DBC1 phosphorylations in the DNA damage response

Human DBC1 (Deleted in Breast Cancer-1; KIAA1967) is a nuclear protein involved in apoptosis, transcription and chromatin structure regulation. A key inhibitory target of DBC1 is SIRT1, a NAD+ dependent deacetylase involved in metabolism, stress response and ageing. We have recently shown that in human cells treated with DNA damage, ATM and ATR kinases phosphorylate DBC1 on Thr454, promoting DBC1 binding to and inhibition of SIRT1, ultimately increasing p53 acetylation and p53 dependent apoptosis. Using the TALEN and the RNA guided/Cas9 systems we have generated DBC1 knock-out U2OS cell lines and re-complemented them with wild type and T454A mutant protein. We are currently investigating in these cells the role of DBC1 and Thr454 phosphorylation in cell cycle checkpoint activation, DNA repair and chromatin structure maintenance, and found that DBC1 deficiency can affect cells viability. Moreover, gene expression profile analyses on these DBC1 knock-out clones are ongoing to determine the potential involvement of DBC1 in transcription regulation. We have additionally carried out mass spectrometry experiments on FLAG-DBC1 immunoprecipitated from U2OS cells before and after DNA damage, and identified three new DBC1 phosphosites and an acetylated residue. We are currently studying the role of these post-translational modifications and of candidate DDR kinases in the regulation of DBC1 function. The findings so far obtained confirm the role of DBC1 in DDR and suggest also new functions for this protein.