Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

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Science  27 Jul 2018:
Vol. 361, Issue 6400, pp. 412-415
DOI: 10.1126/science.aar4199
  • Fig. 1 Mediator and Pol II form transient and stable clusters in living mESCs.

    (A) A superresolution image of endogenous Mediator labeled with Dendra2 in living mESCs. (B and C) Representative superresolved images of transient and stable Mediator clusters and corresponding time-correlated photoactivation localization microscopy (tcPALM) traces. (B) and (C) correspond to areas boxed in blue and yellow, respectively, in (A). (D) Superresolution image of endogenous Pol II labeled with Dendra2 in living mESCs. (E and F) Representative superresolution images of transient and stable Pol II clusters and corresponding tcPALM traces. (E) and (F) correspond to areas boxed in blue and yellow, respectively, in (D). (G and H) Lifetime distributions of Mediator and Pol II clusters, respectively. Scale bars, 1 μm in (A) and (D) and 500 nm in (B), (C), (E), and (F).

  • Fig. 2 Mediator and Pol II clusters colocalize in a transcription-dependent manner.

    Live-cell direct images of (A) JF646-Mediator and (B) Dendra2–Pol II. Yellow arrowheads indicate stable clusters. (C) Superresolution image of Dendra2–Pol II overlaid with a background-subtracted JF646-Mediator image. Insets 1 to 5 show Mediator and Pol II colocalization in clusters. (D) JF646-Mediator and (E) Dendra2–Pol II maximum-intensity projections of a fixed cell imaged by lattice light-sheet microscopy. (F) Overlay of background-subtracted images. Yellow arrowheads indicate clusters identified in the Dendra2–Pol II channel. (G) Scatter plot of the distance D from a Dendra2–Pol II cluster to the nearest JF646-Mediator cluster (n = 143 clusters). Histograms outside the scatter plot show the distances along the x and y axes. (H) Same analysis for clusters identified in the Dendra2-Mediator channel (n = 67). (I and J) Superresolution images of Dendra2-Mediator and Dendra2–Pol II under normal conditions (left) and after 15 min (middle) or 6 hours (right) of incubation in 1 μM JQ1. (K and M) The number of transient Mediator and Pol II clusters per cell in a 2D focal plane as a function of time after JQ1 addition. (L and N) The number of stable Mediator and Pol II clusters per cell in a 2D focal plane. n = 17 to 25 cells and n = 14 to 24 cells at each JQ1 time point for Mediator and Pol II, respectively. (O) DRB treatment and washout experiments. DRB (100 μM) was added at 0 min and washed away after 45 min. Arrowheads indicate stable clusters identified in the JF646-Mediator channel. Black arrowheads in the middle panel (bottom) indicate Mediator clusters that did not colocalize with Pol II clusters. (P) Ratio (top) and absolute number (bottom) of clusters detected in the Pol II and Mediator channels per cell in a 2D focal plane. Nine to 15 cells were analyzed for each DRB incubation time point. The red arrow indicates the addition of DRB, and the blue arrow indicates DRB washout. Scale bars, 2 μm in overview images and 200 nm in insets.

  • Fig. 3 Mediator and Pol II form condensates that coalesce, recover in FRAP, and are sensitive to hexanediol.

    (A to E) Cluster fusion. (A) Maximum-intensity projection of a live cell imaged by lattice light-sheet microscopy. Trajectories of two clusters are indicated. (B and C) Clusters observed at 0 s and fusing at 369 s. (D) Individual time points around the fusion event. Orange and blue arrows indicate the precursor clusters, and the red arrows indicate the fused cluster. (E) Time course of the cluster intensities. a.u., arbitrary units. (F to H) FRAP analysis of clusters. (F) (Top) Images of a JF646-Mediator cell before (0 s) (left), immediately after (1 s) (middle), and 30 s after (right) bleaching. The red box indicates the position of the cluster on which the FRAP beam was focused. The blue box indicates an unbleached control locus. (Bottom) Cropped images as a function of time for both loci. (G) The normalized recovery curve for Mediator (n = 9 cells) yielded a recovery fraction of 60% during the 60-s observation, with a half-recovery time of 10 s. (H) FRAP analysis of JF646–Pol II (n = 3 cells) yielded 90% recovery, with an identical half-recovery time of 10 s. (I and J) Treatment with 10% hexanediol (v/v) gradually dissolved clusters of JF646-Mediator (I) and JF646–Pol II (J). Maximum-intensity projections of epifluorescence z stacks are shown. Yellow arrowheads indicate clusters identified at 0 min. Black arrowheads indicate clusters that disappeared. (K) Average number of clusters per cell (single 2D focal plane) observed in direct imaging as a function of time after hexanediol addition (n = 14 cells for JF646-Mediator, and n = 14 cells for JF646–Pol II). Scale bars, 1 μm (A to D) and 5 μm (F and J).

  • Fig. 4 Mediator clusters dynamically kiss actively transcribing SE-controlled genes.

    (A) Illustration of the working model describing cluster-kissing interaction with a gene locus. (B) Maximum-intensity projection of a cell imaged by using lattice light-sheet microscopy showing colocalization of a JF646-Mediator cluster with the actively transcribing Esrrb gene locus marked by MS2-tagged RNA (white box). (C) Single plane from the z stack after background subtraction. (D) Snapshot images of a Mediator cluster near the actively transcribing Essrb gene locus. Time points listed in (B) to (D) indicate minutes after the start of acquisition. Scale bars, 2 μm in (B) and (C) and 500 nm in (D). (E) Plot of the centroid-to-centroid distance from the gene locus to the nearest cluster as a function of time. (F) Cumulative distribution of distances from the Esrrb locus to the nearest Mediator cluster pooled from six cells (291 time points). The red dashed line in (E) and (F) indicates the colocalization threshold (300 nm). CDF, cumulative distribution function.

Supplementary Materials

  • Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

    Won-Ki Cho, Jan-Hendrik Spille, Micca Hecht, Choongman Lee, Charles Li, Valentin Grube, Ibrahim I. Cisse

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S18
    • Tables S1 to S10
    • Captions for Movies S1 to S3
    • References

    Images, Video, and Other Media

    Movie S1
    Pol II clusters coalescing
    Typical example of Pol II clusters coalescing as discussed in Fig. 3. Maximum intensity projection of full cell nucleus, no background subtracted. Time is indicated in upper right corner. Scale bar 5μm.
    Movie S2
    Mediator clusters coalescing
    Typical example of Mediator clusters coalescing. Maximum intensity projection of full cell nucleus, no background subtracted. Time is indicated in upper right corner. Scale bar 1μm. Two consecutive coalescence events were observed for JF646-Mediator.
    Movie S3
    Esrrb locus and Mediator cluster 3D tracking
    3D rendering of dual color lattice light sheet data showing an Esrrb gene locus labelled by MS2-tagged nascent mRNA dynamically interacting with a Mediator cluster. Scale bar 1 μm.
     
    Correction (9 July 2018): Since First Release, the y-axis label in fig. S5B has been corrected to refer to Pol II rather than Mediator, and minor editorial changes have been made in the legends to figs. S6 and S9.
    The original version is accessible here.

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