GSE183059
Multiscale reorganization of the genome following DNA damage facilitates chromosome translocations via nuclear actin polymerization
Purpose: Evaluate how ARP2/3-coupled DSB mobility affects chromatin organization (Hi-C) and translocation frequency (HTGTS) in mouse embryonic fibroblasts (MEFs) harboring an inducible AsiSI restriction enzyme. MEF Hi-C Methods: Damaged samples were treated with 3 ug/mL doxycycline for 24 hours. For the last 6 hours of doxycycline treatment, 1 ug/mL 4-OHT was used to induce AsiSI translocation to the nucleus, triggering DSBs. Cells were co-treated with DMSO, 100 µM CK-666, or 10 µM NU7441. For DNA-PKcs inhibitor experiments, cells were pretreated with 10 µM NU7441 for 1 hour prior to induction of damage with 4OHT. 2 biological replicates were performed for each experimental condition. MEF HTGTS Methods: WT MEFs and AsiSI MEFs were treated as above. Following 6 hours treatment with dox and 4OHT, cells were lysed. For WASP inhibition, cells were treated with 3 µM wiskostatin following induction of damage. For flag-actin experiments, AsiSI-MEF cells were transfected with Flag–actinR62D–NLS 24 hours prior to induction of AsiSI expression. For cas9 experiments, WT MEF cells were transfected with sgRNA targeting Chr2 48 hours prior to cell lysis. Overall design: Hi-C and HTGTS analysis in MEF cells harboring inducible restriction endonuclease.