Project Summary Despite advances in sequencing, imaging and screening technologies, morphological changes in nuclear structure are still utilized as a common and reliable diagnosis of cancer. Healthy cells invariably have ellipsoid, smooth nuclear shape and distinctive chromatin distribution, while cancerous cells are characterized by irregular, jagged nuclear shape and disrupted chromatin distribution. Relatedly, one of the most commonly mutated proteins in human cancers is a component of the chromatin remodeler BAF complex known as ARID1A. These mutations lose nucleosome sliding activity, leading to altered transcriptome and cancer progression. As a structural scaffold of the BAF complex, many of the mutations in ARID1A that cause cancer result in early truncation and lead to loss of BAF complex assembly. However, driver mutations also exist in the relatively uncharacterized intrinsically disordered regions of ARID1A whose molecular mechanism is unknown. Investigations into nuclear organization have identified the importance of intrinsically disordered regions and phase separation in dictating overall nuclear structure and organization, as well as forming nuclear bodies like the nucleolus, and chromatin compartments like heterochromatin, but until recently we have not been able to control this organization, or link it conclusively to chromatin compartment function. The Brangwynne lab has developed optogenetic tools that allow for control of in vivo biophysical behavior of condensates. As a Life Science Research Fellow through the Mark Foundation for Cancer Research, I have determined the role of chromatin-chromatin crosslinking protein HP1a in determining nuclear shape and mechanics, as well as discovered rules for chromatin inclusion, exclusion, and compaction dictated by phase separated compartments. In this proposal I will expand my research to understand the biophysical changes that occur in these nuclear compartments upon ARID1A mutations that drive cancer, and how this is connected to their disease phenotype. In Aim 1, I will interrogate interactions that dictate how condensates interact with chromatin, and utilize light-triggered phase separation to modulate chromatin compaction and transcription in living cells. In Aim 2, I will determine how the chromatin polymer directs phase separation, and how nuclear stiffness influences nuclear body volume and functional output. In Aim 3, under the guidance of my co-mentor Dr. Cigall Kadoch, I will utilize knowledge gained in Aims 1 and 2 to build a molecular mechanism of BAF complex proteins ARID1A and ARID1B in oncogenesis, including the roles of their IDRs in targeting BAF complex activity and effects of cancer-associated mutations on condensation behavior, sequence targeting and transcriptional output. With the support of my mentors and the greater research environment at both Princeton University and the Dana Farber Cancer Institute, I will have access to unique tools, and will receive training in cancer biology methods, biophysical theory, and next-generation sequencing assays. Together, these aims will provide a new perspective on nuclear organization in cancer that may lead to novel venues of therapeutics.

Project Narrative The functional role of phase transitions in biology are not well proven, but cancers exist that are caused by mutations in or mistargeting of phase-separating chromatin-binding proteins. I will utilize my understanding of these biophysical processes to mechanistically understand this previously undescribed class of oncogenic transformations and work toward building synthetic systems that can rescue defects in chromatin localization, enzymatic functionality and phase behavior of nuclear condensates seen in cancer progression.