Condensates induced by transcription inhibition localize active chromatin to nucleoli

Abstract

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

Keywords: chromatin; compartment; gene fusion; nucleolus; phase separation; rRNA; transcription.

Figure 1.. RNAPII Inhibition Induces CITIs

(A) The effect of THZ1 (1 μM) treatment for 1 hr on the localization of SFPQ and NONO was analyzed by immunofluorescence (IF) in the U2OS cell line. Scale bars, 10 μm. NONO and SFPQ insets (white dashed line) shown at right, with nucleus outlined. (B, C) The effect of treatment with the indicated drugs (DRB 100 μM for 1 hr, Triptolide 1 μM for 1 hr, Actinomycin D 1 μM for 1 hr, CX5461 10 μM for 3 hr, CPT 1 μM for 1 hr) or UV irradiation (30 J m⁻³, fixed 1 hr after irradiation) on the localization of SFPQ was analyzed by IF. Scale bars, 10 μm (B). The quantification of the total area of SFPQ condensates per cell upon treatment with the indicated drugs or UV irradiation is shown as mean with 95% confidence interval (CI) (C). (D, E) The effect of acute degradation of RNAPII or THZ1 treatment on the localization of SFPQ was analyzed by IF after 1 hr auxin induction using the DLD1 mAID-POLR2A-mClover cell line. Scale bars, 10 μm (D). The quantification of the total area of SFPQ condensates per cell is shown as mean with 95% CI (E). (F, G) The effect of THZ1 treatment on the localization of TAF15 (F) or FUS (G) was analyzed by IF. Scale bars, 10 μm. (H) Live-cell imaging of GFP-TAF15 or GFP-FUS between 0 to 60 min after THZ1 treatment. See also Video 1 and 2. Scale bar, 10 μm.

Figure 2.. CITIs are Formed around FC and DFC in Nucleoli

U2OS cells expressing GFP-TAF15 were untreated or treated for 1 hr with THZ1 and stained for GFP-TAF15 (green) and two other proteins associated with different nucleolar compartments or SFPQ. (A) Three-color SoRa imaging of representative cells. The color of each protein is shown on the top. Scale bar, 5 μm. (B) SoRa volumetric renderings of z-stacks of a single representative nucleus. Green, TAF15; magenta, DKC1; blue, NPM1. See also Video 3 and Figure S2M. (C, D) The DKC1 or NPM1 fraction of volume inside CITIs (C) and fraction of NPM1 surface area adjacent to TAF15 or DKC1 (D) were quantified from the SoRa volumetric renderings. (E) Two-color STORM imaging of representative CITIs. Top row shows the individual localizations for each channel as points. Bottom row shows the reconstructed image. Scale bar, 100 nm. (F) Map of localizations of each channel for a representative region of interest (ROI) for Clus-DoC analysis (top row). Map of degree of GFP-TAF15 localizations in the ROI colored by DoC score (middle row). Distribution of DoC scores for GFP-TAF15 to the other channel across 4-6 nucleolar regions per sample (bottom row, mean with SD, n=4-6). Scale bar, 5 μm.

Figure 3.. CITI is a Dynamic Structure

(A) Representative images from the FRAP analysis of THZ1-treated GFP-TAF15 expressing cells. The bleached area is indicated with a yellow square. Scale bar, 1 μm. (B) The relative fluorescent recovery of GFP-TAF15 or GFP-SFPQ after bleaching of condensates (CITIs) or diffused areas (nucleoplasm) was analyzed (mean with SD, n=10 or 11 for data points of the cells from each category, representative results from three biological replicates). The fitted curves for each category are shown as a dotted line. (C) The diffusion coefficient was calculated from the FRAP analysis (mean with SEM, n=3 for data points of three biological replicates). (D, E) The effect of 1,6-hexanediol treatment (1.5%, 15 min) on TAF15 in CITIs was analyzed by IF (D). Scale bars, 10 μm. Since the nuclear size was shrunk by 1,6-hexanediol treatment, the size of CITIs was normalized to the nucleus size (E). The results are shown as mean with 95% CI. (F) The effect of DRB wash out on CITIs was analyzed by IF. Scale bars, 10 μm. The quantification of CITI positive cells at each time point is shown.

