Identification and regulation of DNA damage response pathways in human cancer cells

Maintenance of genome integrity is of importance for prevention of a number of human diseases. The DNA damage response (DDR) orchestrates a network of cellular processes that recognize, signal and repair different types of DNA lesions to preserve genome integrity. Acetylation signaling as a critical pathway that can modulate the DDR by either changing chromatin structure or regulating proteins binding to chromatin. Bromodomain (BRD) proteins contain the primary reader domain of lysine acetylation (i.e. the BRD) that recognizes these signals to facilitate DDR pathways. Understanding how BRD acetylation readers coordinate chromatin-based responses to DNA damage is fundamental to elucidating the mechanism of genome maintenance for preventing the pathogenesis of human diseases. The first part of my thesis focused on identification of BRD proteins involved in the DDR. I screened the recruitment of all human BRD proteins using laser microirradiation. I showed that ZMYND8, one of the damage responsive BRD proteins, was recruited to sites of DNA damage depending on its BRD interaction with H4 acetylations. ZMYND8 interacted with the NuRD complex to transcriptional suppress actively transcribed chromatin and facilitate repair of DNA double-strand breaks (DSBs) by homologous recombination. For my second project, I focused on identifying human genes that destabilize genome integrity by altering the levels of endogenous DNA damage. Mutations arise from increased DNA damage, potentially even with intact DNA repair pathways, which can contribute to the development of cancer. We have collaborated with the Rosenberg lab (Baylor College of Medicine), who have identified a DNA damaging (DD) genes network from E. coli that promote endogenous DNA damage when overproduced. I validated the concept of human DD (hDD) genes and showed that 33 out of 73 human homologs of E. coli DD genes promote endogenous DNA damage when overexpressed in human cells. I determined several of these hDD genes with increased mutation rate. I characterized KCNAB1 and KCNAB2, the potassium channel subunits that promote DNA damage via creation of reactive oxygen species (ROS). Furthermore, I identified many PCNA interactors including DNMT1 that display DNA-damage promoting activity depending on the binding to PCNA. In summary, knowledge obtained from these studies can enhance our understanding of how chromatin associated factors participate in the DDR and how endogenous DNA damage levels are regulated in cells, alterations of which may contribute to genome instability and human diseases including cancer.Cellular and Molecular Biolog