Reciprocal Regulation of DBC1 and CHK2 Functions in the DNA Damage Response

Introduction: human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a nuclear protein with controversial effects on cancer cells and with important roles in the regulation of apoptosis, transcription and histones modifications. Previously we reported that, in response to DNA damage, ATM and ATR phosphorylate DBC1 on T454, promoting the inhibition of the NAD+ dependent deacetylase SIRT1, thus inducing p53 acetylation and apoptosis. Materials and methods: these studies were performed in human osteosarcoma U2OS cell line; in order to monitor SIRT1 activity, p53 acetylation was analysed by western blot while protein-protein interactions were studied by co-immunoprecipitation. We then generated a U2OS-DBC1-/- cell line using the CRISPR/Cas9 system for genome engineering, in order to study the effects of DBC1 on the activity of the checkpoint kinase Chk2. Results: we deepen characterized the molecular mechanism by which DBC1 inhibits SIRT1 and found that, beside ATM/ATR, the DBC1-dependent inhibition of SIRT1 requires also the checkpoint kinase Chk2 and the 11S proteasome activator REGγ. Specifically, in response to etoposide Chk2 phosphorylates REGγ promoting DBC1-REGγ interaction, thus increasing DBC1-SIRT1 binding and SIRT1 inhibition. In addition we report that, upon DNA damage, the catalytic activity of Chk2 is modulated by DBC1, with a final impact on DNA double strand breaks repair. Indeed, in response to etoposide, DBC1 favors Chk2 homodimerization and activation, which leads to KAP1 phosphorylation and DNA repair promotion. Conclusions: we reveal a new physical and functional crosstalk among DBC1, SIRT1, Chk2 and REGγ, giving novel insights on their role in the DNA damage response.