Figure 1. SET8 localizes to DSBs.

A Endogenous SET8 and H4K20me1 accumulate at DSBs in a damage-dependent manner. ChIP-qPCR for SET8 and H4K20me1 was carried on using U2OS cells stably expressing the tamoxifen (4-OHT)-inducible AsiSI endonuclease system. Results are normalized to the input. P < 0.05 (paired t-test for difference in SET8 and H4K20me1 at DSBs in −OHT and +OHT conditions).

B Western blot showing the SET8 knockdown efficiency after siRNA treatment. Whole-cell extracts from U2OS cells treated either with control or siRNA against SET8 (two subsequent siRNA transfections in 48 h) were immunoblotted and probed with the indicated antibodies. Actin was used as a loading control.

C Levels of γH2AX at DSBs are similar in siCON and siSET8 cells (P = 0.5; paired t-test for difference in γ-H2AX in −OHT and +OHT conditions). ChIP-qPCR was done on the same batch of cells as shown in (B).

D, E Increases in H4K20me1 and 53BP1 at DSBs are abrogated in SET8-depleted cells. ChIP-qPCR experiment was performed with anti-53BP1 and anti-H4K20me1 antibodies. Data are shown relative to input (P < 0.05; paired t-test for difference in H4K20me1 and 53BP1 at DSBs in −OHT and +OHT conditions).

F Abrogation of 53BP1 IRIFs upon knockdown of endogenous SET8 cannot be rescued by catalytic deficient SET8. Immunofluorescence for 53BP1 in SET8-depleted U2OS cells (two subsequent siRNA transfections in 48 h) after expression of GFP or siRNA-resistant SET8 form or siRNA-resistant SET8 catalytic dead mutant followed by IR treatment.

G Quantification of rescue experiments shown in (F).

H SET8 colocalizes with 53BP1. U2OS cells expressing GFP-SET8 were sensitized with BrdU and subjected to laser micro-irradiation. Cells were fixed and immunostained for γH2AX and 53BP1 30 min after laser irradiation. GFP-SET8 was visualized by GFP fluorescence.

I Quantification of the extent of colocalization of SET8 with 53BP1 shown in (H).