DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)

DNA Double-Strand Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the tridimensional genome organization around DSBs and its role in DSB repair foci formation. We performed Hi-C experiments before and after DSB induction and upon ctrl or SCC1 depletion, 4C-seq experiments before and after DSB induction and upon cohesin (SCC1) depletion or ATM inhibition. We also performed ChIP-seq of pATM(S1981), CTCF, P-SMC3(S1083), MDC1 and a calibrated ChIP-seq of SCC1 with or without damage. ChIP-chip of SMC3 (S1083) and SMC1 (S966) were used to show the recruitment of these marks on DNA repair foci. ChIP-chip of gammaH2AX was realized upon SCC1 depletion and ChIP-seq of gammaH2AX was realized upon SCC1 or WAPL to show the role of the cohesin complex in gammaH2AX foci formation. ChIP-seq of gammaH2AX was realized upon ATM or ATR inhibition to show that ATM is the major kinase that phosphorylates H2AX at clean breaks in human cells.