Investigating the Function of Canonical Polycomb Repressive Complex 1 Phase Separation in Epigenetic Regulation

Abstract

Polycomb group (PcG) proteins are key developmental regulators involved in gene repression and the maintenance of cell identity. They are a dynamic part of facultative chromatin found clustered in the nucleus as Polycomb bodies. Canonical Polycomb Repressive Complex 1 (cPRC1) is hypothesized to facilitate the silencing of chromatin by compaction and phase separation. The CBX subunit recruits cPRC1 to chromatin and has been shown to contribute to PRC1 condensate formation. The goal of my thesis work was to characterize the phase separation of PRC1 driven by paralogs of the CBX family of proteins and to demonstrate the function of phase separation in PRC1 epigenetic regulation. In Chapter 1, I will introduce PRC1 and its role in gene repression and development. I will also introduce the concept of liquid liquid phase separation and its proposed role in transcriptional control. Additionally, I will describe the many ways in which PcG proteins, such as the CBXs, are misregulated in cancer. In Chapter 2, I will describe my characterization of the CBX4 and CBX8 proteins and their phase separation activity both in vitro and in cells. I will show how positively charged amino acids in the intrinsically disordered regions of these proteins contribute to their condensate behavior and compare the dynamics of CBX4 and CBX8 to CBX2 in cells. In Chapter 3, I will share an adaptation of my submitted manuscript demonstrating that heterologous phase separation can rescue PRC1 function of a CBX2 phase separation mutant in a cancer model system. In Chapter 4, I will summarize the conclusions from my thesis research and discuss the ways in which this work advances the field of epigenetic regulation. I will also explore potential future directions for further understanding the mechanisms of CBX phase separation in Polycomb-mediated gene repression.