Figure 4.. Perturbation of rRNA Processing Induces CITIs

(A) The effect of THZ1 on the rRNA binding of SFPQ was analyzed by RIP (mean with SD, n=3 for measurements from one representative experiment of two biological replicates). The position of the primers for detection of pre-rRNA is shown above. (B) A SFPQ mutant that lacks RNA-binding domains (ΔRRM) was exogenously expressed and its localization was analyzed by IF. It is of note that the ΔRRM mutant did not localize to CITIs formed by endogenous SFPQ/TAF15. Scale bars, 10 μm. (C) The newly-synthesized RNA was labeled by EU in the presence of THZ1 or CX5461 for 1 hr. After biotin labeling and pulldown by streptavidin beads, the pre-rRNA levels in EU-labeled RNA were quantified by qPCR (mean with SD, n=3 for measurements from one representative experiment of three biological replicates). (D) The EU-labeled (1 hr) newly-synthesized RNA was chased for another 1 hr in the presence of THZ1 or CX5461 and analyzed as in (C). (E-G) The effects of THZ1 treatment on the localization of Nucleolin (E), FBL (F), or U3 snoRNA (G) and CITIs were analyzed by IF. Scale bars, 10 μm. (H, I) The localization of SFPQ after knockdown (KD) of Nucleolin, FBL, U3-55K (H) or U3, U8 snoRNAs (I) was analyzed by IF. Scale bars, 10 μm. The quantification of CITI positive cells is shown. (J) The effects of Nucleolin, FBL, or U3 snoRNA KD on the rRNA binding of SFPQ was analyzed as in (A). (K) A model for the relationship between rRNA processing and condensate formation at nucleoli. (L, M) The effect of cold/heat shock, hypoxia, or osmotic stress on TAF15 localization was analyzed by IF (L). Scale bars, 10 μm. The quantification of CITI positive cells is shown (M). (N) The reversibility of CITIs induced by mild cold shock (25 °C) was tested as in Figure 3F.

Figure 5.. CITIs Localize Active Chromatin to Nucleoli upon RNAPII Inhibition

(A) The experimental scheme of the SFPQ/UBF TSA-seq. (B, C) The genome view of SFPQ TSA-seq at the rDNA repeat unit aligned with the log 2 ratio of THZ1 and DMSO (log2 fc) (B). The quantification of signals at the rRNA coding region and intergenic region within the repeat unit is shown in (C). Representative results from two biological replicates are shown. (D) Two representative genome views of UBF TSA-seq aligned with Compartment A/B prediction scores. The log 2 ratio of THZ1 and DMSO is also aligned. The TSA-seq signals were smoothed in a 1-Mb window so that the scale of changes fitted with that of the Compartment A/B profile. Representative results from two biological replicates are shown. (E, F) The effect of THZ1 treatment on the genome-wide UBF TSA-seq signals were analyzed with the scatter plots (DMSO vs THZ1) (E) or the dot plots of the log2 ratio of THZ1 and DMSO (mean with SD, n=15 for data points from individual chromosomes) (F) in either Compartment A or B. (G) The genome-wide UBF TSA-seq signals in TAF15 KD cells at either Compartment A or B were quantified as in (F). (H, I) The log2 ratio of UBF TSA-seq signals (THZ1/DMSO) and compartment A/B prediction scores at ASXL1, SLC32A1, and LINC01370 (H). Colocalization between these gene loci and the DFC region of nucleoli identified by DKC1 were analyzed by FISH in untreated and THZ1-treated cells (I). The degree of localization was calculated by the divergence from background frequency and compared (mean with 95% CI, n=20 for data points from two biological replicates).

