Fig. 2: TERRA R-loops promote telomere clustering.

a Telomere DNA FISH in TERRA knockdown cells (sgTER_1C6, sgTER_1C21) or control cells (sgSense, sgλ2). b Quantification of telomere intensity in U2OS cells expressing RCas9-sgRNAs. Each dot represents a telomere signal of each spot. Bars, medians. n, number of telomere foci. c Histogram showing the population of each telomere spot grouped by its intensity in cells expressing RCas9-sgRNAs. d Telomere clustering decreases in TERRA knockdown cells (sgTER_1C6, sgTER_1C21). Bars, medians of top 5% telomere intensity. n, number of telomere foci. e Quantification of the area of top 5% large telomere foci in cells expressing RCas9-sgRNAs. Bars, medians. n, number of telomere foci. f Immunostaining of BLM and TRF2 in U2OS cells expressing RCas9-sgRNAs. BLM foci at clustered telomeres decrease in TERRA knockdown cells. Arrowheads indicate BLM with big TRF2 foci. g Quantification of BLM foci at clustered telomeres determined by the colocalization of BLM with big TRF2 foci. n, cell number. h Quantification of the colocalization of RPA70 with big TRF2 foci. n, cell number. i Telomere clustering determined by top 5% telomere intensity decreases after BLM knockdown in cells expressing RCas9-sgRNAs. Bars, medians. n, number of telomere foci. j Western blot analysis shows the expression of RNase H1 after siRNA knockdown for 3 days. k Telomere clustering determined by top 5% telomere intensity increases after RNH1 knockdown. Bars, medians. n, number of telomere foci. l Quantification of the number of APB (PML-large TRF2 colocalized) foci after RNH1 knockdown. n, cell number. m Quantification of the number of BLM foci that were colocalized with large TRF2 after RNH1 knockdown. n, cell number. n Quantification of the number of RPA70 foci that were colocalized with large TRF2 after RNH1 knockdown. n, cell number. b–e, i, k Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. g, h, I–n Data of three independent experiments. Bars, mean ± SEM. b, d, e, g, h, i, k–n P-values by two-sided Mann-Whitney test. Mean or median values of each group shown on the top of figures.