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

Abstract

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

Fig. 1.. Mediator and Pol II form transient and stable clusters in living mESCs.

(A) A super-resolution image of endogenous Mediator labeled with Dendra2 in living mESCs. (B, C) Representative super-resolved images of transient and stable Mediator clusters and corresponding tcPALM traces. (D) Super-resolution image of endogenous Pol II labeled with Dendra2 in living mESCs. (E, F) Representative super-resolution images of transient and stable Pol II clusters and corresponding tcPALM traces. (G, H) Lifetime distributions of Mediator and Pol II clusters, respectively. Red bars indicate stable clusters. Scale bars 1 μm in A, D and 500 nm in B, C, E, F.

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

(A) Live cell direct images of JF646-Mediator and (B) Dendra2-Pol II. Yellow arrowheads indicate stable clusters. (C) Super-resolution image of Dendra2-Pol II overlaid with a background-subtracted JF646-Mediator image. Insets 1–5 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-PolII channel. (G) Scatter plot of the distance from a Dendra2-Pol II cluster to the nearest JF646-Mediator cluster (N=143). Insets show histograms of the distances along the x- and y-axis. (H) Same analysis for clusters identified in the Dendra2-Mediator channel (N=67). (I, J) Super-resolution images of Dendra2-Mediator and Dendra2-Pol II under normal condition (left) and after 15 min (middle) or 6hr (right) incubation in 1μM JQ1. (K, 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, N) The number of stable Mediator and Pol II clusters per cell in a 2D focal plane. N=17–25 cells and N=14–24 cells at each JQ1 time point for Mediator and Pol II, respectively. (O) DRB treatment and wash-out 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 2D focal plane. N=9–15 cells were analyzed for each DRB incubation time point. Scale bars 2 μm in overview images, 200 nm in insets.

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

(A-E) Clusters fusion (A) Maximum intensity projection of a live cell imaged by lattice light sheet microscopy. Trajectories of two clusters are indicated. (B, C) Clusters observed at time 0s fusing at time 369s. (D) Individual time points around the fusion event. Orange and blue arrows indicate the precursor clusters, red arrow the fused cluster. (E) Time course of the clusters intensities. (F-H) FRAP analysis on clusters. (F, top panels) Images of a JF646-Mediator cell before (0s, left), immediately after (1s, middle), and 30s after bleaching (right). Red box indicates the position of the cluster on which the FRAP beam was focused. Blue box indicates an unbleached control locus. (F, bottom panels) Cropped images as function of time for both loci. (G) The normalized recovery curve for Mediator (N=9 cells) yielded a recovery fraction of 60% during the 60s observation with a half recovery time of 10s. (H) FRAP on JF646-PolII (N=3 cells) yielded 90% recovery with an identical half recovery time of 10s. (I, J) Treatment with 10% hexanediol (v/v) gradually dissolving clusters of JF646-Mediator (top) and JF646-Pol II (bottom). Maximum intensity projection of epifluorescence z-stacks. 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, N=14 cells for JF646-Pol II).

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 using lattice light sheet microscopy showing colocalization of a JF646-Mediator cluster with 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. (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 N=6 cells (291 time points). Red dashed line in E and F indicates colocalization threshold (300nm). Scale bars 2μm (B,C), 500nm (D).