CONTEXT SPECIFIC ROLE OF H2AK119UB1 IN ESTABLISHING AND MAINTAINING REPRESSION

Polycomb group (PcG) proteins are central regulators of cell identity, maintaining transcriptional repression at CpG island-containing promoters through Polycomb Re- pressive Complexes 1 and 2 (PRC1 and PRC2), which deposit H2AK119Ub1 and H3K27me3, respectively. While the role of PcG proteins in embryonic stem cells (ESCs) has been extensively studied, their mechanisms in di!erentiated tissues remain poorly understood. This is crucial given PcG proteins are frequently mutated in cancer and neurodevelopmental disorders. In this thesis, I investigated PcG function across two complementary contexts, mouse intestinal epithelium and ESCs, uncovering context- specific regulatory rules and providing novel mechanistic insights. In the mouse intestinal epithelium, contrary to findings in ESCs, I discovered that variant PRC1 and H2AK119Ub1 are largely dispensable for maintaining transcrip- tional repression and tissue homeostasis. Instead, canonical PRC1 (cPRC1) complexes containing PCGF2 and PCGF4, along with variant PRC1 containing PCGF1, were essential to sustain PRC2-dependent H3K27me3 deposition and gene silencing. I fur- ther identified compensatory interactions among PCGF subunits, demonstrating that PCGF1 can compensate for some cPRC1 activity, revealing a context-specific regula- tory axis that operates independently of H2AK119Ub1. Complementing these findings, I investigated the establishment of PcG repression in ESCs using a catalytically inactive RING1B (R1B-I53S) model that lacks H2AK119Ub1. I demonstrated that H2AK119Ub1 is essential for de novo formation of polycomb re- pressive domains at CpG island promoters, while active transcription does not block PcG recruitment. Furthermore, my analysis revealed that vPRC1 subunits, particu- larly PCGF1, act as “memory anchors” that guide RING1B-dependent repression and confer target specificity. Lastly, by implementing predictive models in mESCs, I have characterized synergistic activities between PRC and co-repressors to establish a coherent transcriptional profile and regulatory rules for polycomb repression and target specificity. Collectively, my research provides a comprehensive, mechanistic understanding of PcG- mediated gene regulation, bridging in vivo and in vitro systems. By combining inno- vative genetic models and predictive computational frameworks, I uncover how PRC1 subcomplexes, H2AK119Ub1, and PRC2 interact in a context-dependent manner to establish and maintain transcriptional repression. These findings advance fundamental knowledge of PcG biology and related developmental processes.