Matsa, Elena
(2010)
Developing a model system to investigate the epigenetic mechanisms underlying pluripotency in human cells.
PhD thesis, University of Nottingham.
Abstract
Pluripotent human embryonic stem cells (hESCs) are a valuable tool for clinical therapies, drug testing and investigation of developmental pathways. Recently, over-expression of four pluripotency-associated genes (OCT4, NANOG, SOX2, and LIN28) has proven sufficient to reprogram differentiated cells into pluripotent stem cells, potentially alleviating the need for human embryos to isolate hESCs and opening new avenues for the investigation of pluripotency.
This project aimed to generate an in vitro model system to study the epigenetic mechanisms regulating pluripotency transcription factors. hESCs were differentiated into fibroblasts (hESC-Fib) and subsequently reprogrammed to induced pluripotency stem cells (iPSCs) by lentiviral over-expression of human OCT4, NANOG, SOX2 and LIN28. iPSC colonies were positively identified by live staining with the surface marker TRA-1-81 and expanded in culture. They were then further differentiated into a fibroblast line to allow comparison with hESC-Fib. All cells in the model system shared the same genotype and were cultured under similar conditions, enabling unbiased analysis of epigenetic characteristics.
DNA methylation analysis of key pluripotency-genes such as OCT4, SOX2, NANOG, and REX1 by bisulfite sequencing, revealed that these were hypomethylated in hESCs and iPSCs, and hypermethylated in their fibroblast derivatives. A gradual increase in the number of CpGs gaining DNA methylation was observed when hESCs and iPSCs were differentiated into fibroblasts, while TaqMan real-time PCR and fluorescence staining revealed that expression of these genes was inversely related to the levels of DNA methylation in their promoters. The master pluripotency regulators OCT4, SOX2 and NANOG all showed differential methylation in their OCT/SOX binding regions, suggesting a common regulatory mechanism between them. This is, to our knowledge, the first report for SOX2 differential methylation in human non-cancerous cells. Reactivation of REX1 was not found to be necessary for the reprogramming of hESC-Fib to iPSCs, calling for re-evaluation of its role in human pluripotency.
Based on the observation that the DNA methylation levels of pluripotency genes were higher in fibroblast cell lines compared to hESCs and iPSCs, we hypothesised that reduction in DNA methylation could render differentiated cells more permissive to reprogramming. Stable knock-downs of the DNA methyltransferases (DNMTs) DNMT1 and DNMT3A were, thus, performed in hESC-Fib. Knock-down of DNMT1, the most abundant DNMT in hESC-Fib, resulted in global reduction of DNA methylation levels as determined by restriction digests with methylation specific enzymes. Reprogramming of hESC-Fib carrying a DNMT1 knock-down showed a 40% reduction in generation of iPSC colonies compared to untreated controls, perhaps owing to the delay in progression of S phase in the cell cycle caused by DNMT1 knock-down. In contrast, knock-down of DNMT3A resulted in a >80% increase in iPSC colony formation, potentially indicating differences in mechanism of action and specificity between the two DNMT enzymes.
Through this study, we have gained new insights into the epigenetic mechanisms underlying cell phenotype and provided the foundation for further improving reprogramming efficiency.
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