The molecular determinants of Polycomb-mediated gene repression

Thesis

Precise spatio-temporal regulation of gene expression is required for development of multicellular organisms. Combinatorial activity of DNA-binding transcription factors has been proposed to drive lineage-specific transcriptional programmes. However, in addition to this, the chromatin template, on which transcription occurs, has emerged as another central player in the control of gene expression. The Polycomb repressive system represents one of the most extensively studied paradigms for chromatin-based gene regulation. It is comprised of two multi-subunit complexes, which catalyse post-translational modifications of histone tails - Polycomb repressive complex 1 (PRC1) mono-ubiquitinates histone H2A at lysine 119 (H2AK119ub1) and PRC2 methylates histone H3 at lysine 27 (H3K27me3). Elucidating the molecular mechanisms of Polycomb-mediated transcriptional silencing has proven challenging, as both PRC1 and PRC2 exist as an array of protein assemblies with distinct targeting modules and regulatory activities. Therefore, the primary determinants that underlie target site selection, Polycomb chromatin domain formation and gene repression by the Polycomb system have until now remained elusive.

Here, I have exploited combinatorial genetic perturbations coupled with quantitative genomic approaches to overcome the multiplicity of Polycomb complexes and uncover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. In contrast to the prevailing dogma in the field, I demonstrated that canonical PRC1, which binds Polycomb target sites in a PRC2-dependent manner and mediates higher-order chromatin structures, contributes little to gene repression. Instead, through systematic interrogation of how H2AK119ub1 is placed and kept in check in the genome, I discovered a remarkable degree of synergy between variant PRC1 complexes, which is central to deposition of H2AK119ub1, Polycomb chromatin domain formation and gene repression. Importantly, my observations suggested that the repressive activity of variant PRC1 complexes may rely on their capacity to catalyse H2AK119ub1. Using a conditional genetic strategy to inactivate PRC1, I directly tested this possibility and discovered that PRC1 catalytic activity is the central molecular determinant of Polycomb-mediated gene repression. In conclusion, through systematic dissection of the Polycomb system, I have provided compelling evidence for a variant PRC1-dependent logic for formation of Polycomb chromatin domains and repression of Polycomb target genes. Together, my findings place PRC1 and its catalytic activity at the forefront of Polycomb-mediated transcriptional silencing, paving the way for a mechanistic understanding of Polycomb system function.

Authors: Fursova, N

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: genomics; genome engineering; gene regulation; Polycomb; histone modifications; epigenetics; chromatin
• Subjects: Life Sciences; Epigenetics; Biology