Human pluripotent stem cells (PSCs) exist in two distinct states – naïve and primed, which are interconvertible. Naïve PSCs uniquely harbour several desirable properties that primed PSCs do not, including their ability to model human pre-implantation biology and developmentally-regulated epigenetic phenomena in vitro. Naïve PSCs also have an expanded differentiation potential to produce cell types with possible therapeutic applications.

Naïve PSCs are predominantly generated by reprogramming primed PSCs. This project aimed to address a significant gap in our knowledge about the molecular events that underpin reprogramming, which could assist in unlocking the full potential of naïve PSCs. A genome- wide screen identified novel regulators of naïve cell reprogramming, unexpectedly highlighting the Polycomb Repressive Complex 1 (PRC1) subtype PRC1.3. In this project, I sought to understand the role of PRC1.3 in human pluripotency and reprogramming, and determine the PRC1.3-dependent mechanisms that modulate the acquisition of naïve pluripotency.

I used CRISPR-Cas9 to delete the core PRC1.3 complex component PCGF3 in primed PSCs and ascertained that PRC1.3 is dispensable for human primed pluripotency but critical for naïve cell reprogramming. I used ChIP-sequencing to identify genes that gain PRC1.3 occupancy during reprogramming and these genes become transcriptionally repressed as cells successfully reprogramme, suggesting a key gene silencing role for PRC1.3. Deletion of PRC1.3 reduces the global abundance of the PRC1-deposited H2AK119ub1 modification, and levels of PRC2-deposited H3K27me3 also, in primed PSCs.

Using a proteomics approach, I identified a novel, naïve-specific interaction between PRC1.3 and PRDM14, which cooperate in transcriptional repression of PRC1.3 target genes, I also discovered a developmentally-regulated composition shift in the PRC1.3 complex between human naïve and primed PSCs, which was corroborated in early human embryo datasets. This change in PRC1.3 composition does not appear to alter the enzymatic activity of PRC1.3 in vitro.

Overall, I identified that the PRC1.3 complex plays a critical role in human naïve cell reprogramming. The PRC1.3 complex influences the human primed PSC epigenome and is associated with transcriptional repression, with the complex undergoing both intrinsic and exogenous regulation across the cell state transition. These findings advance our knowledge of molecular events that underpin successful naïve cell reprogramming, and this progress could optimise our ability to produce human naïve PSCs, as an in vitro developmental model and a cell type with considerable therapeutic potential.