Figure 6.. Defective RNAPII Transcription Promotes Gene Fusions

(A) The correlation between the indicated gene or complex expression levels and the number of gene fusions per sample was analyzed in the indicated TCGA datasets. The results were shown as the ratio of the average of gene fusions in high to low groups. A significant ratio over 1 indicates that the high expression of the gene of interest is associated with a higher number of gene fusions, while a ratio below 1 indicates that the low expression of the gene of interest is associated with the higher number of gene fusions. The significant correlation is shown in red. See also Table S2. (B) A schematic representation of the system for chromosomal translocation detection using the U2OS AsiSI cell line. The timeline of experiments is shown. (C) The approximate position of MIS12 and TRIM37 on Chr 17 and the log2 ratio of UBF TSA-seq signals (THZ1/DMSO) and Compartment A/B scores at these loci are shown. (D) The effect of THZ1, DRB, or Triptolide treatment on the frequency of MIS12-TRIM37 fusion was analyzed (mean with 95% CI, n=10, 18, 6, left to right, for data points from more than four biological replicates). (E) The approximate position of PIP5KL1, NR6A1, GNE, and LINGO2 on Chr 9 and the log2 ratio of UBF TSA-seq signals (THZ1/DMSO) and Compartment A/B scores at these loci are shown. (F) The effect of THZ1 treatment on the frequency of intra-chromosome gene fusion between Compartment A-A (PIP5KL1-NR6A1, PIP5KL1-GNE) and Compartment A-B (PIP5KL1-LINGO2) was analyzed (mean with SEM, n=6 for data points of independent experiments). (G) The effect of THZ1 treatment on the frequency of inter-chromosome gene fusion was compared between MIS12-ASXL1 and MIS12-SLC32A1 (mean with SEM, n=10, 12, left to right, for data points of independent experiments). (H) The effect of THZ1 treatment on MIS12-RASA3 fusion and MIS12-NGR fusion was analyzed as in (G). (mean with SEM, n=11, 10, left to right, for data points of independent experiments). The approximate position of RASA3 and NGR on Chr 13 and the log2 ratio of UBF TSA-seq signals (THZ1/DMSO) and Compartment A/B scores at these loci are shown left.

Figure 7.. CITIs Mediate Oncogenic Gene Fusions

(A-B) The effect of pretreatment with 1,6 hexanediol (A) or CX5461 (pretreatment at 1 μM for 24 hr) (B) on the frequency of MIS12-TRIM37 fusion was analyzed (mean with SD, n=4 for data points from one representative experiment of three biological replicates). (C-E) The effect of SFPQ KD (C), NONO KD (D), or TAF15/FUS KD (E) on the frequency of MIS12-TRIM37 fusion was analyzed (mean with SD, n=4 for data points from one representative experiment of three biological replicates). (F) Two hypothetical outcomes of gene fusion detection by SFPQ TSA labelling. The total fusion events within the whole nucleus are detected in the input sample, while the fusion events at CITIs are detected in the pull-down sample. (G) Gene fusions occurring in CITIs were detected by SFPQ TSA labelling. The intact/fusion ratio was compared between the input and pull-down samples in untreated and THZ1-treated cells. Three different gene fusions (MIS12-TRIM37, MIS12-LINC01970, ASXL1-SRSF6) were tested (mean with SEM, n=3-4 for data points of biological replicates). (H) A schematic representation of the system for chromosomal translocation detection using the U2OS iCas cell line. The timeline of experiments is shown. (I) The approximate position of ALK and EML4 on Chr 2 and the log2 ratio of UBF TSA-seq signals (THZ1/DMSO) and Compartment A/B scores at these loci are shown. (J) The frequency of EML4-ALK fusion were analyzed by qPCR in the cells transfected with the indicated gRNAs and treated as indicated (mean with 95% CI, n=5-6 for data points from two biological replicates). (K) The effect of FUS KD on the frequency of EML4-ALK fusion was analyzed (mean with SD, n=3 from one representative experiment of three biological replicates). (L) A model for how defective RNAPII transcription leads to localization of Compartment A, but not B, to CITIs and promotes oncogenic gene fusions. See the text for details.