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Title: Mechanisms of Xist-dependent Polycomb recruitment
Author: Almeida, Mafalda
ISNI:       0000 0004 6497 8665
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation, functioning, at least in part, by catalyzing specific histone modifications, H2AK119u1 and H3K27me3, respectively. The hierarchical model of Polycomb recruitment proposes that PRC2 is recruited to target loci by sequence specific factors and/or non-coding RNAs, and that PRC1 recruitment occurs subsequently as a consequence of recognition of H3K27me3 by the PRC1 subunit CBX. However, non-canonical PRC1 complexes, defined by the presence of different PCGF subunits, lack the CBX subunit and are therefore recruited independently of PRC2 activity. Moreover, recent evidence has shown that H2AK119u1 catalyzed by non-canonical PRC1 can directly recruit PRC2, establishing a reverse model of Polycomb recruitment. I analyzed Polycomb recruitment using a classical model system, X chromosome inactivation mediated by Xist RNA. Previous reports suggested that Polycomb recruitment to the inactive X chromosome is initiated by a direct interaction of PRC2 subunits with Xist RNA, and that PRC1 is then recruited via interaction of the CBX subunit with PRC2 mediated H3K27me3. This model has been challenged, since it does not account for all experimental observations. In this thesis, I provide evidence that Polycomb recruitment by Xist RNA is initiated by deposition of H2AK119u1 by noncanonical PRC1 complexes. PRC2 is therefore recruited indirectly, presumably by binding to H2AK119u1. I also show that all non-canonical PRC1 complexes are enriched on the inactive chromosome, albeit with different dynamic behaviors. PCGF3- and PCGF5-PRC1 complexes, which have a slower dynamics on the inactive chromosome as determined by FRAP, are necessary for the initiation of Polycomb domain formation on the inactive chromosome, presumably through direct recruitment by Xist RNA or through an adaptor protein that mediates this interaction. Subsequent enrichment of other non-canonical PRC1 complexes relies predominantly on H2AK119u1 recognition by RYBP/YAF2, which also contributes to the enrichment of the more dynamic fraction of PCGF3- and PCGF5-PRC1 complexes on the inactive chromosome. Altogether, these observations overturn existing models for Polycomb recruitment by Xist RNA, and thereby, provide new insights into the dynamics and mechanisms for Polycomb recruitment by non-coding RNAs.
Supervisor: Brockdorff, Neil Sponsor: Fundação para a Ciência e Tecnologia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available