In progressing the aims of this project, the programme discovered in Aim (i) that the KDM2B/PRC1 protein complex is highly active at placing a modification on chromatin (H2AK119ub1) in vitro and that this activity is regulated by a protein called RYBP (Rose NR, et al, 2016). We also discovered that this complex does not rely on the histone demethylase activity of KDM2B to efficiently deposit H2AK119ub1 (Rose NR, et al, 2016 and Turberfield AH, et al, 2019). Finally, we demonstrated that deposition of H2AK119ub1 and its regulation by RYBP are central to Polycomb chromatin domain formation inside cells and for gene repression (Rose NR, et al, 2016 and Blackledge NP, et al, 2020).
In addressing Aim (ii) we mechanistically dissected Polycomb chromatin domain formation in progenitor cells as a prerequisite for understanding how this system selects and forms Polycomb chromatin domains during cell lineage commitment. We discovered a central role for variant PRC1 complexes and H2AK119ub1 in Polycomb chromatin domain formation and gene repression (Fursova NA, et al, 2019) and showed that this is regulated by a deubiquitylase enzyme called BAP1 (Fursova, et al, 2021). Importantly, this repressive activity did not rely on Polycomb chromatin domains limiting access to chromatin as previously proposed (King HW, et al, 2018). These discoveries are currently being built on to continue understanding precisely how these systems support Polycomb chromatin domain formation and gene regulation during cell lineage commitment (Sugishita, H, et al, in press 2021).
Finally, in addressing the goals of Aim (iii) we have developed new cell-based systems where we can rapidly disrupt Polycomb chromatin domains and observe how Polycomb system function and gene expression is regulated in single cells. This allowed us to show that the repressive nature of Polycomb chromatin domains relies on H2AK119ub1 but not Polycomb-dependent three-dimensional chromatin topologies (Rhodes JDP, et al, 2019 and Dobrinic P, et al, 2020). Importantly, using these approaches and single-particle tracking in live cells, we provide new evidence that Polycomb chromatin domains drive gene repression through H2AK119ub1 counteracting RNA Polymerase II binding and transcriptional burst frequency (Huseyin MK and Klose RJ, 2021 and Dobrinic P, et al, 2020).
