Skip to main content
. Author manuscript; available in PMC: 2019 Aug 25.
Published in final edited form as: Nat Cell Biol. 2019 Feb 25;21(3):311–318. doi: 10.1038/s41556-019-0282-9

Figure 3. H4K20me0 recognition is required for BRCA1-BARD1 recruitment to DSBs, 53BP1 antagonization, and end-resection.

Figure 3

a, High content microscopy of Flag-HA-BARD1 accumulation at IR-induced DNA DSBs. U-2-OS cells expressing Flag-HA-BARD1 WT or ARD 3A were pulsed with EdU, irradiated with 1 Gy and 45 minutes later pre-extracted and analysed by immunofluorescence. Foci number is shown relative to BARD1 WT in mid S. S phase stages were defined as in Fig. 2a. The mean is shown with S.D., n=3 independent experiments. Data points represent the mean of >381 cells. Scale bar, 10 μm. b, c, BRCA1 (b) and 53BP1 (c) accumulation at IR-induced DSBs in U-2-OS cells treated with control (Ctrl) or BARD1 siRNAs and induced with tetracycline (TET) to express siRNA-resistant Flag-HA-BARD1 WT or ARD 3A. Cells were analysed as in a and late S phase cells are shown. Mean shown as ‘+’, whiskers indicate 10-90 percentile; from left (b), n = 1068, 723, 1224, 896, 1420, 627, 902, 597 cells; from left (c), n = 1068, 723, 1224, 896, 1420, 627, 902, 597 cells. d, BRCA1 and 53BP1 foci in single cells in late S phase analysed as in b and c. BARD1 WT n=896; BARD1 ARD 3A n=597. e, High content microscopy of ssDNA measured by BrdU staining under non-denaturing conditions in U-2-OS cells treated with siRNA and TET as in c. Cells with high (top 15%) and low (bottom 15%) H4K20me0 levels (defined by gating on mean intensity) are shown. Cells were fixed 3 hours after IR (3 Gy). Mean shown as ‘+’, whiskers indicate 10-90 percentile; from left, n = 503, 442, 371, 312, 474, 269, 381, 330 and 617, 641, 316, 237, 540, 495, 277, 194 cells. f, High-content microscopy of IR-induced RAD51 foci analysed as in b. Mean shown as ‘+’, whiskers indicate 10-90 percentile; from left, n = 310, 384, 327, 326, 329, 295, 321, 331 cells.