title: Cooperation between two repressive epigenetic systems, DNA methylation and PRC2, in pluripotency and differentiation
Creators: name: Stepanova, Olga
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Pluripotency
subject: DNA methylation
subject: Polycomb
subject: Embryonic stem cells
subject: Epigenetics

description: The epigenome is established during embryogenesis by the acquisition of cell type-specific DNA methylation and histone modification patterns. Curiously, disruption of DNA methyltransferases or Polycomb Repressive Complex 2 (PRC2) components, which establish repressive DNA methylation and H3K27me3 modifications, respectively, does not affect the proliferation and self-renewal of pluripotent mouse embryonic stem cells (ESCs), despite resulting in differentiation defects and lethality during embryonic development. These modifications are deposited in a mutually exclusive manner, with H3K27me3 diffusing in a hypomethylated background and a limited increase in DNA methylation observed in PRC2 mutants. Therefore, a partial compensation mechanism between these two antagonistic modifications might exist to maintain the pluripotent state but becomes lost upon differentiation. In this thesis, I aimed to test this hypothesis by simultaneously disrupting DNA methylation and PRC2 in distinct pluripotent states and embryoid bodies. I inactivated Ezh2, a catalytic component of PRC2, in a cell line where DNA methylation levels can be controlled by doxycycline-inducible suppression of the endogenous maintenance DNA methyltransferase Dnmt1. I systematically compared the outcomes in ESCs cultured under the ground state naïve (2i/LIF) and metastable naïve (Serum/LIF) conditions, focussing on cellular morphology, stem cell identity and transcriptional changes. I identified that simultaneous disruption of DNA methylation and H3K27me3 had a minor effect on transcriptional regulation and stem cell identity in 2i/LIF conditions but led to a major destabilisation of transcriptional networks in Serum/LIF. This led to spontaneous differentiation, as evidenced by morphological changes, loss of pluripotency transcription factor expression and activation of lineage specification genes. Interestingly, the DNA methylation- and H3K27me3-deficient Serum/LIF cells continued to proliferate as a mixed population where differentiating cells co-existed alongside those that retained stem cell identity. This suggests that co-depletion of DNA methylation and H3K27me3 might be sensitising the primed subpopulation of Serum/LIF cells by amplifying stochastically expressed lineage priming factors. In contrast, 2i-induced signalling was sufficient to maintain the ground state naïve pluripotency independently of deficiencies in DNA methylation and H3K27me3. I also demonstrated that repression by DNA methylation and PRC2 has an antagonistic role in pluripotency and differentiation by co-depleting these pathways in embryoid bodies. In addition to morphological changes, the DNA methylation- and H3K27me3-deficient embryoid bodies showed an impaired ability to extinguish pluripotency networks but could induce gene expression programmes associated with early embryonic, endodermal and germline lineages. In summary, my findings suggest that while the epigenome might shape cell fate and lineage specification choices, the underlying transcriptional state plays a major role in maintaining stem cell identity. Additionally, I demonstrated that DNA methylation and PRC2 can partially compensate for each other, as spontaneous differentiation is only observed in double mutants cultured in Serum/LIF. Lastly, my observations highlight the dual requirement for DNA methylation and PRC2 in metastable naïve pluripotency maintenance and establishment of the appropriate lineage specification programmes during differentiation.