Fig 2: 53BP1 forms nuclear puncta.a MDA-MB-231 parental and 53BP1 knockout (KO) cells were treated or not with 500 nM CPT for 6 h, and protein expression was detected. Arrowhead indicates 53BP1. b Representative images of nuclear localization of 53BP1 in MDA-MB-231 cells under normal growth conditions. Single z-plane images were acquired by sequential scanning using confocal microscopy. Square: an example of puncta+ cell; Circle: a cell with three spontaneous 53BP1 foci; Arrow: a mitotic cell. Scale bar is 16 µm. c Percentage of puncta positive cells were analyzed from n = 36 individual images taken from four replicate experiments with a total n = 447 and 373 cells analyzed for parental and KO groups, respectively. Data represent mean values and standard deviation (SD). d Violin plot of 53BP1 puncta number per cell was analyzed from n = 185 and 105 parental and 53BP1 KO MDA-MB-231 cells, respectively. Data represent mean, 25th, and 75th percentiles with the whiskers extending to the minimum and maximum values. e Representative images of 53BP1 puncta and Cyclin A in MDA-MB-231 parental and 53BP1 KO cells under normal growth conditions. The cell cycle stages (G1, S/G2, and M) were determined by a combination of Cyclin A expression levels and DAPI staining patterns. Single z-plane images were acquired by sequential scanning using confocal microscopy. Scale bar is 10 µm. f Correlation between Cyclin A expression levels (expressed as mean fluorescence intensity, F.I.) and 53BP1 puncta number from n = 102 parental MDA-MB-231 cells. Each dot represents one cell. A size at or above 0.680 µm2 (area) (=25% percentile) was considered to be a 53BP1 punctum in the quantitative analyses. Unpaired two-tailed t test using Prism 9.0 was conducted for c and d with 95% confidence intervals, whereas the P-value in (f) was acquired by the Pearson Correlation Coefficient Calculator.

Fig 3: Uncoupling between LLPS and DSB repair of 53BP1.a U-2 OS cells were transfected with GFP-53BP1-C1 for 36 h, treated with 500 nM ATM inhibitor (ATMi) for 12 h, and the number of GFP-53BP1-C1 puncta per cell was counted and presented as Violin plot from indicated number of cells from two independent experiments. Data represent mean, 25th, and 75th percentiles with the whiskers extending to the minimum and maximum values. b 53BP1 KO U-2 OS cells re-constituted with GFP-53BP1 constructs and protein expression was examined. The uncropped blots are provided in the Source Data file. c Representative single z-plane confocal images of 53BP1 puncta in the absence of DNA damage. Quantitation of the percentage of GFP puncta positive cells from n = 18, 28, 33, 31, 30, 37, and 34 images for each group were analyzed from indicated number (in parenthesis) of cells and presented as mean values and SD acquired from at least two independent experiments. d U-2 OS cells from (b) were treated with 3.5 µM bleomycin for 6 h, fixed and stained with the anti-RIF1 antibody. Representative single z-plane confocal images are shown. Green signals in non-transfected cells represent endogenous 53BP1 by anti-53BP1 antibody staining. Square: an enlarged area showing rare co-localization between 53BP1-FL/DR and RIF1 foci, which helps determine this cell being 53BP1-FL/DR puncta positive, but DSB foci negative. Arrow: a cell showing 53BP1-C1 forming liquid droplets, but not DSB foci determined by the puncta size. Scale bar in all images is 10 µm. e Foci or puncta number of RIF1 and 53BP1 from indicated number (n) of cells in (d). Data represent mean values and SD from two independent experiments. Unpaired two-tailed t test was performed in Prism 9.0 with 95% confidence intervals in (a) and (e). In (e), while the 53BP1 KO group was compared to the parental group in the presence of BLEO, others were compared to the 53BP1 KO group in the presence of BLEO for statistical analysis.

Fig 4: 53BP1 puncta depend on heterochromatin.MDA-MB-231 cells were stably depleted of HP1a (a) or HP1ß (d), and protein expression was analyzed. Representative single z-plane confocal images of parental and HP1a (b), HP1ß (e) or HP1? (g) KD MDA-MB-231 cells. Quantitation of 53BP1 puncta rate (c, h) or number (f, i) from three independent experiments was shown. Violin plots of 53BP1 puncta number (f, i) represent mean, 25th, and 75th percentile from indicated number of cells. The whiskers extend to the minimum and maximum values. In c, n = 60, 37, and 31 images were analyzed for n = 309, 252 and 274 parental, HP1a KD and HP1ß KD cells, respectively. In h, n = 23 and 27 images were analyzed from a total of n = 215 and 214 parental and HP1? KD cells, respectively. Scale bar in all images is 10 µm. Data represent mean values and SD in (c) and (h). Unpaired two-tailed t test using Prism 9.0 was performed for c, f, h, and i with 95% confidence intervals.

Fig 5: 53BP1 maintains the repressive state of heterochromatin.ChIP-qPCR of 53BP1 at alpha satellite repeats of mcBox (a) and SATIII (b) regions in HEK293T parental, 53BP1 KD and KD reconstituted with GFP-53BP1-FL/WT cells from n = 3 biological replicates. c Transcript levels of Maj and Min SATs measured by qPCR in parental and 53bp1 KO MEFs from n = 6 biological replicates collected from two independent experiments. d Transcript levels of alpha satellite repeats of heterochromatin from parental and stable 53BP1 KD U-2 OS or HepG2 cells from n = 3 biological replicates. Inset: representative protein expression in U-2 OS cells from two independent experiments. Transcript levels of the alpha satellite repeat SATa (e) and mcBOX (f) from HEK293T parental, 53BP1 KD and KD cells reconstituted with GFP-53BP1-FL/WT from n = 3 biological replicates. g Representative single z-plane confocal images of parental, 53bp1 KO and KO MEFs reconstituted with GFL-53BP1-FL/WT. Squares: enlarged areas. Scale bar is 10 µm. h Violin plot of the number of heterochromatin centers visualized by DAPI from indicated number of cells from at least three independent experiments. Data represent mean, 25th, and 75th percentiles with the whiskers extending to the minimum and maximum values. Data represent mean values and SD in (a–f). Statistical analyses in (d) were compared with parental cells. Unpaired two-tailed t test was done by Prism 9.0 with 95% confidence intervals in (a–f) and (h).