We used mouse embryonic stem cells (ESCs) as a model to study the mechanisms that regulate stem cell fate. Using gene expression analysis during a time course of differentiation, we identified 281 candidate regulators of ESC fate. To integrate these candidate regulators into the known ESC transcriptional network, we incorporated promoter occupancy data for OCT4, NANOG and SOX2. We used shRNA knockdown studies followed by a high-content fluorescence imaging assay to test the requirement of our predicted regulators in maintaining self-renewal. We further integrated promoter occupancy data for Polycomb group (PcG) proteins, EED and PHC1 to identify 43 transcriptional networks in which we predict that OCT4 and NANOG co-operate with EED and PHC1 to influence the expression of multiple developmental regulators. Next, we turned our focus to the PcG protein PCL2 which we identified as being bound by both OCT4 and NANOG and down-regulated during differentiation. PcG proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs. Using multiple biochemical strategies, we demonstrated that PCL2 associates with Polycomb Repressive Complex 2 (PRC2) in mouse ESCs, a complex that exerts its effect on gene expression through H3K27me3. Although PCL2 was not required for global histone methylation, it was required at specific target regions to maintain proper levels of H3K27me3. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics and defects in differentiation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31965 |
| Date | 11 January 2012 |
| Creators | Walker, Emily |
| Contributors | Stanford, William L. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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