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Title: Function of Polycomb group proteins in embryonic stem cells
Author: Parente, Miguel Casanova Vieira
ISNI:       0000 0004 2687 0389
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Embryonic stem (ES) cells exist in a pluripotent state and have the ability to differentiate into all adult cell types. Numerous molecular mechanisms are responsible for both maintaining pluripotency and directing differentiation. One of these mechanisms is epigenetics, the heritable regulation of gene expression profiles without changes to the underlying DNA sequence. This has recently been the focus of a large amount of research. The Polycomb group (PcG) proteins have been identified as major epigenetic regulators. Recent research has shown that PcG proteins are involved in silencing genes in ES cells which play a key role in development and cell fate decisions. PcG proteins function in two main complexes, the Polycomb repressive complexes (PRC), PRC1 and PRC2. Here I explore the mechanisms involved in the regulation of PcG proteins in ES cells and how these proteins contribute to the maintenance of ES cell pluripotency. Analysis of the expression levels of PRC2 genes showed them to be significantly reduced in differentiated cells compared to undifferentiated ES cells. The differences observed in PRC2 expression are due to a regulation at the transcriptional level by key ES cell transcription factors such as Oct4 and Sox2. Interestingly, ES cell lines which are mutant for PcG members failed to silence genes expressed in distinct lineages of the mouse embryo and show a greater tendency to differentiate. In particular, Ring1A/B proteins from the PRC1 complex were shown to be essential for maintaining a poised form of RNAPII at silenced ES cell promoters, thus contributing to the maintenance of pluripotency in ES cells. Recent data has shown that the Polycomblike protein (Pcl) interacts with the canonical PRC2 complex. To further characterise this interaction I have used an affinity purification strategy to isolate novel Pcl2 complexes from ES cells and identify potential interactors and recruitment factors. The affinity purification of Pcl2 confirmed it to be a bona fide member of a PRC2 sub-complex and identified additional interactor proteins which will be further characterised. The knockdown of Pcl2 does not have a severe impact on the stability of ES cells but affects the recruitment of Ezh2 and Suz12 to the promoters of PRC2 target genes leading to a small reduction on H3K27me3 levels but no changes in gene expression. In addition Pcl2 plays a key role in the recruitment of PRC2 to the inactive X chromosome and enrichment of H3K27me3. These observations suggest that Pcl2 plays an important role in PRC2 mediated epigenetic control.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral