Characterizing the role of tumor suppressor phase separation and chromatin organization in maintaining genomic integrity

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ABSTRACT Cells must integrate competing DNA repair pathways and tightly control their chromatin landscapes to maintain genomic integrity. Disruption of these control systems or defects in any one pathway result in a mutational burden with profound physiological consequences to cells and tissues. DNA repair in human cells is primarily performed by two mutually exclusive pathways governed by two different, well- characterized tumor suppressors – non-homologous end joining (NHEJ) by 53BP1 and homologous recombination (HR) by BRCA1. 53BP1 and BRCA1 are examples of intrinsically disordered proteins (IDPs) containing large stretches of low complexity amino acid sequences. 53BP1 undergoes liquid-liquid phase separation to form biomolecular condensates in vitro and at DNA lesions. Further, recent studies hint that 53BP1 condensation also plays a major role in maintaining chromatin organization. Whether BRCA1 has similar phase separation activity has not been established. In preliminary data, I show the very first evidence that BRCA1 phase separates to form biomolecular in cells and in vitro. Chromatin landscapes also play a vital role in maintaining genomic integrity. DNA damage response requires dynamic rearrangements and specific chromatin modifications to elicit rapid recruitment of repair factors. Conversely, repair factors and their complexes can also modify chromatin to drive repair programs. Despite extensive study of 53BP1 and BRCA1 tumor suppressor activities and their repair mechanisms, it remains unknown by what mechanism 53BP1 or BRCA1 condense and what role condensations plays in DNA damage response. Further, the contributions of chromatin architecture to repair pathway selection and chromatin organization within repair condensates have not been elucidated. The goal of this work is to provide me with new training and expertise to address the proposed aims and establish an independent research program. In Aim 1 (K99 phase), I will identify the specific sequence determinants that promote 53BP1 and BRCA1 phase separation and determine the contribution of protein condensates to promoting DNA repair and fidelity in repair pathway selectivity (NHEJ vs. HR). I will test the hypothesis that the magnitude of tumor suppressor condensation contributes to pathway selection. In Aim 2 (K99/R00 phase), I will define the dynamic rearrangement of chromatin in response to DNA damage and how nucleosome clustering and DNA loop compaction contribute to chromatin dynamics in 53BP1- and BRCA1-mediated repair programs. Collectively, this work will address fundamental gaps in knowledge regarding the role of phase separations in genome integrity and uncover new paradigms that underlie tumor suppressor activities.