PROJECT SUMMARY (See instructions): Mammalian cells are continually exposed to environmental toxicants including UV-radiation and various sources of ionizing radiation (IR) threatening genomic integrity, leading to an increased risk of cancer and neurodegenerative disease. Given our constant exposure to environmental toxicants, elucidating fundamental principles of genome integrity maintenance is critical for developing therapeutic interventions for a host of age-related pathologies. In recent years, several chromatin-based events have been shown to be critical mediators of an effective DNA damage response (DDR), however the lack of high-throughput screening methodologies have significantly hampered the identification of chromatin factors essential for DNA repair. To address this, this proposal will use a newly developed high-throughput screening methodology, coupled with a cDNA library of predicted chromatin interactors (“ChromORFeome”), to identify novel chromatin factors involved in DNA repair. During the R00 phase of this proposal, the candidate will build upon the data developed during the mentored (K99) research phase to further understand the importance of a newly identified chromatin-interacting protein, ZNF280A, for the repair of DNA damage. Specifically, this proposal will seek to understand how ZNF280A is regulated in response to DNA damage. This aim will use a range of complementary approaches including protein biochemistry, phospho-proteomics, and mutagenesis analysis to map post-translational modifications on ZNF280A and determine their importance for regulating the function of ZNF280A in DNA double-strand break repair (Aim 1). In addition, this proposal will build upon preliminary data generated during the K99 phase of the proposal which identified novel protein-protein interaction networks, to determine mechanistically how ZNF280A orchestrates DNA repair (Aim 2A) and ascertain whether this contributes to therapy resistance in pancreatic ductal adenocarcinoma (PDAC), where increased expression of ZNF280A correlates with significantly poorer outcome in patients (Aim 2B). In addition, very little is known about how chromatin structure and function is re-established following DNA repair. To begin to explore this, the proposal will utilize our recently developed high-throughput screening methodology to identify novel chromatin factors involved in the late stages of DNA repair and mechanistically characterize how these factors orchestrate the re-setting of chromatin architecture following DNA damage repair (Aim 3). These experimental aims will provide the candidate with data for an early independent publication and preliminary data for R series