Regulation of pairing between broken DNA-containing chromatin regions by Ku80, DNA-PKcs, ATM, and 53BP1

Chromosome rearrangement is clinically and physiologically important because it can produce oncogenic fusion genes. Chromosome rearrangement requires DNA double-strand breaks (DSBs) at two genomic locations and misrejoining between the DSBs. Before DSB misrejoining, two DSB-containing chromatin regions move and pair with each other; however, the molecular mechanism underlying this process is largely unknown. We performed a spatiotemporal analysis of ionizing radiation-induced foci of p53-binding protein 1 (53BP1), a marker for DSB-containing chromatin. We found that some 53BP1 foci were paired, indicating that the two damaged chromatin regions neighboured one another. We searched for factors regulating the foci pairing and found that the number of paired foci increased when Ku80, DNA-PKcs, or ATM was absent. In contrast, 53BP1 depletion reduced the number of paired foci and dicentric chromosomes—an interchromosomal rearrangement. Foci were paired more frequently in heterochromatin than in euchromatin in control cells. Additionally, the reduced foci pairing in 53BP1-depleted cells was rescued by concomitant depletion of a heterochromatin building factor such as Krüppel-associated box-associated protein 1 or chromodomain helicase DNA-binding protein 3. These findings indicate that pairing between DSB-containing chromatin regions was suppressed by Ku80, DNA-PKcs, and ATM, and this pairing was promoted by 53BP1 through chromatin relaxation.


Legends for Supplementary Figures and Tables
Figure S1 -Colocalisation of 53BP1 foci with phospho-H2AX foci.
BJ-hTERT cells were irradiated with 2 Gy ionizing radiation (IR) and fixed at indicated time points. The fixed cells were then subjected to immunofluorescence staining for 53BP1 and serine 139-phosphorylated histone H2AX. Nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). White arrowheads represent paired foci. (a) HE49 cells (normal human primary fibroblasts) in the G0/G1, S, or G2 phase were irradiated with 2 Gy IR, and cells that progressed to mitosis were harvested to prepare chromosome samples. To analyse the chromosomes of cells irradiated in the G0/G1 phase, confluent cells were irradiated and replated at low density to allow the cell cycle to progress, and then mitotic cells were harvested at 48 h after IR. To analyse chromosomes of cells irradiated in the S phase, S phase cells were pulse-labelled with 10 µM bromodeoxyuridine (BrdU) for 30 min and then irradiated. Mitotic cells were harvested at 10 h after exposure to IR. To analyse chromosomes of cells irradiated in the G2 phase, cells were irradiated and mitotic cells were harvested at 4 h after exposure to IR. To avoid bias by IR-induced cell cycle checkpoints, the experiments were performed in the presence of an ATM inhibitor for G0/G1 phase irradiated samples, and both an ATM inhibitor and Chk1/2 inhibitor were used for samples irradiated in the S or G2 phase. Chromosomes of G0/G1 and G2 phase-irradiated cells were stained with Giemsa solution. S phase-irradiated cells were subjected to BrdU staining and centromere/telomere fluorescence in situ hybridization. Only BrdU (+) chromosomes were analysed.
(d) The number of 53BP1 foci in total cells and paired foci(+) cells after exposure to varying doses of IR (BJ-hTERT, G1 phase). Results at 8 h after IR are shown.
(e) Frequency of paired 53BP1 foci in total cells including paired foci(-) cells after exposure to varying doses of IR (BJ-hTERT, G1 phase). Results at 8 h after IR are shown.
(f) Kinetics of the number of the total and paired 53BP1 foci after IR in BJ-hTERT cells (G1 phase).
(g) Kinetics of the number of the total and paired mCherry-BP1-2 foci after IR in BJ-hTERT cells (G1 phase).              Depletion of 53BP1 performed in Figure 7d and e experiments was confirmed.

Figure S12 -Full-length images of western blots of KAP-1 and β-actin.
Depletion of KAP-1 performed in Figure 7d and e experiments was confirmed.

Figure S13 -siRNA sequences
Sequences of siRNAs used in this study are listed.

Table S1 -Paired-foci frequency in BJ-hTERT cells.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S2 -Paired-foci frequency in wild-type and Ku80 -/cells.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S3 -Paired-foci frequency in wild-type and DNA-PKcs -/cells.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S4 -Paired-foci frequency in 2BN-hTERT cells.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S5 -Paired-foci frequency in BJ-hTERT cells depleted of MRE11 or CtIP.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S6 -Paired-foci frequency in AT5BI-hTERT cells.
Shown are the sums of total and paired 53BP1 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S7 -Paired-foci frequency in BJ-hTERT cells depleted of 53BP1.
Shown are the sums of total and paired mCherry-BP1-2 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

cells.
Shown are the sums of total and paired mCherry-BP1-2 foci in euchromatin (EC) or heterochromatin (HC) in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Table S9 -Paired-foci frequency in BJ-hTERT cells depleted of 53BP1 alone or both 53BP1 and heterochromatin-building factors.
Shown are the sums of total and paired mCherry-BP1-2 foci in all cells analysed. The percentage of paired foci in each sample was calculated based on the sums of total and paired foci.

Supplementary movie
Movie S1. A movie of three-dimensional deconvoluted images of paired 53BP1 foci. A three-dimensional foci image was obtained as described in Materials and Methods, and the movie was produced using Imaris 8.0.1 (Zeiss, Germany).