Hallmarks of glucocorticoid receptor condensates involvement in transcription regulation

Abstract

Several proteins necessary for mRNA production concentrate in intranuclear condensates, which are proposed to affect transcriptional output. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates the expression of hundreds of genes relevant to many physiological and pathological processes. As with all members of the steroid receptor family, GR forms condensates of unknown function. Here, we examine whether GR condensates are involved in transcription regulation using Airyscan super-resolution microscopy and nano-antibodies targeting initiation and elongating states of RNA polymerase II (Pol2). We observed subpopulations of GR condensates colocalizing with initiating and, surprisingly, elongating Pol2 foci. The analysis of GR mutants with different transcriptional outputs suggests a correlation between condensate formation capability and transcription initiation. Moreover, the number of GR molecules within initiation and elongation condensates appears to be linked to transcriptional activity. Taken together, our data suggests an involvement of GR condensates in transcription initiation and elongation.

Keywords: Biochemistry; Cell biology.

Graphical abstract

Figure 1

GR condensates spatially correlate with active Pol2 foci (A) Representative confocal and Airyscan images of D4 cells transiently expressing EGFP-GR (green), (top panels). Scale bar, 5 μm. Zoom-in images of a GR condensate (white square) and its intensity profile indicated by the yellow dotted line (bottom panels). Scale bar, 1 μm. The full width at half maximum (arrow) was determined by a Gaussian fitting. (B) Representative Airyscan images of D4-Halo-GR cells labeled with JF549 (green) and transiently expressing nano-antibodies fused to GFP-tagged Pol2 Ser5P or Pol2 Ser2P (magenta). Scale bar, 5 μm. Zoom-in images of the region delimited by the white squares show the overlap of GR condensates and initiation and elongation Pol2 foci (arrows). Scale bar, 1 μm. (C) Colocalization analysis of GR condensates and GFP-tagged nano-antibodies against Pol2 Ser5P or Pol2 Ser2P foci (left and right panels, respectively). The average CCF curve was calculated for each experimental condition (blue line) and compared to that expected for uncorrelated events (dashed line). (D) Relative intensities of the total GR condensates and of those colocalizing with Pol2 Ser5P (initiation) or Pol2 Ser2P (elongation) foci. Data are expressed as means ± SEM. Data information: Datasets are representative of at least three independent experiments. The number of cells (n) was: (A) n_{GFP-GR confocal} = 15 and n_{GFP-GR Airyscan} = 15; (C and D) n_{Halo-GR/GFP-Pol2 Ser5p} = 23 and n_{Halo-GR/GFP-Pol2 Ser2p} = 14. Statistical analysis was performed by Man Whitney’s test. ns = not significant; ∗p < 0.05; ∗∗p < 0.01 and ∗∗∗p < 0.001. See also Figures S1–S4.

Figure 2

Differential spatial distribution of BRD4 and Pol2 foci (A and B) Representative Airyscan images of D4 cells co-expressing mCherry-BRD4 (green) and GFP-tagged nano-antibodies against GFP-Pol2 Ser5P (A, magenta) or GFP-Pol2 Ser2P (B, magenta). Scale bar, 5 μm. Zoom-in images of the region delimited by the white squares show the relative position of BRD4 with respect to Pol2 foci (arrows). Scale bar, 1 μm. (C and D) Colocalization of BRD4 and Pol2 Ser5P (C) or Pol2 Ser2P (D) foci. The average CCF curve for each experimental condition (blue line) and that expected for uncorrelated events (dashed line) are shown Data information: Datasets are representative of at least two independent experiments. The number of cells (n) was: (C) n_{mCherry-BRD4/GFP-Pol2 Ser5p} = 10 and (D) n_{mCherry-BRD4/GFP-Pol2 Ser2p} = 13.

