Dynamics of canonical and variant PRC1 interaction with chromatin at single-molecule resolution (360G-Wellcome-109102_Z_15_A)

Polycomb Repressive Complexes (PRC) 1 and 2 are protein complexes with histone modifying activity that are key to regulation of gene expression in eukaryotic development. PRC1 ubiquitylates H2A at lysine 119 (H2AK119Ub1), while PRC2 methylates H3 at lysine 27 (H3K27), that function together on chromatin, recognising the modification deposited by the other, suggesting a feedback loop. PRC1 complexes can be divided into two groups, canonical and variant, depending on their components. It is proposed that canonical complexes play a structural role in chromatin compaction, while variant complexes are responsible for sampling chromatin. I aim to determine the dynamics of PRC1 recruitment to chromatin at single molecule resolution. I will initially generate mouse embryonic stem cells (mESCs) expressing HaloTag fusion proteins exclusive to canonical and variant complexes, which can be labelled with cell permeable dyes allowing tracking of individual PRC1 molecules in live cells. Recruitment will be disrupted through mutation of specific components, or knockout or inhibition of processes depositing marks they recognise, with the aim of understanding the mechanism underpinning the dynamics of PRC1 recruitment. Generated cell lines will be used for both serum-to-2i transition and mESC differentiation to investigate how the dynamics of PRC1 recruitment change in development.