a. Clustering does not correlate with position in the nucleus. In the DIvA system, given that AsiSI induces a constant number of DSBs, clustering can be inferred by γH2AX foci size (Fig. 4a). Following γH2AX staining, images were acquired (objective X100) and foci were identified using the foci 3D picker plugin (ImageJ). Their Euclidian distance from the center was further computed. Analysis was performed on 19 cells acquired from 3 independent experiments. The scatterplot presented below show that foci size does not correlate with distance to center. b. Circos plots showing statistically significant (p<0.05) interactions induced after 4OHT treatment at selected captured domains (3 controls regions (top panels), 3 domains exhibiting high clustering (middle panels) and 3 domains showing low clustering (low panels)). c. Initial proximity potentiates but is not sufficient for DSB clustering. Number of reads (normalized) were scored for each pairs of 2Mb domains either within the same chromosome (intrachromosomal, left panels) or between different chromosomes (interchromosomal, right panels) before (x axis) and after (y axis) DSB induction, for both BR#1 and BR#2 as indicated. As expected intrachromosomal contacts are enriched in all conditions compared to interchromosomal contacts (see scales). DSB induction triggered increased contact frequencies both within and between chromosomes. Of note, domains that exhibit very low contact frequencies before 4OHT (right panels, low read counts) do not show higher contact after 4OHT suggesting that initial proximity favours clustering. However, loci exhibiting high contact frequency before 4OHT do not necessarily show more frequent contact after 4OHT (left panels see arrows), indicating that although necessary, initial proximity is not enough to sustain clustering.