Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

DNA damage response (DDR) is instrumental for maintaining genome stability and its deregulation predisposes to carcinogenesis, while revealing attractive targets for innovative therapies. Chromatin governs the DNA repair process via the interplay among different layers consisting of DNA, histones post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to follow, during double-strand break repair, chromatin-mediated interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome. In particular, we functionally attribute novel chromatin-associated proteins to non-homologous end-joining or homologous recombination (HR) repair, we reveal susceptibility to PARP inhibitor treatment upon knockdown of ATAD2, TPX2 and EHMT2, and we profile hPTMs at γH2AX-mononucleosomes. Moreover, through the integration of these multiple chromatin layers we identify a potential role for EHMT2-mediated monomethylation of H3K56 in HR. Collectively, our results provide an innovative chromatin-centered view of the DDR process, while representing a valuable resource for the use of PARP inhibitors in cancer.