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Title: Effect of DNMT3B expression on the human pluripotent stem cell methylome
Author: Shukla, Pooja
ISNI:       0000 0004 7228 820X
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2015
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Reprogramming of somatic cells into induced pluripotent stem cells is accompanied by extensive changes in gene expression and epigenetic marks. The single genotype human pluripotent stem cell (hPSC) model system developed in this study overcomes the experimental variability resulting from cell line derivation and culture, and genetic variations which may contribute to the methylation differences observed between human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). On comparing the expression levels of DNA methylation regulating enzymes -DNMTs between HUES7 and HUES7-iPSC cells of the model system, DNMT1 and DNMT3B showed significantly reduced expression in the iPSCs. DNMT3B in particular displayed —50% reduced expression, when compared to HUES7. From methylated DNA precipitation sequencing (MeDIP-Seq) the methylation profiles of HUES7-iPSCs and HUES7 were found to be globally very similar, with the exception of few subtle differences observed in the iPSC methylome which were predominantly hypermethylated. Knockdown of DNMT3B in HUES7 and HUES7-iPSCs resulted in a hypomethylated state of their genome. The hypermethylated differentially methylated regions (DMRs) in HUES7-iPSCs and the hypomethylated DMRs in knockdown HUES7 and HUES7-iPSCs, were unevenly distributed across the chromosomes and found to be largely enriched in gene bodies and intergenic regions, suggesting DNMT3B's affinity to methylate these regions. A more dramatic effect of DNMT3B knockdown was seen in HUES7-iPSCs compared to HUES7, which might largely be due to the normally reduced expression of DNMT3B seen in these cells. Genes affected by DMRs in DNMT3B knockdown HUES7 and HUES7-iPSCs were found unique to them. Three genes FAM19A5, FZD10 and SLITRK2 affected by hypermethylated DMRs in HUES7-iPSCs had their methylation levels restored to those seen in HUES7, on loss of DNMT3B expression, suggesting a role of DNMT3B in maintaining the methylation levels of these regions. Very little correlation was seen between the methylation and transcriptional profiles for both untreated and knockdown hPSCs. In knockdown cells, a large number of differentially expressed genes were identified with respect to their corresponding untreated samples, this indicates a methylation-independent regulatory role of DNMT3B and demands further investigation. Lastly, the functional analysis of the affected genes in knockdown hPSCs highlights their involvement in cardiac, nervous system, and cellular development. Thus, even though the methylation and transcriptional data for knockdown hPSCs show little correlation, the differentially methylated regions might be primed for later de novo methylation event during differentiation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available