Identifying chromatin factors essential for DNA repair using a novel high-throughput screening methodology

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PROJECT SUMMARY 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 mentored (K99) phase of this proposal, the candidate will determine the importance of a newly identified chromatin-interacting protein, ZNF280A, for the repair of DNA damage, identifying specific repair pathways which require ZNF280A (Aim 1). Preliminary data demonstrates that ZNF280A is recruited to sites of DNA damage induced by a variety of sources including ionizing radiation (IR). The candidate will build upon this data 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). Importantly, while in the mentored (K99) phase, the candidate will take advantage of the resources available at Massachusetts General Hospital for professional development, applying these skills through mentoring, data presentation and writing opportunities. During the non-mentored/independent research phase (R00) of the project, technical skills and reagents developed by the candidate during the K99 phase will be used to elucidate the importance of ZNF280A in the 22q11.2 deletion human syndrome. ZNF280A resides at the 22q11.2 locus and preliminary data demonstrates that depletion of ZNF280A results in spontaneous DNA damage. The candidate will therefore investigate the importance of ZNF280A for the resolution of DNA replication stress and determine whether this can mechanistically explain some of the features of 22q11.2 deletion syndrome (Aim 2C). In addition, very little is known about how chromatin structure and function is re-established following DNA repair. Therefore, in the R00 phase the candidate will extend these approaches and utilize the high-throughput screening methodology to identify novel chromatin factors involved in the late stages of DNA repair (Aim 3). These experiments will provide the candidate with data for an early independent publication and preliminary data for R-series grants (R21, R01). Importantly, during the R00 independent phase, the candidate will develop independence from their mentor by addressing key mechanisms underpinning chromatin re-establishment in the late stages of DNA repair - applying these mechanistic studies to explain how genome integrity is preserved despite continued exposure to DNA damaging environmental toxicants.