Extended Data Fig. 8: RASER-FISH analysis of 53BP1-MDs at site-specific DSBs in KIF23 and KIF11 loci.
From: Stabilization of chromatin topology safeguards genome integrity
a, Depiction of a 0.45-Mb TAD from a reference cell line (adapted from Yue laboratory 3D genome browser, see Methods) harbouring the KIF23 gene (top) and a 0.4-Mb TAD harbouring the KIF11 gene (bottom). Sites of CRISPR–Cas9 site-specific DSBs and a position of each RASER-FISH probe are indicated. b, DAPI-stained U2OS cells transfected with Cas9 ribonucleoprotein complexes with control, KIF23-, or KIF11-targeting guide RNAs (gRNA). Arrows indicate examples of mitotic aberrations inflicted by KIF23 and KIF11 knockout. c, d, 3D-SIM of the KIF23-TAD (c) and the KIF11-TAD (d) RASER-FISH probes in cells treated as in Fig. 3a, b but at loci without DNA damage (no 53BP1 signal). Dual-colour FISH probes FP-A and FP-B are located within the same TAD in c; FP-C and FP-D in in two adjacent TADs in d. e, Widefield microscopy of immunostained 53BP1-MDs at the damaged KIF23-TAD locus labelled by FP-B in U2OS and RPE1-hTERT cells 3 h after transfection with KIF23 gRNA–Cas9. Insets (MD1–3) are magnified 53BP1-MDs shown in xy, xz and yz orientations. f, Widefield microscopy of immunostained 53BP1-MDs at the damaged KIF11-TAD locus labelled by FP-C in U2OS cells 3 h after transfection with KIF11 gRNA–Cas9. Insets (MD1–3) were generated as in e. g, 3D-isosurface projections (V1–3) of 3D-SIM images of FP-C- and FP-D-labelled KIF11 TADs after DNA damage induction as shown in Fig. 3b. Scale bars, 5 μm in whole-nucleus images (e, f), 200 nm in insets (e, f) and in c, d, 20 μm in b. Experiments in b–f were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1.
