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Title: Characterisation of PRC1 dynamics and chromatin binding in live cells
Author: Huseyin, Miles Kocur
ISNI:       0000 0004 8507 2280
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Development of complex multicellular organisms depends on precise spatiotemporal regulation of transcription of genes in the genome. Although this regulation is known to depend on sequence-specific transcription factors, chromatin modifying proteins have also emerged as key regulators of transcription in development. Polycomb proteins represent a class of well studied transcriptional repressors which assemble into two main complexes with histone-modifying activity, Polycomb repressive complexes 1 (PRC1) and PRC2. PRC1 exists in mammalian cells as multiple complexes of distinct subunit composition, formed around a catalytic core of an E3 ubiquitin ligase and one of six PCGF proteins, which monoubiquitylate histone H2A. In addition to dictating the level of catalytic activity of the complex, inclusion of a different PCGF protein results in incorporation of different chromatin-binding modules in each complex. Although previous work has established that enrichment of PRC1 at the promoters of target genes is required to achieve repression, how the mechanisms by which PRC1 subunits interact with chromatin contribute to targeting PRC1 to specific sites in the genome remains unclear. In order to advance our understanding of how Polycomb proteins interact with the genome, I have employed live cell imaging and single particle tracking to characterise the dynamics and subnuclear localisation of PRC1. I discover that PRC1 subunits have broadly similar and dynamic behaviour, although a small fraction of molecules exhibit stable binding. Quantification of the numbers of PRC1 subunits reveals they are of relatively low nuclear concentration, despite their role in depositing H2AK119ub1 across the genome. Using systems in which I can remove PRC1 catalytic activity or induce degradation of a core PRC2 subunit, I demonstrate that PRC1 catalytic activity and PRC2 contribute modestly to PRC1 interactions with chromatin. Despite this modest change, I find that both are required for enriching Polycomb proteins at nuclear foci. I investigate the contribution of DNA binding of PCGF1-PRC1 subunit to the dynamics of both PRC1 and PCGF1, and find that it is not required for PCGF1 chromatin binding. I show, furthermore, that PRC1 stable binding and localisation within Polycomb bodies depends on specific PCGF proteins. Together, these observations provide a quantitative characterisation of PRC1 chromatin binding and localisation, and provide insights into the contribution of major chromatin-binding mechanisms to Polycomb dynamics.
Supervisor: Klose, Rob Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available