Figure 3

GR condensates are sensitive to transcriptional modulators (A) Intensity distributions of WT GR and GR407C condensates registered in D4 cells. The right tail of the distributions was fitted with an exponential-decay function (black line) obtaining the parameters reported in Table S1. (B) Representative Airyscan images of D4 cells transiently expressing EGFP-GR (green) and Halo-Med1 labeled with JF549 (magenta). Scale bar, 5 μm. Zoom-in images of the region delimited by the white squares show the overlap of GR condensates and Med1 foci (arrows). Scale bar, 1 μm. (C) Colocalization of GR condensates and Med1 foci. The average CCF curve (red line) and that expected for uncorrelated events (dashed line) are shown. (D) Density of GR condensates in D4 cells expressing EGFP-GR (control) or EGFP-GR and Halo-Med1 (Med1) labeled with JF549. Data are expressed as means ± SEM. (E) Intensity distribution of GR condensates in D4 cells co-expressing EGFP-GR and Halo-Med1 labeled with JF549. The right branch of the distribution was fitted with an exponential-decay function (red line) obtaining the parameters reported in Table S1. To facilitate comparison, the fitting curve obtained for GR in the absence of Med1 is also shown (gray dotted line). (F) Representative Airyscan images of Dex-stimulated D4 cells expressing EGFP-GR (green) registered without DRB (control condition), after incubation with DRB (DRB) and after drug removal (Wash). Scale bar, 5 μm. (G) Density of GR condensates in D4 cells in control condition, after DRB incubation (DRB) and after drug removal (Wash). Data are expressed as means ± SEM. (H) Relative intensity of GR condensates in D4 cells in control condition, after DRB incubation (DRB) and after drug removal (Wash). Data are expressed as means ± SEM. Data information: Datasets are representative of at least three independent experiments. The number of cells (n) was: (A) n_EGFP-GR = 46 and n_EGFP-GR407C = 37; (C) n_{EGFP-GR/Halo-Med1} = 46; (D–E) n_EGFP-GR = 46 and n_{EGFP-GR/Halo-Med1} = 46; (G-H) n_EGFP-GR = 46 and n_EGFP-GR+DRB = 70. Statistical analysis was performed by Student’s t test or unpaired t-test with Welch’s correction. ns = not significant; ∗p < 0.05; ∗∗p < 0.01 and ∗∗∗p < 0.001. See also Figures S5 and S6 and Table S1.

Figure 4

GR mutants associate with initiation and elongation condensates in a distinct manner (A) Representative Airyscan images of D4 cells co-expressing Halo-GRdim or Halo-GRtetra labeled with JF549 (green) and GFP-tagged nano-antibodies against each of the phosphorylated forms of Pol2 (magenta). Scale bar, 5 μm. (B) Colocalization analyses of the condensates of the GR variants GRdim (orange lines, top panels) and GRtetra (green lines, bottom panels) with Pol2 Ser5P (light color tones, left panels) and Pol2 Ser2P (dark color tones, right panels). The average CCF curve for each experimental condition and that expected for uncorrelated events (dashed line) are shown. (C) Quantification of the nuclear area occupied by the condensates of GR-variants and colocalizing with Pol2 Ser5P (initiation) or Pol2 Ser2P (elongation) foci. Data are expressed as means ± SEM. (D) Nuclear area occupied by receptor’s condensates colocalizing with Pol2 Ser5P (top) or Pol2 Ser2P (bottom) foci as a function of the total density of condensates of GR-variants. (E) Relative intensity of the condensates of GR-variants colocalizing with Pol2 Ser5P or Pol2 Ser2P foci. Data is expressed as means ± SEM. Data information: Datasets are representative of at least three independent experiments. The number of cells (n) was n_{Halo-GR/GFP-Pol2 Ser5p} = 23, n_{Halo-GR/GFP-Pol2 Ser2p} = 14. n_{Halo-GRdim/GFP-Pol2 Ser5p} = 31, n_{Halo-GRdim/GFP-Pol2 Ser2p} = 27, n_{Halo-GRtetra/GFP-Pol2 Ser5p} = 25, n_{Halo-GRtetra/GFP-Pol2 Ser2p} = 25. Statistical analysis was performed by Mann–Whitney test. ns = not significant; ∗p < 0.05; ∗∗p < 0.01 and ∗∗∗p < 0.001. See also Figures S2 and S7.