Fig 1: Telomeric R-loops drive LLPS formation. (A–E) Telomerase-positive BI and telomerase-negative TBI fibroblasts were transduced with lentivirus encoding RNase H1-GFP (RNH1–GFP). Fibroblasts expressing ectopic RNH1 were selected with puromycin. (A) (Top) Number of APBs per cell. More than 100 cells each were scored. (Bottom) Western blot analysis to assess deletion and creation of the Brca2F11 allele after 4-OHT treatment. The effect of Brca2 depletion and/or RNH1–GFP overexpression was assessed. The same blot was re-probed with anti-PML and anti-POLD3 antibodies. (B) Representative fluorescent images showing APB in TBI fibroblasts with or without RNH1–GFP expression in the presence (+) or absence (–) of Brca2. PML, anti-PML immunofluorescence (green); telomere, T-FISH (red). Enlarged images of APBs showing the telomere clustering from the white square are shown below. Scale bar, 5 μm. (C) Effect of R-loop formation and LLPS in G2 telomere synthesis. TBI fibroblasts expressing RNH1–GFP were transduced with lentivirus expressing TRF1–mCh (M), –FUS (F) and –DDX4 (D), respectively, then subjected to G2 telomere synthesis assay. The graph showing the percentage of TBI cells with >3 EdU-positive telomeres is from three independent experiments. More than 100 cells each were scored. (D) Effect of G4 stabilization in telomere R-loop accumulation. BI or TBI fibroblasts, treated with 4-OHT (–) or left untreated (+) to deplete Brca2, were exposed to 5 μM G4 stabilizer pyridostatin (PDS) for 2 h. Cells untreated with PDS were included as control. The percentage of cells with >5 R-loop-positive telomeres is marked. More than 120 cells each were scored. (E) Effect of PDS and/or RNH1 on APB formation. More than 100 cells each were counted. (F) HeLa LT TERC KO cells were transfected with siLuc (control) or siBRCA2, and simultaneously transfected with mCherry- or RNH1–mCherry-expressing constructs. (Top) Representative images of R-loop (green) and telomere (red) co-localization. Representative images of PML (green) and telomere (red) co-localization. Enlarged images from the white arrow are shown. Scale bar, 2.5 μm. (Bottom) Quantification of R-loop-positive telomeres and APBs from images on the top. More than 120 cells each were scored. (G) Model for how the absence of BRCA2 instigates ALT-like telomere synthesis. Brca2 depletion provokes stabilization of G4, and subsequently increases TERRA-R-loops. R-loops trigger LLPS at telomeres, which contain the protein complex required for BIR (6). All graphs are the result of three independent experiments. *P< 0.0001, Student's t-test (mean ± SEM).
Fig 2: Telomeric R-loops are required for telomere H3K27 tri-methylation inside the liquid condensaste.(A) (Left) BI (+mTR) and TBI (–mTR) fibroblasts depleted of Brca2 (–) or left untouched (+) were subjected to ChIP against H3K27me3, H3K9me3 and histone H3, followed by hybridization with telomere probes. (Right) Relative H3K27me3 level at telomeres, normalized to histone H3. Four different BI and TBI MEFs, respectively, originating from different animals were employed in the analysis. (B) (Left) BJ fibroblasts transfected with siLuc (control) or siBRCA2 were subjected to ChIP against H3K27me3 and histone H3. (Right) Abundance of H3K27me3 at telomeres, normalized to H3. The graph is the result of three independent experiments. (C) RNH1-expressing BI and TBI fibroblasts, in the presence or absence of Brca2, were subjected to ChIP against anti-H3K27me3 and -H3 antibodies, respectively, followed by hybridization with telomere probes. (Right) Level of H3K27me3 at telomeres, normalized to histone H3. The data represent three independent experiments. (D and E) BI and TBI fibroblasts were depleted of PRC2 core components EZH2, SUZ12 and EED proteins via siRNA transfection, in the presence (+) or absence (–) of Brca2. (D) Depleting the PRC2 complex abolished telomere LLPS. More than 100 cells each were counted. (E) Depletion of PRC2 markedly reduces telomeric R-loop generation. More than 100 cells each were counted. (F) Oligonucleotide pull-down assay to analyze the PRC2-recruiting region in telomere R-loops. Cell lysates were incubated with the indicated biotinylated oligonucleotides and subjected to western blot analysis with the indicated antibodies. RNA oligonucleotides (CCCUAA)8 and (UUAGGG)8 represent TERRA antisense and TERRA, respectively. The hybrid of (TTAGGG)8 + (CCCTAA)8 represents a double-stranded telomere, and (UUAGGG)8+ (CCCTAA)8 represents DNA:RNA hybrids (R-loops). (UUAGGG)8+ (CCCTAA)5 and (UUAGGG)8 + (CCCTAA)3 represent DNA:RNA hybrids with three and five repeats of exposed TERRA, respectively. (UUAGAG)8 + (CCCTAA)3 and (UUAGAA)8 + (CCCTAA)3 represent non-G4-forming sequences. (G) Illustration of the telomeric chromatin remodeling essential for ALT-like telomere synthesis inside the liquid condensate. TERRA RNA protruding from the R-loop recruits the PRC2 complex, which catalyzes H3K27me3 at telomeres. All results are from three independent experiments. *P< 0.0001, Student's t-test (mean ± SEM).
Supplier Page from Sino Biological, Inc. for Mouse RNH1 Gene Lentiviral ORF cDNA expression plasmid, C-GFPSpark tag